<pubnumber>540982026</pubnumber>
<title>Pesticide Assessment Guidelines: Subdivision G, Product Performance</title>
<pages>431</pages>
<pubyear>1982</pubyear>
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<abstract></abstract>
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PB83-153924
PESTICIDE ASSESSMENT GUIDELINES - SUBDIVISION G: PRODUCT PERFORMANCE
B.A. Schneider
U.S. Environmental Protection Agency
Washington, D.C.
September 1982
U.S. DEPARTMENT OF COMMERCE
National Technical Information Service
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.bdivision G - Product Performance
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September, 1982
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Bernard A. Schneider, Ph.D.
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Office of ?esticide Programs
U.S. Snviror-mental Protection Agency
Washington., D.C. 20460
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Robert K. Hitch
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As a guideline for the Federal Insecticide, Fungicide and Bodenticide Act (FIFRA),
Subdivision G provides guidance to the registrant on developing product performance
data that the Agency may require to demonstrate the effectiveness of their presticide
product in controlling the pests rpecified in the claims on their product label. This
subdivision is organized into section series which describe efficacy data for
specific types of pesticides used on distinct classes of pests. Guidance.on test
methodology and protocols to support label claims are also presented. Discussions
concerning when the submission of efficacy data is required are also presented as a
foundation for the regulatory, pesticide data requirements (40 CFR Part 158). Submission
of efficacy data is generally waived, except for products claiming control of pest
microorganisms that pose a threat to human health and whose jjresence cannot be observed
by the user, including microorganisms infectious to man in the inanimate environment
and in situations where the Agency may require data on a case-by-case basis.
Subdivision G constitutes one volume of a twelve-part FIFRA guideline series
published by the National Technical Information Service.
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PB83-153924
EPA 544/19*82-026
November, 1982
PESTICIDE ASSESSMENT GUIDELINES
SUBDIVISION G
PRODUCT PERFORMANCE
by
Bernard A. Schneider, Ph.D.
Benefits and Field Studies Division
Office of Pesticide Programs
Guidelines Projects Manager
Robert K. Hitch
Hazard Evaluation Division
Office of Pesticide Programs
Technical Support Team
.W. Audia
W. Campbell
S. Duffy
T. ElIwanger, Ph.D.
C. Grable, Ph.D.
D• Guse
D. Hansen
P. Button
W. Jacobs, Ph.D.
D. Jenkins
R. Matheny
R. Michell, Ph.D.
S. Palmateer
D. Peacock
W. Phillips, Ph.D.
D. Portner
J. Touhey
U.S.- Environmental Protection Agency
Off-ice ,of Pesticides and Toxic Substances
Washington,.; DvC . -.20460'-;.
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Discussion on
Subdivision G Product Performance
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DISCUSSION ON
. SUBDIVISION .G PRODUCT.PERFORMANCE "'.
TABLE OF CONTENTS
FOREWORD ' vi
I. PHILOSOPHY AND GENERAL POLICIES AFFECTING SUBDIVISION G 1
II. SCOPE AND ORGANIZATION OF SUBDIVISION G 5
III. GENERAL ISSUES 9
IV. INDIVIDUAL TEST ISSUES 15
V. USDA COMMENTS AND EPA RESPONSES 23
VI. FIFRA SCIENTIFIC ADVISORY PANEL COMMENTS AND EPA RESPONSES 28
ii
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SUBDIVISION G PRODUCT PERFORMANCE
TABLE OF CONTENTS
Section
OVERVIEW, DEFINITIONS, AND GENERAL CONSIDERATIONS
90-1 Overview: product performance 32
90-2 Definitions 36
90-3 General considerations 37
90-30 Acceptable methods ' 49
EFFICACY OF ANTIMICROBIAL AGENTS
Subseries 91A: PUBLIC HEALTH USES 50
91-1 General requirements 50
91-2 Products for use on hard surfaces 52
91-3 Products requiring confirmatory data 57
91-4 Products for use on fabrics and textiles 59
91-5 Air sanitizers 63
91-6 Products for processing and industrial uses 65
91-7 Products for control of microbial pests associated with
human and animal wastes ' 65
91-8 Products for treating water systems 66
91-30 Acceptable methods 70'
Subseries 91B: NON-PUBLIC HEALTH USES 84
91-51 General considerations 84
91-52 Products for use on hard surfaces 86
91-53 Products for use on fabrics and textiles 87
91-54 Products for processing and industrial uses 89
91-55 Products for control of microbial pests associated with
human and animal wastes 93
91-56 Products for treating water systems 94
91-57 Antimicrobial agents sold only for formulation use 96
EFFICACY OF AQUATIC PEST CONTROL AGENTS
92-1 General considerations 97
92-2 Aquatic herbicides 98
92-3 Swimming pool algicides 104
92-4 Industrial cooling water microbicides 105
92-5 Pulp and papermill water systems microbicides 108
92-6 -Secondary oil recovery systems microbicides . 10.9
92-7 : AntifoulijNj biocides .,..,.,..•.. ... .. . ;.. - 109
92-10 Footnotes to S«ries. 9.2 Sections ... : 111
.92-20 .Acceptable..methods ..'•.•--; '. 112
iii-
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"EFFICACY OF FUNGICIDES AND NEMATICIDES
93-1 General considerations 130
93-2 Definitions 130
93-3 Performance standards: acceptable levels of pest control 130
93-4 Products for use against above-ground plant pests 131
93-5 Products for use against soil-borne plant pests 132
93-6 Products for post harvest use on fruits and vegetables 133
93-7 Products for use as grain preservatives 134
93-8 Products, for use as seed treatments 135
93-9 Products for use on ornamental and flowering plants 136
93-10 Products for use on bulbs/ corns, and tubers . 136
93-11 Products for use on trees 137
93-12 Products for use on turf 137
93-13 Treatments for wood and wood products 138
93-14 Treatments for industrial materials and equipment 139
93-15 Products for control of mold and mildew on surfaces 140
93-16 Products for control of organisms producing mycotoxins (Reserved) 140
93-30 Acceptable methods 141
EFFICACY OF TERRESTRIAL HERBICIDES, PLANT REGULATORS, DESICCANTS,
AND DEFOLIANTS
94-1 Overview 166
94-2 General considerations . • 166
94-3 Terrestrial herbicides 173
94-4 Plant regulators 178
94-5 Desiccants 189
94-6 Defoliants 193
94-30 Acceptable methods 196
EFFICACY OF INVERTEBRATE CONTROL AGENTS
95-1 General considerations 230
95-2 Foliar treatments . 232
95-3 General soil treatments 242
95-4 Lawn and turf treatments 245
95-5 Outdoor woody ornamental plant treatments 247
95-6 Greenhouse floricultural treatments 251
95-7 Shade tree and forest land treatments . 255
95-8 Livestock, poultry, fur, and wool-bearing animal treatments 262
95-9 Treatments to control pests of humans and pets , 262
95-10 Mosquito, black fly, nonbiting midge, and biting midge
(sand fly) treatments 264
95-11 Premises treatments . 267
95-12 Structural treatments 271
95-13 Stored product treatments 274
95-14 Fabric treatments . . " 276
95-30 Acceptable methods 279
iv
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EFFICACY OF VERTEBRATE CONTROL AGENTS
96-1 General considerations 287
96-2 Fish control agents 289
96-3 Aquatic amphibian control agents 291
96-4 Terrestrial amphibian and reptilian control agents 293
96-5 Avian toxicants 295
96-6 Avian repellents 297
96-7 Avian frightening agents •300
96-8 Mole toxicants 303
96-9 Bat toxicants and repellents 306
96-10 Commensal r.odenticides 307
96-11 Rodenticides in orchards .'•''• •'-. 310
96-12 Rodenticides on farm and rangelands 313
96-13 Rodent fumigants 316
96-14 Rodent repellents on tree seeds 318
96-15 Rodent repellents on cables 320
96-16 Rodent reproductive inhibitors 323
96-17 Mammalian predacides 327
96-18 Domestic dog and cat repellents 329
96-19 Browsing animal repellents 333
96-25 References to §§ 96-2 thru -19 337
96-30 Methods and protocols 340
.v
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FOREWORD
As a guideline under the Federal Insecticide, Fungicide and Rodenticide
Act (FIFRA), Subdivision G provides guidance to registrants on developing
product performance data that the Agency may require to demonstrate the
effectiveness of their pesticide product in controlling the pests specified
in the claims on their product label. Submission of efficacy data is
generally waived, except for products claiming control of pest microorganisms
that pose a threat to human health and whose presence cannot be observed
by the user, including microorganisms infectious to man in the inanimate
environment and in situations where the Agency may require data on a
case-by-case basis. The waiver of efficacy submission does not relieve
registrants of their responsibility for marketing products effective for
their proposed claims. Under certain situations when the Agency will
request submission of efficacy data, this subdivision provides the registrant
with guidance, procedures, and test protocols useful for evaluating
product performance.
Subdivision G is a nonregulatory companion to 40 CFR Part 158,
Data Requirements for Registration. Public comment on Subdivision has
been taken in a series of public meetings, the last of which was held
in July, 1982. Data requirements established by 40 CFR Part 158 are
discussed in Subdivision G so that it can be read as a complete
package and so that product performance testing procedures can be
explained in their proper context.
vi
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I. PHILOSOPHY AND GENERAL POLICIES AFFECTING SUBDIVISION G
A. Philosophy
This subdivision of the pesticide registration guidelines concerns
the effectiveness of pesticide products. Data developed under this
subdivision are designed to assure that the pesticide product will control
the pests listed on the label and that unnecessary pesticide exposure
to the environment will not occur as a result of the use of ineffective
products. Specific performance standards are used to validate the efficacy
data in the public health related areas and include disinfectant uses to
control microorganisms infectious to man in any area of the inanimate
environment.
Use of these guidelines on product performance by registration
applicants, coupled. with Agency evaluation of- the consequent data submittals
serve two major purposes: protection of the environment and assurance of
consumer benefit through public health protection to purchasers and users
of pesticide products. Environmental protection would be achieved by
making certain that applications of pesticides are fully and adequately
effective for their intended purposes, so that:
( 1 ) Undue pollution of the environment ( and the consequent
environmental exposure) does not result from the use of:
(a) Ineffective products or ineffective active ingredients;
(b) Excessive or insufficient amounts and rates of
pesticides to achieve the desire effects;
(c) Excessive or insufficient frequency of pesticide ap-
plications ;
(d) Inappropriate timing of applications, as, for example
too early or too late, or out of season;
(e) Impractical product mixtures containing certain
active ingredients, which are rarely, if ever, needed
under most pest control circumstances; and
(f) Unnecessary use in areas or situations, or on sites
where pest control is not needed.
(2.). -*:The^ -aser! s t applicator ' s.) time, labor, . eqit;iipment , and
: ' .Qnargy. .rie&d&l-tQ '"achieve the desired effect; are most effi-
' • ciently used; this concurrently constitutes a major benefit
...: toward . improved, safety, for both huaana and the environment,
. . "•; .-'since : reduced opportunity then exists ,.f-pr accidents-.,. : needless
,.-... ..•..vexposures., bioaccumuiatipn , -increased, animal tolerance to
-.r, .; pesticides., and other similar hazards. ." .. . ..
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In situations where efficacy data.is still required to be submitted,
consumers benefit because the use of proper testing procedures will help
insure that label claims are meaningful and truthful, and that the product
label instructions are relevant and practical for safe and effective use
of each product.
B. Waiver of Data Requirements Pertaining to Efficacy.
A detailed discussion of the expansion of the efficacy data waiver
appears in the preamble to the proposed Section 3 revisions as Part
162 Subpart A - Registration, Reregistration and Classification Procedures
(40 CFR Part 162). This discussion is quoted below in its entirety:
"As previously discussed, in 1979 the Agency issued regulations
implementing serveral provision of the 1978 FIFRA amendment (44 FR 27933,
40 CFR, Part 162; May 11, 1979). Among the provisions implemented was
the efficacy data waiver authority provided by Sec.3 (c)(5) of FIFRA.
The Agency defined in §162.18-2(d) the circumstances when efficacy data
were required to be submitted as a matter of course. Other requirements
that efficacy data be submitted were generally waived.
"The Agency proposed in the Regulations for Registration,
Reregistration and Classification Procedures (47 FR 40659, 40 CFR, Part 162,
September 15, 1982) to extend-the-efficacy data waiver to additional use
patterns, and it will include all registration actions, both conditional
and unconditional. As stated in its previous waiver, the Agency's primary
mandate under FIFRA is to evaluate the health and safety aspects of '••
pesticides. Experience under the previous waiver policy indicates that
there have been few complaints to the Agency of nonefficacious products
being marketed, and the Agency is confident that its efficacy data waiver
has occasioned little, if any, serious user dissatisfaction.
"The Section 3 Regulation §162.18-2(d)(2) concerning efficacy data
is proposed to be revised as follows:
"(1) Efficacy data. (i) Efficacy data, in accordance with
Subdivision G of the Registration Guidelines, for each product that
bears a claim to control pest microorganisms (except bacteria, pathogenic
fungi, or viruses living on or in other animals) that pose a threat to
human health and whose presence cannot readily be observed by the user,
including, but not limited to, microorganisms infectious to man in any
area of the inanimate environment.
"(ii) Efficacy data, in accordance with Subdivision G of the
Registration Guidelines, for each product for which a new or added use
is proposed, if the product contains an active ingredient some use of
which has been suspended, cancelled, or is the subject of a Notice issued
under Section Series 162.IK a)(3)(ii) and the risks, identified in the
Notice or suspension/cancellation action, may reasonably be anticipated
as a result of the new use. •
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"(iii) Efficacy data requested by the Agency for any product,
registered or proposed for registration, when:
"(A) A lack of efficacy has been reported for it;
"(B) The Agency needs such data to evaluate benefits of the pesticide
(or of alternative pesticides) when substantial risks have been identified;
or
"(C) Factors exist that make submission of such data necessary or
desirable to support the presumption that it is efficacious.
"(iv) Products which are inefficacious may violate PIFRA section
3(c)(5)(A). Applications to register products which do not meet the
•requirements of the Act will be denied; registrations of products
themselves may be deemed misbranded per FIFRA section 2(q)(l)(A) and
section 12(a)(l)(E) or (F).
' "Those products for which an efficacy data requirement was continued
in 1979 were products which, if they lacked efficacy, could potentially
have significant public health effects, such as mosquito control products,
rodenticides, certain other invertebrate and vertebrate control agents,
and antimicrobial products. The Agency now believes that because many of
the "public health" use patterns identified at that time are more of an
aesthetic and nuisance problem than one of public health and are- adequately
covered, in any case, by other regulatory mechanisms offering assurance
that the products are efficacious, and because the efficacy of products
for other of these uses is adequately discernible by the user, marketing
of inefficacious products is unlikely. The public health authorities of
states and localities, for example, have the expertise to determine the
efficacy of a product used for rodent control. Mosquito control districts
offer similar expertise with respect to mosquito control products.
"Several State pesticide regulatory agencies continue to require
efficacy data to evaluate the pesticide under conditions of use within
their States. The State Cooperative Extension Services use such data in
making recommendations to growers within the State. Efficacy data are
particularly important to the State in administering registrations for
special local needs under FIFRA Sec. 24(c), and in determining suitable
pesticides for use under the emergency exemption provisions .of FIFRA Sec.
18.
"The Agency is proposing to extend its current waiver to efficacy
data for all uses of pesticides except those where control cannot
reasonably be observed or determined by the user and..lack...of control
.results,.in-a.i-clear. •adverse,, health effect..... Eff.ic.acy .data .wo.uld.ctantanue
toi. be ..required for, products bearing" claims <for control of pest microorganisms
that pose a threat to human health and whose presence., cannot be readily
' observed", by ,the-user, including.., but not limited to, • niicr-oo-rganisms ..- .. .-. •,...
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infectious to man in any area of the inaminate environment, and'the
products claiming control of mycotoxin-producting fungi. All other
efficacy data requirements would normally be waived. The specific
uses that require efficacy data are specified in Part 158 - Data
Requirements for Registration in §158.32 with references to the testing
methodology and protocols in Subdivision G - Product Performance. The
level of label claims is described in Subdivision H - Labeling Guidelines
for Pesticide Use Directions (§§ 100-106) and guidance is also given to
test methodology needed to support these claims when necessary.
"The Agency expect and believes that registrants will ensure that
their products are efficacious when used in accordance with label directions
and commonly-accepted pest control practices. Under the statute, the
registrant still has the responsibility to ensure a product satisfies its
label claims. The Agency would take corrective action on a product
including, when necessary, enforcement or cancellation actions, since the
registrants must still comply with the law. In addition, pesticide
producers are aware that they are potentially subject to damage suits by
the user community if their products prove ineffective in actual use.
Such litigation can be damaging to the company's reputation and future
sales. It is in a company's own best interest to continue high quality
efficacy data development and to market only products demonstrated to be
effective.
In cases where efficacy data review is still required, the Agency
determines that: label claims for control of pests or obtaining specific
plant/ animal responses are verified by scientific evidence; label directions
for use are consistent with commonly recognized practices of pesticide
use; and label directions for use are supported by scientific evidence
based on testing of the pesticide under the most suitable conditions for
predicting product performance under the variety of use conditions likely
to be encountered.
"Under this proposal, the Agency retains the right to require the
submission of efficacy test data or other evidence, on a case-by-case
basis, for any pesticide product registered or proposed for registration,
for which a lack of efficacy has been reported, for evaluation of product
benefits when product risks are substantial, or when other factors exist
.which make .submission of such data necessary or desirable to support the
presumption that it is efficacious. If there is evidence (such as a
significant rise in complaints from user groups, scientific societies,
trade associations, or the general public) to establish that this
regulatory relief policy is being abused, the Agency would reconsider its
waiver policy. The Agency is building links to various organizations
that are knowledegeable of efficacy matters through a product performance
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information network. Also, the Agency is actively pursuing the
establishment of formal relations with various departments, such as the
U.S. Department of Interior's Fish and Wildlife Laboratory in Denver to
conduct rodenticide surveillance and with professional organizations
such as the National Pest Control Association and American Hospital
Association to aid in efficacy evaluation when the surveillance network
or other sources indicates the need."
II. SCOPE AND ORGANIZATION OF SUBDIVISION G
A. Scope
The data generated by the product performance studies described in
these guidelines are used by the Agency, when applicable, along with
other data, to assess the efficacy of pesticide products as part of
making the determination as to whether EPA should register the product.
The guidelines also represent the procedures found to be useful in the
Agency1s review of product performances when efficacy data submission is
requested on a case-by-case basis.
These proposed guidelines specify:
(1) The conditions under which each particular data requirement
is applicable to a pesticide product;
(2) The performance standards for'acceptable.testing, stated
with as much specificity as the current scientific disciplines
can provide; and
(3) The information to be included in a test report.
The guidelines also indicate when applicants should consult with the
Agency before initiating certain tests, and when the submission of efficacy
data is waived. In addition, each section series provides useful examples
of acceptable protocols for conducting product performance testing.
B. Section Series.
The Agency has greatly expanded the areas covered in these guidelines
since the 1975 proposal (40 FR 26802 June 25, 1975). This subdivision is
organized into seven series of sections, one covering overview, definitions,
general considerations, and the other six series of sections covering
guidance on efficacy data for specific types of pesticides used
on distinct classes of pests. These sections contain guidelines and
information .needed; by the Agency to. evaluate.efficacy and establish
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limitations for specific use patterns. Subdivisions G'and H.'(Labeling
Requirements for Pesticides and Devices) have parallel organization to
aid in locating label claims, and supporting performance standards and
test methodology when applicable.
Basic considerations which apply to all sections except the antimicrobial
series (§§ 91-1 through -9) appear in the Overview (§ 90-1) and General
considerations (§90-3). Included in the Overview and General considerations
sections are the efficacy data waiver policy, definitions, general
considerations on test standards, plot sizes, geographical distribution,
application methods, dosage rates, experimental design, adverse effects,
assembly of these reports, and related information for submittal to the
Agency. .
Sections 91-1 through -8, Efficacy of Antimicrobial Agents,
include data requirements on sterilizers, disinfectants, virucides,
certain fungicides (those to control fungi pathogenic to man or domestic
animals), sanitizers, and bacteriostats. .These section are concerned
with public health related uses only. Section 91-30 provides recommended
methods for satisfying data requirements as well as supplemental recommendations
for expanding methods for specific claims and use patterns. Subseries
9lB are concerned with providing guidance on non-public health uses
only. Among the target pests which are covered in these section series
are those posing a potential health hazard to man or animals, or the
ones causing spoilage, deterioration, or the production of offensive odors
in substrates in which they grow. "
Sections 92-1 through -7, Efficacy of Aquatic Pest Control Agents,
includes testing and performance guidance on pesticides used in aquatic
environments, such as aquatic herbicides, swimming pool algicides,
industrial cooling water microbicides, pulp and papermill water system
microbicides, secondary oil recovery system microbicides and antifouling
paints. Section 92-30 lists acceptable test methodologies and references
which serve as a guide for developing suitable test methods. This section
series concerns efficacy data for use pattern of pesticides all of which
are routinely waived for submittal.
Section 93-1 through -16, Efficacy of Fungicides and Nematicides,
includes testing and peformance guidance for fungicides and nematicides
used to control above-ground plant pests and soilborne pests; disease and
deterioration organisms on post-harvest fruits and vegetables, grains,
seeds, ornamental plants (including bulbs, flowers, and trees), turf
areas, wood and wood products (to prevent rot); industrial materials and
equipment (to prevent deterioration, mold, and staining by fungi to
products such as fabrics); and mold and mildew on surfaces. Section 93-30
contains acceptable test methods for evaluating control of certain fungal
pests-on.inanimate surfaces, polymeric materials, and plant parasitic
nematodes. This section series provides guidance for developing data on
non-public health uses of pesticides, all of which are routinely waived
for submission except for .products claiming control of myootoxin-producing
fungi. .
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Section 94-1 through -6, Efficacy of Terrestrial Herbicides, Plant
Regulators, Desiccants and Defoliants, includes guidance on these types
of pesticides. The section on plant regulators provides data guidance on
products claiming increased yields, flowering, fruitset, berry size of
seedless grapes, and products enhancing abscission, inhibiting tobacco
sucker growth, inhibiting apple scald, etc. Guidance for herbicides,
desiccants, and defoliants are also arranged by crop and use. Section
94-30 contains references to herbicide evaluation and to specific
conditions for evaluation of each type of plant regulator discussed in
the guidelines. This section series contains efficacy data for use
patterns of pesticides, all of which are routinely waived for submission.
Sections 95-1 through.-14, -Efficacy of Invertebrate Control Agents,
includes data guidance for invertebrate pesticides which are substances
or mixtures of substances intended for preventing, destroying, or repelling
invertebrate animals declared to be pests, including any member of the
class Insecta, and other allied classes in phyla Arthropoda and Mollusca
(including but not limited to spiders, slugs, sowbugs, centipedes, and
snails), but excluding the class Nematoda (nematodes) of the phylum
Nemathelminthes. These target pests are present at a variety of sites,
including households, building premises, agricultural and ornamental crops,
in or on livestock, on humans and pets, wood structures, and in stored
products. Section 95-30 contains references to acceptable test protocols.
This section series concerns data for use patterns of pesticides, for
which product performance data are routinely waived for submission.
Section 96-1 through -19, Efficacy of Vertebrate Control Agents
includes data guidance for vertebrate pesticides which are substances or
mixtures of•substances intended for preventing, destroying, repelling, or
mitigating any vertebrates including fish, amphibians, reptiles, birds,
and any wild and domestic mammals (except man). Vertebrate pesticides
include oral,.dermal, and inhalation toxicants; irritants; repellents
(odor, taste, or tactile reaction); chemical frightening agents;
anaesthetizing chemicals; and reproductive inhibitors. The sections of
acceptable methods are composed of two parts: one containing references
on supplementary information about test procedures (§96-25), and the
other containing acceptable methodologies (§96-30). This section series
concerns use patterns of pesticides, for which product performance
data are routinely waived for submission.
C. Organization of Sections within Subdivision G.
1. General requirements or considerations. Each section series on
specific pesticides begins with a general requirements or general
considerations section. This section discusses scope, definitions,
general data considerations, and general performance standards pertinent
to: the .specific types of pesticides. The section also emphasizes any
.information, that.differs-from or supplements the information ,in
Section seriea/90-i'.through,3.'-on Overview, Definititwis, ,and General ..
Considerations. . '> . ' ' ' . * ' ' '••".. ... -."•'- ;
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2. Section subdivisions. Most of the Section series'contain-sections' • .
which cover products used on or in specific sites (e.g.-," hard surfaces,
swimming pools, turf, crops, orchards, and industrial cooling water
systems). Other section series are organized according to organism (e.g,.
avian toxicants, fish control agents, mole toxicants, and soil-borne
pests). cFurther subdivisions of these sections are bases on sites where
the pests are found.
3. Suggested performance standards. Many of the individual sections con-
tain performance standards. A performance standard represents the lowest
level of product performance which would normally be acceptable for
protecting the public health, when required, or for economic control of
a specific sites for a given pest or pest combination. The proposed
performance standards are usually expressed as percentages of pest control
and serve as general guidance. Public health related label claims to
be supported should meet the specific performance standards. If these
standards are not met, the registrant may choose to use a lesser label
claim or put a disclaimer on the label that the product did not meet EPA
standards for effectiveness.
4. Effectiveness paragraphs. Each section describes the criteria used
to determine the effectiveness of the product in preventing, destroying,
repelling, or mitigating a pest? accelerating or retarding the rate of
growth of a plant or insect or otherwise altering the behavior of the
pest or host organism; or defoliating plants or artifically accelerating
the drying of plant tissues. Effectiveness 'as determined by experiments
could also be designed to obtain information on adverse effects. Adverse
effects and hazards.to man and the environment are discussed in :Subdivisions
D, E, F, J, K, L, M, and N. In addition to the adverse effects specifically
evaluated by other subdivisions, this subdivision provides guidance on
evaluations of other kinds of adverse effects such as deteriorated food
quality, discolored and weakened fabrics, unsightly residues on plant
foliage, reduced crop palatability, increase in harmful nontarget organisms,
and presence of dead pest organisms as a potential food source for domestic
or wild nontarget organisms.
5. Sections on Acceptable Methods. Each of the section series for
specific types of pesticides contains a section on acceptable methods.
Each of those sections discusses specific test methods and provides
references to published literature which gives the registrant specific
examples of acceptable or related test protocols. Much of the literature
also aids in developing test protocols and in reporting details on
evaluation of tests and experimental designs and on statistical
evaluations. Several of the cited references are methods developed by
consensus organizations such as those from American Society for Testing
and Materials Committee E-35 on Pesticides; Association of Official
Analytical Chemists; and from the Agency's Manual of Biological Testing
Methods for Pesticides and Devices as an aid to the registrant.
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9
III. GENERAL ISSUES
A. Test Standards.
This part of the discussion concerns certain issues which have arisen
with respect to test standards governing methodologies. The issues
discussed here concern general test standards which apply to meeting data
specifications of several kinds of tests.
Issues involving technical aspects of individual tests standards are
discussed in part IV of this Discussion.
1. Test substance. Final tests to support the effectiveness of a
product are usually conducted with the formulation proposed for registration
and frequently with the product in the same packaging intended to be used
commercially. This latter test is especially important for pesticides
marketed and applied directly from containers or container-devices. End-
use tests using the formulated product are required in cases where
formulated products found to be effective in laboratory tests are
ineffective when packaged in commerical quantities, for a variety of
reasons, including synergism, antagonism, physical incompatibility with
inerts, short shelf-life, chemical reaction with a component added at
repackaging, or improper functioning of containers which also serve as
application devices. Moreover, a product initially effective at the user
level may suddenly become ineffective or unusable because of a change in
the can liner coating, product emulsifier, solvent, or other component
used. Mixtures of two or more pesticides in a single formulation may
react chemically, be physically incompatible (producing a useless product),
or be mutually antagonistic or synergistic for effectiveness or
adverse effects.
2. Minimum effective dose (MED) and effective dosage range (EDR).
In § 90-3 General considerations, paragraph (b)(3) it is stated that the
applicant demonstrate the minimum effective dose and the effective dosage
range. [See the definitions in § 90-2(a) and (f).] These data are useful
in risk/benefit considerations where a determination of lower dosage
rates may, in some cases, allow a reduction in adverse effects or
environmental contamination while still providing acceptable levels of
pest control.
The need for MED and EDR would not apply to the section series 91-
Efficacy of Antimicrobial Agents, for two principal reasons:
(1) For many disinfectants, the important information concerns
whether or not total elimination of the target organism(s) is achieved,
rather than an MED or.EDR; and '•'... ' ' - : , .
(.?> .for :many,antimicrobial product applications, the .extent and
.influence of. Aincontrollable factors (such as level and type of contamination,,
.zesistasc/e .of •'m^creorganiSffis^ -rtature .,and..cotsfigust.tipa.. of .§utf-ace;./affd .
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soil load) are too variable and .great for an MED or DER to have any
significant value.
(3) Plot Size. Many questions arose on the proposed guidance
concerning size of the test plot or site. Several people were of the
opinion that the Agency usually would request test, plot sizes larger than
one acre, even up to hundreds of acres, and they felt that this would
invalidate most of the data developed from experiments on plots smaller
than an acre. The Agency feels that small plots, properly relicated and
well designed, provide very useful and valuable data. The Agency encourages
small plot testing since considerably less of the pesticide would be
placed in the environment than if testing were performed on larger plots.
In addition, larger plots may occasionally increase the potential for
hazard to wildlife.
B. Effectiveness Evaluations.-
1. Suggested performance standards. The product performance guidelines
suggest specific performance standards for several different areas.
These standards would benefit the Agency and the consumer because they
would ensure that the products are useful and will control the pests
indicated on their labeling. The performance standards have been discussed
extensively at the Agency and are considered to represent suitable guidances
at this time. Many of the performance standards have been routinely
used by industry and the Agency for many years. In some instances,
experts throughout the country were contacted to develop some of the
performance standards, as well as the Agency's own scientists at head-
quarters and at laboratories and field stations.
Some of the suggested performance standards are based on comparisons
of the effectiveness of products to the effectiveness of standard reference
chemicals. Often the performance standards are very explicit as to
whether the comparison involves the amount of chemical per acre, reduction
in pest levels, different target pest, or adverse effect to the crop.
Some people have commented that a performance standard based on percent
reduction of pests is appropriate but are concerned that applicants at
times would be encouraged to use-increased number of target pests in the
test area to misrepresent the derived benefit.
The performance standards are useful for guidance purposes and would
be applied flexibly. EPA recognizes that the level of control derived
from a single pesticide dosage varies with each pest and site combination
and a number of other factors, including the user group; the geographic
region; crop grading and quarantine standards; users of the treated commodity;
the anticipated level of pest population to be encountered by users;
climatic conditions; soil textures; crop cultivars; and, in some instances,
comparisons with existing control measures.
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Comments already received have questioned the general performance
standards in Section series 93- Efficacy of Fungicides and Nematicides,
and 94- Efficacy of Terrestrial Herbicides, Plant Regulators, Desiccants,
and Defoliants. Both of these section series suggest standards of 70%
pest control. The performance standard is useful primarily to distinguish
between products having barely adequate effectiveness and those having
fully adequate effectiveness. The Agency may use this distinction to
determine the appropriateness of acceptable label claims. Accordingly, when
a product's performance is somewhat below the proposed performance
standard, the registrant could label his product with a lesser degree of
control claims, such as "aids in control," "partially controls," or
"suppressses," as opposed to "control" or kills claims.
In certain cases, less than 70% control may be acceptable, for
example, when the target pest is particularly resistant or when no suitable
alternative or more effective pesticide exists. Such cases would need
supporting background information, when applicable.
A suggested general performance standard of 70% control is not
adequate for some pest problems. For example, when human safety is
involved, as in the case of many disinfectant uses, the Agency generally
would require close to or not less than 100% control. Comments received
from USDA recommended general 70% performance standards, but presumably
these were recommended only in relation to pesticides for use in crops.
Actually, 70% control for pests of humans and pets would not be totally
unacceptable from a nuisance control standpoint, but it would provide
essentially no protection of health to humans or domestic animals. To
support the Agency's mandate to protect health and through persuasion of
arguments submitted by the Chemical Specialties Manufacturing Association
(CSMA), certain pesticide performance standards were raised from 90% to
95% control to meet acceptable levels of control that could be tolerated
by humans. EPA was persuaded by these comments, for example, that 90%
control of some populations of mosquitoes would not be high enough,
since the uncontrolled mosquito biting rate of 50 per minute would be
reduced only to 5 bites per minute, a level which would still be unacceptable
to the consumer.
Also, in section series 96- on Vertebrate Control Agents, the suggested
use of a general 70% performance standard for reduction in pests was
considered to be inappropriate. For example, if 70% of the woodchucks
in a given area were killed each year, there would still be the same
potential number the next year due to their reproductive capacity.
Control of other animals such as foxes and birds presents a similar
problem.
With rodenticides, a 50% reduction in population control in
.actual use could leave a massive population not controlled and capable of
quickly replacing, • the animals/killed.. Therefore:, percent .reduction :in .pest
damage is.Often-.;,a;.<better.. rating fac.tor: to use..jto,,evaluate .damage potential .' •':
to orchards, .-crops, 'and. homes than, percentage of animals killed.
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The suggested performance standards are generally 'based .on current • ...
agricultural'practices in the United States. Such standards do not
presently take into account performance under any of several integrated
pest management (IPM) practices. Such programs do not generally result
in unacceptable levels of control, but may involve lower rates or less
frequent .applications of pesticides. Use of pesticides in such programs
should meet the suggested performance standards when evaluated in conjunction
with the nonchemical practices that are an integral part of directions
for use in IPM. Evaluations would be expected to take into account
those situation such as when predators further reduce pest populations
below the levels attained with the pesticide used alone. Periodic revisions
of these guidelines will provide opportunity for greater attention to
any such programs that become commonly recognized practices.
By utilizing Subdivision H and Subdivision G guidelines, the registrant
would be provided an opportunity to make his own judgements regarding
claims to be made on the pesticide product label. These claims could
predetermine specifically what the Agency expects as to effectiveness of
his products. The suggested peformance standards would provide breakpoints
to aid in the determination of what the registrant will claim in his
label in relation to results of testing to meet the data requirements.
Registrants can distribute portions of these guidelines to cooperators
and contractors who could than more easily supply the exact information
needed to support product registration.
2. Mode of action. These guidelines recommend that a description of
the pesticide's mode of action be submitted or referenced if published.
Commenters have expressed concern as to the amount of sophisticiation
necessary for the studies to report on mode of action. Since the mode
of action depends upon the individual chemical, use pattern, and type of
product, the Agency decided that stating the general mode of action is
adequate, e.g. that the substance acts as repellent as opposed to a
nerve toxin. Although it in not intended that molecular studies on
modes of action be required, the molecular mode of action, if known, should
be reported.
3. Rating scales. Fating scales are often used to assess pest damage
or pest control, and the guidelines often specify that such scales be used
as long as each value in the scale is fully defined. Several commenters
were concerned over reporting results using only the rating scales
described in the draft test protocols, since many other rating scales are
used in evaluating pesticide product performance. The agency feels that
rating scales to assess factors such as quality, fabric damage, or algae-
growth, etc., would also be acceptable, provided that adequate explanation
for each rating system is given. When the test specifies a certain
rating system, then the specified system should be used; however, additional
rating systems may also be used and results reported, if the registrant
so desires.
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4. Benefits data. Data submitted to the Agency should also demonstrate
the benefit of the product to the user. While the Agency will not make
lack of essentiality a criterion for denying registration of any pesticide,
this does not mean that benefits information should not be submitted when
it is required. Benefits derived from pesticides may be prevention of
storage rots, decrease in disfigured fruits, yield increases, decreases
in crop damage, decrease in disease incidence (e.g., Rocky Mountain
spotted fever) prevention of fabric staining, prevention of lumber staining,
etc. If the product is not effective and no benefit results from its
use, the use of the chemical would merely add undue pollution to the
environment. For further discussion of benefit data, see §90-3 General
considerations.
5. Testimonials. Section 90-3 General considerations statas that
testimonials by experts regarding the efficacy and limitations of
particular products would be acceptable as part of the data submission.
Several commenters are of the opinion that the submission of testimonials
would be of limited value because it is usually possible to obtain positive
testimonials, and because most registrants would not be prompted to submit
negative testimonial letters on their products. In addition, they
commented that accepting testimonials is likely to create a burden on the
Agency, since the credentials of the persons submitting the testimonials
would have to be examined. The opinions of such experts may also be too
biased by their position or economic relationship with the registrant.
However, the Agency will determine the value of each testimonial
submitted based on its merits.
C. Product Not Mentioned in the Guidelines.
The Agency has not provided protocols for the types of products
which have never been submitted for registration. Accordingly, certain
uses of products are not mentioned in the proposed guidelines. It should
not be concluded that such products do not have to be registered, nor
does it mean than there are no data requirements. Rather it only means
that a number of current or unforeseen future uses of products are not
covered because the methodology for testing these products has not been
adequately developed. The Agency1s Product Managers should be contacted
directly about requirements for products not mentioned in the guidelines,
since the efficacy data requirements for these products will handled on
a case-by-case basis.
Also, in development of these guidelines, the Agency anticipates
that, with further new product development the Agency policy and guidance
may well change to reflect the new development. The guidelines will be
revised-.1:o incorporate new advances in science and. technology, new pesticide
uses., and. 'changes in methods, development. These iguidelines are. meant to
be dynamic.; and-:the Agency will, strive to keep current with the state of
the sciences involved.. " •
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D. Separation of Agricultural Products from Household'Products.
One comment received stated that the guidelines fail to' -recognize
the difference between agricultural-commercial pesticides and household
products.. However, the Agency disputes this point. The guidelines are
organized so that any person who is at all familiar with pesticides and
their uses can distinguish between the guidance designed for agricultural
products and those for household products. For example, this separation
of agricultural and household products in the invertebrate area is
evident in § 95-7 shade tree and forest land treatments vs. § 95-11
premises treatment.
E. Organization of Antimicrobial and Fungicide Sections.
Several commenters were concerned over the separation of nonpathogenic
fungi, especially mold and mildew (§ 93-15) from the antimicrobial section
series (91). They felt that all non-agricultural uses of antifungal
products and algicides should fall into section series 91- Efficacy of
Antimicrobial Agents. At present, all or most combination-use products
have different directions for use and site of application due to the
inherent differences between mold and mildew pest problems and antimicrobial
pest problems. The Agency recognizes that there are numberous approaches
that could be used in presenting the information contained in the
guidelines, one logical approach seems to be grouping the information
according to general type of pest organism, since the testing and labeling
considerations primarily relate to the type of pest organism and nature
of the pest problem, and only secondarily to the sites of application and
other factors. Additionally, this type of-approach complements the scope
of expertise of scientists developing, using and evaluating the data.
The Agency would like to know what specific types of problems have
occurred as a result of handling mold and mildew and antimicrobial products
separately (as reflected in the guidelines). This has been the policy
for some time and the Agency is not aware of any important problem which
warrants changing this practice.
F. Toxicity Test Recommended for Animal Repellents.
Some commenters questioned the need for toxicity tests data on rats
for animal repellent products designed to repel other vertebrate animals
(but not rats). See, for example/ the rat acute oral LD50 studies in §96-
18 Domestic dog and cat repellents, and in §96-19 Browsing animal repellents
(designed to repel deer, elk, and lagomorphs).
First, the toxicity tests on rats will give an indication of the
lethal dose for the target species. The LD50 value will allow EPA to
determine whether the dose level which effectively repels the target
species is close to the lethal dose. If the Agency concludes that there
is not an adequate margin of safety between the repellent dose and the
lethal dose, further testing may be needed.
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Second, the rat studies are less expensive and easier to perform for
toxicological tests than studies involving larger target species. Also,
the toxicity methodology for large mammals is not yet as well developed
as it is for rats, and the legal problems associated with large animal
studies can be avoided. .
A third reason for the toxicity test is to aid in determining the
mode of action of the repellents [e.g., whether it works as a poison or
through tactile response (by rapid inflammation of tissue)]. An
understanding of the mode-of-action would aid in developing more accurate
labeling.instructions and performance standards.
IV. INDIVIDUAL TEST ISSUES.
A. Simulated Use or Actual Use Studies for Antimicrobial Agents.
In section series 91 Efficacy of Antimicrobial Agents, more emphasis
is placed on the use of test methods which more closely reflect actual
conditions of use. This new emphasis would be particularly valuable for
methods to support claims for "one-step" cleaner-disinfectants, surgical
instrument sterilizers and disinfectants intended for re-use, and residual
bacteriostatic claims for treated surfaces or materials. As a result of
this new emphasis, the following problems would likely be alleviated:
- (1) Dependence on zone-of-inhibition methods that are not
related to actual use situations;
- (2) Dependence on many standard methods such as AOAC procedures
employed for disinfectant-type products, which do not ade<juately represent
the manner in which these products are intended or claimed to be used;
- (3) Dependence on standard methods which do not address such
factors as presence of organic soil, varying exposure period, or re-use
of prepared solutions;
- (4) Propensity for label claims and directions on certain products
not to be restricted to those which could be actually supported by the
standard test methods.
B. Uniformity of Test Microorganisms, Carrier, and Batch
Replication Required for Testing Antimicrobial Agents.
The test standards which have applied in the past to the performance
.of efficacy studies on antimicrobial products have been revised for these
guidelines,to-.provide greater uniformity and reliability in testing. In
the past, the number of...'carxiexs (.i.e., '.surfaces .sach as porcelain, ' ,
stainless steel.; -and. surgical suture loops on which microorganisms are
•-•deposiliecll regjiixed -for: basic efficacy ••t-es-ting. of disinfectants- varied . .
.fraa: 3.0-;'to'':6&,: depending;on>;whether .-the organisms' was -considered: pr-itaary •
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or secondary for either the hospital or general level of disinfectant.
This subdivision provides that all basic efficacy data for disinfectants
will be conducted using 60 replicates per type of carrier per test
microorganism per sample. Also, one of the three microorganisms required
in the testing of general disinfectants was not specified. The guidelines
now provide that only two specified test microoganisms/ Staphylococcus
aureus and Salmonella choleraesuis, be required for efficacy testing for
general disinfectants. For hospital disinfectants, S. aureus, S. choleraesuis,
and Pseudomonas aeruginosa are required, as was the case previously.
Finally, a separate 60-day shelf-life stability sample of the product was
originally required to be tested in addition.to three different freshly-
prepared -batches. In the current guidelines, three samples of the product,
representing 3 different batches (one of which must be at least 60 days
old), would be required; this new requirement would eliminate the previous
need for a separate shelf-life stability study. The current, simplified
requirements would result in fewer replications being performed in the
case of limited and general disinfectants, and would provide the reliability
indicated in the Use dilution method and the AOAC Germicidal spray products
test. More replication is required for hospital disinfectants because
greater confidence is needed so that these products will achieve their
important function.
The Agency has received several comments regarding the data requirements
to support sterilization claims. Tests to provide the basic efficacy
data for sterilizers are also to be conducted using 60 replicates per
type of carrier per test organism per sample. This requirement is
analogous to the requirements for disinfectants, and provides for greater'
reliability in assessing the effect of the two types of carriers specified
in the AOAC Sporicidal test. The performance standard of no failures in
60 replicates to demonstrate efficacy of products intended as sterilizers
is indicated in this AOAC method. The specific performance standard for
Sporicidal activity is not considered in the guidelines, since the sporicide
category has been eliminated, as explained in paragraph C (below).
C. Sporicide Category of Antimicrobial Agency Eliminated;
For purposes of the guidelines, the Agency proposes to treat sporicides
and sterilizers alike. It appears that most consumers consider "sporicidal"
and "sterilizing" activity to be synonymous. In the past, the Agency has
accepted a lower level of efficacy for sporicidal products than for
sterilizers. This situation could have potentially hazardous consequences
if users expected sterilization and obtained only sporicidal control.
Therefore, in order to avoid the marketing of products with claims which
might be misunderstood, the Agency will henceforth deem "sterilizers"
and "sporicides" to be synonymous; products not killing all organisms,
including spore-forming bacteria, may be deemed "disinfectants" but would
not be deemed "sporicides."
D. Use of Neutralizers in Microbiological Assay Systems.
Several commenters suggested that evidence to show that the neutralizer
mentioned in § 91-l(b)(3) inactivates the active ingredient(s) and does not
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possess antimicrobial activity per se should not be required where there
are known standard neutralizers for the active ingredients in the
formulation such as those documented in the literature or cited in the
AOAC method.
Most of the standard neutralizers are designed for a specific type
of active ingredient. Because most antimicrobial products are complex
formulations containing several active ingredients, the use of a neutralizer
designed for one active ingredient may not effectively neutralize the
formulation. Additionally, there is evidence that standard neutralizers
such as letheen broth possess antimicrobial activity against some bacteria.
For these reasons, the Agency has concluded that evidence of neutralization
of each antimicrobial formulation is required. Citation of existing data
on the identical formulation would be acceptable in lieu of actual
submission of the test data and would satisfy the intent of the requirement.
E. Status of Ingredients in Antimicrobial Products as Active or Inert.
Comments were received from the public stating that the requirement
for the AOAC Phenol Coefficient method to determine the status of
questionable ingredients as active or inert [see § 91-l(b)(10)] was
inappropriate. The Agency agrees that more flexibility should be provided
in this area and this section has therefore been revised to allow other
types of testing which may be mutually agreed upon by the applicant and
the Agency. It should be pointed out that it was not the intention of
the Agency to require this testing routinely to determine the active or
inert status of ingredients in antimicrobial pesticides, but rather to be
responsive, to numerous requests by applicants who want a basis for establishing
that an ingredient, heretofore considered active, is actually an inert
ingredient.
F. Antimicrobial Terminology
A number of comments were received from the public regarding the
definitions in section series 91. The Agency did not intend to use the
classical definitions for all of the terms because the definitions are
designed specifically for registration activity. Also, many of these
definitions are similar or identical to those set forth in the Act and at
§ 162.3 of the FIFRA sec. 3 regulations. Since the terms are primarily
important for'label claims, § 91-l(c) has been moved to Subdivision H
(Labeling Guidelines for Pesticide Use Directions) at § 101-l(d).
G. Sample Requirements for Testing Products Used as Laundry Additives.
Several commenters proposed.reducing the number of fabric swatches
required..for;demonstratingdisinfection. CSee §: 91-4(a) (1.).(.!.)• (C.):.] The
Agency agrees ..with, this suggestion, 'and has. reduced the number of. fabric
.swatches £rom 60 .to 9. This proposal has been adopted on'the basis that
.the amount 'of replication originally proposed is''unnecessary where fabric
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swatches-, rather than hard surface carries, are involved. EPA assumes
that it is easier to disinfect fabric surfaces than hard surfaces. This
revision should allow for considerable reduction in the amount of required
replication. If subsequently submitted data does not confirm this
assumption, the Agency may consider modifying the requirement to increase
the number of swatches.
Comments were also received questioning the need to test both the
fabric and the laundry water. Commenters felt that testing the wash water
is redundant and serves no purpose, since the fabric swatches will retain
some of the wash water anyway; if the organisms are not killed, the water
residual in the fabric will indicate this. However, the concentration of
antimicrobial content in or on the fabric may differ from that found in
the water and give different results. The Agency feels that testing the
wash water is necessary to show whether organisms eluted from contaminated
fabric into the water can or cannot recontaminate the clothes or contaminate
the clothes of the next user. This testing is required to prevent public
health problems in commercial and coin-operated washing machines where
cross-contamination between users may be a problem.
H. Standard Carpet Test Samples and Microorganisms.
The Agency recognizes the diversity in carpet samples and wants the
carpet intended to be tested to be fully identified in the test report.
Characteristics such as the pile fiber type, pile yarn weight'of finished
carpet, pile density, and tuft height should be included. [See §91-
4(b)(l)(i).] These characteristics may affect the performance of the
product. Commenters have suggested that the Agency supply specific test
carpet standards, but the Agency rejects this comment until more information
on suitable test standards is developed. Industry cooperation in developing
suitable test carpet standards, such as through CSMA, AOAC, or ASTM,
would be useful to testers, users, and evaluators of carpet sanitizers.
A commenters has suggested that Pseudomonas aeruginosa is a poor
selection as a carpet organism because of its low recovery counts after
drying. This statement is contrary to information the Agency currently
has on this organism. The Agency welcomes additional test information
to expand upon any problems associated with using P_._ aeruginosa as a
test organism for carpet sanitizer testing, and would also welcome
suggestions for any other suitable or superior organism for this test.
I. Air Sanitizer Test Methodology.
Recent public comments on test requirements in § 91-5 Air Sanitizers
have suggested that there is methodology now available that provides
specific test standards to evaluate air sanitizers, such as: air sampling
procedures, suitable type of air filtration, suitable exhaust systems,
use of Micrococcus ly.sadeikticus as a test organism, specific glycol
concentrations, proper time frames for sampling, and acceptable types of
air samplers. The concern by certain other commenters :about the need
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for certain standards could be evaluated once the Agency evaluates such
standards. The Agency therefore invites submittal of test protocols and
related information on air sanitizer testing.
J. Use of Mixed Cultures in Testing Products for Processing and
Industrial Uses.
Commenters have disputed the need for identifying each test organism
as a contaminant in metal-working fluids, and for the separate testing of
bacteria and fungi inocula in the test protocols. In response to these
comments, the organism identification was reduced to the level of genus
only. And for mixed cultures, where there is evidence that both bacteria
and fungi are normally present and necessary to (re)produce the spoilage
problem [as in fuels, §91-6(d)], a mixed inoculum is appropriate and
should be used; where mixed cultures are not normally the case [as in
metal-working fluids, § 91-6(c)], mixed cultures should not be used and
the organisms should be tested separately.
Bacterial and fungal mixed cultures are special cases. When the
applicant can justify the use of mixed bacteria/fungal inoculum as
necessary to (re)produce a specific spoilage problem, then the use of
such mixed cultures can be considered on a case-by-case basis.
A list of representative organisms in metal-working fluids has been
developed by the American Society for Testing and Materials.(ASTM) E35-15
Subcommittee on Antibacterial and Antiviral Agents and may prove to be useful.
K. Water Purifier Units and Bacteriostatic Water Treatement Units.
Section 91-8(a)(3) would delineate tests standards and requirements
for water treatment units. The- current Agency policy with respect to
regulation of water treatment units was first explained in the Interim
Requirements for Registration of Bacteriostatic Water Treatment Units for
Home Use, published in 41 FR 32778, August 5, 1976, and in an unpublished
EPA document entitled "Interim Standards for Water Purifiers." The
scientific premises upon which these test procedures were based have been
reexamined in view of these units and expected product performance under
actual use conditions.
- 1. The "Interim Standards for Water Purifiers," which was
intentionally directed solely to the pesticidal use of
silver, now has limited significance because data submitted
more recently by registration applicants showed that silver
will not purify water at the maximum allowable concentration
50 pbb in effluent water.
';.•'•;.'. - 2..; :TJte gbiicy explained in the "Interim. Requirements,for Registra-
•••";; .;' . :; .' ' -ticn ©£' Bacfe&f.ibstat:ic"water. Treatment 'Units for: Home Use" . -
'.. •'' '.,_.:was' based on the assumption that extensive growth ;of sapro-
" • -phytic bacteria trapped within tfa« filtering .medium during
_' . j ..;..'",'sta.gn.atiqn .periods could• pose ,a,;p6ten.tial bacteriological,
:.;;- ••'/ •.'"•'; "ptobieja. of ./public -health, significance.---Scieistific evidence
'.-'.: _.>•.' published^since:!-that.tike lias .'not documented <or "substantiated"
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this assumption. Data.developed as a result of issuance of
this "Interim Requirement Notice" have failed to demonstrate
either a bacteriological problem or a valid product function
other than an assumed aesthetic value. Under the present efficacy
data waiver policy (see Part I.B. of this discussion), effectiveness
data are being waived for bacteriostatic water treatment units
since only an assumed aesthetic value can be associated with
these products.
If, however, scientific information indicates that a potential
health hazard exists because bacteria-infested water filter
units are used to process potable water, the following must
be taken into consideration before an adequate testing program
can be undertaken:
(1) The level of saprophytic bacteria that would be considered to
be of public health significance in potable drinking water
must be defined.
(2) The level of antimicrobial activity required to eliminate a
potential health hazard would have to be at least at the san-
itizing level; the inhibitory level of activity (bacterio-
statis) would not be adequate.
M. Antimicrobial Agents Intended for Treatment of Municipal
Drinking Water.
The Agency, under FIFRA, does not exert primary regulatory control
over antimicrobial chemicals used to treat municipal water supplies for
the purpose of rendering them microbiologically potable. The use of such
products is regulated by EPA, in cooperation with local jurisdictions,
under the Safe Drinking Water Act (PL 93-523). For example, chlorine is
commonly used to disinfect water supplies, but no tolerance (under the
Federal Food, Drug, and Cosmestic Act) has been granted or approved for
chlorine as a pesticide in this pattern of use. The organizations within
the Agency responsible for the pesticide and the drinking water programs
will coordinate their efforts to provide safe and efficacious treatment
of municipal drinking water. Antimicrobial agents intended for treatment
of municipal water supplies will be registered for such use if they are
certified and approved by the EPA Office of Drinking Water.
N. Antimicrobial Agents Sold Only as Manufacturing-use Products.
In § 91-9, the Agency formally provided requirements for efficacy
testing of manufacturing-use products intended for incorporation into
antimicrobial formulations. The purpose of these requirements would be to
ascertain whether such products have any intrinsic value as antimicrobial
agents. Comments already received have questioned the usefulness of such
testing, especially in view of the efficacy waiver policy [see § 90-l(a)]
which requires data submittal only for pesticides intended for public
health uses. Manufacturing-use products Obviously do not have a direct
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impact on public health, since they are not sold as such for an end use;
they are, however, used in making pesticide formulations whose uses are
designed to protect public health. Since testing data are already
required for such formulated products intended for public health end
uses, the Agency agrees that submission of efficacy data for manufacturing-
use products is not necessary under the efficacy waiver policy.
0. Test Standards for Insecticide Premises Treatments.
The test standards for insecticides to be used in houses and other
similar premises (see § 95-11) are based on comparison of the test
substance with either the official test aerosol (OTA.) or the official test
insesticide (OTI). Submitted comments have questioned these test standard.
First, the OTA standard contains freons which are no longer acceptable for
use in pesticide products, and consequently is an inappropriate reference
standard. The OTI standard, which was changed from DDT to a pyrethrums
mixture, can be very difficult to adjust for dosage mortaility, and this
is not very practical. Also, results in actual premises tests vs. laboratory
tests often show significant discrepancies for new compounds.
Many universities have rapidly-expanding programs in urban entomology
where adequate methodology is being developed. However, the procedures
on premises treatements included in these guidelines are still widely
recognized and provide guidance until researchers develop more accurate
test methods. The Agency invites the public to submit information on new
methodology in this area, and on the OTA and OTI standards and potential
substitutes.
P. Field Tests vs. Lab Tests for Structural Treatments.
Section 95-12 Structural Treatments would apply to invertebrate
control pesticides used in controlling pests such as wood-destroying
beetles, carpenter ants, and termites. This section would need data
derived from field testing. Comments already received have recommended
that EPA accept laboratorys efficacy studies for purposes of evaluating
the performance of pesticides intended for control of wood-destroying
insects. At the present time, however, the Agency feels that the field
tests for structural treatments would give the best estimate of efficacy,
since laboratory testing cannot duplicate field conditions in these
cases, and for the efficacy of soil toxicants for termite control.
However, others feel that laboratory test methods offer good screening
tools for termite toxicants and have certain other advantages; for example,
relative effectiveness of a product in controlling more than one pest can
often be more easily evaluated in laboratory tests, variables are reduced, .
and specific species can be used in lab tests.
It.-should ;be. noted, that. some, species of termites do .not colonize in soil
'-but..instead in..the wood, of housing and other .structures, and:, some in •
other nonr-soil .sites. -Therefore, test protocols for such species will
need to be developed, that differ from the .field stake.method: described..in
these guidelines,./ As suitable.new. test .procedures;.are -developed,: the ...
• protocols.wdJil- -be.-,incorporated into these .guidelines:. , , ....-• •
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Q. Public Comments on Test Methodology.
Through dispersal of early drafts of this subdivision, the Agency has
already received comments from the public, and takes this opportunity to
express appreciation for such submittals. Comments from organizations
such as ASTM (American Society for Testing and Materials) and CSMA
(Chemical Specialities Manufacturers Association) have been particularly
useful in correcting and updating portions of the guidelines. However, to
utilize fully some of the proposed changes in methodology, rationales and
documentation supporting suggested changes should also be submitted. This
could be done following publication of these guidelines. This supporting
information will be reviewed and evaluated by Agency scientists in the
process of making changes and improvements in the guidelines.
More supporting information on the following subjects already commented
on is of special interest to the Agency:
1. Sanitizers - Carpet: Method No. 13 in § 91-30.
2. Sanitizers - Non-food contact surfaces: Method No. 8 in § 91-3.
3. Disinfectants - Efficacy against viruses (hard surfaces):
Method NO: 5 in § 91-30.
4. Disinfectants - Swimming pools: Method No. 14 in § 91-30.
5. Air Sanitizers: § 91-5.
6. In-can paint preservatives: § 91-54(a).
7. Metal-working fluids: § 91-54(b).
8. Fabric mildew fungistatic test: Method No. 1 in § 93-30.
9. Use dilution mildew fungicidal test: Method No. 6 in § 93-30.
10. Fabric, cordage, and fiber (rot, decay, mold and mildew method):
in § 93-30.
R. Test Methods Needed.
The Agency is very interested in receiving copies of test methods that
may ultimately be included or referenced in these guidelines. Also,
references are requested that provide useful background information.
Information on the following areas would be especially welcomed:
1. Test methods for egg sanitizers (see § 91-6);
2. Test methods on water purifier units (see § 91-8);
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3. Test methods for controlling Legionaire's Disease bacteria in
industrial water systems.
4. Test methods for controlling organisms that produce mycotoxins.
5. Test methods for controlling animal - or human-parasitic nematodes.
V. USDA COMMENTS AND EPA RESPONSES
FIFRA Sec. 25 (a)(2)(A) requires the Administrator to provide the
Secretary of Agriculture with a copy of any proposed regulation at least
60 days prior to signing it for publication in the Federal Register.
This provision is to permit the Secretary opportunity to comment on the
proposal, and he did so for Subpart G, I, and J of the guidelines proposed
in 1979, proposed when they were being developed as regulations. Since the
present version of Subdivision G has been edited to reflect product
performance recommendations and guidance the reader should consult CFR 40,
Part 158 for current data requirements. References to performance
standards in the Agency's response to USDA and SAP should be viewed as
"suggested performance standards" for guidance purposes only. Specific
comments of Dr. Flamm on Subpart G are published below. The page numbers
mentioned in Dr.. Flamm's comments refer to pagination in the June 22, 1979
draft reviewed at the time. For purposes of brevity in this addendum,
each USDA comment is followed immediately by insertion of the corresponding
EPA responses. ..-..•
United States Department of Agriculture
Office of Environmental Quality
Washington, D.C. 20205
August 6, 1979.
Mr. Edwin L. Johnson (TS-769)
Deputy Assistant Administrator for Pesticide Programs
U.S. Environmental Protection Agency
Washington, D.C. 20460
Dear Mr. Johnson: Thank you for your July 5, 1979 letter, transmitting
copies of EPA's proposed "Guidelines for Registering Pesticides in the
United States." The guidelines transmitted include Subpart G - Product
Performance; Subpart I - Experimental Use Permits; and Subpart J - Hazard
Evaluation: Nontarget Plant and Microorganisms.
We are pleased that the U.S. Environmental Protection Agency is making
progress in the development of, guidelines for registering pesticides in
the United-States.. These guidelines will be of value to those wishing
to pursue registration of pesticides. ' .
•' OSDft cursory comments on each of the subpat^ts are-, attached.. .We .- . • . •
.- aspect. t^-;-h^e\'a<idijtional.-:eonHnen-ts during :the. 9.0-day •comment. per.iod.;after
':-tther. guidelines.-are published- in the-.Federal .Register.. • . V. ;,.'.....,. ••-.
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We waive the 30-day waiting period requirement for publication in the
Federal Register after receipt of USDA comments as you requested in your
letter.
We hope you will find our comments constructive. We look forward to continued
cooperation with the U.S. Environmental Protection Agency as the development
of these important registration guidelines progress.
Sincerely,
Robert C. Riley (for
Barry R. Flamm,
Director
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USDA Comments on Draft of Subpart G
Subpart G - Product Performance
There is a wide disparity in performance standards between the three
major classes of chemicals of interest to us. While we agree with the
general concept of performance standards and think that they should be
included in the guidelines, we feel that .insecticide performance standards
should be similar to the herbicide and fungicide and nematicide performance
standards. In some instances, the insecticide performance standards may
be beyond current scientific technology.
We also fail to understand how the insecticide performance standards
were developed. Some of the major insects are not covered in the
performance standards. There are different standards for some insects
within a crop for no apparent reason. It appears that the performance
standards for insecticides were decided in an arbitrary and capricious
manner. We prefer to see an across-the-board performance standard of
70% as for the herbicides and fungicides and nematicides.
[EPA Response: The Agency is pleased to note the Department's
concurrence in the general concept of performance standards for pesticides.
Such standards, when utilized in direct relation to the tests procedures
recommended in the appendices to these guidelines, should prove a major
protection to the environment and the consumer, as explained more fully
in the preamble parts I A and II B.
The Department is correct that performance standards for some section
series are more explicit than for certain other section series. The
Agency feels that more specific standards for some kinds of pest control
products would be more useful, particularly in the case of fungicide,
insecticides, and vertebrate control agents. Sections for the latter two
groups already contain such specific standards. Perhaps general standards
would be more suitable for products which control wide varieties and
numbers of organisms at one time, such as preemergence herbicides. It
would seem naive, however, to set, say 70% performance standards for
products designed to control, for example, headlice, brown rot of peaches,
nematodes in strawberry nursery stock, wood-rotting organisms, aflatoxin
organisms infesting grains, rats, poison ivy, poisonous weeds, ticks,
and pests of fabrics. A performance standard requiring only 70% control
for uses such as these would not be tolerated by the user, consumer, or
the marketplace. Buyers would never accept most food commodities receiving
such a low level of control. Yet there are instances within each of
these disciplines where 70% control might be tolerated and be considered
reasonable acceptable, such a premergence control of crabgrass, control
of tomato and potato .late, blight and control .of algae in lakes. Thus.,.
more specific performance standards linked to specific uses .-or site, pest
• combinations would, seem to be. more appropriate direction, rather than
•more general standards.. ••' '••...
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With specific reference to comments concerning the invertebrate
control sections, (series 16,3.9.5)., the'insecticide-performance, standards
used were supplied by experts in their respective disciplines throughout
the United States. These experts include State, Federal, university, and
industry scientists. Some of the performance standards were written in
the American Institute of Biological Sciences (AIBS) performance series
on efficacy test methods as part of an EPA contract to AIBS. (See Appendix
to section series 163.95 (95-30(b)) for details of the test methodologies
and standards).
In the areas of invertebrate control agents, such as control of
mosquitoes, fleas, lice, ticks, biting flies, cockroaches, fire ants,
wasps, hornets, poisonous spiders, scorpions, centipedes, and bedbugs, a
performance standard of only 70% would be totally unacceptable. No
significant public health protection would occur if a 70% performance
standard were required in these cases. Similarly, 70% control for many
insects attacking crops would result in commodities that would be unsalable
and could neither be stored nor transported satisfactorily.
The Agency has explained in the preamble and emphasizes again that
THESE PERFORMANCE STANDARDS MUST BE INTERPRETED PRINCIPALLY IN DIRECT
RELATION TO THE TESTS AND TEST STANDARDS AS INDICATED IN THE GUIDELINES
and should not be interpreted solely as applicable directly to the
commercial or marketing areas. The latter may often demand higher performance
standards than those mentioned in these guidelines. These performance
standards should be used as suggestions for guidance-purposes only.
The Agency welcomes USDA's able assistance in developing a list of
pesticide uses and site/pest combination with corresponding realistic
performance standards.]
Percentage reduction of a pest population is a good indication of
efficacy, but does not entirely address the problem of crop protection.
For instance, if there was a pest population of 10,000 individuals
attacking a crop and the performance standard is 90% then 1,000 of these
pests are still remaining which would be enough to cause economic damage.
If the pest population was only 1,000 then 100 would be remaining and 100
may not be enough to cause economic damage to the crop.
[EPA response: The Agency recognizes this fact and discusses this
problem in the preamble part II B. Generally, the percent reduction of a
pest population is avoided whenever other more suitable measurements can
be used instead, such as percentage of infested fruit, reduction in number
of eggs present, reduction in the number of holes in leaves (or nuts or
bolls), reduction in number of lesions on animals, and reduction in
percent lodging.]
[§ 163.90-3 General Requirements]
Geographic distribution of tests is a good concept to ensure that
the product will perform under a wide variety of climatic geographical
conditions. However, we 'hope that EPA will use discretion with these
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requirements and not request data where the pest does not exist or the
crop is not grown in significant acreage.
[EPA response: The Agency accepts the comments and now states in
§163.90-3(b)(7) that the pesticide must be tested in each major geographic
area where the pest is known to exist and .be of importance, or where the
crop is grown in significant acreage. When a pest control program is
intended for only one locality where the pest is known to exist in
significant amounts, test data from that locality are usually sufficient.
Further instructions regarding testing in various georgraphic areas are
located in the individual section series on efficacy.]
Steps should be taken to assure that the guidelines will not impede
the development of data to register minor uses of pesticides and specialty
chemicals such as attractants, hormones, pheromones or pathogens for use ..
in IPM programs.
[EPA response: Specific standards for attractants, hormones,
pheromones, and biological pesticides will be provided for in sections of
the guidelines to be published in the future (Subpart M). As stated in
the preamble at part II B, the performance standards are generally based
on current agricultural practices in the United States,-and are to be
used as suggestions for guidance purposes only.
These standards do not take into account performances under any of
the several integrated pest management (IPM) practices. However, if
pesticides are to be considered as part of an IPM program, they must be
sufficiently dependable to control designated pests, even though the
amounts, frequencies, and timings may differ significantly from
pest control under a non-IPM program. Therefore, it would seem logical
to use pesticides that easily achieve satisfactory non-marginal pest
control if one is to be confident of their use in an IPM program. Thus,
the argument.for lower performance standards for pesticides to be used in
IPM program is not really very credible.
Periodic revisions of these guidelines will provide opportunity for
greater attention to any such programs and any further developments as they
become recognized practices. (See also preamble Part II. D.) The Agency
will welcome comments on any means by which IPM standards could or should
be incorporated into these guidelines.)
The Agency .has .updated information on IPM by adding to the
Subdivision H discussion and to § 105-l(c), which states that "Labels for
foliar treatments, soil treatments, greenhouse treatments, shade and forest
treatments, uncultivated and non-agricultural area treatments, must
include a statement which informs the user to consult with the local
extension service or state university for information on economic threshold
levels.,, timings, "and. pest..management programs." . .
'.. :This is to-alert such .users, .to the. possibilities :of Integrated Pest
Management Programs, which may cover agricultural and:.npn^-agrioultural.
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On page .244, reference is made to'"Regardless of the site for this
performance evaluation, the equipment, the equipment operation and
adjustment, and procedure...must be identical to those employed in the
spray drift evaluation study described...in Subpart J." It is impossible
to have "identical" situations for this type of study. Also, our comment
on Subpart J indicates that additional study is needed before finalizing
these guidelines.
[EPA response: This sentence, which now appears in § 163.94-
2(a)(5)(i), was changed to state that "aerial application performance
evaluation SHOULD BE RUN IN CONJUNCTION WITH spray drift evaluation
studies as described in section series §163.126 of Subpart J." As indicated
in the Department1s comments on Subpart J, the Agency is presently involved
in discussions with the Department concerning spray drift studies.]
VI. FIFRA SCIENTIFIC ADVISORY PANEL COMMENTS AND EPA RESPONSES
FIFRA Sec. 25(a)(2)(A) requires the Administrator to provide the
FIFRA Scientific Advisory Panel a copy of any proposed regulation at
least 60 days prior to signing it for publication in the Federal Register.
This provision is to permit the Panel opportunity to comment on the impact
of this proposal on health and the environment. The specific comments of
the Panel and the Administrator's reponses thereto on the June 22, 1979
draft are published below. In this instance, comments were supplied by
Dr. H. Wade Fowler, Executive Secretary of the Panel, to Edwin Johnson,
Deputy Assistant Administrator for Pesticide Programs. For purposes of
brevity in this discussion, each-Panel comment is'followed^immediately.by
insertion of the corresponding EPA response set off in brackets. -Page
numbers mentioned in Panel comments refer to the pages of the June 22,
1979 drafts under review at the time of the formal meeting on July 19-20,
1979. During this meeting, the proposed good laboratory practices sections
of Subpart F were also reviewed by the Panel; however, all mention of the
Subpart F material was deleted from the Panel's comments reproduced below
to avoid confusion with the proposed Subpart G, I, and J presented and
discussed in this issue of the Federal Register. The Panel's comments
presented in this discussion consist of their general comments on this
Subpart.
United States Environmental Protection Agency
Office of Pesticides and Toxic Substances
Washington, DC 20460
October 22, 1979.
Subject: Review of proposed Guidelines for Registering
Pesticides in the United States
From: Dr. H. Wade Fowler, Jr.> Executive Secretary
FIFRA'Scientific Advisory Panel
To: Deputy Assistant Administrator for Pesticide Programs
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The PIFRA Scientific Advisory Panel has completed review of Subparts
G, I, and J of the Guidelines for Registering Pesticides in the United
States. The Review was completed in open meetings held in Arlington,
Virginia, during the period July 19-20, 1979. •
Attached is a report of finding by the Panel.
The report was delayed due to a shift of office resources for
resolution of Panel business relating to conclusion of the RPAR1s on
2,4,5-T and Silvex. The secretary regrets the delay and sincerely hopes
that the delay did not significantly impede progress with proposed
rulemaking on the Guidelines. Please convey our special thanks to Dr.
Preston, Mr. Jordan and all members of the EPA staff who participated
in the meeting for an excellent briefing on the important features of the
proposed rulemaking documents.
The Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA)
Scientific Advisory Panel completed review of several additional Subpart
of Proposed Guidelines for Registering Pesticides in the United States
during open meetings held in Arlington, Virginia, during the period of
July 19-20 1979. The following specific subparts were reviewed during the
meeting:
1. ..
2. Subpart G. Product Performance.
3. Subpart I. Experimental Use Permits
4. Subpart.J. Hazard Evaluation: Nontarget Plants [and
Microorganisms].
Maximum public participating has always been encouraged at all
meetings of the FIFRA Scientific Advisory Panel. In respect to this
session of the Panel, a Federal Register Notice was published on July 3,
1979. In addition, telephonic notices and special mailings were sent to
the general public who had previously expressed an interest in activities
of the Panel. Written statements relative to the Proposed Guidelines,
Subpart G, Product Performance, were received from Huntington Laboratories,
Inc; In addition to comments by EPA staff, informal comments were received
from the general public and representatives of the pesticide industry.
The superb efforts of Dr. William H. Preston, Jr. and other technical
staff of EPA, who presented various Subparts of the Guidelines to the
Panel are worthy of special recognition. The Panel wishes to commend the
Agency for being exceptionally cooperative in their working relationship
with the Panel in all matters dealing with the proposed Guidelines. This
has materially aided in the expeditious review of a large amount of
technical material. Additionally, the Panel wishes to recognize the value
of. preliminary review of massive documents such as Subpart G, Product
Performance (676 pages). Introduction of. the document to the Panel and
the public in Houston, Tessas,<'during, a special subcommittee meeting in
."November .1978 made-it'possible to..focus attention on. a limited number of. "
central .issues and avoid lost time in discussions emanating from problems
of commun-i-cation. relative' to issues easily .resolved by Agency officials. •".
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In consideration of all matters brought out in the meeting and a
careful review of the proposed rulemaking document, the Panel submits the
following 'report: • .
The FIFRA Scientific Advisory Panel is of the opinion that the...
subparts of the Guidelines (Subparts ...G, I, and J) submitted to the
Panel as proposed rulemaking during the Twenty-fourth meeting of the
Panel, July 19-20, 1979, deals effectively with the intended procedures
with a limited number of specific exceptions and recommendations:
II. Subpart G. Product Performance. This subpart is concerned
with the effectiveness of pesticide products.
A. Section 163.95-1, Efficacy of Invertebrate Control Agents. The
use of specific figures of required percent mortality and/or percent
control is meaningless, and may impede adoption of Integrated Pest
Management (IPM) programs. Panel Comments and Recommendations: The
Panel wishes once again to object unanimously to the inclusion of specific
figures for required percent mortality and/or percent control under the
individual pests discussed in Section 163.95-1 "General Requirements for
Invertebrate Control Pesticides." Very strong and virtually unanimous
objections to this meaningless exercise were raised at the Houston
Subcommittee Meeting (November 1978) by both the Panel and the attendees
at this meeting. Inclusion of these precise and often unobtainable
figures can only serve to cause endless discussion and contention between
registrants and EPA. Moreover, the precise mortality or control data is
dosage-related, is affected by genetic susceptability or resistance of
pest races, by climatic conditions, and by method of application. Under
Integrated Pest Management (IPM) it may be desirable to obtain lower
degrees of control, e.g., 50-70% mortality, than are generally indicated
in the Guidelines. This is important to protect food sources for beneficial
insects. Major changes in this procedure have been made in the revised
guidelines under Section 163.93-3, Performance Standards Acceptable Levels
of Pest Control (for Fungicides and Nematicides) where the phrase, "the
product must provide, under moderate to severe pest pressure, at least
70% control of the pest organisms or their symptoms, (compared with
untreated controls)." Lesser claims, such as, "aids in control" or
"suppresses" may be made if less than 70% control of the plant disease is
obtained. Another appropriate phrase would be, "the product must provide
good commercial control of the pest organisms or their symptoms." This
phrase would apply specifically to either standard pesticide usage or to
IPM usage, The Panel recommends that this more appropriate, and clearly
much more acceptable, terminology be applied to Section 163.95-1, "General
Requirements for Invertebrate Control Pesticides" and elsewhere in the
guidelines where appropriate.
[EPA Update: It should be noted again that these SAP comments
reflect a 1979 draft of Subpart G 'written for proposed regulations. This
current Subdivision G is written' for guidance purposes only, and the
performance standards are to be used as.suggestions in areas covered by the
waiver of efficacy data submission policy (See Part I.B. of this Discussion).]
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[EPA response: Responses to the same comments made by the U.S. Department
of Agriculture appear in Part V of this discussion. A more extensive
explanation of performance standards is now provided in the preamble Parts
. I C 3 and II B 1. Attention should be focused specifically on the
interpretation of performance standards: they should be utilized in
conjunction with the test standards and recommended test procedures of
the Appendices and should not be interpreted exclusively as applicable to
the commercial arena.
The suggested test procedures require applications at the rate or
rates specified on the label of the proposed product. Such rates are the
ones expected to perform satisfactorily regardless of genetic susceptibility,
pest resistance, climatic conditions, and methods of application if not
already specifically limited on the pesticide label.
The Agency has also addressed comments relating to IPM in Part V of
this discussion indicating the it would deal separately with the special
considerations required for IPM, biological pesticides, pheromones,
hormones, attractants.
The Agency is particularly concerned about the inadequacy of general
performance standards such as 70% control for all plant disease and pest
control products, and is hoping to obtain during the public comment period
more specific and realistic performance standards in these areas.
The Panel's suggestion that the performance standard by "the product
• must provide good commercial control of pest organisms or their symptoms"
is not particularly helpful, since such a criterion would likely require
much higher percentages in performance standards than are provided in the
proposed guidelines, percentages that would often be unobtainable using
the test procedures suggested in the Appendix. Commercial marketing
procedures would not likely tolerate much less than 98-99% pest control
or control of pest symptoms for most crop produce that must be shipped
long distances or stored for more than very short periods.]
B. Section 163.96, Efficacy of Vertebrate Control Agents. The use
of specific figures for required percent mortality and/or percent control
is meaningless, and may impede adoption of Integrated Pest Management
(IPM) programs.. Panel Comments and Recommendations: The Panel reiterates
the general comments and recommendations previously outlined for Section
163.95, Efficacy of Invertebrate Control Agents.
[EPA response: The Agency reiterates its position as explained under
A and in EPA update (above), in Part V of this discussion, and in the
preamble Parts I C 3 and II B 1.]
. [End of 'comment.'] .
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SUBDIVISION G — PRODUCT 'PERFORMANCE '•'•'•
Series 90: OVERVIEW, DEFINITIONS, AND GENERAL CONSIDERATIONS
§ 90-1 Overview; product performance.
(a) General concepts. (1) The term "product performance" refers to
all aspects of a product's effectiveness and usefulness. Typically, any
evaluation of product performance is conducted in light of expressed and
implied labeling claims or recommendations concerning pests, sites, methods
of application, application equipment, dosage rates, timing and number of
applications, use situations,•nature and level of pest control, duration
of pest control, compatibility with other chemicals, benefits and/or adverse
effects of product use, compatibility of common practices associated with
the sites, active ingredient status of chemicals in the formulation, and
equipment.
(2) Typically, initial laboratory, greenhouse, or small plot field
testing is conducted to determine the effectiveness of a substance to
control or kill specific pest organisms, or to produce desired effects on
plants or plant parts, and also to determine whether the substance has
sufficient pesticide potential to warrant larger scale testing.
(3) Effectiveness and usefulness of the proposed product is further
proven through advanced large-scale laboratory tests, field tests, in-use
tests, or simulated-use tests by procedures which closely approximate actual
use and which employ typically-used application equipment.
(4) All advanced tests must address those factors which would normally
be encountered in the use patterns claimed for the product. These factors
would depend on the type of pest and site to be treated, and may include:
specificity, degree, and duration of pest control; impact of climate on
chemical residuals and bait acceptance; nature and extent of spray coverage;
adverse environmental effects such as bioaccumulation (see Subdivision N),
and toxicity to beneficial nontarget organisms (see Subdivision E, F, J, L,
and M); increase in population levels of other pests of the target site
resulting from control of predatory or competitive microorganisms, or
interference with the performance of other pesticides; or any other factors
which would establish the safe, effective use of the product.
(5) Except as provided in 40 CFR Part 172, an experimental use permit
must be obtained to cover test trials involved in use of a pesticide that
is not registered with the Agency and test trials involving a new use of a
previously registered pesticide. (See Subdivision I for further information
on experimental use permits.)
(6) The Agency may require additional'information on benefits when
a product does not achieve the performance standards, when exorbitant rates
are used to achieve.the performance standards, or when exceptionally low
levels of effectiveness.are attained.
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(b) FIFRA mandate and waiver policy. To register a substance
as a pesticide product, FIFRA Sec. 3 requires an applicant to submit data to
show that the product will perform its intended functions without causing
unreasonable adverse effects on the environment. Performance tests are
necessary, therefore, to show that products to perform their intended
function. Hawever, § 3(c)(5) of the amended FIFRA provides that "in con-
sidering an application for the registration of a pesticide, the Administra-
tor may waive data requirements pertaining to efficacy, in which event the
Administrator may register the pesticide without determining that the
pesticide's composition is such as to..warrant proposed claims of efficacy."
As previously discussed, in 1979 the Agency issued regulations
implementing several provisions of the 1978 FIFRA amendments (44 FR 27933,
40 CFR, Part 162, May 11, 1979). Among the provisions implemented was the
efficacy data waiver authority provided by Sec. 3(c)(5) of FIFRA. The
Agency defined in § 162.182(d) the circumstances with efficacy data
were required to be submitted as a matter of course. Other requirements
that efficacy data be submitted were generally waived. The Agency is
proposed in the Regulations for Registration, Reregistration and
Classification Procedures (47 FR 40659, 40 CFR, Part 162, September 15,
1982) to extend the efficacy data waiver to additional use patterns, and
it will include all registration actions, both conditional and unconditional.
As stated in its previous waiver, the Agency's primary mandate under FIFRA
is to evaluate the health and safety aspects of pesticides. Submission of
efficacy data.will be waived for all uses of pesticides except those where
control cannot reasonably be observed and determined by the user and lack
of control results in a clear adverse health effect; or certain pesticides
under suspension or cancellation orders, or in cases where lack of efficacy
is reported; or in evaluation of benefits when substantial risks have been
identified; or when factors exist that make submission of such data necessary
to support the presumption that it is efficacious. The specific uses that
require efficacy data are specified in 40 CFR Part 158 - Data Requirements
for Registration § 158.32 with references to the testing methodology and and
protocols in Subdivision G - Product Performance. The level of label
claims are described in Subdivision H - Labeling Guidelines for Pesticides
Use Directions (§§ 100 thru 106) and guidance is also given to test methodology
needed to support these claims when necessary. Details of the extension
of the efficacy waiver are:
(1) With regard to public health uses, the Agency has determined
that a threat to human health exists when the presence of the
pest organisms cannot be readily observed by the user. Such
uses include, the following ones listed and product performance
data are, therefore, required to be submitted on products having:
(A) Uses of antimicrobial agents intended to control pest
••:'•: ... •microorganisms. (.except bacteria, .pathogenic, fungi,, or
viruses, living on. or in man or other, animals') that pose, a threat
to human health and whose presence cannot readily be observed
,'• . ' ..- by. the'user, including, but not limited to,, microorganisms .
r.\\.;; 'infectious to .man in- :any area- o-f •ehe--i&atrifflate :environment-;• . ' ;
y.'G:B;)'* Uses -of -fungicides .intended for .control o:f organisms that .••'.' . -
." - -product mycotoxins* .'''..- : .
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(ii) With regard'to certain pesticides under suspension or
cancellation action or subject to a Notice of Special Review
under Section 162.11(a)(3)(ii), the Agency has determined that
product performance data are required to be submitted to
support any new or additional uses of products that are:
(A) Already under suspension or cancellation action by the
Agency for reason that some uses cause human or environmental
safety hazards, provided that the risks identified in the
suspension or cancellation action also apply to the new uses;
or
(B) Already subject to a Notice of Special Review, provided that
the risks identified in the Notice also apply to the new or
additional use(s).
(2) For those products for which the Administrator will ordinarily
waive the requirement for submittal of efficacy test data as indicated in
paragraph (b)(l) of this section, the Agency reserves the right and
authority to require, on a case-by-case basis, submission of such efficacy
data or other test data for any specific product, registered or proposed
for registration, whenever the Agency deems that such data are necessary
to make proper evaluations for a decision as to acceptability for
registration or continued registration.
(i) With regard to conditions where the Agency may request efficacy
data but not be limited to these causes for any product,
registered or proposed for registration such as when:
(A) A lack of efficacy has been reported for it; or
(B) The Agency needs such data to evaluate benefits of the
pesticide (or of alternative pesticides) when substantial
risks have been identified; or
(C) Factors exist that make' submission of such data necessary
or desirable to support the presumption that it is
efficacious.
(ii) Thus, the guidelines in this Subdivision shall be used by
registration applicants as efficacy testing standards in
conjunction with label claims and efficacy data reporting
requirements when efficacy data must be submitted to support
registration applications; in addition, the guidelines should
also be Used to provide helpful guidance on testing to
determine the claims arid directions for use on labeling to
products for which efficacy data submittal is waived.
(c) Relation of general considerations to specific considerations.
(1) The provisions contained in § 90-3 General considerations apply to
most pesticides except antimicrobial-type pesticides. To understand the
product performance considerations fully, the registrant"should read
§ 90-3 along with the appropriate sections dealing with specific kinds of
products. Because of their unique and complex use patterns, antimicrobial
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type pesticides are covered in §§ 91-1 through 91-8 which contain both
general and specific requirements for these pesticides.
(2) The actual test procedures will vary according to the character-
istics of the chemical, the type of formulation, the target pest, the use
patterns, the methods and timing of application, and many other factors.
But certain basic techniques are essential, and these are discussed in
§ 90-3 General considerations: product performance. The relationship of
test results to label directions for use and limitations is described in
§90-3 and in sections on specific uses elsewhere in this subdivision. The
applicant or registrant should study these appropriate sections as inte-
grated parts of the general and specific guidelines for Subdivision H -
Labeling Guidelines for Pesticide use Directions.
(3) Identification of explicit methods in these guidelines for all
uses is not practical, because an acceptable test for one product or use
may not be valid to support another product intended for the same use, or
for the same product intended for even a slightly different use. Examples
of acceptable methods are provided in specially designated sections of the
guidelines, but the applicant or his testing agent must be responsible for
the validity of any particular test or tests employed.
(d) Relation of Subdivision G to other subdivisions of the guidelines.
(1) Relation to 40 CFR Part 158 Subparts A and B. The registration
applicant is referred to CFR 40 Part 158 — Data Requirements for Registration
for general provisions and policies pertaining to registration data require-
ments -including policy on flexibility in relation to. deviation from test
standards and acceptable protocols, when tests are required (§158.32).
and requirements for additional data.
(2) Relation to Subdivision H. The registration applicant is referred
to Subdivision H for guidance on pesticide labeling. Label claims in
Subdivision H should meet the performance standards in Subdivision G when
applicable. An important objective of the testing programs described in
Subdivision G is to develop sufficient data to support appropriate and
adequate efficacy related labeling claims and directions for use. The
applicants should read the general and appropriate specific sections of
Subdivision H before initiating efficacy testing in support of Subdivision G.
(3) Relation to Subdivision I. An experimental use permit may be
required for trials involving use of a pesticide that is not registered
with the Agency and test trials involving a new use of a previously regis-
tered pesticide. These permits are issued in advance of proposed field
studies after submitting proper applications for the Permits. For specific
details and information on Experimental Use Permits, see Subdivision I.
. . (4). .Relation to. Subdivision J. The registration applicant is referred
• to Subdivision. ,J. Hazard Evaluation: Nontarget.Plants for data, guidance
on pesticides that:contact plants through direct applicatio-n or through
movement of pesticides in the environment, and submiss-io.n of. data on adverse
phytotbxic .effects, to .nontaiget plants both: within, the target area..and.
..outside the,'target'area.,"•'•'.''.' "•"'./..'''' ' • 'J.i.-,.^...^ ,; '• :'''':: .'•''• ... . '... ' ...
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§ 90-2 .Definitions* .-• • "• . ...-.-
Terms used in this subdivision shall have the meanings set forth in
FIFRA, at § 162.3 of the FIFRA Sec. 3 regulations, at § 302, and at
§ 60-2. In addition, for the purposes of this subdivision:
(a) The term "effective dosage range" or "EDR" refers to the range
of dosage levels beginning with the lowest dosage capable of achieving the
level of control specified by the applicable performance standard for the
least taxing conditions under which it will be used (e.g., pest levels,
soil types, water conditions, geographical and climatological conditions,
etc.), and ending at the losest dosage required to achieve the specified
level of of control under the most taxing conditions under which it will
be used.
(b) The term "effectiveness" refers to a product's ability to control
the specific target pest or produce the specified plant or animal response
when the product is applied in accordance.with the label directions direc-
tions, precautions, and limitations of use. The term "effectiveness", as
used in this subdivision, is synonymous with the term "efficacy".
(c) The term "full coverage", as used in common agricultural practice,
refers to a volume of spray applied to plants to the point of runoff or drip.
(d) The term "large scale plot" refers to any plot large enough to
permit the use of typical commercial application equipment (when such equip-
ment is needed for pesticide application). . :
(e) The term "low volume" or "LV", as used in common agricultural
practice, refers to a total volume of spray applied broadcast at more than
0.5 gallon but less than 5.0 gallons per acre (more than 1.89 liters but
less than 18.93 liters per hectare) I/ or less than a full coverage spray.
(f) The term "minimum effective dosage" or "MED" refers to the' lowest
dose level at which the test substance achieves the level of control speci-
fied by the applicable performance standard.
(g) The term "performance data" refers to any data pertaining to pesti-
cide effectiveness and usefulness.
(h) The term "serial application" refers to the label recommended use
of a pesticide on a site before or after application of another pesticide to
that site, such that the presence of one of the pesticides may affect the
effectiveness and usefulness of the other.
(i) The term "ultra low volume" or "ULV", as used in common agricul-
tural practice, refers to a total volume of 0.5 gallon or less per acre
(1.89 liters or less per hectare) broadcast. 1/
I/ For further information, refer to: the American Society of A^ricul-tural
Engineers' Pesticide Application Subcommittee of the Agricultural Chemical
Application Committee. 1978. Uniform;terminology for' pesticide spcaxing
ASAE Handbook, ASAE-S327 (Agricultural Engineers Yearbook), p. 313.
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37 .
§ 90-3 General considerations.
(a) Scope of considerations. (1) The registration applicant is reminded
that certain efficacy data submittal requirements are waived as described
generally in § 90-l(b) of this subdivision. Therefore, while the other para-
graphs in this section and the other sections in this subdivision establish
requirements concerning the methodology of efficacy testing and the content
of test reports, they do not independently establish any data submittal
requirements.
(2) The standards contained in this section apply generally to all
studies in this subdivision [except for antimicrobial agents (§§ 91-1
through -8)], unless another section of this subdivision contains a specific
standard on the same subject. In such a case, the specific standards in
the other sections shall apply to the conduct of that particular study.
(b) Test standards. (1) Personnel. All testing and evaluation should
be done under the direction of personnel who have the education, training,
and/or experience to perform the testing and evaluation in accordance with
sound scientific experimental procedures. The Agency may require resumes of
personnel who have performed, supervised, reviewed, or evaluated the testing.
(2) Test substance. (i) The test substance shall generally be the
formulated product.
(ii) In addition to or in lieu of data otherwise mentioned by this
subdivision, the Agency may require, after consultation .with the applicant,
data derived from testing to be conducted with:
(A) An analytically pure grade of an active ingredient with or
without radioactive tagging;
(B) The .technical grade of an active ingredient;
(C) The representive technical grade of an active ingredient;
• (D) The inert ingredient of a pesticide formulation;
(E) A contaminant or impurity of an active or inert ingredient;
(F) A plant or animal metabolite or degradation product of an active
or inert ingredient;
(G) The end-use pesticide product;
(H) The end-use pesticide product plus any recommended vehicles and
adjuvants;
(I) Any additional ^substance, which could act .as.a synergist to the
product ,for which'registrations is sought; or '••''"".•'•'•'• •;'.'. :
(J.)- Any combination of substances metttio-hed in paragraph (b.)-(2JCii.)
:of this. section:. .. \, •..••'.-..•.• : -. .'-•'"•'.-:.•'' ..'.••'•
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(3) Dosage rates, (i) Typically, the test substance should be tested
at various dosage levels including the dosage rates associated with the
proposed use. Dosage rates should be tested as requested by each section.
Special attention should be paid to treatment rates on food crops in rela-
tion to the tolerance or proposed tolerance. Additional guidance on selec-
tion of test dosage rates may be found in the specific discipline sections
of this subdivision.
(ii) The test program (sum total of preliminary and final stage tests)
should establish clearly the effective dosage range (EDR) or the minimum
effective dosage (MED) as appropriate for the uses involved. The develop-
ment of an EDR, rather than a single MED, is encouraged, whenever feasible
and practical, because the EDR permits at least some of the users the
opportunity to use rates other than the maximum rate needed to cover all
use situations.
(4) Serial applications. The label use directions may specifically
direct serial applications of different products, such as when tank mixing
is impractical. These directions should be supported by tests designed to
compare the effectiveness and usefulness of application of each product
alone with applications of the products applied serially. Special emphasis
should be directed toward determining whether or not a minimum time interval
between applications of the respective chemicals is warranted.
(5) Package mixtures. These products contain more than one active
ingredient. Data are needed to establish the efficacy of each active
ingredient in a package mixture. However, since it is the efficacy of the
.formulated product/use-pattern combination that is to established, testing
as part of an experimental use permit program is usually conducted with
the package mixture only. In many instances one or more of the separate
active components will have been previously registered as a single-component
product. In such cases, these data may be included as part of the data
base for the package mixture when suitable comparability data have' been
developed to demonstrate that each active ingredient is effective and safe
to use on the target site regardless of whether it is used alone or in the
package mixture. Note; package mixtures which result in a significant
amount of inappropriate or unnecessary usage (dosages, certain active
ingredients, inappropriate timing, and unnecessarily high number of applicat-
ions) of one or more of the active ingredients are not acceptable.
(6) Tank mixes. Product labeling which implies or recommends mixing
products in the spray tank before application should have acceptable suppor-
ting data as described below.
(i) Directions for tank mixing of products should-be supported by per-
formance data on each component (of the proposed mixture) tested separately
as well as data on the mixture used at the dosage rate(s) specified for each
pest indicated. The combined minimum and combined maximum dosage rates of
each product in the tank mixture should be tested. [Guidance pertaining
to tank mixtures for environmental fate data appear in § 164-4(4) of Sub-
division N. ]
(ii) The components of a pesticide tank mixture should be physically
and chemically compatible. Evaluations of physical compatibility should be
conducted using maximum rates of each component in minimum recommended
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39
volumes of diluent per acre or hectare, demonstrating effects of order-of-
component-addition to the tank, and evaluating effects of water hardness,
pH, and temperature on separation, suspendability , and sprayability . Where
compatibility is questionable from static tests, actual testing should be done
which should employ constant agitation of the mixture as in most commercial
field sprayers. Note ; tank mixes which result in a significant amount of
inappropriate or unnecessary usage (dosages, certain active ingredients,
inappropriate timing, and unnecessarily high number of applications) of
one or more of the active ingredients are not acceptable.
(7) Adjuvants. Products with labeling which allows. or recommends
the additions of separately packaged adjuvants to the -spray tank should be
supported with data indicating their benefits (if claimed) and any detri-
mental effects (such as increased crop phytotoxicity) which may result
from their addition to the herbicide, plant regulator, desiccant, or
defoliant. The only adjuvants actually permitted for use with a
pesticide will be those adjuvant brand names or defined adjuvant classes
specifically named on the pesticide label. The adjuvant rate or range
of rates should be named on the pesticide label. The adjuvant rate or
range of rates should be indicated on the pesticide label, and should be
supported with data on efficacy and any detrimental effects. If a range
of adjuvant rates is recommended, the maximum and minimum rates within
that range should be evaluated in conjunction with the intended pesticide
product. (Additional details on adjuvants are discussed in § 94-2 (a) (9)).
(8) Geographic distribution of tests. Pesticide products marketed
on a nationwide basis may be used under a wide variety of conditions. The
pesticide should be. tested in each major geographic area where the pest is
known to exist and be of importance, or where the crop is grown in signifi-
cant acreage. When a pest control program is intended for only one locality
where the pest is known to exist in significant amounts, test data from that
locality are usually sufficient. Further instruction regarding testing in
various geographic areas are located in the individual section series on
efficacy.
(9) Test design and statistical procedures, (i) General test design.
Sound statistical designs and procedures are necessary to assure that valid .
and appropriate statistical analyses of the data can be performed and that
any observed statistically significant differences are attributable to the
respective pesticide treatments, and that such pesticide treatments provide
the expected pest organism response. Direct comparison of group means of
treated sites and untreated control sites is usually sufficient for evalua-
ting treatment effects. The test results, however, for most pesticidal pur-
poses, should show more than just statistically significant differences
between treated and control sites: the differences should generally be of a
magnitude which meets or exceeds the performance standards described in each
of the subsequent sections of . this subdivision. [See paragraph (d.) of this
section .fe.r ^ , more; ^information. on performance standards.] For useful refer-
ences on- test -design, and . statistica-i procedures, ; -see references .2-r5: in
§. 90-30. ".'•"-• ..•"; .. ; . • .- -; :. . ':. V :"''•;- •
(;li.):: ••/-; Mul-fcLjjle, sifcey.aitd, pest. -target ^combina.'faioris..- ' When- mo-£e-, 'than.
or .s-ite is:.' involved, in- -pesticide applications., separate "tests-'are
usually necessary. tp,.:eyaluate product .performance. again-S-t^each .kind 6-f-;j>est
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40
or each kind of plant under each set of variables or use conditions. For
vertebrate control agents, however, it is preferred that efficacy be evalu- "
ated on one pest species at a time. If more than one method of application
is to be employed (such as, for example, air and ground sprays, drenches
and injections, or impregnation and surface coating), experiments should be
designed to obtain the required data for each method, on or in the same
experimental sites, and if separate evaluations can be made, their respec-
tive levels of control can be assessed during the test.
(iii) Replicates. Generally, the number of replicates necessary to
demonstrate treatment differences will depend upon several factors, such
as variability of test organisms and materials (crops, pests, application
equipment, soil conditions), magnitude of treatment effects, and the
desired statistical confidence level.
(iv) Sampling procedures. Sampling procedures should assure that all
of the characteristics of the test population to be measured are represented
in the samples. The size and number of samples necessary for reliable esti-
mates will vary mainly with the level and uniformity of the organism or the
effect to be measured as well as the size and precision of available equip-
ment. For example, entire replicates may need to be harvested to make
accurate yield estimates or to measure plant growth responses when low pest
populations are present, while representative portions of each plot will
probably be sufficient for measuring high density pest populations.
(v) Considerations for crop test designs. In designing the test, all
variables, both uncontrollable (such as so'il texture and microclimate) and
controllable (such as irrigation, cultivation, pruning, fertilization,
cultivar, and test product application) should be considered. Care should be
taken to duplicate carefully all controllable variables (other 'than test
product application) on all treated and untreated plots.
(A) Plot sizes. . Plots should be large enough to reflect actual use
conditions and to allow representative application techniques, which may
include commercial application equipment. Small plots, such as a single
10-ft (3.3m) row of vegetable and forage crops, and single-branch or single-
tree plots of fruits, are ordinarily insufficient to support valid conclu-
sions about product performance. Factors such as the crop grown, equipment
used, expected incidence of the pest, need for residue samples, yield data,
and quality studies should be considered in selecting the size of field
plots. The plots should be located within a field so as to be representative
of conditions throughout the field. Areas of fields such as borders and
atypical wet or dry locations must generally be avoided, unless these are
the optimum areas for pest damage. Ifore specific guidance as to adequate
test plot sizes is provided in the sections on specific pesticide types
which follow this subpart.
(B) Crops or sites treated. A representative number of the major
cultivars of crops should be represented in the tests. Cultivars should
be more extensively utilized as a test variable to demonstrate adverse
responses in tests. .
(C) Climatic factors. Th« testing schedule should be designed to per-
mit evaluation of the effectiveness of pesticides applied under different
environmental- conditions such as low versus 'moderate and high precipition,
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41
cool and cloudy versus normally hot and sunny conditions, and low versus
high humidity and dew formation, as appropriate to the product use.
(D) Edaphic (soil) and other substrate factors. The effectiveness of
a pesticide product can often be influenced by the type of substrate to
which it is applied. Therefore, testing procedures should be designed to
evaluate product performance on those surfaces or substrates intended for
treatment. A number of variables relating to soil, such as soil temperature,
texture, fertility, pH, organic matter content, moisture content, tillage
practices, irrigation, and crop rotation schedules, measurably influence
performance of soil-applied pesticides. Accordingly, field tests should be
designed, as appropriate, to assess the potential effects of the pertinent
variables. Data to determine safety to crops planted in the treated area
the same season and in following seasons should be submitted. [See § 121-1
of Subdivision J on phytotoxicity and §§ 165-1 and -2 of Subdivision N.]
(E) Spray volume. The volume of spray is another important variable
affecting the performance of pesticides because it directly relates to the
distribution and coverage obtained on the target site. When appropriate,
testing procedures must be designed to determine the acceptable range of
spray volume to be used with the intended application equipment. Special
emphasis should be placed on obtaining data on the minimum and maximum
spray volumes when a specific amount of product is intended for use in a
range of spray gallonages.
(P) Timing of applications. Test reports should specify the time at
which treatment was begun, duration of exposure (if applicable), and inter-
vals between succeeding applications. For example, data on crop treatments
should include.the following information (when applicable) in relation to
timing of applications: •
(1) Date(s) of treatment(s) and harvest.
(2) Treatment time in relation to number of days before or after
planting, plant emergence, or harvest.
(3_) The stage of growth of the crop when treatment was made.
M) The stage of growth or expected appearance of the pest at treat-
ment time.
(J5) Duration of exposure to pesticide treatment.
(<j) Treatment-to-observation interval(s).
(G) Seasons, for pesticides used outdoors where variations in clima-
tological conditions alter efficacy, the seasons during which the tests
were conducted shall be reported.
!. (c),' .Suggested performance' .standards. (IT When efficacy data submissions.
.are hot. ^waived, performance standards will .represent the levels'of product
performance that axe. exhibited by .pesticide^ ,o.n .specific site/pest combinations
.and considered/acceptable' for registration,,purposes. .These • standards, however .
afe not,.always-absolute or. .inflexible.. ' .Labeling .statements-.^-claims)
described in Subdivision H are based on' the performance standards.and
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42
utilize tests in Subdivision G. Data showing deviation from the standards,
however, may prompt the Agency to request additional information on benefits
(e.g., increased yields, unblemished fruits, reduction in nuisance pest
levels, etc.), such as from the use of high rates to achieve standards
greater than stated in the guidelines, or from low levels of effectiveness
in relation to wasteful applications and unnecessary pollution of the
environment.
(2) The suggested performance standards alone do not reflect the entire
perspective of what is expected regarding a pesticide's effectiveness.
To meet a performance standard, a pesticide would frequently be expected
to control pests when they are at levels that cause economic damage (such
as to crops). Demonstration of (say) 70% control when pest numbers are
far below economic injury levels would not be adequate proof of efficacy.
(3) Suggested performance standards are usually expressed as percentages
of pest control, or percentages of other intended responses, calculated
from measurements made on treated plots compared with those made on untreated
control plots. Reliance only on untreated control plots, however, is not
always sufficient or appropriate, particularly when testing on very large
areas against mobile pests, when testing dog repellents, or when conducting
tests in mobile substrates, e.g., herbicides applied to moving bodies of
water. In such cases, a product may be.tested against some other base,
such as against another formulation or chemical of known efficacy, or by
comparison to pest levels or damage measured before and after the test.
For most pest control patterns, efficacy data should be obtained under a
full range of pest severity conditions, with particular emphasis on the
maximum pest severity likely to be encountered by users.
(d) Adverse effects. To the extent possible, efficacy tests should be
designed to evaluate possible adverse effects resulting from use of the
pesticide. The following are examples of adverse effects which should be
considered:
(1) Phytotoxicity. Details of data .submitted on phytotoxicity are
provided in Subdivision J of these guidelines, but the following explanation
may serve as an introduction for those persons developing product performance
data. A good test design providing for dosages higher than necessary for
pest control on -plants will allow an estimate of the adequacy of the margin
of safety between effective pesticide,levels and those which may injure the
plants intended to be protected. Phytotoxicity is usually measured in terms
of chlorosis, malformed plant parts, leaf burning, plant wilting, stunting
(reduced height), reduced stand, and death. For certain uses, some injury
can be tolerated (depending.upon the reversibility of effects, or on economic
or aesthetic factors), but all injuries should be evaluated and reported. Ac-
cordingly, the lack of observable phytotoxic effects should also be reported.
(2) Effects on quality of commodities and inanimate objects. Test
programs should be designed to evaluate adverse pesticide effects on treated
commodities and surfaces, such as discolored and weakened fabrics, deterior-
ated food quality, decrease in wool quality, milk production, and unsightly
residues on plant plan-t foliage. Taste panel, tests, color determinations,
blemish counts, livestock palatability trials, or other similar measures,
should be considered for incorporation into the test program, depending
upon the end use of the commodity. For information on sensory methods of
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43
evaluation (flavor, odor, taste, palatability , and repellency) , see reference
1 in § 90-30.
(3) Yields and other effects. Such determinations will aid in
advanced planning in the test design. Pesticide treatments may decrease
yield, reduce crop quality, or so alter the normal ripening or maturing
process that economic problems arise in harvesting. Data should address
the absence or the extent to which such effects occur.
(4) Effects on wildlife and aquatic organisms. Refer to Subdivisions
E and L of these guidelines for test requirements to evaluate adverse
effects on wildlife and aquatic organisms. Observations and evaluation of
efficacy test results should include relevant information about possible
increase in harmful nontarget organisms as a result of the pesticide use
and application/ as well as possible increase in beneficial organisms to
intolerable levels, and possible adverse effects of presence of pesticides.
Treated dead and dying pest organisms as potential food for domestic or
wild nontarget organisms.
(e) Test descriptions and data reporting. (1) Extant of report.
When applicable, systematic and complete descriptions of the tests employed
and accurate reporting of data derived from laboratory tests and field
tests to support label claims for performance of a product or mixture may
be essential for proper Agency review and evaluation. Both English and
metric units should be given for all rates and measurements for laboratory
tests; English units. alone are sufficient for field data, but both measurements
may be supplied. Application rates expressed as "ppm" must indicate the
basis (weight/weight or weight/volume) .
(2) Assembly of report. Considerations for assembling the reported
efficacy data to expedite Agency review of the detailed report include:
(i) An index of the test reports arranged primarily according to the
general types of performance data and secondarily by the site/pest combina-
tions on the label. Additional subdivisions based on methods of application,
soil textures, geographic areas, or other pertinent variables are encouraged
wherever it is feasible and will facilitate an evaluation of the data. It
is recommended that numbered tabs be used to identify the individual test
reports .
(ii) Tabular summaries of the data. It is recommended that each hori-
zontal line (or series of several lines) be equivalent to a test, and that
columns reflect the major test variables being reported, such as those
details listed in paragraph (e) (3) of this section. These summaries should
be organized primarily according to site/pest combinations and secondarily
according to pertinent variables, such as methods of application, soil tex-
tures, or test locations. The purpose of summary tables is to present a
.condensed and simplified overview of the scope of the test, .program and the
.level of. product. .performance obtained.* In order to. achieve :. maximum/. utili-
zation of space in. tables* the use of abbreviations , acronyms,, or defined
codes as ,wel,i as the grouping, of several tyj?e.s .of information, within columns
is. encouraged:...;/-. ..... . . . . . "-.''-.•':•.'
"('iii'J C Summarize'^ •£Qte'!i&S±o--aS related" to' label -claims >' frata analyses ...
and evaluations ,sliouid;;be included. . );•'"'•/ '-. •"••'•, -. • ("•"''
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44
(3) Details of report. All details of the tests should be reported,
giving particular emphasis to variables'that relate to the label directions,
limitations, and precautions. Such details may include:
(i) Personnel data. Names, positions/ and addresses of persons who
conducted and supervised the tests should be reported. Names of all persons
who recorded or generated data for the tests should be made part of the
record (not submitted but held as records) along with the dates when the
items of data were recorded.
(ii) Test substance. (A) Identification should be made of the test
substance, including chemical name, molecular structure, and qualitative
and quantitative description of its chemical composition as required by
Subdivision D of these guidelines.
(B) Manufacturer and lot sample numbers of the test substance should
be reported.
(C) Type of formulation and content [percent and, for liquids, pounds
per gallon (kg/1)] of active ingredient should be reported. When a product
is diluted before or during application, the report should specify the quanti-
ties and identification of each diluent.
(D) For many pesticide products, data on similar formulations may be
used to supplement data on the specific formulation when the comparability
of the formulations has been demonstrated. This procedure is not acceptable,
however, for vertebrate control agents because very slight differences in
formulation may cause marked differences in efficacy due, principally, to
the highly developed sensory perception of many vertebrate pests.
(iii) Testing period. Report dates during which each test was con-
ducted.
(iv) Method of application. The methods and types of pesticide
placement, such as surface, sub-surface (as in soils), or incorporated,
shall be reported. Descriptions of surface placements should indicate the
method of application, such as wiping, soaking, mopping, dusting, painting,
spraying, trail-building, broadcast seeding, or scoop placement.
(A) If the surface is to be sprayed, details on spray volume, such
as ultra low volume (ULV), low. volume (LV), concentrate, or conventional
.full coverage spray, should be given in terms of volume per unit area.
(B) For sub-surface methods, descriptions should indicate whether done
by furrow placement, side-dressing, injection, or burrowbuilding. When
describing injection methods, such information as spacing, number, and
arrangement of chisels with respect to the row and depth of injection should
be given.
(C) Descriptions of incorporated methods of application should indicate
whether done by mixing, drenching, or impregnating. [For the latter case,
pursuant to § 162.4(b) and (c) of the FIFRA Sec. 3 regulations, impregnated
articles for which pesticidal claims are made or implied as to protection
of other surfaces or objects are considered to be pe'sticides.] Details' on
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45
the methods and equipment used and depth of incorporation may be requested
for agricultural applications to soils.
(D) Information detailing the type of application, such as row (furrow
placement, band, or side-dressing) or broadcast, bait boxes, swath placement,
pressure-treated, or soaked, should accompany the test report data. Pesticides
applied as row or band treatments should specify the band width and the amount
of material used per unit of linear row distance, and the amount of material
per acre (or hectare) and the row spacing. When per acre (hectare) figures
are included in test reports for row or band treatments, the report should
specify whether these figures represent the "actual" amounts of pesticides
applied or the "broadcast equivalent" rates. ......
(v) Equipment. (A) The types of equipment used,should,be reported.
This may include such items as mistblowers, cyclone seeders, hydraulic
ground sprayers, artificial perches, dusters, burrow builders, soil
injectors or incorporators, aircraft systems (specify whether fixed wing
or helicopter), metering devices, smoke generators, rodent guns, impregnators,
and aerosol dispensers.
(B) For mistblower applications, information on spray volume, air
velocity, swath width, distance from nozzles to the target, and angle of
air flow from the vertical should be given.
(C) Ebr pesticide application through irrigation systems, the infor-
mation includes: the types of systems used, such as sprinkler (stationary
or mobile), furrow, drip, or flood; the quantity of water applied; description
of the pesticide metering devices; pesticide concentrations in water
samples collected at various points throughout the system; and any spatial
arrangement of crops (if applicable).
(D) When pesticides are to be mechanically incorporated into soil,
information should be reported on the equipment used, speed and depth of
operation, number of passes over the treated area, and intervals between
repeated incorporations (if applicable). Sufficient information on
mechanical incorporations into soils or other growth media used at sites
other than fields may be needed.
(vi) Dosage rate. (A) The dosage rate expressed as active ingredient
and formulated product should be reported. Dosages should be reported in terms
similar to the following: amounts per unit of surface area, per unit volume
of solvent or diluent, per unit volume or weight of commodity, per unit
.volume of space, per unit area and depth (acre-foot or hectare-meter), per
linear distance of crop row, per animal, per unit weight of animals, and
the length of time of spraying and the distance from the target surface (as
for certain pressurized products).
(B) When other pesticides are applied in the test area, the rates of
application for each, product, the. .identification..of-..pesticides used, and
the .timing of 'application should be reported.. ,.
(C.)l,Spray, volumes should be included: in the.data.reports.
• •;-.(vi-i-);• Description of .application' •s'i'te.r--..•(•A;)'.-. The, 'site of .application. •• ••
should be reported. -.- ; ' . -:! ,_• .- .....:.
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46
(B) The following information should be taken into consideration in
describing'the sites, contact pesticides are applied to the pests themselves
or to plant parts for which plant regulator, desiccant, or defoliant activity
is intended. Residual pesticides are applied to substrates or surfaces
which will in turn be contacted by pests.- The specific site of application
required for effective use of residual pesticides may be related to feeding
or behavioral habits of insects and animals, characteristics of plants, mode-
of-action of the pesticide, or the location of infection sites of plant
pathogenic fungi and bacteria. For example, dogs establish urinary scent
posts, and certain repellents should be applied to these areas to be effective;
or, as another example, since mites feed principally on the undersides of
plant leaves, nonsystemic miticides should be applied to both surfaces to
control those feeding and those crawling around. The site of application
may also be directly related to the mode-of-action of the pesticide. A
systemic insecticide may, for example, be applied to foliage or the root
system so it can be transported to various parts of the plant where it will
kill or repel feeding insects. A.plant regulator may be sprayed on the
foliage and cause the desired response at parts of the plant, that were not
directly sprayed. A herbicide may be applied to the soil, be absorbed by
the roots, and be translocated through the stem to the foliage where it
exerts its pesticidal action.
(C) Texture of soil and its organic matter content should be reported if
applicable to the pesticide usage. In situations where pesticides are inten-
ded to act through soil or in burrows, conditions such as tilth, compaction,
drainage, moisture, mineral content, temperature, and pH should be reported.
(D) Dimensions of test plots or sites and number of replicates should
be reported. The type of experimental design used such as detailed descrip-
tion and diagram of the experimental test area should be reported.
(E) Number and length of crop rows, row spacing, and plant spacing
within rows, if applicable, should be reported.
(F) If crops or crop sites are treated, a statement regarding cultivar
name and other distinguishing characteristics (e.g., level of pest suscepti-
bility) should be reported.
(G) When buildings are treated with pesticides, the number of rooms,
their dimensions, and their spatial arrangements should be reported.
(viii) Geographic areas. Geographic areas (state, county, and town)
where the tests were conducted, and the rationales for selection of these
sites, should be reported.
(ix) Climatic factors. Critical- environmental conditions at applica-
tion time, such as precipitation, temperature, sunlight, humidity, and
wind velocity, should be reported. Abnormal climatic conditions may occur
within a given area which cannot be considered in the test design but
these may markedly affect results. Such conditions and effects observed
should be reported in the discussion or conclusions. . .
(x) Pest populations and crop stage. Target pest population levels
at the beginning of treatment, at periodic intervals after treatment, and
at the end of the test'.period should be reported when .applicable. The
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47
growth stages of the pests and host plants should be reported. Crop growth
stage should be referenced to the number of days before or after planting,
emergence, or specific development stage or to its height. Whenever
possible, the general level of the pest problem being tested should be
characterized (light, moderate, severe, or similar phraseology).
(xi) Cultural practices. When applicable, information is needed
on cultural practices that may affect pesticide application to crops
because of their impact on product performance. Where applicable, the
report must include information on seedbed preparation, seed planting
depth, cultivation practices, and supplemental irrigation. Additional
details on irrigation practices as an experimental••vari.able--'are discussed.
in paragraph (e)(3)(v)(C) of this section.
(xii) Observation times. The interval between treatments and obser-
vations for pest control should be reported. Observations for efficacy and
adverse effects should be made at intervals which indicate minimum response
times and duration of effects. Dates observed and percent control of
specific pests or plant responses to growth regulators, desiccants, or
defoliants compared to untreated controls and to commercial pesticides
(if used as standards) should be reported.
(xiii) Unusual events. Pertinent comments regarding effects test con-
ditions on performance should be reported, particularly when they adversely
affect the level of product performance or would invalidate the test data
obtained.
(xiv) Mode of pesticide entry, movement, and action. A description
of the mode of action and movement of the pesticide (e.g., translocation,
tenacity, redistribution through rain) should be submitted or referenced
when known. -For a pesticide used to control vertebrate animals, the report
should note how the pesticide enters the pest organism, such as by body con-
tact, inhalation, oral ingestion, or by any combination of these routes.
(xv) Statistical procedures. A description of the statistical proce-
dures used in the test design and analysis should be submitted.
(4) Performance evaluations. (i) A special section of the test
report should be devoted to product performance evaluations. The following
are examples of systems that may be used to evaluate the submitted data:
(A) A rating scale (or percent) showing performance related to effi-
cacy and commercial acceptability as a rating score. Descriptive criteria
for each numerical value if a rating scale should be presented.
(B) Dose-response data for all site/pest combinations for which
registration is proposed.
;. .: (C) .. Clearly defined..statements of benefits, .such as increased yields,
unblemished.fzuits^ reduction in nuisance pest levels, reduced.disease
.incidence, fewer rat bites., .to be derived .from the pesticide use. The
applicant should,. indicate ;what lie considers, to :>e.. a commercially, acceptable;.
level of pest control. .- : :- ';: ••:•'.:-.-• _..".;': ' '.'• '''•:,.,"-^.---:----'-: ;/ "•'••• . >• "••V"i" •'
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48
(ii) Refar to Subdivision J for reporting phytotox.icity. Report
other adverse effects such as spotting of paint, weakening of cloth or"
fibers, presence or odors of dead pest organisms, secondary poisoning, j
increase of nontarget species to intolerable levels, and similar adverse
or undesirable results*
(f) Supporting statements. An applicant may submit written statements
of opinion regarding the efficacy and limitations of a particular product,
when expressed by individuals reasonably expert in observation and having
experience with repeated use of such products. Evidence of the expert's
experience should accompany such statements. Testimonials or letters of
recommendations from individuals with less than the qualifications described
in this paragraph are not acceptable as support for effectiveness claims.
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49
§ 90-30 Acceptable methods.
Reference 1 is useful in developing test procedures on sensory methods
of evaluation. These include test procedures on flavor, odor, taste, pala-
tability, and repellency. [See paragraph (d)(2) of this section.]
References 2-5 are useful in developing sound test designs and employ-
ing statistical procedures for evaluations of product performance. [See
paragraph (b)(8) of this section.]
1. Anonymous. 1968. Manual on Sensory Testing Methods. STP 434. Amer.
Soc. for Testing & Materials, 1916 Race St., Philadelphia, Pa. 19103.
82 pp.
2. Oochran, W.G., and G.M. Cox. 1957. Experimental Designs, 2nd Ed.
John Wiley and Sons, Inc.: N.Y. 611 pp.
3. LeClerg, E.L., W.H. Leonard, and A.G. Clark. 1966. Field Plot Tech-
nique. 2nd Ed. Burgess Publ. Co.: Minneapolis, Minn. 373 pp.
4. Snedecor, G.W., and W.C. Cochran. 1969. Statistical Methods. 6th Ed.
Iowa State College Press: Ames, Iowa. 593 pp.
5. Steel, R.G.P., and J.H. Torrie. 1960. Principles and Procedures of
. Statistics. 2nd Ed. McGraw-Hill Book Co., Inc.: N.Y. 481 pp.
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50
Series 91: EFFICACY OF ANTIMICROBIAL AGENTS
Subseries 91A: PUBLIC HEALTH USES
§ 91-1 General requirements.
(a) Scope. Section series 91-1 to 91-8 contains requirements for
testing and performance of antimicrobial pesticide products with public
health uses for which efficacy test data are required to be submitted to
support registration. These include all antimicrobial products intended
to control microorganisms infectious for man in any area of the inanimate
environment where these microorganisms may present a hazard to human
health. The label claims for an antimicrobial product determine whether
or not it is considered to be related to human health. Refer to Subdivi-
sion H, §§ 101-l(b),(c),(d), and § 101-30 (Label Identification of Health-
Related and Non-Health Related Claims for Antimicrobial Agents) for
additional information on the relationship between label claims, human
health considerations/ and performance requirements for antimicrobial
products. For those uses of antimicrobial pesticide products which are
identified as not directly related to human health, guidance for testing
and performance are provided in Subseries 91B:' Non-Public Health Uses.
(b) General testing requirements. To fully, comprehend the data
required to demonstrate the effective performance of -various types of ..•'-'
antimicrobial pesticides, the applicant should understand the following
basic information. This information is critical to the development and
submission of appropriate data.
(1) Test substance. Unless otherwise .specified, antimicrobial
pesticides must be tested on the formulation to be offered for sale and,
in some cases (e.g./ pressurized sprays) with the product in the same
packaging intended to be marketed. The manufacturer must also submit
effectiveness data to show that he can consistently reproduce the for-
mulation (batch replication), as well as to show that the product will
retain its effectiveness for a minimal period of storage under average
conditions to which it is likely to be'exposed (shelf-life stability).
(2) Test methods. The product must be tested in accordance with the
proposed directions for use. Section 91-30 contains citations for AOAC
test methods referred to in this section. These methods, and supplemental
modifications for registration testing, as well as other suggested test
protocols or criteria, are entered separately in the § 91-30 or are
described individually in the following sections.
(3) Neutralizers. In testing the efficacy of any antimicrobial
product, appropriate neutralizers should be employed in the microbiological
assay'system, and evidence must be submitted to show that the neutralizer
employed inactivates the active ingredient(s) and;does not possess.any
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51 "
antimicrobial activity itself. In lieu of chemical neutralization, it must
be documented that appropriate subculture techniques have been employed
that preclude residual carryover of active ingredient(s). (See § 91-30(e)
Supplemental recommendation No. 7.)
(4) Duration of testing. All products tested by methods referenced
in the § 91-30 may be tested at the exposure periods prescribed in those
methods. However, if the product is to be represented in labeling for use
at exposure periods shorter or longer than those specified in the method,
the method must be modified, in a manner acceptable to the Agency, to
reflect the deviation in exposure intended. (See § 91-30(e) Supplemental
recommendation No. 1. Refer also to Labeling Guidelines for Pesticide
Use Directions, Subdivision H,
§§ 101.)
(5) Reuse of product. Efficacy data must be developed to substantiate
label directions and claims in regard to the number of days or the number
of single applications a prepared use solution of an antimicrobial product,
such as (but not limited to) surgical instrument sterilizers or surgical
instrument disinfectants, can be used or reused before a fresh solution
must be prepared. (Refer also to § 91-30 Supplemental recommentation No.
5 and Subdivision H §§ 101.) Such data must show retention of the claimed
level(s) of antimicrobial activity in the use solution after repeated
microbial and other appropriate challenges for the period of time or
number of times specified in .the directions for use.
(6) Variations in testing. The protocol for testing, test methods,
and basic test elements will vary according to the'type of product, type
of substrate to be treated, proposed use-pattern,, label claims, "directions
for use, and other factors peculiar to that product. In many cases,
specific requirements (including such elements as replication) can be
provided only after consideration of all these factors. Before initiation
of in-use or simulated-use studies, the proposed test protocols may be
submitted for review and comment by the Agency. Complete details of all
testing procedures and test/control results must be submitted. (Refer to
§ 91-30 for guidance.)
(7) Hard water claim. Any product that bears label claims for
effectiveness in hard water must be tested by the appropriate method in
synthetic hard water at the level claimed. [See § 91-30(e) Supplemental
recommendation No. 3].
(8) Organic soils. Any product that bears label claims for effective-
ness as a "one-step" cleaner-disinfectant, cleaner-sanitizer, or in the
presence of light or moderate organic soils must be tested by the appropri-
ate method in the presence of a representative organic soil, such as 5%
blood serum. [Refer to § 91-30(e) Supplemental recommendation No. 4.]
Additional organic material .need not be incorporated into those procedures ,•
,where at least 5% blood serum is already present, in the inoculum,to be
dried on. the surface. When a product is recommended for certain patterns
. of. ,use where the organic soil claimed, is .of a specific -type (soich as soap .
film residue-•oC'rhaica. water scum.)., .the .product .must .be-, tested in/the.;presence:
'b'f.'-'that specific.:pEganic.soil:«...... „...<.•-•.;•. ...•>,•>«.•; •...••••••*•••'• ' •'•'.•':.'.••- •
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(9).-Validation of efficacy. The Agency reserves the option to perform
its own tests for validation of efficacy of products selected on a case-by-
case basis. If the applicant or registrant is notified by the Agency that
samples are required for Agency testing, such samples shall be sent to EPA
Microbiology Laboratory at the following address: Building 406, Agr. Res.
Center - East, Beltsville, Md. 20705.
(10) Test failure. Failure of a product to meet the specified testing
or performance requirements constitutes evidence that the product is unlikely
to be effective as claimed in actual use. Refer to § 101-1(c) of Subdivision
H for alternatives which can be considered in such cases.
(c) Terminology. Because of the variety of microorganisms to be
controlled and the different claims and many use patterns of antimicrobial
products, uniform label terminology and a common understanding of a few
key words are important to a program for evaluating product performance.
Therefore, the principal labeling terms used to describe antimicrobial
activity and performance are defined in § 101-l(d) of Subdivision H.
§ 91-2 Products for use on hard surfaces.
(a) Sterilizers. The following'apply to all products represented in
labeling as sporicidal or sterilizing agents:
(1) Test standard. The'AOAC Sporicidal:Test (§ '91-30 .Recommended .
method No. 1) is required. Sixty carriers representing each of two types
of surfaces (porcelain penicylinders and surgical silk suture loops) are
required to be tested against spores of both Bacillus subtilis (ATCC 19659)
and Clostridium sporogenes (ATCC 3584) on 3 samples representing 3 different
batches, one of which is at least 60 days old (240 carriers per sample, or
a total of 720 carriers). Any sterilizing agent (liquid, vapor, or gas)
which is recommended for use in a specific 'device must be tested by the
AOAC Sporicidal Test in that specific device and "according to the directions
for use.
(2) Performance standard. Killing on all of the 720 carriers is
required. Data submitted to support sterilizing claims will be subject to
validation by tests conducted in the Agency's Microbiological Laboratory
before the product submitted for registration will be considered acceptable.
(b) Disinfectants (limited efficacy). 'When a disinfectant is recom-
mended in labeling for use against a specific major group of microorganisms
only (such as Gram-negative or Gram-positive bacteria), it is considered
to have only limited effectiveness, and consequently, only limited value
as a disinfectant. The following requirements apply to such products:
(1) Test standard. Limited disinfectant activity must be substan-
tiated with efficacy data derived from either the "Association of Official
Analytical Chemists (AOAC) Use-Dilution.Method for water-soluble powders
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53
and liquid products {§ 91-30 Recommended method No. 2), or the AOAC Germi-
cidal Spray Products Test (§ 91-30 Recommended method No. 3) for spray
products. Sixty carriers are required on each of 3 samples, representing
3 different batches, one of which is at least 60 days old, tested against
Salmonella choleraesuis (ATCC 10708) for effectiveness against Gram-negative
bacteria, or Staphy lo coccus aureus (ATCC 6538) for effectiveness against
Gram-positive bacteria.
(2) Performance standard. The product must kill the test microorgan-
isms on 59 out of each set of 60 carriers to provide significance at the
95% confidence level.
(c) Disinfectants (general or broad-spectrum efficacy). When a
disinfectant is represented in labeling as having a broad spectrum of'
activity, more extensive testing is required. The following requirements
apply to such products.
(1) Test standard. Sixty carriers on each of 3 samples, representing
3 different batches, one of which is at least 60 days old, must be tested
against both S_._ choleraesuis and S. aureus . [Employ the AOAC Use-Dilution
Method (§ 91-30 Recommended method No. 2) or the AOAC Germicidal Spray
Products Test (§ 91-30 Recommended method No. 3).]
(2) Performance standard. Same as in paragraph (b)(2) of this section.
(d) Disinfectants (hospital or medical environment efficacy). (1)
Test standard. The following apply when a disinfectant is recommended in
labeling for use in hospitals, clinics, dental offices, or any other medical -
related facility: Sixty carriers are required on each of 3 samples, repre-
senting different batches, one of which is at least 60 days old, tested
against each of the following: S. aureus, s. choleraesuis , and Pseudomonas
aeruginosa (ATCC 15442). [Employ the AOAC Use-Dilution Method (§ 91-30
Recommended method No. 2), or the AOAC Germicidal Spray Products Test
(§ 91-30 Recommended method Mb. 3).]
(2) Performance standard. Same as in paragraph (b)(2) of this section.
(e) Fungicides (pathogenic fungi). These requirements apply to
disinfectants which bear additional label claims of effectiveness against
fungi pathogenic to man:
(1) Test standard. Effectiveness of liquid disinfectants against
pathogenic fungi must be supported by efficacy data derived from each of
2 samples representing 2 different batches using the AOAC Fungicidal Test
(§ 91-30 Recommended method No. 4) against Trichophyton mentagrophytes
(ATCC 9533 is suitable).
(2) Performance. standard. Killing of. all fungal spores is required.
'. (3) Alternative/ test standard. Alternatively, the AOAC
.Method, modified to confozm'with appropriate elements in the .AOAC Eungicidal
Test., may be. employed. If .the product is intended for "use as .a. spray, the
:AQAC • GermiaidaL . Spray Products- Test must be employed.. Ten carriers on, each
of 2 samples representing .2 different batches must be"" employed in the test..
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54
(4) Alternative performance standard. Killing of the fungal spores
on all carriers in required.
(f) Virucides. These requirements apply to disinfectants which bear
additional label claims of effectiveness against viruses. Most virucidal
pesticides- are intended for use on dry inanimate surfaces. For this reason,
acceptable virological data are usually developed by carrier methods.
(1) Test standard. The test must simulate in-use conditions. The
specific virus to be tested must be inoculated onto hard surfaces, allowed
to dry, and is then treated with the product according to the directions
for use on the product label. Each specific virus against which effective-
ness is claimed must be treated. To demonstrate virucidal activity of a
product against dried viruses on inanimate surfaces, petri dishes, glass
slides, stainless steel cylinders, or other appropriate test surfaces may
be used. The test surface must show a recoverable virus titer of at least
104 particles per surface. Virucidal performance must be demonstrated for
each of two batches of product, each batch to employ a single surface
specimen in the test. (Refer to § 91-30 Recommended method No. 5.)
(2) Performance standard. Inactivation of virus must be demonstrated
at all dilutions when no cytotoxicity is observed in the assay system, or
at all dilutions above the cytotoxic level when it is observed. The data
must demonstrate at least a 3-log reduction in viral titer for both samples
when cytotoxicity is present. The calculated viral titers must be reported
with the test results.
(g) Tuberculocides. The .following requirements apply to disinfectants
which bear additional label claims of effectiveness as tuberculocides: .
(1) Test requirements. Effectiveness against Mycobacterium tubercu-
losis must be substantiated with data derived on 10 carriers by the AOAC
Tuberculocidal Activity Method [II. Confirmative In Vitro Test for Determin-
ing Tuberculocidal Activity (§ 91-30 Recommended method No. 6)] against
M. tuberculosis var. bovis (BCG) for each of 2 samples representing two
different batches of a liquid product under test. If the product is a
spray, the procedure must be modified to conform with the AOAC Germicidal
Spray Products Test using the media, test culture, and other elements
described in the AOAC Tuberculocidal Activity Method.
(2) Performance standard. Killing of the test microorganisms on all
carriers, and no growth in any of the inoculated tubes of two additional
media, is required.
(h) Phenol coefficient(s). Data from this test are required only
when permitted phenol coefficient claims are made in labeling of disinfec-
tants. [See § 101-3 (k) of Subdivision H for those labeling claims which
are permitted.]
(1) Test standard. Phenol coefficients for Salmonella typhi (ATCC
6539), the only official test organism, .and'for any additional Gram-negative
or Grampositive asporogenous bacteria must be determined by the AOAC Phenol
Coefficient Method (§ 91-30(e) -Recommended method No.7) on each of two
samples representing 2 different batches against each bacterium.
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55
(2) Performance standard. The phenol coefficient is a numerical
value that compares the bactericidal concentration of a disinfectant to
the bactericidal concentration of pure phenol. This numerical value is
obtained by dividing the greatest dilution of disinfectant killing S^
typhi in ten minutes/ but not in five minutes/ by the greatest dilution
of phenol showing the same results. Since phenol coefficient values are
usually an unreliable index as to the effective use-dilution of a
disinfectant product, the AOAC Use-Dilution Method must be employed to
determine the actual disinfecting efficacy of a product, and its effective
use dilution.
(i) Additional microorganisms. The following requirements apply to
disinfectants which bear label claims against specific microorganisms
other than those named in the AOAC Use-Dilution Method, AOAC Germicidal
Spray Products Test, AOAC Fungicidal Test, and AOAC Tuberculocidal Activity
Method (see § 91-30 Recommended methods Nos. 2, 3, 4 and 6), and not includ-
ing viruses [see paragraph (f) of this section for virucides] .
(1) Test standard. Effectiveness of disinfectants against additional
specific microorganisms must be determined by either the AOAC Use-Dilution
Method (§ 91-30 Recommended method No. 2) or the AOAC Germicidal Spray
Products Test (§ 91-30 Recommended method No. 3), as appropriate, on 10
carriers for each of 2 samples representing 2 different batches against
each microorganism.
(2) Performance standard. Killing of the test microorganisms on
all carriers is required.. Plate count data, on appropriate culture media,
must be submitted on each test miroorganism to demonstrate that a concen-
tration of at least 10^ microorganisms survive the carrier-drying step
to provide meaningful results at the 95% confidence level. [Refer to
§ 91-30(e) Supplemental recommendation No. 6.]
(j) Sanitizers (for non-food-contact surfaces). The following
requirements apply to products bearing label claims for effectiveness as
sanitizers for inanimate hard surfaces other than those which come in
contact with food or beverages (e.g., floors, walls, furnishings).
(1) Test standard. These products must be tested by a protocol
incorporating the basic elements outlined in § 91-30 Recommended method
No. 8.
(2) Performance standard. The results must show a reduction of at
least 99.9% in the number of each test microorganism over the parallel
control count.
(k) Sanitizing rinses (for previously cleaned food-contact surfaces).
.The following requirements apply to any product with a label recommendation
for treatment, of previously-cleaned -food- contact surfaces (e.g. ,. eating and
' drinking .utensils and: foocl ^processing equipment) as a terminal sanitizing.
rinse.. Antimicrobial agents applied to food— contact surfaces are defined
as incidental: food additives under the -Federal RKjd, Drug, and,. Cosmetic
.Act.., as amended. (:2i;U:.S«C»: 201 et seq»>, aJid ; require a:food additive regu-
lation.-or--an- ^ei^^eIon,.from;s'u^::r'egulation%''--Rex:aWftett
f a potable
water '/rinse after treataent does not preclude this requirement.. ••
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56
(1) Halide chemical products. Efficacy of sanitizing rinses formu-
lated with iodophors, mixed halides, and chlorine-bearing chemicals must
be demonstrated by data derived from the AOAC Available Chlorine Germicidal
Equivalent Concentration Method (§ 91-30 Recommended method No. 9).
(i) Test standard. Data from one test on each of 3 samples/
representing 3 different batches, one of which is at least 60 days old,
against Salmonella typhi (ATCC 6539) are required.
(ii) Performance standard. Test results must show product concen-
trations equivalent in activity to 50,_ 100, and 200 ppm of available
chlorine. (The reference standard in sodium hypochlorite.)
(2) Other chemical products. The efficacy of sanitizing rinses
formulated with quaternary ammonimum compounds, chlorinated trisodium
phosphate, and anionic detergent-acid formulations must be substanitated
with data derived from the AOAC Germicidal and Detergent Sanitizers Method
(§ 91-30 Recommended method No. 10).
(i) Test standard. Data from the test on one sample from each of 3
different batches, one of which is at least 60 days old, against both
Escherichia coli (ATCC 11229) and Staphylococcus aureus (ATCC 6538) are
required. When claims for the effectiveness of the product in hard water
are made, all required data must be developed at the hard water level
claimed.
(ii) Performance standard. Acceptable results must demonstrate a
99.999% reduction in the number of each test microorganism within 30 .
seconds. The results must be reported according to the actual count and
percentage reduction over the control.
(1) Residual bacteriostatic activity of dried chemical residues on
hard inanimate surfaces. Bacteriostatic claims are permitted only
against microorganisms identified as causing economic or aesthetic problems
(e.g., odor-causing bacteria) in the presence of moisture; but not for
public health uses. Testing and performance guidance for non-public
health uses are provided in Subseries 91B.
(m) Residual self-sanitizing activity of dried chemical residues on
hard inanimate surfaces. The following requirements apply to products
which bear label claims to provide residual self-sanitizing activity (i.e.,
significant reduction in numbers of infectious microorganisms which may be
present or subsequently deposited) on treated surfaces that are likely to
become and remain wet under normal conditions of use.
(1) Test standard. Each test must include the following basic elements:
(i) It must be based upon an adequately controlled in-use study or
simulated in-use study employing as test microorganisms those target
pathogens that are likely to be encountered in the environment in which
the product is to be used.
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57
(ii) Inocula of the test microorganisms at a sufficient concentration
to provide at least 101* survivors on the parallel control surface must be
employed for initial and subsequent challenges.
(iii) The residue on the treated surface(s) must be activated by the
addition of moisture in a manner and over an exposure period identical to
the use pattern for which the product is intended.
(iv) Quantitative bacteriological sampling must be conducted at
frequent and regular intervals.
(v) The same type(s) of surface without the treatment must be-employed
in the test and inoculated in a manner and over an exposure per-iod .identical
to the use pattern for which the product is intended.
(vi) The environmental conditions, such as relative humidity and
temperature, employed in the test must also be reported; these must be the
same as those which are likely to be encountered under normal conditions
of use.
(5) Performance standard. For residual self-sanitizing claims, it
must be demonstrated that at least 99.9% reduction in the numbers of test
microorganisms occurred on the treated surface(s) over that of the parallel
control surface(s).
§ 91-3 Products requiring confirmatory data.
(a) Specific situations. Confirmatory data (supplemental to the basic
reference data) are required in certain situations in which an applicant
intends to utilize previously submitted basic efficacy data to support an
application or amendment for registration of a product. Confirmatory data
are required on an applicant's own finished product to demonstrate his ability
to produce an effective formulation. There are three commonly encountered
situations in which an applicant is permitted to use previously submitted
basic efficacy data, and submit only confirmatory data to support the regis-
tration of the antimicrobial product. These three specific situations and
the corresponding required confirmatory data are described below. These
specific confirmatory data are not applicable to any other categories of
antimicrobial products. For use patterns other than those indicated below,
required confirmatory data will be determined by the Agency on a case-by-
case basis. •
(b) Product formulations which are identical or diluted forms of a
registered product. In this .situation, the product proposed -for .registra-
tion 'has. formulation,, claims, and recommendations for/use identical to. those
of .a product already registered and manufactured by .another registrant. The
proposed product'. is .merely repackaged., relabeled; or : is :,a .simple dilution.
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. (1) Data standard. A document substantiating that the product is
formulated by the registrant for another applicant must be submitted.
Specific references to the supporting data developed for the original
product are also required.
(2) Test standard. If the product is a pressurized spray, all
materials and devices must be shown to be identical to those utilized by
the basic registrant. Furthermore, the filler packaging company must be
the same for both products. If this identity cannot be substantiated,
the applicant must submit complete efficacy data by the appropriate test
method, which will be based upon the claims made for the product. [See
paragraphs (b) thru (g) of § 91-2 of this Subdivision for test and
performance standards.] Specific references to supporting data developed
for the original product are also required.
(3) Performance standard. All documentation submitted must be
appropriate and correct.
(c) Duplicated product formulations. In this situation, the formula-
tion for the product proposed for registration duplicates a formulation
which is registered, but the two products are not manufactured (or packaged)
by the same company. The chemical composition, label claims, and recommen-
dations for use are identical (in substance) to .those accepted for the
original registered product. See paragraphs (e) through (h) of this section
for the test standards and performance standards for'the confirmatory data
that are to be submitted. Specific references to supporting data developed
for the original product are also required. .
(d) Minor formulation change in a currently registered product. In
this situation, the change in the formulation is relatively minor, e.g.,
change in the level of an inert ingredient. The label claims and recommen-
dations for use are identical to those accepted -for the registered formula-
tion. See -paragraphs (e) through (h) of this section for the test standards
and performance standards for the confirmatory data that are to be submitted.
Specific references to supporting data developed for the original product
are also required.
(e) Disinfectants (limited efficacy). (1) Confirmatory test standard.
Ten carriers on each of two samples representing 2 different batches must be
tested against either Staphylococcus aureus or Salmonella choleraesuis,
depending upon the microorganism against which the activity of the product
is limited. [See paragraph (b) in § 91-2 of this subdivision.] The AOAC
Use-Dilution Method for liquids, or the AOAC Germicidal Spray Products Test
for spray products, must be used with the same modifications employed for
the original referenced data. Certification is required specifying that all
parts and materials used in manufacturing the container for -pressurized spray
disinfectants are identical to those specified by the basic manufacturer.
(2) Confirmatory performance .standard. Killing of the test microorgan-
isms on all carriers is required.
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(f) Disinfectants (general or broad spectrum efficacy). (1) Confirma-
tory test standard. Ten carriers on each of two samples representing two
different batches must be tested against both S. aureus and S. choleraesuis,
using the AOAC Use-Dilution Method for liquids, or the AOAC Germicidal Spray
Products Test for spray products, are required with the same modifications
employed for the original referenced data. Certification is required specify-
ing that all parts and materials used in manufacturing the container for
pressurized spray disinfectants are identical to those specified by the basic
manufacturer.
(2) Confirmatory performance standard. Killing of the test microorgan-
isms on all carriers is required.
(g) Disinfectants (hospital or medical environment efficacy). (1)
Confirmatory test standard. Ten carriers on each of two samples represent-
ing 2 different batches are required against each of the following: S^
choleraesuis, £•_ aureus, and Pseudomonas aeruginosa. The AOAC Use-Dilution
Method for liquid products, or the AOAC Germicidal Spray Products Test for
spray products, must be used with the same modifications employed for the
original referenced data. Certification is required specifying that all
parts and materials used in manufacturing the container for pressurized spray
disinfectants are identical to those specified by the basic manufacturer.
(2) Confirmatory performance standard. Killing of the test microorgan-
isms on all carriers is required.
(h) Sanitizers (for previously cleaned food-contact surfaces). (1)
Confirmatory test standard. One test on one sample, with or without hard
water (depending on label claims), is required using either: The AOAC
Germicidal and Detergent Sanitizers Test (§ 91-30 Recommended method No.
10) against Escherichia coli for quaternary ammonium compounds, chlorinated
trisodium phosphate, and anionic detergent-acid-formulations; or the AOAC
Available Chlorine Germicidal Equivalent Concentration Test (§ 91-30 Recom-
mended method No. 9) against Salmonella typhi for iodophors, mixed halides,
and chlorine-bearing chemicals.
(2) Confirmatory performance standard. See paragraphs (k)(l)(ii) and
(k)(2)(ii) in § 91-2 of this subdivision for acceptable results.
§ 91-4 Products for use on fabrics and textiles.
(a) Laundry additives. The following requirements apply to antimicro-
bial products which bear label recommendations for use in the treatment of
laundry (as a. pre-soak, treatment or in household and commercial laundry
operations.) to provide various levels of: antimicrobial activity including
disinfection,.sanitizatioh, or .residual self-sainitiza'tion> :. :', •' •'"•••
(.1.) Disinfecting pre-soak treatment. - Effectiveness of .products., for
"one—stejp" cleaning, and .-'disinfect-ing ;osf- hard, -surfaces -{.in.,the. presence
of- "moderate" amoan-ts. of organic' soaD-.-.may1 ;be '-extrapolated to disinfecting. : :
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of soiled fabrics by total immersion in the use solution prior to routine
laundry operations. The requirements are as follows:
(i) Test standard. The AOAC Use Dilution Method (§ 91-30 Recommended
method No. 2) modified to include organic soil (§ 91-30(e) Supplemental
recommendation No. 4) must be employed in accordance with § 91-2(b)(l),
(c)(l), or (d)(l).
(ii) Performance standard. Same as § 91-2(b)(2).
(2) Disinfecting laundry additives (non-residual). The following
requirements apply to products which bear label claims as disinfectants
'for use in automatic or manual washing machine operations.
(i) Test standard. A suggested protocol, published by Petrocci and
Clarke in the Journal of the Association of Official Analytical Chemists,
is referenced in the § 91-30 Recommended method No. 11. That protocol is
a simulated in-use study. However, an actual in-use study utilizing washing
machines may be employed. The following basic elements must be incorporated
in either study:
(A) The test microorganisms are Klebsiella pneumoniae (ATCC 4352)
and Staphylococcus aureus (ATCC 6538). If the product is intended for
use on hospital linens, it must be tested against the test microorganism
Pseudomonas aeruginosa (ATCC 15442).
(B) Propagation of cultures, fabric-to-water ratios, test materials,
water temperatures, exposure time, subculture media, and the basic procedures
must be the same as those specified in the Petrocci and Clark protocol.
(C) Tests must be conducted with 3 samples representing 3 different
product batches, one of which is at least 60 days old. Each sample must
be tested with 9 fabric swatches against each of the test bacteria.
(D) The method employed must be designed to include testing of both
the fabric and laundry water (5 ml from the automatic washer, or 0.5 ml
from the simulated washing device) in individual widemouth jars containing
subculture media and neutralizers. The laundry water-to-media volume
ratio must not exceed 1:40. •
(E) Growth or no-growth must be recorded after a 48-hour incubation
period.
(ii) Performance standard. There must be no growth in the fabric
subcultures and no growth in the subcultures from the laundry water with
each test microorganism.
(3) Sanitizing laundry additives (non-residual). The following
requirements apply to products which bear label claims as sanitizers for
use in automatic or manual washing machine operations.
(i) Test standard. The same type of studies referred to in paragraph
(a)(2)(i) of this section must be employed for evaluating the efficacy of
laundry additives intended to sanitize laundry, with the following exceptions:
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(A) Each of the product samples must be tested with 3 cloth swatches
against each of the required test bacteria.
(B) Quantitative bacteriological assays must be conducted and the
plate counts reported for the cloth swatches and laundry water.
(ii) Performance standard. At least 99.9% reduction in bacteria over
the control count for both laundry water and fabric must be demonstrated
against each test microorganism.
(4) Self-sanitizing laundry additives (residual). The following
requirements apply to products which bear label claims to provide residual
self-sanitizing activity (i.e., significant reduction in numbers of infec-
tious microorganisms which may contaminate the items) on treated fabrics
when used in automatic or manual washing machine operations (usually in
the final rinse). Label claims for residual antimicrobial activity on
laundered materials or articles can only be considered in those situations
when such materials are likely to become and remain wet (for example,
diapers, and bed linens of incontinent persons) under normal conditions of
use and storage between launderings.
(i) Test standard. A suggested protocol published by Petrocci and
Clarke (referenced in § 91-30 Recommended method ifo. 11) is acceptable
for treating the fabric. The basic elements outlined in the protocol of
the "Quantitative Procedure" of the American Association of Textile Chemists
and Colorists (AATCC) Test Method 100-1974 (see § 91-30 Recommended method
No. 12) employing Staphylococcus aureus (ATCC 6538) and Klebsiella
pneumoniae (ATCC 4352) are acceptable for evaluating the residual antimicro-
bial activity. However, 3 samples representing 3 different product batches •
must be tested, and the following modifications to the method must be
incorporated:
(A) Use a sufficient number of swatches placed exactly on top of each
other so that they completely absorb 1 ml of inoculum which is prepared to
contain at least 10^ microorganisms/ml;
(B) The number of swatches used per jar must be reported;
(C) Incubation of the swatches must be at 20-21°C (68-70°F);
(D) Quantitative bacteriological assays should be performed at the
following time intervals: 0, 30 min., 1-hr, 2-hr, 3-hr, 6-hr, and 24-hr.
Consideration can be given to fewer or different time intervals, depending
on the label claims, on a case-by-case basis.
(ii) Performance standard. Eor residual self-sanitizing claims
against pathogenic microorganisms, the reduction of each test microorganism
must be at least 99.9% over the "0-time" control and the parallel untreated
inoculated control. - . .
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(5) Bacteriostatic laundry additives (residual). Bacteriostatic
claims are permitted only against microorganisms identified as causing
economic or aesthetic problems (e.g., odor-causing bacteria); but not for
public health uses. Testing and performance guidance for non-public
health uses are provided in Subseries 91B.
(b) Carpet sanitizers. The following requirements apply to products
bearing label claims for effectiveness as carpet sanitizers.
(1) Test standard. Sanitizers for pre-cleaned carpeting must be
tested by a protocol incorporating the basic elements of § 91-30 Recommended
method No. 13. I£ the product is intended to be represented in labeling
as a "one-step" cleaner-sanitizer, the method must be modified by including
an appropriate soil with the bacterial inoculum. The following requirements
must be met:
(i) Three product samples representing 3 separate batches, one of
which is at least 60 days old, must be tested against Staphylococcus
aureus (ATCC 6538) and Enterobacter aerogenes (ATCC 13048) with 2 different
types of representative synthetic carpeting, such as acrylic and polypropyl-
ene tufted-loop types. If the product is intended for use in hospitals or
medical institutions, it must also be tested against Pseudomonas aeruginosa
(ATCC 15442). If the product is also intended for use on wool carpeting,
an additional representative sample of WDO! carpet must be tested; otherwise,
the label must bear a disclaimer for use on wool. All type carpet samples
tested must be fully identified by the pile fiber, pile yarn weight of
finished carpet, pile density, and tuft height. Adequate controls must
demonstrate that bacteriostatic agents in the carpet pile or backing do
not interfere with the tests results.
(ii) The amount of solution applied to the sample carpeting in the
tests must be determined and extrapolated to obtain the amount of the use
solution of product to be applied to carpeting (volume per unit area) as
stated on the label.
(2) Performance standard. A 99.9% reduction of test bacteria over
the scrubbed control count must be demonstrated.
(c) Mattresses and upholstered furniture. The use of gases or vapors
is currently the only effective and practical means of treating mattresses,
upholstered furniture, pillows, and similar objects to sterilize, disinfect,
or sanitize against microorganisms. The following requirements apply to
products bearing such label recommendations.
(1) Test standard. Simulated-use studies in which artificially-
contaminated articles of this type are employed must be performed to demon-
strate the level of effectiveness intended, as follows:
(i) Each test article must be inoculated throughout the entire article;
(ii) Samples must be taken or withdrawn randomly from the entire
treated article and cultured for microbial growth;
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(iii) An adequate control using a similar untreated article must be
employed in such a study;
(iv) The test protocol, including such elements as replication, test
microorganisms, etc., will vary with the level of effectiveness intended
and the directions for use of the product, but the basic elements described
above must be incorporated in any test protocol;
(v) A complete description of the test protocol employed must be sub-
mitted.
(2) Performance standard. (i) Same as § 91-2(a)(2) for sterilizing;
(ii) Same as § 91-2(b)(2), (c)(2), or (d)(2) for disinfecting;
(iii) Same as § 91-2(j)(2) for sanitizing.
(d) Impregnated self-sanitizing fabrics and textiles. The following
requirements apply to products intended for treatment of fabrics and textile
materials, usually during the manufacturing process, to provide durable
residual self-sanitizing activity (i.e., significant reduction in numbers
of infectious microorganisms which may be subsequently deposited on the
finished item) in the presence of moisture or wet contamination.
(1) Test standard. The test standard is the same as in § 91-2(m)(l)
of this subdivision, employing as test and control surfaces the treated
and untreated fabric material or finished items, as appropriate.
(2) Performance standard. Same as in § 91-2(m)(2) of this subdivision.
(e) Impregnated bacteriostatic fabrics and textiles. Bacteriostatic
claims are permitted only against microorgainsms identified as causing
economic or aesthetic problems (e.g., odor-causing bacteria); but not for
public health uses. Testing and performance guidance for non-public health
uses are provided in Subseries 91B.
§ 91-5 Air sanitizers
(a) General. (1) These requirements apply to products bearing label
claims for treatment of air to temporarily reduce the numbers of airborne
microorganisms. There is considerable evidence that glycol vapors produce
significant decreases in numbers of viable airborne bacteria under relatively
wide conditions of relative humidity and temperature when properly and contin-
uously dispensed by a. vaporizing device so as to maintain suitable concentra-
tions in..the .airof. --enclpsed- spaces. . .. ; • . , v . .
(2.)- With (dispensers for the intermittent treatment of. air, such'as
pressuri.zed aerosols, several1 investigators have shown .that glycols -(tri-
et'hylene, diprcfpyiene.-, '.• bir prtSp;ylene;<gtycol.) ;at.concenttratiofis. e>i• -5%', CRT. more •
in such formulations will temporarily reduce numbers of- airborne bacteria :
when adequate •amounts.are dispensed under, relatively ideal conditions..'
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(3) For. other types of products intended for the treatment of air,
claims for "reducing numbers of airborne microorganisms will be considered,
providing supporting experimental data are submitted to justify such claims.
(b) Test standard. No standard method for evaluating air sanitizers
has been adopted. Proposed testing protocols for studies of this kind may
be submitted for review and evaluation by the Agency prior to initiation
of the test.
(1) Glycol products. For products containing at least 5% glycols
(triethylene, dipropylene, and/or propylene glycols), quantitative chemical
determinations must be performed, using an air sampling device, to show the
concentration of glycol vapor achieved with the product, used as directed,
in an enclosed experimental room or chamber.
(2) Other products. For products other than those specified in para-
graph (b)(l) of this section, quantitative microbiological assays must be
performed, using an air sampling device, to show the level of reduction of
viable microorganisms achieved with the product, used as directed, in an
enclosed experimental room or chamber. The primary test bacteria are
Staphylocoecus aureus (ATCC 6538) and Klebsiella pneumoniae (ATCC 4352).
If the product is intended for use in hospitals or medical environments,
Pseudomonas aeruginosa (ATCC 15442) must also be tested.
(3) The methodology employed, such as spraying and sampling procedures,
and the environmental conditions in the room or chamber, such as temperature,
relative humidity, etc., must be reported. Raw data, as well as any statis-
tical or graphical interpretation.of the results, must be included in the.
reports.
(c) Performance standard. (1) Glycol products. The results must
show that adequate glycol vapor concentrations (50% saturation or more)
are achieved in the air of the test enclosure.
(2) Other products. The results must show a viable count reduction
of at least 99.9% over the parallel untreated control, after correcting
for settling rates, in the air of the test enclosure with each of the
required test bacteria.
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§ 91-6 Products for processing and industrial uses*
(a) Bacteriostatic preservatives. Currently, only non-public health
claims are permitted for products to control microorganisms recognized as
causing economic or aesthetic problems in processing and industrial uses.
Testing and performance guidance for these non-public health uses are
discussed in § 91-53 of this subdivision. Examples of these uses include:
(1) Deterioration of water-based paints, metalworking fluids, and
other industrial products;
(2) Eouling of kerosene-based fuels (including jet aviation fuel),
diesel fuels, and heating oils;
(3) Bacterial growth and spoilage in cane or beet sugar processing.
(b) (Reserved.)
§ 91-7 Products for control of microbial pests associated with human and
animal wastes.
. (a) Treatments for toilet bowl and urinal surfaces. The following
requirements apply to products bearing label claims as disinfectants or
sanitizers for toilet bowl and urinal surfaces.
(1) Disinfectants. (i) Test standard. Products recommended for
disinfection of surfaces of toilet bowls and urinals must be tested by the
AOAC Use-Dilution Method (for liquid products), or the AOAC Germicial Spray
Products Test (for spray products), in accordance with the requirements
outlined in § 91-2(b)(1),(c)(1) or (d)(l) of this subdivision. Note that
the contained bowl water (approximately 96 fl. oz.) must be taken into
consideration in determining the approprate use dilution for testing.
(ii) Performance standard. Same as in § 91-2(b){2) of this sub-
division.
(2) Sanitizers. (i) Test standard. Products recommended for
sanitization of toilet bowl and urinal surfaces must be evaluated by an
appropriate protocol similar to the sanitizer test for non-food contact
surfaces specified in § 91-2(j) of this subdivision (.§ 91-30 Recommended
method No. 8). However, the test microorganisms must be the same as
those outlined in § 91-2(b) and (c.) of this subdivision in accordance
with the areas of use referred to therein.
(ii.) Performance standard. The results must show a reduction of at
•'leiast 99.9% of,.each, test microorganism over the-parallel control., count.
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(b) Sanitizers for toilet and urinal bowl water^ The following
requirements apply to products bearing label claims as sanitizers for
toilet and urinail bowl water.
(1) Test standard. The product must be tested by a simulated-use
study incorporating all of the following basic elements:
(i) The product must be added to samples of the bowl water from three
toilets or urinals at the use concentration, employing the recommended
method of dispensing. Untreated control samples from the three toilets or
urinals must also be included.
(ii) When the product is automatically "metered" or dispensed in
some other fashion into the bowl water (or urinal trap), the consistent
accuracy of the concentration dispensed and maintained must be documented.
(iii) Ino'cula containing representative pathogenic Gram-positive
and Gram-negative test bacteria must be added to the treated and control
samples of the bowl water from each of the toilets or urinals to provide
a concentration of at least Id"* colony-forming units per ml.
(iv) Microbial counts of the treated bowl water and the control bowl
water must be conducted at a minimum of three exposure intervals, in addi-
tion to a "0-time" control.
(2) Performance standard. The reduction of each test microorganism
must be at least 99.9% over the "0-time" control and the parallel untreated
inoculated control.
(c) Bacteriostatic treatments -for self-contained toilet systems,
vomitus absorbents, and bird and 'animal cage litter. Bacteriostatic claims
are permitted for such uses aga'inst microorganisms identified as causing
economic or aesthetic problems (e.g., odor-causing bacteria). Testing and
performance guidance for non-public health uses are provided in Subseries
91B.
91-8 Products for treating water systems.
(a) Drinking water for humans. (1) - Public water supplies. Municipal
drinking water must meet the requirements of the Safe. Drinking Water Act
(Public Law 93-523).
(i) Test standard. Evidence must be submitted that the chemical
intended for use as a drinking water disintectant has been tested under
the auspices of the Office of Drinking Water (WH-550) of the Agency.
(ii) Performance standard. Documentation must be submitted that
the chemical tested as above has been accepted for use as a drinking water
disinfectant under the auspices of the Office of.Drinking Water .of the
Agency.
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(2) Emergency water supplies. The following requirements apply to
chemical additives such as solutions, powders, or tablets intended for
emergency disinfection of small quantities of drinking water of questionable
potability by the general public in the absence of bacteriological monitor-
ing facilities:
(i) Test standard. Controlled simulated-use studies which represent
actual use conditions must include the following basic elements :
(A) Representative levels of organic and inorganic soil contamination;
(B) Various water temperatures;
(C) The specific dosage and exposure period recommended for the
proposed product;
(D) A variety of representative test microorganisms, including cysts,
bacteria and viruses ; and
(E) Quantitative determination of the level of microbial contamination
of the water before and after treatment.
(ii) Performance standard. The treatment must eliminate all test
microorganisms from the water.
(3) '. Water treatment units. (i) Water purifier units. Any unit
intended for the treatment of raw water to eliminate the potential health
hazard posed by microorganisms is identified as a water purifier. The
unit may rely on physical filtration (pesticidal device), or chemical
treatment (pesticide), or a combination thereof, to achieve the intended
purpose of purifying microbiologically non-potable water by eliminating
water-borne pathogens in the water itself. Those units, such as submicron
membranes and absolute filters, which rely solely on a physical means of
removal of microorganisms from water, are identified under the Act as
devices, and are subject to regulation but not registration. The test
requirements indicated below are for the units containing an antimicrobial
chemical (pesticide) .
(A) Test standard. Controlled in-use or simulated-use studies for
the water purifier unit must be conducted under conditions representing
its actual use employing a defined actual or simulated raw water source
containing a high level of microbiological pollution, including waterborne
cysts, bacteria, and viruses. The test design will vary with different
types of units and patterns of use, but must include such basic elements as :
(1) Representative levels of organic and inorganic soil contamination;
(.2) Vario.us ,water temperature's.; " ;
: (:3) •: Documen:ta:tib.h of: -the • antiniipzo.bial concentration-.found 'in the test
•system; •:. • : . . •:v/;f::":- •. :: •'.:':'.-.:. •• . • , .. ..... -.-.•'. •' • " • •' ' -• . '
level....
of the .water before .and' after, passage through the unit;'.. _••:•„. .....
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(5) Documentation of the duration of effectiveness or effective
capacity of the unit before a replacement is necessary.
(B) Performance standard. The treatment must eliminate the microbial
pollution from the water.
(ii) Bacteriostatic potable water treatment units. Only bacterio-
static claims are permitted for such units against microorganisms identifed
as causing aesthetic problems (e.g./ objectionable tastes, odors, and the
like). Testing and performance guidance for non-public health uses are
provided in Subseries 91B.
(b) Drinking water for poultry and livestock. Non-drug claims for
treatment of poultry and livestock drinking water with antimicrobials to
provide disinfection, sanitization, or bacteriostasis are not considered
to be directly related to human health. However, such products require
establishment of a tolerance from EPA under the Federal Food, Drug, and
Cosmetic Act. Efficacy testing and performance guidance on non-public
health uses are provided in Subseries 91B.
(c) Swimming pool water. The following requirements apply to products
bearing label claims for swimming pool water disinfection. Numerous factors
influence the concentrations necessary for disinfection of swimming pool
water in practical applications: number of swimmers in the pool; frequency
of use; frequency with which water is changed; general weather conditions;
and types and degree of organic contamination of the water by the swimmers
themselves (e.g., suntan lotions and oils) and by various debris. Therefore,
.a two-phased study (presumptive laboratory testing and confirmatory field
testing) is required. • \ •
(1) Test standard. (i) Laboratory test. Presumptive efficacy of
swimming pool water disinfectants may be indicated with data derived from
the AOAC Method for Water Disinfectants for Swimming Pools (§ 91-30 Recom-
mended method No. 14), or with slight modifications (e.g., pH) thereof,
against both Escherichia coli (ATCC 11229) and Streptococcus faecalis (PRO).
(ii) Field test. In addition to the laboratory test requirements
referred to in paragraph (c)(l)(i) of this section, confirmatory efficacy
data shall be derived from in-use tests under an Experimental Use Permit
in at least two swimming pools. The tests must be conducted for an entire
swimming season (4 to 12 months). Reports must include (but are not limited
to) the following information concerning the test pools:
(A) The daily bather load.
(B) The design of the pool, the recirculation and filter system, and
water capacity.
(C) The amount and identification of all chemicals added daily to the
swimming pool water (including the time, site, and method).
(D) The range-of chemical characteristics of the swimming pool water,
such as: pH, nitrogenous substances, metals, and hardness.
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(E) The physical characteristics of the swimming pool water, includ-
ing temperature and clarity, determined at least daily*
(F) Meteorological data, including air temperature, rainfall and
number of hours of sunlight (determined daily) for outdoor pools.
(G) Bacteriological monitoring, conducted daily, in accordance with
the Suggested Ordinance and Regulations Covering Public Swimming Bools of
the flinerican Public Health Association. (See reference in § 91-30 Recom-
mended method No. 14).
(H) The concentration of the antimicrobial agent in the swimming pool
water monitored daily at the same time-intervals that the bacteriological
assay samples are obtained.
(I) The method that the product user will employ for monitoring the
level (concentration in ppm) of antimicrobial agent contained in the pool
water.
(2) Performance standard. (i) Laboratory test. The lowest concen-
tration of the test germicide providing results equivalent to those of the
sodium hypochlorite control is the lowest concentration of the product that
can be considered effective.
(ii) Field test. The product must meet all of the efficacy require-
ments outlined in Suggested Ordinance and Regulations Covering Public
Swimming Pools of the American Public Health Association.
(d) Control of Legionnaires' disease bacteria in industrial water
systems. (Reserved)
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§ 91-30 Acceptable methods.
(a) Scope. This section provides recommended methods for satisfying
most efficacy data requirements for public health uses of antimicrobial
agents. It also provides supplemental recommendations for modifying or
expanding the recommended methods to obtain additional data necessary to
support certain specific claims and/or special patterns of use.
(b) General. Depending upon the type of antimicrobial agent, target
microorganism, and site to be treated, all tests must address those factors
that would normally be expected to be encountered in the use pattern
intended for the product, such as: the method of application? the nature
of the surface, item, or substrate to be treated; the presence or absence
of soil or other interfering conditions; temperature; exposure period;
and the number of times or duration of time that the use solution can be
used or re-used. The actual test procedure to be employed will vary
according to the characteristics of the product, the target pest(s) and
the pattern of use intended. Specification of methods in these Guidelines
for all conceivable public health uses is not feasible, and the applicant
must be responsible for the validity of the test method selected to
substantiate efficacy. The applicants should assure themselves that the
selected method is current and applicable to the product and use(s) pro-
posed for registration. Reference to Labeling Guideines for Pesticide Use
Directions, Subdivision H, should be made concurrently with consideration
of product performance of antimicrobial agents. • •
(c) Reporting of data. Systematic and complete descriptions of
the tests employed and results obtained are 'essential for proper review
and evaluation of product performance by the Agency. All test reports
must include identification of the testing laboratory or organization,
when and where the tests were conducted, and the name of the person(s)
responsible for the conduct of the tests.
(1) Recommended methods. • When the recommended methods (such as
standard AOAC tests) are employed to develop efficacy data, certain minimal
information must be provided in the test report. • The report must include,
but is not limited to, the following:
(i) Test method employed, and any modifications thereto;
(ii) Test microorganisms employed, including identification of the
specific strain (ATCC or other);
(iii) Concentration or dilution of product tested and how prepared;
(iv) Number of samples, batches, and replicates tested;
(v) Preparation date of each product batch (individually formulated
preparation of the product); .
(vi) Phenol resistance of test microorganisms (actual'test results)
when specified in the methods;
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71
(vii) Identification of all material or procedural options employed,
where such choice is permitted or recommended in the test method selected
(for example, growth media, drying time for inoculated carriers, neutralizer
and/or subculture media, secondary subculturing);
(viii) Complete report of results obtained for each individual repli-
cation;
(ix) Any control data essential to establish the validity of the test.
(2) Modification of recommended methods. Where recommended methods
are significantly modified to support specific claims and/or use for a
product, the protocol employed for modifying the test must be provided in
specific detail with the test report. The applicant may submit the proposed
modification for review and evaluation prior to initiation of the test. ' '
(3) Other methods. When recommended methods, or modifications-thereto,
are not employed to develop efficacy data (such as actual inuse or many kinds
of simulated-use testing), complete testing protocols must be submitted with
the test reports. All materials and procedures employed in testing must be
described in a manner consistent with original research reports published in
technical or scientific journals. Where references to published reports or
papers are made, copies or reprints of such references should be provided
with the test reports. Proposed testing protocols for in-use or simulated-use
studies of this kind may be submitted for review and evaluation by the Agency
prior to initiation of tests.
(d) Recommended methods. (See the Supplemental recommendations for
modifications appropriate for the intended pattern of use.)
(1) Sterilizers.
Horowitz, William, ed. Sporicidal test - official final action.
Official Methods of Analysis of the Association of Official Analytical
Chemists. Current Edition. Association of Official Analytical
Chemists, Washington, D.C.
Also see: Beloian, A. 1978. Report on disinfectants. J*_ AOAC 61:372.
(2) Disinfectants water soluble powders and liquid products (Hard
surfaces). . - .
Horowitz, William, ed. Use-dilution method - official final action.
Official Methods of Analysis of the Association of Official Analytical
Chemists. Current Edition. Association of Official Analytical Chemists,
Washington, 0. C.
(.3) Disinfectants - Spray products (Hard surfaces).
Horowitz, William, ed. Germicidal spray products - official final
action. ~ Official Methods of .Analysis. 6-f the .Association of' Official
Analytical Chemists. .Current 'Edition.. Association of Official
*•....' Analytical Chemists, Washington, D.C. . . . . . "': ..•-..••-. ...,-•-
image:
72
(4) Disinfectants - Efficacy against pathogenic funcfi (Hard surfaces).
HDrowitz, William/ ed. Fungicidal test - official final action.
Official Methods of Analysis of the Association of Official Analytical
Chemists. Current Edition. Association of Official Analytical
Chemists, Washington, D.C.
(5) Disinfectants - Efficacy against viruses (Hard surfaces).
(Proposed method prepared by Registration Division, Office of
Pesticide Programs, EPA, 1976)
The Agency will accept adequate data developed by any vixological tech-
nique which is recognized as technically sound, and simulates, to the extent
possible in the laboratory, the conditions under which the product is
intended for use. For virucides,whose use-directions identify the pesticide
as one intended for use upon dry, inanimate, environmental surfaces (such as
floors, tables, clean and dried medical instruments, etc.), carrier methods,
which are modifications of either the AOAC Use Dilution Method (for liquid
surface disinfectants) or the AOAC Germicidal Spray Products Test (for sur-
face spray disinfectants), must be used in the development of the virological
data. To simulate in-use conditions, the specific virus to be tested must
be inoculated onto hard surfaces, allowed to dry, and then be treated with
the product according to the directions for use on the product label. If
the product is intended to be represented as virucidal in the presence of
organic soil ("one-step"), an appropriate organic soil, such as 5% blood
serum, must be included with the inoculum. Additional organic material need
not be incorporated into those procedures where at least 5% blood serum is
already present in the virus suspension used as the inoculum.• The product
must be tested against a recoverable virus titer of at least 104 from the
test surface (such as petri dish, glass slide, cylinder) for a 10-minute
exposure period at room temperature. The virus is then assayed by an
appropriate virological technique. The protocol for viral assay must pro-
vide the following information:
(i) The virus recovered from a minimum of 4 determinations per each
dilution in the assay system (tissue culture, embryonated egg, animal
•infection, or whatever assay system is employed).
(ii) The activity of the germicide against the test virus from a
minimum of 4 determinations per each dilution in the assay system.
(iii) Cytotoxicity controls: the effect of the germicide on the assay
system for a minimum of 4 determinations per each dilution.
(iv) Any special methods which were used to increase the virus titer
and to detoxify the residual germicide.
(v) The ID-50 values calculated for each assay.
(vi) The test results shall be reported as the reduction of the virus
titer by the activity of the germicide (ID-50 of the control less the ID-50
of the test system), expressed as log 10, and calculated by a statistical
method (Feed and Muench, 1938; Litchfie^d and Wilcoxon, 1949; as examples).
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73
(vii) For virucidal data to be acceptable, the product must demonstrate
complete inactivation of the virus at all dilutions. When cytotoxicity
is observed in the assay system (as in Tables 1, 2, and 3, below), at least a
3-log reduction in titer assay system must be demonstrated. The calculated
viral titers must be reported with the test results. A typical laboratory
report of a single test with one virus (recovered from a treated surface)
involving a tissue culture, therefore, would include the details shown in
the following tables:
Table 1. EXAMPLE OF HYPOTHETICAL TEST RESULTS DEMONSTRATING VIRUCIDAL ACTIVITY
Dilution - Virus - Virus - Cytotoxicity
inoculated Disinfectant1 Control1. - Control
10""1 TTTT I It I TTTT
10-1 TTTT ++++ TTTT
10-2 TTTT -M-++ . TTTT
ID"3 TOOO ++++ . TOGO
10~4 OOOO -M-H- OOOO
10 ~5 OOOO -H-H- OOOO
10 ~6 OOOO +-H-O OOOO
10~7 OOOO OOOO OOOO
10~8 OOOO OOOO OOOO
1 Recovery of virus from surfaces demonstrated by cytopathogenic effect,
fluorescent antibody, plaque count, animal response, or other recognized
• acceptable technique.
Note: T = toxic; + = virus recovered; O = no virus recovered.
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74
Table 2. CALCULATION OF THE TISSUE CULTURE INFECTIVE DOSE 50% (TCID50*)
Accumulated Values
-4
Dilution
inoculated
10-1
10-2
10 ^3
10 ~4
10-5
10~6
10-7
ID-8
No. infected/
No. inoculated
4/4
4/4
4/4
4/4
4/4
3/4
1/4
0/4
No.
infected
4
4
4
4
4
3
1
0
No . not
infected
0
0
0
0 . .
0
1
3
4
No.
infected
24
20
16
12
8
4
1
0
No. not
infected
0
0
0
0
0
1
4
8
No. infected/
no. inoculated
24/24
20/20
16/16
12/12
8/8
4/5
1/5
0/8
Percent
infected
100
100
100
100
100
80
20
0
*TCID50 = 10
6.5
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75
Table 3. CALCULATIONS OF THE TISSUE CULTURE LETHAL DOSE 50% (TCLD5Q*)
Accumulated Values
>•••
Oil lit ion • No. toxic/
inoculated . No . inoculated
• ;• ip-i 4/4
10~2 4/4
:iQ~3 1/4
ld~4 0/4
; Ip"5 0/4
.: 10-6 0/4
•1Q-7 ' 0/4
; ld-8 0/4
No.
toxic
4
4
1
0
0
0
0
0
No . not
toxic
0
0
3
4
4
4
4
4
NO.
toxic
9
5
1
0
0
0
0
0
No. not
toxic
0
0
3
7
11
15
19
23
No. toxic/
No . inoculated
9/9
5/5
1/4
0/7
0/11
0/15
0/19
0/23
Percent
toxic
100
100
25
0
0
0
0
0
*TCLD50 = 102'7; therefore, virus inactivation = TCID5Q - TCLD5() = 103'8 log 10. Claims
for virucidal activity for a product must be restricted to those viruses which have actually
been tested.
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76
(viii) Also see:
Litchfield, J.T., Jr., and F. Wilcoxon. 1949. A simplified method of
evaluating dose-effect experiments. J. Pharm. Exp. Therapy 96:99-115.
Reed, L.J., and H. Muench. 1938. A simple method of estimating 50%
endpoints. Amer. J. Hygiene 27:493-497.
(6) Disinfectants - Efficacy against Mycobacterium tuberculosis (Hard
surfaces).
Horowitz, William, ed. Tuberculocidal activity - official final action.
II. Confirmative In-Vitro Test for Determining Tuberculocidal Activity.
Official Methods of Analysis of the Association of Official Analytical
Chemists. Current Edition. Association of Official Analytical Chemists,
Washington, D.C.
(7) Disinfectants - Phenol coefficients.
Horowitz, William, ed. Phenol coefficient - official final action. Official
Methods of Analysis of the Association of Official Analytical Chemists.
Current Edition. Association of Official Analytical Chemists, Washington,
D.C.
(8) Sanitizers - Non-food contact surfaces.
(Proposed method prepared by Registration Division, Office of Pesticide
Programs, EPA, 1976).
To substantiate the sanitizing claims for a product, the applicant must sub-
mit data to show that the product, when used as directed, will substantially
reduce the numbers of test microorganisms on a treated surface over those
on an untreated control surface. The following protocol may be utilized:
(i) The product must be tested against each test bacterium on each
representative surface depending on the uses proposed on the label. The
test microorganisms are Staphylococcus aureus (ATCC 6538) and Klebsiella
pneumoniae, aberrant, (ATCC 4352). Enterobacter aerogenes (ATCC 13048) may
be substituted for K. pneumoniae. Representative test surfaces include,
but are not limited to: glass, metal, unglazed or glazed ceramic tile, or
vitreous china. The propagation of cultures and the use of subculture
media and other related equipment shall be as specified in Sec. 4.001 and
4.002 of the Official Methods of Analysis of AOAC, 12th ed. (1975).
(ii) Determine the count of bacteria in an 18- to 24-hr broth culture
and add a 0.01- to 0.03-ml quantity of the broth culture by spreading on
1 x 1 in. square of test surface using a bacteriological loop. If the
product is intended to be represented as a "one-step" cleaner-sanitizer, an
appropriate organic soil, such as 5% blood serum, must be included with the
inoculum. The square shall be dried for 20-30 min. in a bacteriological
incubator at 30 to 37° C. A "zero time" bacterial numbers recovery test
must be performed to show the efficiency of the recovery-process and must
be reported. The "zero time" test shall show the loss in viability that
image:
77
occurred during the drying. Apply the product to the test surface as
directed on the label. Run parallel tests on the formulation with active
ingredient(s) omitted in an identical manner to serve as the controls. If
such a control solution is not suitable, use sterile distilled water con-
taining 0.01% isooctylphenoxypolyethoxyethanol (9-10 moles oxyethylene,
e.g., Triton X-100). After a suitable time interval, recover test organ-
isms by washing the squares with adequate agitation in appropriate media
or dilution fluid containing appropriate neutralizers. Make plate counts
on appropriate nutrient agar containing the same neutralizers by the pour
or spread plate technique. Exposure time intervals between zero time and
5 min. must be tested for the product as well as for the untreated controls.
(ill.) -The results must show a bacterial reduction of at least 99.9%
over the parallel control within 5 min.
(iv) Also see:
Horowitz, William, ed. Official Methods of Analysis of the Association
of Official Analytical Chemists. Current Edition. Association
of Official Analytical Chemists, Washington, D.C.
(9) Sanitizing rinses - Food contact surfaces (Halides).
Horowitz, William, ed. Available chlorine germicidal equivalent
.concentration - official final action. Official Methods of Analysis
.•'•'-• of the Association of Official Analytical Chemists. Current
• Edition. Association of Official Analytical Chemists, Washington,
• '_ / '• D.C. ';• -.-.'.--,•.• ; '. • . •;..-.• . '••'.-,. y. .'..
(10) Sanitizing rinses - Food contact surfaces (Non-halides).
Horowitz, William, ed. Germicidal and detergent sanitizers official
final action. Official Methods of Analysis of the Association of
Official Analytical Chemists. Current Edition. Association of
Official Analytical Chemists, Washington, D.C.
(11) Disinfectants and sanitizers - Laundry additives.
Petrocci, A.M., and Paul Clark. 1969. Proposed test method for
antimicrobial laundry additives. J. AOAC 52:836-842.
(12) Residual self-sanitizers - Laundry additives.
AATCC Committee RA31. 1974. AATCC Test Method 100-1974 (9. Quantitative
Procedure) (Reference or Confirmatory Test). Pp. 264-265 in Tech-
nical Manual of the American Association of Textile Chemists and
Colorists. Volume 50. American Association of Textile Chemists
'.and. Colonists,. Research Triangle Park., N.C. .
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78
(13) Sanitizers - Carpets. . • , . ,,
N
(Proposed method prepared by Registration Division, Office of
Pesticide Programs, EPA, 1976; revised 1981)
(i) Special equipment and materials. (A) Carpet mounting board.
Mount a piece of 1/8-in. (0.3 cm) tempered hardboard, tempered surface up,
on a 16 x 16-in. (40.6 x 40.6 cm) base of 3/4-in. (1.8 cm) thick marine
plywood, with 3/4-in. (1.8 cm) brads.
(B) Cutting equipment. 2 x 2-in. (5.1 x 5.1 cm) squares of 1/4-in.
(0.6 cm) acrylic plastic with 3/32-in. (0.24 cm) holes in the center as
templates, and a sharp knife with replaceable blade.
(C) Scrub brushes. 1 1/4 x 3 1/2-in. (4.2 x 8.9 cm) surgical hand
brush with 5/8-in. (0.6 cm) nylon bristles.
(D) Extraction bottles. 8-oz. (236.6 ml), widemouth, round, poly-
propylene bottles with screw caps (Naigene 2105 or equivalent) containing
10 stainless steel penicylinders and 100 ml of appropriate neutralizer
broth. Similar style glass bottles may be used, but care must be taken to
prevent breakage during shaking.
(E) Spray device. Adjustable spray atomizer modified to feed from a
calibrated test-tube or bottle. A Model 15 DeVilbiss atomizer on a 2r^sz.
(59.2-ml) bottle graduated with 10-ml marks may be used.
(P) Carpet. If the product. Is intended for use on commercial grade
carpeting, two representative carpets^ such as acrylic and polypropylene
tufted-loop type must .be tested. No carpeting is available to serve as a
standard. If the product is intended for use on wool carpeting, a repre-
sentative wool sample must additionally be tested. All carpet samples
tested must be fully identified, and the pile fiber type, pile yarn weight
of finished carpet, pile density and tuft height must be reported. Adequate
controls must demonstrate that bacteriostatic agents in the carpet .pile or
backing do not .interfere with the test results.
(ii) Test cultures and media. (A) Test bacteria. Use Staphylococcus
aureus (ATCC 6538) and Enterobacter aerogenes (ATCC 13048). If the product
is intended for use in hospitals, Pseudomonas aeruginosa PRD-10 (ATCC 15442)
must additionally be tested. . • .
(B) Nutrient Agar B. AOAC Methods, sec. 4.023 (a)(2).
(C) Phosphate buffer dilution water. AOAC Methods, sec. 4.023 (f).
(D) Double strength neutralizer broth.' Ibr phenolic based products,
Letheen broth [AOAC Methods, sec. 4.001 (d)(3)] plus an additional 0.7 g
lecithin (Azolectin) and 5 g polysorbate 80 (Tween 80) per liter may be
used; or a defoaming neutralizer consisting of nutrient broth [AOAC methods,
sec. 4.001 (a)] plus 1.0%-Pluronic. 2'5R2 (Meroxapol 252') has been suggested.
In the case of halogen or heavy metal based products,. 0.1% sodium thio-
glycolate and 0.01% isooctylphenoxypol:ye;thoxyethanQl ('.Tr-Jton ;X-100')' ;in:
phosphate .buffer (pH 7<2') may be used. •• '-:.• '•••' .,
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79
(E) Neutralizer plate count agar. Tryptone glucose extract agar [AOAC
Methods, sec. 4.037 (a)] plus 0.7 g lecithin (Azolectin) and 5 g polysorbate
80 (Tween 80) per liter.
(iii) Bacterial inoculum. Prepare French square culture bottles with
nutrient agar B and test bacteria (AOAC Methods, Sec. 4.026). Prepare
standardized bacterial stock suspensions by washing growth from bottles and
adjust to a density of 10 x 109 bacteria per ml with phosphate buffer
dilution water (AOAC Methods, sec. 4.026).
(iv) Procedure. (A) Cut the carpet into 8 x 12-in (20.3 x 30.5 cm)
pieces. With the aid of the 2 x 2-in. (5.1 x 5.1 cm) template, cut six 2
x 2-in. squares (2 rows of 3-squares per row) from the backing side of the
carpet, leaving at least 4 in. (10.2 cm) between the center of each square.
The preferred method is to leave about 1/8 in. (0.32 on) of backing intact
at each corner of each cut square so that the entire piece of carpeting can
be sterilized and inoculated without separation. Mark the pile surface in
the center of each test square with a waterproof marking pen with the aid
of the hole in the center of the template. Cover the pile surface of the
carpeting with aluminum foil and fold over edges to secure. Steam sterilize
and dry. Only carpet that has been determined to be free from residual
bacteriostatic activity on the pile or backing, following autoclaving, shall
be used. A seeded agar plate overlay technique should be used for this
determination.
(B) Dilute the standardized bacterial stock suspensions, prepared as
in paragraph (d)(13)(iii) of this section, with phosphate buffer dilution
water containing 0.01% isooctylphenoxypolyethoxyethanol to a concentration
10 x 10^ bacteria-per ml. Inoculate the previously marked center of each
cut square with 0.1 ml of the bacterial suspension. (Retain the bacterial
suspensions for determination of inoculation numbers). Dry inoculated
carpet in an incubator at 35-37°C for 60 min. with the foil wrap loosely
in place.
(C) Condition brushes by immersing the bristles in separate containers
(15- cm glass petri dishes or equivalent) of diluted test solution and a
control solution without the active antimicrobial ingredient(s) for 15 min.
(If such a control solution is not available, use sterile distilled water
containing 0.01% isooctylphenoxypolyethoxyethanol). Fasten 2 pieces of
innoculated carpet (each containing 6 test squares) onto the carpet mounting
board by nailing each corner with upholstery tacks, and with the foil wrap-
ping positioned so as to protect the controls during spraying and scrubbing
with the test solution. Place the board in a biological hood or glove box.
A simple safety chamber can be constructed from a large plastic bag.
(D) Determine the amount of test solution intended to be applied to
one piece of the carpeting containing 6 spots of dried bacterial inoculum
[96 sq. in. or 2/3 sq. ft. (.244 sq. cm.)] and subtract approximately 15
ml which will be applied later in the brushing procedure. Apply the pre-
•de.termined amount of diluted test solution at room temperature uniformly .
by metered spray to one piece of the test .carpet. Shake .excess test solu-
tion from a .conditioned brush.and transfer to .a fresh.dish containing 100
ml. of test, solution at rbom temperature. Dip bristles of., brush and transfer
the retained test .solution to an inoculated spot on the sprayed carpet.
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80
Scrub the spot for 30 sec. using 30 circular clockwise strokes and 30
circular counterclockwise strokes. A circular area of pile approximately
3 in. (7.6 cm) in diameter around each spot must be covered by this treat-
ment. Moderate to heavy pressure should be applied downward on the brush
to work the solution to the base of the pile. Repeat dipping of brush into
test solution and scrubbing procedure until each of the 6 spots is treated.
The brush dipped into the solution no more than 6 times will deliver about
15 ml of solution to the carpet. Do not exceed this amount. Record the
total volume of solution applied by spray and brush. Allow the treated
carpet piece to remain at room temperature for 60.min. for partial drying
of the treated areas.
(E) While the piece of carpet treated with the test solution is
drying, spray the non-active control solution at room temperature onto
half of the other (control) piece of carpet so as to cover 3 of the 6
spots of dried inoculum. Position the aluminum foil over the remainder.
Spray an amount equivalent to half of the amount of sprayed test solution.
Scrub the 3 wet spots in the same manner as the test carpet. The remaining
3 spots are unscrubbed controls to determine the numbers of bacteria which
survived drying of the inoculum. Care must be taken not to wet or scrub
over the unscrubbed control area. Allow the scrubbed and unscrubbed con-
trols to remain at room temperature for 60 min. as with the test piece.
(F) Following the 60-min. drying periods, cut each 2 x 2-in. test
square free with flamed forceps and knife. Transfer each square of carpet
to a separate extraction bottle of neutralized broth. Shake each extraction
bottle vigorously for at least 1 min. to free the'bacteria from the carpet
fibers. Determine the number of viable bacteria in each sample bottle by
plating duplicate dilutions in neutralizer place count agar. Similarly
determine the number of viable bacteria in 0.1 ml of the suspension used for
inoculating the carpet. Also incubate all broth extraction bottles to deter-
mine whether neutralization of the test sample was achieved.
(G) Determine the percent reduction of viable bacteria by the test
solution by comparing the number of survivors from each treated test square
against the average viable count from the scrubbed control squares. An
average viable count of at least 1.0 x 106 bacteria from the extracted
unscrubbed control squares is necessary for a valid test.
(v) Also see:
Iforowitz, William, ed. Official Methods of Analysis of the Association
of Official Analytical Chemists. Current Edition. Association
of Official Analytical Chemists, Washington, D.C.
(14) Disinfectants - Swimming pools.
Horowitz, William, ed. Water disinfectants for swimming pools official
final action. Official Methods of Analysis of the Association
of Official Analytical Chemists. Current Edition. Association
of Official Analytical Chemists, Washington, D.C.
image:
81
(i) Also see:
Joint Committee on Swimming Fools of the A.P.H.A. in cooperation with
the O.S.P.H.S. 1964. Suggested Ordinance and Regulations
Covering Public Swimming Pools. The American Public Health
Association, New York, N.Y.
(e) Supplemental recommendations. When an antimicrobial agent is
intended for a use pattern that is not reflected by the test conditions
specified in the recommended methods indicated above, one or more test
conditions specified in the method must be modified and/or supplementary
data developed in order to provide meaningful results relative to the
conditions of use. The following information is critical to the develop-
ment and submission of appropriate data.
(1) Exposure period. The exposure period required for an antimicrobial
agent to be effective may be shorter or longer than the exposure period
specified in the recommended method. A modification to provide a shorter
exposure period is restricted by the manipulative limitations inherent in
the method, while a modification to provide a longer exposure period is
restricted by the practical considerations of the use pattern.
(2) Type of surface. When an antimicrobial agent is intended to be
effective in treating a* hard porous surface, some of the above recommended
methods may be modified to simulate this more stringent condition by
substitution of a porous surface carrier (such as a porcelain penicylinder
or unglazed ceramic tile) for the non-porous surface carrier (stainless
steel cylinder or glass1 slide) specified in the method. In addition,
control data, described below in Supplemental recommendation No. (6), must
be developed to assure the validity of the test results when this modifica-
tion of the method is employed. Since the use of a porous surface would
simulate the more stringent test condition, demonstrated efficacy on porous
surfaces would suffice 'to support an analogous claim for efficacy on non-
porous surfaces as well,. In no case may a surface carrier which represents
a less stringent condition be substituted for a surface carrier which is
specified in the Recommended method.
(3) . Hard water. • The above recommended methods may be modified to
demonstrate the effectiveness of an antimicrobial agent in hard water.
The hard water tolerance level may differ with level of antimicrobial
activity claimed. To establish disinfectant efficacy in hard water, all
microorganisms (bacteria, fungi, viruses) claimed to be controlled must be
tested by the appropriate Recommended method at the same hard water tolerance
level. Refer to Recommended method Mo. 10 for the method of preparing hard
water.
(4) Organic :soil. An antimicrobial agent identified as a "one-step"
.cleaner-disinfectant., cleaner-sanitizer, or one intended to be effective
in. the presence of., organic soil must be tested for efficacy by the appro-
.priate methpd(s) .which have been modified to include a .representative
organic -soil''such as • 5% blood.serum- ..A suggested procedure :to simulate
in-use conditions' where the :antimicrobial /agent -is intended-, to; treat dry
inanimate- sWfaces with:.-an. organic soil, load'involves-contamination of. the--**
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82
appropriate ^carrier surface with each test microorganism culture containing
5% v/v blood serum (e.g., 19 ml test microorganism culture + 1 ml blood
serum) prior to the specified carrier-drying step in the method. Control
data, described below in (6), must also be developed to assure the validity
of the test results when this modification is incorporated into the method.
The organic soil level suggested is considered appropriate for simulating
lightly or moderately soiled surface conditions. When the surface to be
treated has heavy soil deposits, a cleaning step must be recommended prior to
application of the antimicrobial agent. The effectiveness of antimicrobial
agents must be demonstrated in the presence of a specific organic soil at
an appropriate concentration level when specifically claimed and/of indicated
by the pattern of use. A suggested procedure for incorporating organic soil
load where the antimicrobial agent is not tested against a dry inanimate
surface, such as the AOAC Pungicidal Test, involves adding 5% v/v blood serum
directly to the test solution (e.g., 4.75 ml test solution + 0.25 ml blood
serum) before adding 0.5 ml of the required level (5 x 10^/ml) of conidia.
(5) Re-use. The recommended methods indicated in this section are
designed to demonstrate efficacy of a freshly prepared antimicrobial solu-
tion intended for a single application. When the same use solution is
intended for repeated applications, testing must be conducted in accordance
with a test protocol specially designed to demonstrate retention of the
claimed level(s) of antimicrobial activity in the use solution after repeated
microbial and other appropriate challenges (such as organic soil or hard
water) and stress conditions (such as inadvertant or incidental dilution
inherent in the use pattern) over the period of time or number of times
specified in the directions for use.
(6) Microorganism survival after drying on a hard surface, (i) Quan-
titative determinations of the microbial concentration on the untreated con-
trol carrier after drying are required in order to determine the validity of
the test results obtained with the treated carriers when the recommended
methods indicated above are modified to include such elements as:
(A) Test microorganisms not specified in the method;
(B) Substitution of a porous surface (e.g., porcelain penicylinder,
unglazed ceramic tile) for the specified nonporous surface (stainless steel
cylinder, glass slide); and/or
(C) An organic soil load.
(ii) The detailed protocol for this testing must include:
(A) Preparation of inoculum;
(B) Application of inoculum to the carrier;
(C) The time/temperature and relative humidity conditions for drying
the microorganisms on the carrier;
(D) The technique for removal of the microorganisms from the carrier;
and
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(E) The specific assay procedure indicating such details as replication,
subculture media/diluents, and the incubation time/temperature conditions for
the enumeration procedure employed.
(iii) The test results must include the individual counts obtained by
the method.
(7) Neutralization. for each antimicrobial product, procedures must
be employed that will preclude residual effects of the active ingredient(s)
in the subculture medium. A specific medium capable of neutralizing the
antimicrobial effects of a product (whenever one is known) should be employed
prior to the microbiological assay. Some of the recommended methods described
in this section rely solely upon the selection of an appropriate subculture
medium to neutralize the antimicrobial effects of certain general types of
chemical compounds (active ingredients). (Refer to § 91-30 recommended
method Mb. 7). Hswever, to document the absence of residual effects of
the active ingredient(s) in the subculture medium, the following testing
is necessary:
(i) Secondary subcultures must be performed to demonstrate that anti-
microbial effects were overcome; or
(ii) At the conclusion of the incubation period specified or employed
in the method, the primary culture medium with test carrier must be inocu-
lated with approximately 10 microorganisms/ml of the specific bacterial
species under test (documented'by actual plate counts) and reincubated for
the specified period to demonstrate that the subculture medium was capable
of supporting bacterial growth.
(8) Batch replication for modified tests. Where the required batch
replication has already been performed and accepted for a product registra-
tion with unmodified tests by the recommended methods, additional testing
at the same use concentration under modified conditions (e.g., different
exposure period, presence of organic soil or hard water, porous surface
carrier, etc.) may be conducted with reduced batch replication, as follows:
(i) For basic efficacy claims [e.g., sterilizers, § 91-2(a); disin-
infectants, § 91-2(b), (c), or (d); sanitizers, § 91-2(j) or (k)], two
samples, representing two different batches instead of three.
(ii) For supplemental efficacy claims [e.g., fungicides, § 91-2(e);
virucides, § 91-2(f); tuberculocides, § 91-2(g)], one sample instead of
two.
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Subseries 91B: NON-PUBLIC HEALTH USES
§ 91-51 General considerations.
(a) Scope. Sections 91-51 through -56 contain information concerning
testing and performance of antimicrobial pesticide products for uses which
are not directly related to hi""*" health. These uses include control of
odor-producing bacteria, bacteria causing spoilage, deterioration, or foul-
ing of materials such as paint or industrial fluids, and microorganisms
infectious only for animals, where product failure against the specified
pests would not have human health consequences. Pursuant to the efficacy
data waiver provisions of Section 3(c)(5) of FIFRA, and § 162.18-2 of the
FIFRA sec. 3 regulations, efficacy test data for these uses are not gener-
ally required to be submitted to support product registration [See § 90-l(b)].
Also, refer to § 101-Kb) ,(c), (d), and § 101-30 of Subdivision H for additional
information concerning the relationship between label claims, human health
considerations, and performance requirements for antimicrobial products.
Requirements for testing and performance for those uses of antimicrobials
which are identified as directly related to human health are provided in
§§ 91-1 through 91-8 of this series. Labeling guidance for all uses of
antimicrobial pesticides, both health-related and non-health related, are
contained in §§ 101-1 through -16 of Subdivision H.
(b) General testing considerations. (1) In-use tests. Generally,
demonstration of effectiveness of antimicrobial products in controlling
microorganisms which are aesthetically or economically undesirable may be
accomplished by establishing a correlation between successful control of
the pest problem (e.g., odor, spoilage, fouling) and limitation of numbers
of the target microorganisms at the site under actual conditions of use.
In-use tests can be considered for any product of this kind on a case-by-
case basis. However, field tests under an experimental use permit (refer
to Subdivision I) are prescribed as a requirement only for the following
non-public health uses:
(i) Antimicrobial fuel additives [see § 91-53(c)] .
(ii) Antimicrobial additives for sugar mills [see § 91-53(d)].
(iii) Antimicrobial additives for poultry and livestock drinking
water [see § 91-55(a)].
(2) Simulated-use tests. Except for the uses indicated in paragraph
(b) of this section, simulated use laboratory tests can usually be con-
sidered as acceptable alternatives to actual in-use tests. Simulated-use
tests should be designed to include the following basic elements:
(i) Identified test microorganisms (at least to the generic level)
associated with the pest problem at specified site(s).
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(ii) Appropriate surface(s)-or substrate(s) which support growth of
the test microorganisms under the environmental conditions (e.g., temperature,
relative humidity) which simulate the in-use situation.
(iii) Adequately replicated test systems consisting of material inocu-
lated with the test microorganisms and treated as directed with the anti-
microbial product, together with parallel inoculated untreated controls.
(iv) Periodic observations on the presence or absence of the pest
problem (e.g., odor, spoilage) which should include chemical, physical, or
olfactory measurements.
(v) Parallel quantitative sampling techniques (e.g., agar plate counts)
to enumerate the test microorganisms at appropriate intervals.
(vi) Conduct of the tests for a period of time which is recommended or
required in actual use.
(3) Tests designed for public health uses. Effectiveness of anti-
microbial products for certain uses in controlling microbial pests which
are aesthetically undesirable (e.g., odor-causing bacteria) can often be
extrapolated from the same kinds of efficacy tests required for public
health uses (e.g., disinfectants, sanitizers, residual self-sanitizing
treatments; see §§ 91-1 through -8 of this series) except for substitution
of appropriate test microorganisms. Efficacy test data must be developed
and submitted in accordance with human health uses (see §§ 91-1 through -3
of this series) when effectiveness is claimed or implied in labeling
against microorganisms infectious for both man and animals. This is
necessary to assure minimal protection of persons in contact with the
animal environment. Qualified label claims against animal pathogens only
would not generally require submission of specific test data against
those microorganisms. When necessary [see § 162.18-2(d)(3)(ii) of the
FIFRA sec. 3 regulations] , the tests and performance criteria would be the
same as those indicated for public health uses (§§ 91-1 through -8) except
for substitution of appropriate test microorganisms.
(4) Qualitative screening tests. Qualitative data developed by
presumptive screening tests, such as phenol coefficient tests, nutrient
broth inhibition tests, or zones of inhibition on seeded agar or streak
plates, are not considered to be of value in providing meaningful results
that can be associated with end-uses of antimicrobial products and are
unacceptable as documentation of efficacy for end-use claims. However,
qualitative tests of this kind are acceptable to document potential or
presumptive value of antimicrobial pesticide products intended only for
formulation purposes (see § 91-57].
(5) Test substance. Unless othewise specified, products should be
tested on the formulation as offered.for sale and in accordance with the
proposed directions :for. use. . •
06) tteutrg-Liz&rs. In testing the efficacy of any antimicrobial
•product, appropriate..neufcralizers should .be. employed in the..microbio-logicai,'.' ''
assay system, and evidence obtained to show that the:' neutralizers employed
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inactivate the active ingredient(s) and-do not possess any antimicrobial
activity themselves. In lieu of specific evidence of chemical neutraliza-
tion, it must be documented that appropriate secondary subculturing tech-
niques have been employed that preclude residual effects cf active ingred-
ients in the assay medium. [Refer to § 91-30(e)(7).]
(7) Test variations. The protocol for testing will vary according
to the type of product, type of substance to be treated, proposed use
pattern, label claims, directions for use, and other factors peculiar to
the specific product. In many cases, specific recommendations (such as
the amount of replication) can be determined only after consideration of
these factors. Refer to § 91-30(e) for guidance on some common test modi-
fications (e.g., hard water, organic soil).
§ 91-52 Products for use on hard surfaces.
(a) Disinfectants (animal health). The following apply to all products
represented in labeling as disinfectants for animal premises and equipment,
including veterinary uses, farm uses, kennels, pet shops, zoos, and household
pet areas. x
(1) Control of microorganisms 'infectious for both man and animals;
Public health uses. The efficacy data waiver.provision § 90-l(b) is not
applicable to microorganisms which are infectious for both man and animals.
Unless disinfecting, germicidal, or bactericidal claims are specifically
qualified as intended against animal and veterinary pathogens only, animal
and veterinary premises disinfectants must be supported by basic efficacy
data developed4and submitted in' accordance with the requirements for public
health uses.
(i) Test standard. Same as § 91-2(b)(l), (c)(l), (d)(l), or (g)(1)
of this series.
(ii) Suggested performance standard. Same as § 91-2(b)(2), (c)(2),
(d)(2), or (g)(2) of this series.
(2) Control of microorganisms infectious only for animals: Non-public
health uses. The efficacy data waiver provision § 90-l(b) is applicable
to microorganisms which are infectious only for animals. However, the
efficacy tests appropriate for such supplemental efficacy claims are the
same as those which are required for public health uses, except for substitution
of specifically claimed animal pathogens as. test microorganisms.
(i) Test standard. Same as § 91-2(e)(l), (f)(l), (h)(l), or (i)(1)
of this series, using specifically claimed animal pathogens as test micro-
organisms.
(ii) Suggested performance, standard. Same as § 91-2{e') (2), (f) (2),
(h)(2), or (i)(2) of this series. -
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(b) Odor control treatments (non-residual). The following apply to
products represented in labeling as non-residual treatments to kill or
reduce the number of odor-causing bacteria.
(1) Test standard. Same as § 91-2(b)(l), (c)(l), or (j)(l) of
this series, except that pure culture isolates of identified odor-causing
bacteria must be employed as test microorganisms.
(2) Suggested performance standard. Same as § 91-2(b)(2) or (c)(2)
of this series for claims to kill odor-causing bacteria; same as § 91-2(j)(2)
of this series for claims to reduce the number of odor-causing bacteria.
(c) Odor control treatments (residual). The following apply to
products represented in labeling as residual treatments to reduce the
number of odor-causing bacteria or bacteriostatic odor control 'in the
presence of moisture.
(1) Test standard. Same as § 91-2(m)(l) of this series, except
that pure culture isolates of identified odor-causing bacteria must be
employed as test microorganisms.
(2) Performance guidance. Same as § 91-2(m)(2) for claims to
reduce the number of odor-causing bacteria; for bacteriostatic odor
control claims, the numbers of test microorganisms recovered from the
treated surfaces should be less than the number recovered from the parallel
control surfaces and no greater than the "0-time" control.
§ 91-53 Products for use on fabrics and textiles.
(a) Laundry additives. The following applies to antimicro-
bial products which bear label recommendations for treatment of laundry for
odor control.
(1) Odor control pre-soaking treatments (non-residual). The require-
ments for products recommend to kill odor-causing bacteria on soiled fabrics
by total immersion in the use solution prior to routine laundry operations
are as follows:
(i) Test standard. Same as § 91-4(a)(1)(i) of this series, except
that pure culture isolates of identified odor-causing bacteria must be
employed as test microorganisms.
(ii) Suggested performance standard. Same as § 91-4(a)(1)(ii) of this
series.
(.2.) Odor control laundry additives (non-residual). The following apply
to products which bear label claims to kill or reduce the number of odor-
causing bacteria when.used in automatic or manual washing.-machine-.-operations
are .as-follows: •., .... • -. . . • . . '
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(i) Test standard. Same as § 91-4U) (2) (i) or (a)(3)(i) of this
series, except that pure culture isolates of identified odor-causing
bacteria, must be employed as test microorganisms.
(ii) Suggested performance standard. Same as § 91-4(a)(2)(ii) for
claims to kill odor-causing bacteria; same as § 91-4(a)(3)(ii) for claims
to reduce the number of odor-causing bacteria.
(3) Odor control laundry additives (residual). The following apply to
products which bear label claims as laundry treatments to reduce the number
of odor-causing bacteria or provide bacteriostatic odor control on treated
fabrics in the presence of moisture when added to washing machine operations
are as follows:
(i) Test standard. Same as § 91-4(a)(4)(i) of this series, except
that pure culture isolates of identified odor-causing bacteria must be
employed as test microorganisms. If claims are made for controlling devel-
opment of ammonia odors from urine on laundered fabrics, Proteus mirabilis
ATCC 9240 is required as the test microorganism and urea J/ must be added
to test swatches.
(ii) Suggested performance standard. Same as § 91-4(a)(4)(ii) of this
series for claims to reduce the number of odor-causing bacteria; for
bacteriostatic odor control claims, the numbers of test microorganisms
recovered from treated swatches should be less than the numbers recovered
from the parallel control swatches and no greater than the "0-time" control;
and for ammonia control claims, ammonia production should be delayed for
the time period claimed.
(b) Carpet treatments. The following apply to products bearing
label claims as carpet treatments to reduce the number of odor-causing
bacteria.
(1) Test standard. Same as § 91-4(b)(l) of this series, except
that pure culture isolates of identified odor-causing bacteria should be
employed as test microorganisms.
(2) Suggested performance standard. Same as 91-4(b)(2) of this series.
(c) Mattresses and upholstered furniture. (1) Gases or vapors.
The use of gases or vapor is currently the only effective and practical
means of treating entire mattresses, upholstered furniture, pillows, and
similar objects to kill or reduce the number of odor-causing bacteria.
The following apply to products bearing such label recommendations:
(i) Test standard. Same as § 91-4(c)(l) of this series, except
that pure culture isolates of identified odor causing bacteria should be
employed as test organisms.
_1/ See: Latlief, M.A., M.T. Goldsmith, and J.L. Stuart. 1951. Germicidal
and sanitizing action of quaternary ammonium compounds on textiles; preven-
tion of ammonia formation from urea by Proteus mirabilis. J. Pediatr. 39:
730-737.
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89
(ii) Suggested performance standard. Same as § 91-2(b)(2) or (c)(2)
for claims to kill odor-causing bacteria; same as § 91-2(j)(2) for claims
to reduce the number of odor-causing bacteria.
(2) Liquids. The use of liquid products applied by mechanical or
pressurized spray for treating mattresses, upholstered furniture, pillows,
and similar objects is an effective means of reducing the number of odor-
causing bacteria on or in the ticking only. The following apply to products
bearing such label recommendations:
(i) Test standard. Same as § 91-2(j)(l) of this series, employing
ticking material instead of hard surface carriers as the test and control
surfaces, and employing pure culture isolates of identified odor-causing
bacteria as test microorganisms.
(ii) Suggested performance standard. Same as § 91-2(j)(2).
' (d) Impregnated fabrics and textiles. The following apply to products
intended for treatment of fabrics and textile .materials, usually during
the manufacturing process, to provide durable residual antimicrobial activity
for reducing the number of odor-causing bacteria or bacteriostatic odor
control on treated surfaces in the presence of moisture.
(1) Test standard. Same as § 91-2(m)(l) of this series, employing
treated and untreated fabrics or fabricated items instead of hard surface
carriers as the test and control surfaces, and employing pure culture
isolates of identified odor-causing bacteria as test microorganisms.
(2) Suggested performance standard. Same'as § 91-2(m)(2) of this series,
for claims to reduce the number of odor-causing bacteria; for bacteriostatic
odor control claims, the numbers of test microorganisms recovered from
treated surfaces should be less then the numbers recovered from the parallel
control surfaces and no greater than "0-time" control.
§ 91-54 Products for processing and industrial uses.
(a) In-can paint preservatives. Antimicrobial products which bear
claims for use as preservatives in paint formulations are pesticides, and
should meet the requirements .indicated below. Paints containing preservatives
are not pesticides unless pesticidal claims are made or implied.
(1) Test standard. Products proposed for use in preserving water-
based paints should show effectiveness in controlling spoilage or deterioration
caused .by bacteria in at .least two representative paint formulations in which
the product is;intended £or use. .Tests should be.-carried o^t.in -at, least three
replicates of each of the two paint formulations.using pertinent microorgan-
isms and adequate controls. Actual bacterial isolates '(identified, at least
to. gen-us.)yf£-0ffl; spoiled paint- and/or; ATCC. paint .spoilage .bacteria; should be
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-90
employed as test inocula. Mixed bacterial and fungal''inocula are not-- I ...
acceptable-in demonstrating'bacterial deterioration. Efficacy data should
be derived from simulated-use type tests with quantitative bacteriological
sampling and concurrent observations of paint quality. Both test and con-
trol samples should be tested for a period of six months to one year. The1
test protocol, including such elements as frequency of repeated bacterial
challenge, is contingent upon the intended preservative use pattern.
(2) Suggested performance standard. The data should show control of
bacterial growth and control of bacterial-caused deteriorative (physical
and chemical) changes in the treated paints during the test period. The
data from control paints should show not only survival of test bacteria,
but also significant growth and resultant deteriorative (physical and
chemical) changes.
i
(To) Metalworking fluids. The following apply to products bearing
label claims for preservation against bacterial growth and deterioration
in metalworking fluids.
(1) Test standard. The product should be tested in one identified
representative metalworking fluid formulation for each type (e.g., emulsi-
fiable oil, semi-synthetic fluid, synthetic fluid) in which the product is
recommended for use, and at the fluid-to-water ratio recommended in labeling.
Three replicate tests should be carried out on each metalworking fluid formu-
lation using appropriate controls. Each metalworking fluid formulation should
.be inoculated with a minimum of three different test bacteria. Each of the
test bacteria should be identified at least to genus level. It should be docu-
mented that each of the test bacteria has been isolated, from spoiled metal-
working fluids of the'type(s) in which the prduct'will be tested or bias
been successfully employed to induce spoilage of such fluids in other tests.
Either single, pure cultures of bacteria or a mixed bacterial inoculum may
be employed. However, a mixed culture inoculum of bacteria and fungi is
not acceptable. Although the control of microbial growth in metalworking
fluids involves fungi as well as bacteria, fungal growth should be considered
as a separate, though related control problem. Refer to §§ 93 (Efficacy
of Fungicides and Nematicides) for information regarding the control of
fungal growth. Each of the test bacteria should be present in the inoculum
at a concentration at least 10^ viable cells per ml of metalworking fluid.
The tests should be carried out at a temperature of 25-28°c for periods of
time with dosage amounts and intervals, and with fluid make-up procedures
that are consistent with the recommendations for use on the label. Quanti-
tative bacteriological sampling should be conducted with concurrent observa-
tions of fluid quality. Reinoculation with the test bacteria at regular
intervals (e.g., weekly) to simulate repeated contamination/challenge to
the system is necessary. The metalworking fluid in the control should be
subjected to.the same procedures.
(2) Suggested performance standard. The test should demonstrate control
of deteriorative changes and inhibition of bacterial growth in metalworking
fluids treated with the proposed product as recommended in labeling. The
tests should also demonstrate, in metalworking fluids not treated with the
proposed product, not only survival, but significant bacterial growth and
resultant deteriorative changes. The results should include a report of
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91 .
the physical or chemical changes observed in the fluids being tested.
(c) Antimicrobial fuel additives. The following apply to products
bearing label claims for control of bacterial growth in kerosene based
fuels (including jet aviation fuels) subject to water contamination, and
diesel fuels or heating oils stored in metal tanks. With aviation fuel
additives, the Federal Aviation Administration (FAA) should be consulted as to
the acceptability of the additive from the standpoint of certification for
particular airframes or engines.
(1) Test standard. (i) Laboratory test. The following basic
elements should be incorporated into a presumptive laboratory test. A micro-
biological assay using Bushnell-Haas media plus the fuel (the fuel-to-liquid
media ratio should be equivalent to that found in the field under actual condi-
tions of use) inoculated with a mixed culture of bacteria and fungi (identi-
fied at least to genus) isolated from contaminated fuel and treated at the
concentration recommended on the label. These data would presumptively
determine the efficacy of a product.
(ii) Field test. (A) Aviation fuel additives. After presumptive
efficacy is established as indicated in paragraph (c)(l)(i) of this section,
products proposed for use in engines and/or airframes of aircraft should
be field-tested according to the requirements specified in FAA Advisory
Circular AC 20-24A, dated April 14, 1967, under an experimental use permit
issued by the Agency. When an additive has not been certified for use in
a particular aircraft engine and/or airframe, a disclaimer for such use
must appear on the label.
(B) Other fuel additives. Any other proposed uses (diesel fuels, heat-
ing oils) would require field-derived efficacy data under an experimental
use permit issued by the Agency after presumptive efficacy is established
as indicated in paragraph (c)(l)(i) of this section.
(2) Suggested performance standard. The product should be shown to
inhibit microbial growth in the presumptive laboratory test, and control
the problems associated with microbial growth in the fuel systems employed
in the field test. Federal Aviation Agency certification is required for
aviation fuel additives.
(d) Antimicrobial additives for sugar mills. The following apply to
products bearing claims for control of bacterial growth in sugar mill
processes. Because cane-sugar and beet-sugar mills differ both in plant
design and processing procedures, actual in-use testing should be conducted
in both types of mills when products are recommended in labeling for use
in both types.
(1) Test standard, (i) Laboratory test. Laboratory data showing
the effectiveness of the product in inhibiting the growth of or reducing
the number of.representative Leuconostoc mesenteroides isolated from spoiled
cane or beet sugar'pressing should be provided. . .
(ii) Field east. 3ased on these data and on label recommendations,
in-use..testing should, be :coftducfced in. at .least--one cane-sugar and/or one beet-
sugar ,mill..uadef<;ran,experimental use permit to -demonstrate the efficacy Of
the-product when used.-as.-directed. -The. basic elements which should be
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incorporated in the test protocols generally employed in -the sugar mills
should include the following: all chemical.assays (e.g., Brix, invert, sugar,
lactic 'acid); all bacteriological assays based on plate counts, standard
dilution methods, or other methods recognized as suitable by the industry
(indicating time intervals and points of location in the systems where
assay samples were taken); visual or other suitable rating of the control
of bacterial slime accretion in the mill system; identification by genus
and species if possible) of the isolated microorganism(s) which utilize
sucrose; and the control treatment. The control treatment may be substi-
tuted with published information providing bacteriological data from un-
treated or inadequately treated systems, along with comparative bacteri-
ological data from a comparable sugar mill treated with a formulation
already registered for this use. Test reports should include, but are not
limited to, the following: weight of raw cane or beets processed per unit
time; product feed rate and/or concentrations used; the point or points in
the mill system of product addition; location(s) and dates of the tests;
and names (and titles or positions) of persons conducting the tests.
Prospective registrants are reminded that a food-additive regulation or
exemption from the requirement of such regulation under the Federal Food,
Drug, and Cosmetic Act must be established before a product of this type
can be registered.
(2) Suggested performance standard. The laboratory test should show
that the product inhibits the growth of Leuconostoc mesenteriodes. The
field test data should show the application of product according to label
directions permits efficient operation of the mill system by reducing
dextran deposits caused by the growth of sucrose-utilizing bacteria (i.e.,
L. mesenteroides) and that by maintaining the microbial population at an
acceptable level, an increase in the yield of sucrose is realized due to
the reduction of inversion losses. .
(e) Miscellaneous preservative uses. In accordance with § 162.4(a)
and (b) of FIFRA sec. 3 regulations, products that are recommended in label-
ing for use as non-food commodity preservatives are pesticides. Preserva-
tives commonly bear claims to control bacterial spoilage or deterioration
in such commodities as paper coatings, adhesives, plastic formulations,
ceramic glazes, grouts, floor wax emulsions, gaskets (paper, felt, cork,
rubber, vinyl), films and foams of polyvinyl and polyurethane, dextrin-based
inks, photographic solutions, laundry starch, and colloidal graphite. Such
products should be tested in each commodity claimed to substantiate effective-
ness as a perservative. In accordance with § 162.4 (c) of FIFRA sec. 3 regu-
lations, the preserved commodities themselves are exempt from registration.
(1) Test standard. Efficacy data should be derived from simulated-
use tests with identified (at least to genus) spoilage bacteria. The tests
should be carried out in triplicate using untreated controls with each commod-
ity for a period ranging from several days to a year, depending upon the
intended end use. Quantitative bacteriological sampling and concurrent
observations of commodity quality should be performed.
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93
(2) Suggested performance standard. For an effective treatment, the
results should show inhibition of bacterial growth by quantitative techniques
that can be related to colony-forming units with those microorganisms that
have been isolated from the specific deteriorated substrate. Deterioration
of the substrate in the untreated controls should be demonstrated, and the
integrity of the treated substrate should be maintained and protected. The
type of spoilage or deterioration which occurs in the untreated substrate
should be described and documented.
§ 91-55 Products for control of microbial pests associated with human and
• animal wastes .
(a) Self-contained toilet systems. Since it is ordinarily impractical
to disinfect or sanitize human excrement in self-contained toilet systems by
treatment with antimicrobial chemicals, the only pesticidal value attribut-
able to such treatment is bacteriostatic odor control. The .following
apply to products bearing such label claims or recommendations.
(1) Test standard. Controlled in-use or simulated-use studies
should be conducted comparing self-contained toilet systems treated with
the bacteriostatic chemical with identical systems without the chemical.
Quantitative bacteriological assay techniques, which can be related to
colony-forming units, should be conducted periodically to evaluate inhibition
of growth of the natural microflor^ contained in the waste of the treated
system, when compared with growth in the untreated system. The test and
control systems should be subjected to similar usage to provide meaningful
data. The test protocol should incorporate a sampling schedule consistent
with the time interval over which bacterial growth control is intended.
Olfactory determinations comparing the development of odors in the test
and control phases of the study should be performed simultaneously with the
bacteriological determinations. The test should be conducted with an adequate
control on each type of toilet system for which the product is intended for
use.
(2) Suggested performance standard. The study should show that the
product is effective in preventing the development of offensive odors
during the time period that such control is intended. Bacteriological
assays should demonstrate the inhibition of growth of microorganisms in
the test system.
(b) . Toilet bowl and urinal surfaces. The following apply to products
bearing label claims to kill or reduce the number of odor-causing bacteria
on toilet bowl and urinal surfaces. &
(..!)•• .Test standard. Same as § 91-2(.b|(l.) (c) (.1) or >-(j.Hl). of this
series;, except; that pure; culture isolates of; identified odor-causing
bacteria, should, be employed as test microorganisms.-". Note that the contained
bowl water (approximately 3.qts. or 96 fl. oz.) should be taften into* consi-
deration in determining the appropriate use. dilution' to-'..be. tested^ "
•toilet, bowlsv . '. - '.:','/ : '-.•'-.• •-'•;.-.-'
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94
(2) Suggested performance standard. Same as § 91-2(b)(2) or (c)(2)
o£ this -series for claims to kill odor-causing bacteria; same as § 91-2(j)(2)
of this series for claims to reduce the number of odor-causing bacteria.
(c) Toilet and urinal bowl water. The following apply to products
bearing label claims to reduce the number of bacteria or bacteriostatic
control for odor, slime, or discoloration in toilet bowl water.
(1) Test standard. Same as § 91-7(b)(l) of this series, except
that pure culture isolates of identified odor-, slime-, or discoloration-
producing bacteria must be employed as test microorganisms.
(2) Suggested performance standard. Same as § 91-7(b)(2) of this series,
for claims to reduce the number of bacteria; for bacteriostatic claims,
the numbers of test bacteria recovered from the treated water should be less
than the numbers from the parallel control and no greater than the "0-time"
control; and. for slime, odor or discoloration control claims, such problems
should be delayed for the time period claimed.
(d) Bird and animal cage litter treatments. The following apply to
products intended for application to or incorporation in pet bird and
animal cage litter for bacteriostatic odor control in the presence of
urine or wet fecal contamination.
(1) Test standard. Controlled in-use or simulated-use test should be
performed to show the following:
(i) Numbers of bacterial contaminants in treated and untreated litter
after initial deposition of actual bird and/or animal .excrement and at
periodic intervals thereafter (including repeated challenges with additional
excrement) for the time interval recommended for use of the litter.
(ii) Olfactory assessment of the degree of odor control achieved over
the same interval.
i
(2) Suggested performance standard. The numbers of bacterial contaminants
in the treated litter should show a reduction over those in the untreated
control, and the development of offensive odors should be reduced or delayed
in the treated litter over the time interval claimed.
(e) Treated vomitus absorbents. The following apply to products
intended for bacteriostatic odor control during clean-up and disposal of
vomitus removed from inanimate surfaces.
(1) Test standard. Controlled in-use or simulated-use tests should
be performed to show the following:
(i) Numbers of bacterial contaminants in treated and untreated absor-
bent after initial deposition of actual vomitus and at periodic intervals
thereafter for the time period recommended or claimed for use of the absor-
bent to control odor.
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(ii) Olfactory assessment of the degree of odor control achieved over
the same period.
(2) Suggested performance standard. Same as paragraph (d){2) of this
section.
§ 91-56 Products for treating water systems.
(a) Drinking water for poultry and livestock. The purpose of the
antimicrobial treatment of poultry and livestock drinking water should be
clearly defined in labeling. Treatment of drinking water for the purpose
of providing medication for animals, and/or implied claims of disease
control, identify the product as a drug, and required approval by the Pood
and Drug Administration. The standards for products represented in
labeling for treatment of poultry or livestock drinking water for pesticidal
benefits (disinfection, sanitization, bacteriostasis) are considered below.
Such products require a pesticide tolerance from the EPA under the Federal
Food, Drug, and Cosmetic Act.
(1) Test standard, (i) Laboratory tests. Presumptive efficacy
of poultry and livestock drinking water disinfectants or sanitizers may be
established with data derived from the AOAC Method for Water Disinfectants
for Swimming Pools (§ 91-30 Recommended method No. 14 in § 91 of this series)
or with slight modifications thereof, against Escherichia coli (ATCC 11229)
and Streptococcus faecalis (PRO). Presumptive efficacy for chemicals in-
tended to provide bacteriostasis may be substantiated with any of several
presumptive microbiological screening tests (e.g., minimal inhibitory con-
centrations derived/ from a broth tube-dilution type method, and zones of
inhibition derived from a seeded agar cup plate type method).
(2) Field tests. Based on these data, controlled quantitative, micro-
biological studies should be designed to demonstrate the level of efficacy of
the product in poultry or animal drinking water under actual conditions of
use. Field-derived data should be developed under an Experimental Use Permit
demonstrating the efficacy of the product when used as directed. Test
conditions will vary with the level of effectiveness claimed, types of
microorganisms to be controlled, application techniques for treating the
water, treatment intervals, water dispensing system, type of animal facility,
organic load, and other factors related to the proposed use.
(2) Suggested performance standard. The laboratory test should show .
elimination, reduction, or inhibition (i.e., disinfection, sanitization,
bacteriostasis) of the test bacteria. Acceptable results for the field
test will depend upon the .level of activity claimed for specific, use conditions.
(b) Potable water treatment units.; Any unit intended for physical
and/or chemical treatment of microbiologicaLly..potable wa-ter from a
•municipal, treatment facility to Remove undes-ira&le.taste odbrsv-chemic.als',''"
•or other .aesthetically objectionable properties, -i-s.-identi-f-ied as a potable
water treatment unit. A substrate such'as activated charcoal (with or
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without a bacteriostratic agent) is incorporated into the unit for this
terminal processing treatment of potable water prior to consumption.
Since the requirements of the Safe Drinking Water Act do permit municipally-
treated drinking water to contain a limited number of harmless "saprophytic"
bacteria which are commonly recognized contaminants of water, an antimicro-
bial agent is sometimes incorporated in a potable water treatment unit to
provide bacteriostatic activity against these contaminants. Only potable
water treatment units containing a bacteriostatic agent are under the
purview of the Act.
(1) Test standard. Controlled, simulated-use studies for the
potable water treatment unit should be conducted under 'conditions representing
actual use, employing a defined municipally-treated water source. The test
design of the study, which will vary for different types of units and
patterns of use, should include the following basic elements:
(i) Evidence that the function of the potable water treatment unit
(without a bacteriostatic agent) is impaired and/or adversely affected by
identified microbial contaminants present in municipally-treated water, re-
sulting in a recognized aesthetic problem (e.g., undesirable tastes or odors);
(ii) Quantitative determination of the level of microbial contamina-
tion in the test water before and after passage through the control (without
a bacteriostatic agent) and test units;
(iii) Documentation of the bacteriostatic agent concentration found in
the test system; and
(iv) Evidence of the effective capacity, .or duration of effectiveness
of the bacteriostatic agent in controlling the contaminants responsible
for the identified problem occurring under simulated in-use conditions.
(2) Suggested performance standard. The effective capacity or duration
of effectiveness of the bacteriostatic agent incorporated in a potable
water treatment unit should be established by meaningful results that can
be associated with actual in-use conditions. The data should demonstrate
that microbial contaminants in municipally-treated water cause a recognized
aesthetic problem (e.g. undesirable tastes or odors) in the control units
without a bacteriostatic agent, and that such problems are prevented or
delayed in the test units with the bacteriostatic agent.
§ 91-57 Antimicrobial agents sold only for formulation use.
(a) Type of data. The manufacturer (or registrant) of a technical
chemical intended for this type of use should submit presumptive evidence of
intrinsic value as antimicrobial agent. Examples of the types of presump-
tive tests acceptable are the following: minimal inhibitory concentrations
derived from a tube-dilution type method, and zones of inhibition derived
from a seeded agar plate type method.
(b) (Reserved). •
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Series 92: EFFICACY OF AQUATIC PEST CONTROL AGENTS
§ 92-1 General considerations.
(a) Overview. A wide range of pesticides are used directly in aqueous
environments or are intimately associated with them. These pesticides are
used both in man-made systems, such as industrial cooling systems and swim-
ming pools, and in natural aquatic areas, such as lakes and ponds. Applying
pesticides to water can magnify unreasonable adverse impacts on man and the
environment because contamination can extend to surface waters, ground waters,
and aquifers used for, or in the production of, drinking, irrigation, or
industrial process.water. Subsequent contamination of plants and animals may.
lead to undesirable reduction of a species and economic losses. Bioconcen-
tration of pesticide residues in the food chain or direct ingestion of
pesticides by organisms in water may represent potential long-term hazards
to man and the environment.
(b) Scope. Testing and performance guidance for the following products
are stated in this section: aquatic herbicides, swimming pool algicides,
industrial cooling water microbicides, pulp and paper mill water microbicides,
secondary oil recovery systems microbicides, and antifouling paints.
Mosquito larvae control agents are discussed under § 95-10 of this subdivision,
and fish toxicants and fish repellents are discussed in § 96-2 of this
subdivision. Applicants should also read the aquatic pest control agent
label development discussions in § 103-1 of Subdivision H that relate to
the specific pest, site, and proposed use-pattern.
(c) Considerations involved in efficacy testing. Guidance for establishing
efficacy of aquatic pest control products discussed below in sections
under specific product types (e.g., aquatic herbicides, antifouling coatings),
are based on the following considerations:
(1) The intended or expected use of a pesticide-treated water.
(2) Dissolved and particulate constituents in the treated water, which
may increase or decrease pesticide effectiveness.
(3) Those physical characteristics of the system which will affect
volatility, absorption, dissolution, frequency, and efficiency of contact
with the pest(s) to be controlled.
(4) Those physico-chemical characteristics of the water to be treated
(such as temperature, pH, and hardness) which may affect efficacy.
(5) Application method(s), to enable the Agency to evaluate the
practicability and potential selectivity of the proposed use, and to aid in
the determination of proper classification of the. pesticide .according to .
§ 162.11(c.) of the FIFRA Sec. 3 regulations.. '
(6.) 'Expected;, concentrations of pesticide-in treated water as a result
•'a£- treatment. -at. the recommended, dosage (.s), to. enable the Ageltcy -to-..-evaluate*' '
'the, .potential for nontar-get-..effects and .to., aidin-: the...determination, of
proper' use classification. , • . . . -..-••••
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(7) Phytotoxicity to crops or other nontarget vegetation expected to
be exposed to the pesticide or to the pesticide-treated water, including
total exposure of crops resulting from use in irrigation water, type of
irrigation system, type of soil in which crop is grown, and distance of
treatment site from crop.
(8) Indirect effects on nontarget organisms, including oxygen deple-
tion and potential for resultant fish kills that may occur with use of
aquatic herbicides.
(d) Relationship to use pattern. The test methodologies are organ-
ized according to use pattern. Different methodologies may be discussed
even though a product is used to control one type of pest, such as algae
in several sites. Differences between pests, sites, or application tech-
niques can affect the efficacy of a product. Accordingly, in most cases,
data demonstrating the efficacy of a product will be acceptable only if
such data are derived from the methodology appropriate to that product's
specific use pattern.
§ 92-2 Aquatic herbicides.
(a) Scope. This section provides guidance for pesticides designed
to control aquatic macrophytes (mosses and vascular plants), ditchbank
plants, and algae. Efficacy data concerning control by pesticides in
swimming pools, industrial cooling water systems, pulp and papermill water
systems, secondary oil recovery systems, and on ship or boat bottoms, as
well as other underwater surfaces, are discussed in §§ 92-3, -4, -5, -6,
and -7.
(b) General data conditions. The following conditions should be
met in order to establish efficacy of aquatic herbicides and algicides
covered in this section (§ 92-2) and effects of these pesticides on the
environment, whether they are intended for use in standing or moving waters,
adjacent to water, or in a location expected to contain water at a later
time. Additional conditions which pertain to specific categories are
covered as follows: standing water, moving water, ditch bottoms, and ditch-
banks [paragraphs (c), (d), (e), and (f), respectively, of this section].
Field testing should include a geographical distribution of plot sites that
reflects the general climatic and water quality variations expected in the
locations where the product is proposed for use. Not all such environmental
variations can be anticipated or tested, but sites shall include those which
have various water hardness, pH, temperature, turbidity, alkalinity, and
weed infestations (both in types and numbers of weeds). Field tests should
be designed to allow statistical comparison of weed control between treated
and untreated ("control" or "check") plots. Whenever possible, a known
"standard" treatment should be included in the testing. For example, when
testing a new algicide, copper sulfate -may be used for a standard. For
these comparisons, the n-umber and kind, (.species) of target weeds present
in the control and treated plots should be similar in extent and type of
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weed, in relative species abundance, and in water quality. The design of
such plots is discussed in the Acceptable Method No. 3 § 92r-20(d). (Efficacy
Testing Methods for Aquatic Herbicides in Standing Waters). In all cases,
the following data should be reported for each test site.
(1) Description of test site. This includes the following: (i) Type
of aquatic site, such as lake, pond, reservoir, ornamental pool, irrigation
ditch, and geographic location of site (i.e., state, county, town).
(ii) Size (area and depth, or volume, or length and width of the
treated areas, and of the whole site), as is appropriate to the type of
application and the type of target weed(s).
(iii) Number of replicate treated plots and control plots.
(iv) Water quality, including pH, temperature, hardness, alkalinity,
salinity, turbidity, conductivity, and dissolved oxygen.
(v) Soil texture, including that of soils along the immediate shore-
line or ditchbank and the submersed soil where the target weeds are present
(with the percent organic material in the soil also reported). (Acceptable
methods and soil texture classifications are found in the Walkley-Black
Procedure in Soil Sci. 63:251, 1947, and the Soil Survey Manual, U.S. Dept.
Agr. Handbook No. 18, 1951, Fig. 1, and Soil Sci. Soc. Amer. 26:305-317,
1962.)
(2) Description of vegetation present at test site. Description
should include the vegetation present and any organisms that affect product
performance. The following shall be reported:
(i) Target weed species (common name if available, and scientific
name), and weed density (number or amount per unit area or per unit volume,
as appropriate).
(ii) Other vegetation (common name or scientific name).
(iii) Growth stage of target weeds, such as seedling, preflowering,
or heading stage.
(iv) Other appropriate observations relating to target weeds, such as
the presence of a heavy coating of epiphytic algae on the leaves of the
target vascular plants.
(3) Methods of herbicide application. Information should include:
(i) Target site where the herbicide was applied (for example, to weed
foliage, to surface of water, to bottom of water body, into water, to
ditchbank, or -to shoreline).
• ' ';:'•• ' -
••.. (ii.)' Description of the equipment used to apply the herbicide (e.g.,
groundr-spraying device, pumping .device, .boat, blower, helicopter, or fixed-
wing.aircraft). . . •
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(iii) Description of any water level changes used in conjunction with
the herbicide application, such as drawdown operation or drainage of convey-
ance system, including the extent of water level change, the.time of the
change in relation to the herbicide application, and:the duration of the
change in water level.
(iv) The timing of the application in relation to the calendar date
and the stage of growth of the target weeds.
(4) Dosage of herbicide. The dosage ranges tested should be broad
enough to determine the minimum efficacious dosage and the effective range
for each target weed claimed. In addition, the toxicity to desirable non-
target vegetation should be determined if it is within the dosage ranges
tested. (Refer to Subdivision J of these guidelines for guidance and
acceptable methods in determining phytotoxicity to nontarget plants.) The
following conditions should also be known:
(i) The amount of product and active ingredients should be expressed in
units appropriate to the method and placement of the application, such as
weight per unit area, weight per unit volume, or the weight per unit flow-
volume, and, in every case, the concentration of active ingredient in the
water.
(ii) Sufficient variety of soil textures and water quality conditions
should be used to determine the effective minimum dosage or dosage range for
the particular soil and water, conditions likely to be encountered in the
geographical area where the use is proposed. The broader the geographical
area of proposed use, the more tests will be needed. Generally, in any
one locality or geographical area (for example, _the southeastern United
States), a minimum of three testing sites will be needed, but six or more
are strongly recommended. The acceptability of the number of tests will
ultimately depend on the quality of the data.
(iii) If adjuvants such as surfactants, "spreaders", or "stickers"
are required for proper efficacy, appropriate adjuvant control plots (with-
out herbicide) should be included to distinguish the possible physiological
or phytotoxic effects of the adjuvant alone.
(5) Limitations on water use for irrigation. If label claims intend
that the product will be used to treat water that will be a source for
irrigation water, or to treat ditchbanks in irrigation systems (including
drainage ditches), then the following are necessary:
(i) Test(s) should be conducted to determine if phytotoxic effects of
the herbicides occur on crops normally expected to be irrigated with treated
water, and to determine the highest concentration of the product in water
which will not injure crops or desirable vegetation expected to be exposed.
(See § 92-20(c) for references on methods for determining phytotoxicity
of aquatic herbicides to irrigated crops).
(ii) To evaluate potential phytotoxicity to crops as a result of
cumulative effects of herbicide residue in irrigation water, the following
information for anticipated crop practices are needed: frequency of
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irrigation, amount of water per irrigation treatment, total acreage treated
per crop season, and concentration of herbicide in irrigation water when
treated water reaches crop.
(iii) Concentrations of the active ingredient in water as result of
proposed application should be predicted, as well as the decline of that
concentration with time and/or distance of flow. Appropriate statistical
analyses of the data and development of depletion curves are essential.
(See § 92-20(a) for references on dissipation analysis methods;
also, see §§ 164-1 through -5 of Subdivision N.)
(6) Results of aquatic herbicide treatment(s). In this subparagraph,
guidance for reporting results of tests on any aquatic herbicide are stated
first. Additional guidance for aquatic macrophytes and algicides are
discussed in (ii) and (iii) of this subparagraph (6).
(i) Tests results for all aquatic herbicides should include.:
(A) The observation dates, with the time interval(s) between initial
applications and subsequent observations;
(B) The plant species controlled, and the duration of control for
each species in days, weeks, or other time period;
(C) Comparison with "standard" herbicide treatment plots (if used);
(D) Herbicide phytotoxic effects on target weeds and other vegetation
in the test plot. The indicative visual phytotoxic symptoms of the target
weeds and the time of their appearance are particularly important if
subsequent herbicide treatment(s) or the manipulation of water levels or
water flow is dependent upon the knowledge of the effectiveness of initial
applications of the herbicide. Examples of terminology for such symptoms
appear in § 102-2 of Subdivision H;
(E) Results of crop phytotoxicity tests (if crops are expected to be
exposed to treated water), and the maximum level of the herbicide tolerated
by crops expected to receive treated water, including any limitations of
the type of irrigation and on timing of irrigation after water treatment;
and
(F) Changes in water quality following herbicide treatment and weed
control. [This includes those analyses specified under paragraph (b)(l)(iv)
of this section.]
(ii) Test results for aquatic macrophytes (including ditchbank plants)
should also include the extent of weed infestation before and after herbicide
treatment(s) in both control and treated.plots, and the calculated percent
weed control for each weed species. Data should be based on appropriate
measurement's.), .of. weed species population density and may include dry or
wet weight of weeds> .length of weeds., and height of weeds.,-depending tupon
the type of control and.the-type of weed. =
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(iii) Test results for algae should also Include: . .-: •
(A) Amount of growth present (for example/ density per unit area or
per unit volume of water) before and after treatment(s). Methods for
evaluating algae control are discussed in § 92-20{d) Efficacy Testing
Methods for Aquatic Herbicides in Standing Waters; and
(B) Changes in the degree of infestation of other aquatic plants fol-
lowing target algae control.
(c) Specific data guidance for standing water. In addition to the
guidance given in paragraph (b) of this section, the following are needed:
(1) Dosage of herbicide, (i) For applications to aerial foliage,
the amount of herbicide used per unit area, or per total spray volume, or
both.
(ii) For applications where effectiveness is dependent upon concentra-
tion in the water, the dosage used in amount per volume [e.g., hectare-meter
(acre-ft.)], and the concentration in water (e.g., parts per million) result-
ing from the application.
(2) Hydrological features. Those hydrological features which can
alter herbicide usefulness, such as thermoclines (depth and steepness),
inflows which may reduce initial concentration levels, and wave action.
(d) Specific data guidance for moving and flowing waters. Herbicides
that are applied directly to moving water (for example, canals,, ditches,
irrigation systems, streams, rivers) are included here. In addition to the
guidance given in paragraph (b) of "this section, the following are needed:
(1) Dosage of herbicide. (i) For herbicides whose effectiveness is
related to concentration in water, the amount of herbicide per unit volume
of water per unit time and its dissipation with time. [See § 92-20(b) for
information on method.]
(ii) The most effective concentration and contact-time combination(s)
for the target weed.
(2) Flow characteristics. The flow rate (flow volume) in the convey-
ance system (e.g., cubic feet per second).
(3) Construction material. The type of construction of the treated
conveyance system (for example, earthen or concrete-lined).
(4) Previous and subsequent comparisons. Duration of weed control
and comparison with weed infestation during previous years, when known.
(5) Untreated control. Untreated plots should be located upstream
from treated plots at a sufficient distance to preclude pesticidal effects.
If there is no appropriately similar site upstream, comparison before treat-
ment and after treatment should be made.
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(e) Data guidance for ditch bottoms. For treatments to canal or
ditch bottoms/ the following should be reported, in addition to paragraph
(b) of this section:
(1) Weather conditions. Type and amount of precipitation, and general
weather conditions between application time and time when water is turned
into the canal.
(2) Draining interval. Time interval between drainage and application,
and between application and time when ditch or canal is refilled.
(3) Amount of control. The length (meters, kilometers) of canal
treated, and length of achieved weed control in the canal.
(4) Herbicide residue. The concentration of the herbicide in water
after it is turned into the drained canal, if the water is to be used for
irrigation purposes. (See § 164-2 of Subdivision N for analysis techniques.)
Other guidance related to use of treated irrigation water are described
under paragraph (b)(6) of this section.
(f) Specific data guidance for ditchbanks. For ditchbank treat-
ments, the extent of water overlap, if any, should be stated so that the
amount of exposure of water can be calculated and correlated with the
analyses of concentrations in the water.
(g) Performance standards. Normally, the degree of aquatic weed
control that is acceptable depends upon the particular use site and use
pattern. The calculation of percent weed control should be based on weed
biomass, height (or length) of weeds, number of weeds, or amount of surface
area covered by weeds. Other assessments may be utilized if they reflect
beneficial effects of weed control. When percent weed control is based on
comparisons of untreated plots to treated plots, or to pretreatment weed
infestations in treated plots, a minimum of 70% control is desirable.
Lesser effectiveness may be acceptable, provided that appropriate qualifying
terminology is used on the label. [See § 102-2 of Subdivision H. Specific
standards for performance are stated below for the indicated uses.
(1) Algae control. For laboratory testing using pure-culture methods
to obtain presumptive efficacy data, a minimum of 70% control is needed.
To support algicidal claims, subcultures should not show growth within 21
days after inoculation into fresh medium. For field tests, the following
degree of control should be obtained:
(i) Prevention of growth (bloom) by at least 80% compared to untreated
plots, or
(ii) Reduction of existing growth (bloom) by at least 70%, or
(,iii.)- Indirect performance results, such .as improvement of volume-flow
in canals or ditches by at least 50%., reduced clogging of pump screens by
at least.70%, and. :reduced clogging of drip-irrigation systems by 95%.
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(2) Aquatic macrophyte control, (i) Standing water. -A minimum of
70% control as measured by weed count, biomass, length, area, or other
appropriate value. For floating weeds, the percent cleared water may be
used as an evaluation, and ordinarily should be at least 70% of the treated
area.
(ii) Moving water. At least 70% control of submersed or emersed
weeds, or an increase in flow-volume by at least 50%.
(3) Ditchbank weeds. At least 70% control and lesser control will
be acceptable only if appropriate qualifications are stated on the label.
§ 92-3 Swimming pool algicides.
(a) Scope. Two types of tests are used to.demonstrate effectiveness
of swimming pool algicides to control algae: laboratory tests using pure-
culture methods [see paragraph (b) of this section], and confirming tests
[see paragraphs (c) and (d) of this section] in swimming pools or simulated
swimming pools. If an experimental permit is needed, data from laboratory
pure-culture testing is needed. For-tests to determine efficacy of
pesticides to disinfect or control disease organisms in swimming pool water,
see § 91-8(c) of this subdivision. : . •
(b) Laboratory tests. Laboratory procedures are necessary to assess
the algistatic versus algicidal capabilities of these products. An accept-
able laboratory pure-culture method is given in § 92-20(e) Pure-culture
Technique for Evaluating Algicides.
(c) Confirming in-use tests. Data developed from tests conducted by
either of the following methods are acceptable.
(1) In-use pools. Along with adequate laboratory data, confirming
efficacy data are recommended from in-use tests in two or more properly main-
tained swimming pools of different sizes located in different geographical
regions of the country. The tests should be conducted for a period of not
less than 30 consecutive days under climatic conditions occurring during
the swimming pool season. Reports should include but are not limited to the
following additional information on the condition of the test pool:
(i) The design and capacity of the pool.
(ii) The pool use and bather load.
(iii) The amount of product added to swimming pool water and
the time of addition.
<iv) The'range of chemical characteristics of the swimming pool water,
such as pH, nitrogenous substances, alkalinity, hardness, and amount of
residual disinfectants and stabilizer(s).
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(v) The physical characteristics of the swimming pool water, including
temperature, color, and clarity.
(vi) Metarological data, including air temperature, rainfall, and
number of hours of sunlight, which may affect results.
(2) Simulated pools. In lieu of the in-use tests, acceptable confirm-
ing data may also be obtained from tests conducted in simulated swimming
pools artificially inoculated with algae. Consultation with the Agency
about procedures and test species prior to undertaking these tests is
strongly recommended. An acceptable simulated swimming pool method is pro-
vided in f 92-20(f) Method for Evaluating Algicides in Simulated
Swimming Pools.
(d) Suggested performance standards. (1) Pure-culture tests. For
pure-culture testing, the acceptable level of performance is 70% control
of algae in primary cultures for 3 weeks. (See § 92-20(e)).
(2) In-use pools. For in-use swimming pool testing, the level of
performance should equal or exceed that of a standard chlorine treatment.
(3) Simulated pools. For simulated-swimming pool testing, the
acceptable level of performance must be equal to or better than a chlorine
standard at 1.0 to 1.5 ppm for isocyanuric acid-stabilized pools or at 0.6
to 1.0 ppm for non-stablized pools. (See § 92-20(f)). j
§ 92-4 Industrial cooling water microbicides.
(a) Scope . Products intended to control microbiological slimes in
cooling water systems include those with algicidal, bactericidal, and
fungicidal activity. Microbiological slimes consist of microorganisms
(algae, bacteria, and/or fungi) and their secretions, plus embedded debris.
(b) Definitions . The following definitions and explanations are
particularly pertinent to the understanding of this section:
(1) Bactericide. As used in this section, this term is restricted to
products used in cooling water for controlling, bacterial slime, and must
not be construed to include products represented or defined as disinfectants
described in §§ 91 (Efficacy of Antimicrobial Agents).
(2.) Slowdown (bleed-off) . This term means: water discharged from the
system to -control concentrations of salts or other impurities in the cir-
culating water. .Slowdown is measured, in volume p^r unit time.
(3.): Cloged reeirculatJJiq- systems . ..This term means: a .system of
"•cOntinuouS'ly •;recirt:ula.ting water, in a. heat, exchanger, tha.t. .may .be-. -cooled by
"a-ir, mechanical -refrigeration., or a separate open ^coolings-waller. -system. •
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(4) . Makeup water. This term means: water added to the circulating
water system to replace that lost because of blowdown (bleed-^off'), drift,
evaporation, leakage, and overflow.
(5) Microbicide. As used in this section, this term means: any
substance or mixture of substances which effectively reduces the number of
algae, bacteria, and/or fungi.
(6) Once-through systems. This term refers to systems where water is
taken from some primary source, used for cooling, and then either discharged
to waste or used for other purposes.
(7) Open recirculating systems. This term refers to systems where a
major portion of the water used for process cooling is continuously recycled
to some type of evaporative cooling device, such as a cooling tower, to
remove the heat picked up in the process, and is then used again for process
cooling.
(c) Data guidance. (1) Laboratory testing. Laboratory data should
be submitted to show presumptive effectiveness of the product in controlling
algae, bacteria, fungi or combinations thereof when field testing is used
to evaluate product performance.
(i) Standard laboratory techniques should be used to determine minimum
concentrations of the product which will kill or prevent growth of target
organisms. The viability of pure culture algae -should also be determined by
a technique involving subculture into untreated medium. (See § 92-20(e)).
Laboratory evaluation of bacteria and fungi shall consist of determining
minimum effective static and cidal concentrations of product-on unshaken
liquid cultures.
(ii) The following test species should be employed: bacteria: Pseudomonas
spp. and Bacillus mycoides (ATCC); fungi: Aspergillus niger (ATCC); algae:
Chlorella pyrenoidosa, Euglena gracilis, and Scenedesmus obliquus. (For
algae, see § 92-20(e) for methods and growth media).
(d) Field testing. Tests should be made in at least 2 different climatic
locations for a period of not less than 30 days. Test systems should be
representative of the more severe conditions that the product is intended
to control.
(1) Reports should include, but are'not limited to, the following:
(i) Dosage applied at each treatment and contact period necessary for
control of organisms.
(ii) Interval between treatments.
(iii) Visual or other suitable ratings of the nature and amount of
microbial growth. The rating pcale shall be defined, and the commercially
acceptable level of control should be indicated.
(iv) Total volume.in the system.
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(v) percent blowdown, if applicable, per unit of time.
(vi) Water characteristics, such as pH, hardness, temperature; identi-
fication and estimate of concentration of any anticorrosion inhibitors in
the system; identification of any antimicrobial chemicals that were used
immediately before or during the test period.
(vii) Engineering data, if available and if appropriate for comparative
purposes, reflecting the relative efficiency of operation of the system, such
as heat transfer readings, head pressures, and down time. Baseline data on
each are necessary for comparative purposes.
(viii) Microbial counts before and after each treatment to support the
claimed interval(s) between applications and to indicate the degree of control
obtained.
(ix) For solid products such as briquettes and tablets, data must be
submitted showing the rates of dissolution of the product in water at various
temperatures and flow rates.
(x) Where and when the tests were conducted.
(xi) Background and training of person(s) conducting test.
(2) Foreign data. Similar field data developed in foreign countries are
acceptable if the data have been obtained from two cooling water systems
where climatological conditions and the microorganisms encountered are similar
to those found in cooling water systems in the CJnited States.
(3) Laboratory evaluation of cooling tower bacteria and fungi. In lieu
of field testing, water samples from operating cooling towers containing
indigenous microorganisms can be tested by subculture and plated onto surface
medium for enumeration. Algicidal or algistatic activity will be determined
by specified laboratory testing. See ASTM Method E-645: Test for Efficacy of
Microbicides Used in Cooling Systems. \/
(4) Laboratory model systems. Laboratory methods for efficacy evalua-
tion of cooling system microbicides will be considered on an individual
basis. Registrants are advised to submit such proposed laboratory methods to
the Agency for review and approval prior to their use in developing support
data for registration.
(d) Suggested performance standards. (1) Laboratory testing. For
laboratory methods used in lieu of field tests, a minimum of 90% bacterial
kill within a 3-hour contact period with the product is desirable. Growth
of fungi should be prevented. Performance standard for algae is 70% control
of algae growth in primary cultures for 3 weeks. (See § 92-20(e) Pure-culture
Technique for Evaluating Algicides.)
(2) Field testing. ' Application of the'praduct'-according to label
directions during field tests should permit efficient operation of cooling
. systems by controlling biological fouling so that proper compressor head
pressures•and temperature differentials are. maintained.
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108
§ 92-5 Pulp and papermill water systems microbicides.
(a) Residues in paper. Treatment of pulp and papermill process water
with pesticides may result in residues in finished paper or paperboard.
Residues occurring in finished paper products intended for use in contact
with food must be covered by food additive regulation or by an exemption
from the requirement for such regulation. (Refer to § 63-1.)
(b) Data guidance. Laboratory data should be developed to show the
effectiveness of the product against both bacteria and fungi by the ASTM
"Tentative Methods of Test for Efficacy of Slimicides for the Paper Industry."
2/ Two individually formulated samples of the microbicide developed be tested
separately, in duplicate, against bacteria and against fungi, for a total
of four tests. For solid formulations, data should be developed showing the
rates of dissolution of the product in water at various temperatures and
flow rates. Field data from 2 operating mills in the United States will be
acceptable. Similar data developed in foreign countries are acceptable if
the data have been obtained in 2 mills where climatological conditions and
the microorganisms encountered are similar to those found in mills in the
continental United States.
(c) Suggested performance standards. (1) Bacterial control. The
Agency will accept the performance standard described in the ASTM "Tentative
Methods of Test for Efficacy of Slimicides for the Paper Industry" for
bacterial control.
(2) Fungal control. The performance rating system for control of fungi
assessed by the ASTM "Tentative Methods for Test for Efficacy of Slimicides
for the Paper Industry" is as follows:
0 - no growth
1 - scant, or questionable growth
2 - poor growth
3 - good growth
.4 - excellent growth
The level of performance can be indicated with the use of a standard refer-
ence microbicide.
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§ 92-6 Secondary oil recovery systems mlcrobicides.
(a) Methodology. The American Petroleum Institute (API) "Recommended
Practice for Biological Analyses of Subsurface Injection Water Tests" 3/
contains an acceptable method for assessing the efficacy of products against
Desulfovibrio desulfuricans, Bacillus cercus, and Pseudomonas fluorescens.
The bacteriostatic and time-kill test must be carried out in duplicate.
(b) Suggested performance standard. The product tested as specified
by the API method must prevent growth of test bacteria.
§ 92-7 Antifouling biocides.
(a) Scope. Antifouling coatings fall into two main usage categories:
marine and freshwater. Many marine paints will not perform satisfactorily
in fresh water. To some degree, the reverse is also true. The main testing
I emphasis has been in the marine environment. To some extent, marine and
| freshwater paints are used to prevent fouling on substrates other than
[. boats or ships: surfaces on submersed equipment such as irrigation wiers,
[ power plant.intake or outflows, and pipe lines, are often protected in both
I; environments by antifouling coatings. But the major use of these coatings
£ is on ship and boat bottoms. Control of all fouling, be it algal or
t invertebrate, is desired. All non-biocidal aspects of coatings, such as
f color and friction reduction, are outside the scope of pesticide regulation.
i (b) Testing considerations. (1) General. Sward (1972) 4/ points
,: out that tests should start early in the spring, and that panels should be
} exposed at widely-separated geographical points. The USNI (1952) 5/ advises
[ that panel tests are the most valuable and oldest of the testing methods
;. used, but must be conducted in locations when the larvae of fouling organisms
are present throughout the year. Panels coated with an antifouling paint
i of proven performance should be included in all tests to serve as "standards".
! Since some variation in standard performance will occur depending upon condi-
i tions at each test location, such standard antifouling paint panels are
j; essential for comparison purposes. The use of standard panels coated with
!' U.S. Navy Formula 121/63 (MIL-P-15931B) antifouling paint is strongly advised.
[ The use of a nontoxic untreated "control" panel is required to properly
[ evaluate the fouling potential. The standard panel gives a known performance
[ rating and can be used as a high performance standard. The untreated panels
j' give a survey of the fouling community that would, be noted if no toxicant
[ were present in the paint under test, or if the toxicant were ineffective.
I (2) Data guidance. Antifouling paints or coatings should be tested
[ : for periods of. one or more "seasons" in the locations of intended .usage,
| depending upon .in-tended, label claims. The length, of a "season" may vary
[ ' from six months to one year, depending on the location of the test site.
f '• The. information.-and,-data that1:'should,be .recorded.:and, reported;-are. enumerated
• '• ' -below:.. .-••"•'• ..-.•"-. .'-... • •-•'.. ..••.•-..•.-.:-•.".:•••'. ..." ';V •'. .
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110
(i) Substance of test panels, i.e., wood, steel, aluminum, or other
material.
(ii) Painting system used for undercoat (including panel preparation).
(iii) Method of application of paint being tested (brush, spray, or
roller).
(iv) Wet or dry film thickness, weight of film applied, and/or number
of coats.
(v) Drying or curing time before immersion at test site.
(vi) Formulation used, including percent active ingredient.
(vii) Panel size and number of replications. A minimum of four test
panels per condition or paint to be tested should be employed, i.e., four
standard panels, and four untreated control panels. The surface area of
each panel should contain a minimum of 46208 sq mm (72 sq in.), or be at
least 152 by 304 mm (6 x 12 in.).
(viii) Location of test site.
(ix) Conditions encountered during test period (such as average water
temperature, salinity, pH, hardness, turbidity, and sediment).
(x) Application date(s).
(xi) Immersion date and method of exposure when:. < -; . ' •':.
(A) Suspended from a float, a fixed support, recording depth at low-
est tide.
(B) Partially immersed, for evaluating waterline fouling.
(xii) Termination data and period of acceptable control (effective
life of coating).
(xiii) Monthly readings of fouling during the "season" of paint expo-
sure. Identity of algae, barnacles, tunicates, or other fouling organisms
as to genus and species is not necessary; however, if such identification
is possible, it should be reported. Readings should include data enumerating
the discrete numbers of individual organisms or the percent area occupied
by colonial forms for each panel observed and for each type of organism.
Control and standard panels should be rated in a manner identical to that
for test panels.
(3) Evaluation of data. Fouling rating: for each observation a Foul-
ing Resistance (F.R.) and an Antifouling Film Rating (A.F.) should be devel-
oped. Acceptable rating systems are presented as follows^:
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Ill
(i) Surface free of attached fouling organisms = score of 100.
Substract from 100 the total number of individual organisms present,
or the percent area covered by colonial forms, for each panel
observed. For example:
Barnacles - 5 each 5
Tunicates - 2 each 2
Algae - 7 percent 7
(Total) 14
100
-14.
Fouling Resistance (F.R.) = 86
(ii) For test films, the Antifouling Film .Rating (A.F.) is developed
by subtracting the percentage of the surface showing apparent defects
(pits, cracks, checks, peeling, flaking, etc.) from 100. A film with no
defects scores 100.
(4) Suggested performance standard. For an antifouling coating to be
considered acceptable, the test panels at the end of the test should still
show at least 85% freedom from attached fouling organisms. Panels rated
under 85% "F.R." for the time period claimed by the label are considered
failures.
§ 92-10 Footnotes to Series 92 sections.
\/ Copies of E-645 may be obtained from the American Society for Testing
and Materials, 1916 Race St., Phila., Pa. 19103.
2/ Copies of the current test procedures may be obtained from the American
Society for1 Testing and Materials, 1916 Race Street, Philadelphia, Pa.
19103: E-599 for fungal method, and E-600 for bacterial method.
V API RP 38, 3rd Ed., Dec., 1975. Copies of this test procedure may be
obtained from the'American Petroleum Institute, Division of Production,
300 Corrigan Tower Building, Dallas, Tex. 75201.
4/ Sward, G.G., ed. 1972. Paint for Marine Environment. Pp. 478-485 in
Paint Testing Manual, 13th Ed. Amer. Soc. for Testing Materials, 1916
Race St., Phila.., Pa. 19103. . . ' . :
5/. Anon., 1952. Marine Fouling and. its Prevention. o.S. Naval Institute,
.Annapolis, Md. 2,1402, . . . • '
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§ 92-20 Acceptable methods.
(a) Introduction. The discussions that follow describe acceptable
methodologies for obtaining efficacy data in support of registration appli-
cations' 'for aquatic pest control products. Where methods are specifically
delineated, the Agency has judged that these procedures will provide the
necessary data for evaluation of efficacy. In other cases, reference is
made to published literature as a guide for developing acceptable test meth-
odologies. More than one type of referenced study may have to be utilized
to meet the efficacy data requirements as stated in the guidelines, on
Aquatic Pest Control Agents. The uses of aquatic pesticides are so varied
that it is impractical to provide detailed testing protocols for many
product types; however, if the cited references are consulted and the data
referenced in these guidelines and those on labeling requirements
(Subdivision H, §§ 102-1 to -6 are carefully read, the applicant will be
able to carry out acceptable testing. If there are questions regarding a
particular test procedure that the applicant intends to use, the applicant
or tester should confer with the Agency prior to testing. Testing to meet
both general and specific data guidance is discussed in the following
paragraphs.
(b) Dissipation analysis methods. (1) General. The intent of these
studies is to obtain in-use field data on the concentration of herbicides
in water that will contact crops when the herbicide is applied in any of
the following sites:
(i) Directly to or in water .used as an irrigation source.
(ii) Drained irrigation conveyance systems.
(iii) Drawdown impoundments used for holding water intended for irri-
gation.
(iv) Ditchbank applications to irritation water conveyance systems or
shoreline application to irrigation water holding impoundments.
(v) Multi-use aquatic sites for which irrigation is an expected use.
(2) Use of data. The dissipation behavior of herbicides applied to
sites (i) through (v), mentioned in paragraph (a) above, should be known to
accurately assess the potential phytotoxicity of the herbicides to crops
irrigated with treated or exposed water. The information is used in con-
junction with testing of crop phytotoxicity. [See paragraph (c), "Methods
for Testing Crop Phytotoxicity of Aquatic Herbicides"] . The data derived
from these tests are used to develop appropriate precautionary labeling to
insure that aquatic herbicides will be used without injury to crops. For
this reason, the dissipation testing must allow the prediction of crop
exposure to herbicides as a result of the treatment.
(i) Data guidance for application of herbicides to standing waters,
or shorelines thereof:
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113
(A) The time (hours, days, weeks) necessary for herbicide concentrations
to decline to pre-application levels at the site of application and at the
closet point of intake into the irrigation system;
(B) The minimum distance from application site to point of intake into
the irrigation system;
(C) For drawdown application, the minimum time required for concentra-
tion of herbicides in re-elevated water to decline to preapplication levels
and the minimum distance from application site, if any, to point of intake
into irrigation system.
(ii) For application to irrigation ditchbanks or water in irrigation
conveyance systems:
(A) The time (hours, days, weeks) required for herbicide concentration
in the water to decline to pre-application levels at the point where maximum
levels are reached and at the point where treated water first contacts crops;
(B) The minimum safe distance from application site to point where
treated water first contacts crops.
(iii) For application to drained irrigation conveyance systems:
(A) The minimum time required for herbicide levels in reintroduced water
to decline to pre-application levels in water prior to draining at the site
of application and at the point of first contact with crops;
(B) The minimum safe distance, if any, from application site to point
of first contact of reintroduced water to crops.
(iv) Acceptable methodologies. Simple and direct methods for determin-
ing the dissipation of several phytotoxic chemicals in flowing water are
published (Bartley, 1967, 1969; Chancellor et al., 1957; Demint et al., 1970;
Demint, 1971; Frank et al., 1967; Nelson et al., 1969). Some further
theoretical considerations of herbicide applications to moving water, and
some examples of dissipation data are found in O'Loughlin and Bowmer (1975).
Examples of field analysis for dissipation in standing waters are found in
Grzenda et al., (1966), Hiltibran et al., (1972); Simisiman and Chesters
(1976); and Yeo (1967). Only minimal research has been carried out on the
behavior and fate of herbicide residues that occur in flowing water as a
consequence of treating the soil in drained canals or other temporarily dry
waterways (Comes et al., 1975, 1976; Frank et al., 1967, 1970; Smith et al.,
1975). Except for the work reported by Nelson et al., (1969) and Bartley
(1957, 1959), little has been done to determine the roles played by factors
other than dilution in the disappearance of phytotoxic chemicals in flowing
water. The effects of sediment and.other forms of particulate matter have
been reported (Coats et al. 1966; Tucker et,al., 1967; Adams (1973); Kahn
01974);'.it is recommended .that these latter references be consulted for
information on possible effects .from sediments and particulates in. treated
'water. : -• ': •' : • - . ..- - : • ' . . •.
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114
References.
Adams, Russell S. 1973. Factors influencing soil adsorption and bioactiv-
ity of pesticides. Residue Rev. 47:1-54.
Hartley, T.R. 1967. progress Report on Evaluation of Copper for Aquatic
Weed Control and Herbicide Residues on Irrigation Systems. U.S. Dept.
Int., Bur. Reclam., Report No. WC-32.
Hartley, T.R., and N.E. Otto. 1964. Progress Report of 1963 Field Evalua-
tions on Antifouling Materials for Algae Prevention. U.S. Dept. Int.,
Bur. Reclam., Div. Research. Water Conservation Report No. WC-18.
1967. Progress Report on Antifouling Materials for Algae Preven-
tion. U.S. Dept. Int., Bur. Reclam. Report No. WC-30.
Chancellor, R.J., A.V. Coomns, and H.S. Foster. 1957. Control of aquatic
weeds by copper sulfate. Proc. 4th Brit. Weed Control Conf. 4:80-84.
Coats, G.E., H.H. Fvmderburk, Jr., J.M. Lawrence, and D.E. Davis. 1966.
Factors affecting persistence and inactivation of diquat and paraquat.
Weed Res. 6:58-66.
Cochrane, D.R., J.D. Pope, Jr. H.P. Nicholson, and G.W. Bailey. 1967.
The persistence of silvex in water and hydrosoil.' Water Resour. Res.
3:517-523.
Comes, R.D. P.A. Frank, and R.J. Demint. 1975. TCA in. irrigation water
after bank treatments for weed control. Weed Sci. 23:207-210.
Comes, R.D. V.F. Bruns, and A.D. Kelley. 1976. Residues and persistence
of Glyphosate in irrigation water. Weed Sei. 24:47-50.
Demint, R.J. 1971. Use of dye as a model of herbicide dissipation in
irrigation water. Proc. West. Soc. Weed Sci. 34:35-51.
Demint, R.J., P.A. Frank, and R.D. Comes. 1970. Amitrole residues and
rate of dissipation in irrigation water. Weed Sci. 18:439-442.
Eichelberger, James W. and J.J. Lichtenberg. 1971. Persistence of pesti-
cides in river water. Environ. Sci. Tech. 5:541-544.
Frank, P.A., R.H. Hodgson, and R.D. Comes. 1967. Residue of two herbicides
in water in irrigation canal-bank treatment for weed control. Weed Sci.
18:687-692.
Grzenda, Alfred R. H.P. Nicholson, and W.S. Cox. 1966. Persistence of
four herbicides in pond water. J. Amer. Water Works 58:326-332.
Hiltibran, Robert C., D.L. Underwood, and J.S. Fickle. 1972. Fate of
diquat in the aquatic environment. Univ. .111. Water Research Kept. No.
52. 45 pp. (Available through the Nat. Tech. Informat. Serv., U.S. Dept.
Commerce) ' "
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115
Kahn, S.U. 1974. Humic substances reactions involving bipyridylium herbi-
cides in soil and aquatic environments. Residue Rev. 52:1-26.
Nelson, J.L., V.F. Bruns., C.C. Coutant, and B.L. Carille. 1969. Behavior
and reaction of copper sulfate in an irrigation canal. Pest. Monit. J.
3:186-189.
O'Loughlin, Emmett M., and K.H. Bowmer. 1975. Dilution and decay of aquatic
herbicides in flowing channels. J. Hydrol. 26:217-235.
Simisiman, G.V., and G. Chester. 1976. Persistence of diquat in the aquatic
environment. Water Res. 10:105-112.
Smith, A.E., R. Grover, G.S. Emmond, and B.C. Korven. 1975. Persistence and
movement of atrazine, bromacil, monuron, and simizine in intermittently-.
filled ditches. Can. J. Plant Sci. 55:809-816.
Tucker, B.V., D.E. Pack, and J.N. Ospenson. 1967. Adsorption of bipyridylium
herbicides in soil. J. Agri. Food Chem. 15:1005-1008.
Yeo, R.R. 1967. Dissipation of diquat and paraquat, and effects on aquatic
weeds and fish. Weeds 15:42-46.
Zepp, Richard G., N.L. Wolfe, J.A. Gordon, and G.L. Baughman. 1975. Dynamics
of 2,4-D esters in surface water. Environ. Sci. and Tech. 9:1144-1150.
(c) Methods for determining phytotoxicity of aquatic herbicides to
irrigated crops. (1) Introduction. There are no standard protocols for
testing the toxicity of herbicide-treated irrigation water. However, some
general, acceptable practices should be used as follows: field testing is
usually conducted in two steps, small-plot testing using metering devices
to control the level of herbicide in irrigation water, and large-scale
in-use testing that normally requires an experimental use permit.
(i) Small-plot tests. The small-plot testing should include the fol-
lowing procedures:
(A) Use of replicated plots (preferably at least in triplicate) for
each dosage (concentration) tested and for each crop type or variety, as
appropriate.
(B) Sufficient dosage ranges to bracket the normal concentrations
expected to be present when the product is used at the maximum dosage. Thus,
concentrations should include 3x to 4x the maximum expected concentration
.down to a concentration where no effect on the crops is observable.
(.C) Appropriate control or check plots (no herbicide .in irrigation water)
must.be used .and must include at least duplicate plots for each crop type.
.(D) Irrigation, schedules, and methods should be as similar, as possible
'•.to.:, those .used..in. full-scale crop practices./This .includes;.the. normal, irriga-
tion-practices in reiati'on to planting"development (growth) of .crops.
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(E) Metering devices and methods should be accurate enough to maintain
the desired concentration within 10% from beginning to end...
(ii) Large-scale tests. Large-scale testing should be made at actual
application sites in the field and include the types of crops and irrigation
methods that will be used in the areas where the product is to be used. The
irrigation schedules must be recorded as well as the amount of water per
irrigation.
(2) Reporting effects on crops. In both the large-scale and small plot
tests, the condition of the crops must be noted at the various growth stages,
including any recovery from initial phytotoxic effects. Appropriate evalua-
tion of crops include crop yield and quality. The yield may be measured in
volume, weight or extracted product (e.g. Brix value for viticulture). For
seed production, seed viability must be assayed to determine the percent
germination during storage life.
(3) Example method references. Initial small pilot procedures such
as those used by Bruns and Kelly (1974, 1975) are acceptable for obtaining
experimental use permits for full scale tests. Other acceptable methods
are found in the references below.
References.
Bartley, T.R. 1967. Progress Report on Evaluation of Copper for.Aquatic
Weed Control and Herbicide Residues on Irrigation Systems. U.S. Dept.
of Interior, Bureau of Reclamation, Report No. WC-32.
1969. Copper Residues on Irrigation Canal. Weed Sci. Soc. Amer.
Abstr. No. 98.
Bruns, V.F., J.M. Hodgson, and H.F. Arle. 1972. Response of several crops
to six herbicides in irrigation water. U.S. Dept. Agr., Agr. Res. Serv.
Tech. Bull. No. 1461. 29 pp.
Bruns, V.F., B.L. Carille, and A.D. Kelley. 1973. Responses and residues
in sugarbeets, soybeans, and corn irrigated with 2,4-D or Silvex-treated
water. U.S. Dept. Agr. Agr. Res. Serv. Tech. Bull. No. 1476. 31 pp.
Bruns, V.F., R.R. Yeo, and H.F. Arle. 1964. Tolerance of certain crops to
several aquatic herbicides in irrigation water. U.S. Dept. Agr., Agr.
Res. Serv. Tech. Bull. No. 1299.
Bruns, V.F., and A.D. Kelley. 1974. Effect of sprinkler irrigation with
dylene-treated water on six crops. U.S. Dept. Agr., Agr. Res. Serv.
Tech. Bull. No. 796.
Bruns, V.F., and A.D. Kelley. 1975. Responses and residues in certain
crops irrigated with water containing Glyphosate. U.S. Dept. Agr., Agr.
Res. Serv. Tech. Bull. No. 812.
Smith, R.J., Jr. -1974.. Responses of rice to post-emergence treatments or
propani'l. 'Weed Sci. 22:563-568.
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(d) Efficacy testing methods for aquatic herbicides in standing waters.
(1) Acceptable test plots; The following types of test plots may be
used for some types of aquatic plants as described below.
(i) Whole pond treatments. Whole pond treatments have been used for
many years and most closely approximate some actual use conditions for
aquatic herbicides. First, it is difficult, if not impossible, to obtain
comparable replicates. Even adjacent ponds separated by narrow dikes and
i located on identical soils frequently react in dissimilar manners when
I treated with herbicides. In addition to making replication difficult, this
j lack of uniformity makes comparisons between treated and untreated ponds
i difficult. Usually a comparison of the same pond before and after treatment
is more meaningful than comparing a treated pond with an untreated pond,
but even this fails to account for population variation with time.
I (ii) Open plots. Open plots are treated areas in large bodies of water
i not enclosed or contained by any artificial means. Open plot treatments
[ have been used successfully under a variety of situations and in some cases
| closely simulate in-use conditions. A comparison of open plot treatments
with whole pond and plastic-enclosure treatments can be found in 3 papers by
I Walker (1964, 1964, 1965). There are major differences between whole-pond
; tests and open-plot tests. The first is a problem of dilution of the
} herbicides by the surrounding untreated waters, which indicate that size of
! the plot is very important. Although many investigators have used smaller
| sized plots, open plots should be at least 0.1 hectare to get reliable
j results. Even with this size plot, border effects might extend to the center
j of the plots. Data should be taken, from the center of the treated areas.
| For open plots of 0.1 hectare or smaller, standard treatments with pesticides
I of known effect must be included for comparison with the pesticide being
| tested. Second, with open plots one must also be cognizant of water movement
' from currents, tides, and wave action, which might carry the herbicide, or
i plants, out of the plot boundaries. Because of the plant-drift problem,
i open plots are not suitable for unattached algae or for free-floating macro-
I phytes. On the other hand, open plots are suitable for emersed and marginal
i plants, and to a lesser extent for floating-leafed species. One advantage
I over whole pond treatments is that, with many plots in one body of water,
t plot-to-plot variation is less.
i . .
1 (iii) Enclosed plots. Enclosed plots are areas in larger bodies of
water which are "sealed off" from the rest of the water mass, usually by
| plastic .film. Enclosed plots have been used by various investigators in
j the past (Gallagher et al., 1968; Walker, 1964, 1965). They offer the
smallest, most reproducible type of plots available for use with naturally-
occurring plants which have not been disturbed or transplanted and a natural
bottom which has been disturbed very little, if at all. They can be quite
small because there is no dilution of the herbicide by the surrounding
untreated water.. This is a distinct advantage over the open-plot method.
.Also, because of their smaller size [5 x 5 .ft (1.52 x 1.52 m.) used by
Gallagher et al.: 1968J, visual estimation of herbicidal effectiveness is
simpler.and possibly.more accurate. A major, concern with enclosed plots is
to. assure that the.plastic is, not. permeable to the herbicide and that the
/herbicide .is. n6t. adsorbed .ayiabsorbed by the particular type,-of plastic • '
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being used. A tight seal at the bottom is also important. The technical
difficulties of assuring a tight seal without disturbing the site indicate
that this method is less than ideal.
(iv) Artificial pools. Above-ground, plastic-lined or concrete pools
offer a convenient system for testing potential aquatic herbicides. The
method is described in detail by Lawrence and Blackburn (1962). The same
care must be taken as with enclosed plots to assure the herbicide is not
adsorbed or absorbed by the plastic liner. The advantage of artificial pools
is that a wide number of variables can be studied under semi-controlled
conditions, and a wide spectrum of weeds can be tested. Extrapolation of
the results of artificial pool tests to natural bodies of water may not be
valid. The substrate added to the pool is "artificial" and may not resemble
natural pond or lake sediments in either chemical composition or physical
structure. Some degree of testing by either the whole pond or open plot
method should be done to confirm the results of artifical pool tests.
(2) Application methods. The appropriate method of application depends
on formulation, size of plots, label directions for use, and, possibly,
rate of application. It is important that the method be described in
detail, that the method results in accurately-applied amounts of pesticide,
and that the method give reasonably uniform distribution of pesticide
throughout the test area. The latter is most important with certain
chemicals and with types of vegetation to which spray is directly applied,
such as floating mats of algae, free-floating plants, emergent vegetation
and floating-leafed species controlled by liquid spray applied to the top
surface of leaves.
(3) Evaluation of results. A number of methods have been developed
for measuring aquatic plants. One or more of the following should be
selected for evaluation of treatments and untreated control plots. The
evaluation technique must be appropriate for the type of plant and the
nature of the test.
(i) Colorimetric and spectrophotometric techniques. Chlorophyll de-
terminations by spectrophotometric methods are widely used for determining
single-celled phytoplankton and small plankton colonies of algae. Any
methods detailed by Golterman (1971) and by Weber (1973), such as the tech-
nique given in § 92-20(e) may be used. No chlorophyll technique is
acceptable for macrophytes, and such techniques are generally unsatisfactory
for filamentous algae, since gravimetric techniques are better when sampling
is adequate. Visual estimation of water color and/or turbidity is not
normally sufficient for measuring degree of control under field conditions.
However, when used in conjunction with cell counts, data obtained with the
use of a Secchi disc will be acceptable for phytoplankton evaluation.
(ii) Standing crop methods. There are a number of simple quantitative
and semi-quantitative sampling techniques available involving collection of
plants with rakes, with samplers or by hand (Weber, 1973). These techniques
lend themselves to gravimetric determination of standing crop. Dry weight
is preferred over wet weight and, due to high surface deposits of inorganic
sediments, ash-free dry weight is morei reliable than conventional dry weight
image:
119
(Stanley et al., 1976). An appropriate ash weight technique is given by
Weber (1973) in the section on plankton. The greatest disadvantage with
gravimetric methods is the difficulty of obtaining an adequate number of
samples due to the erratic occurence of aquatic plants. In a dense, uni-
form stand, 3 to 5 samples may be adequate for a dominant species, but
less common kinds of plants may require 30 to 40 samples for an adequate
measure of standing crop, and rare species will be impossible to quantify
gravimetrically (Livingston et al. 1976). Sampling is particularly diffi-
cult for free-floating macrophytes and for floating mats of algae. A
gravimetric productivity method has been developed which uses wooden floats
to protect duckweeds from drift (Rejmankova, 1973). Similarly, artificial
substrates with algal growth could be used to simulate periphyton which
have not broken free to form floating mats (Weber, 1973). Consultation
with the Agency is advised prior to the use of methods such as these latter
two.
(iii) Population counting methods. Direct counts of cells or colonies
of plankton may be made microscopically according to the methods outlined
by the American Public Health Association, Inc. (1965) or by Weber (1973).
On the other extreme, large emersed plants can be counted individually.
However, in many cases, prolific asexual reproduction and intricately inter-
twined population masses make it impossible to distinguish and count individ-
ual members of a population.
(iv) Transects and surface cover methods. Transects are among the
several terrestrial sampling methods that- are adaptable to aquatic plants
(Wood, 1963). Both line intersect and transect belt have been used suc-
cessfully (Kvet and Ondok, 1973; Taylor, 1971). Estimates of surface area
coverage in quadrats randomly or systematically located along a transect
can be performed more rapidly than standing crop measurements. With the
many replicates, which are possible due to the ease of performing the
estimate, this technique is as reliable as gravimetric techniques. When
casually applied, this method is similar to visual estimates discussed in
the following section. The report of evaluations using transects shall
include details such as type of transect belt or transect-quadrat, random
and systematic selection methods (e.g., transects located randomly within
the site and quadrats located systematically based on depth along the
transect), and nature of count (at surface of water or surface of substrate).
(v) Visual estimation methods. The majority of tests are evaluated
by visually estimating the reduction in vigor and stand density of each
species present. When this method is used, more "samples" are required to
reduce variation than with more quantitative techniques. Whenever possible,
observer bias should be minimized by pooling results of a number of inde-
pendent observers or by periodically "standardizing" estimates against more
reliable quantitative techniques. Visual estimation is not acceptable for
phytoplanktons and periphyton populations, for which more precise methods
are available. . (See American Public Health .Association, Inc., 1971; and
Methods of Hydrobiology Fresh Water,.Biology by J. Schwoerbel, 1970..)
(vi) Underwater records. Several' of the techniques discussed above
c"ah. benef i't'by: the-, use. of scuba for; eya-koatlon of-submersed-, .plants (.Timmons,
' 19.7.6; Wood, • 1963).. Underwater recording equipment, has been developed for
image:
120
performing transect and quadrat counts while completely submersed (Fag.er et
al., 1966), and a quantitative phototechnique has been developed that can
be used in clear water (Johnston et al., 1969). Scuba is especially bene-
ficial for careful collection of deeply submersed standing crops (Stanley
et al., 1976). If scuba diving observations are used, the report should
clearly indicate so.
(vii) Photographic techniques. Aerial photography techniques, already
a well developed evaluative technique for terrestrial locations, has been
widely used for emersed and marginal aquatic plants (Shima et al., 1976)
and can be applied to free-floating and floating-leafed species (Tarnocai
and Kristof, 1976). Supported by ground truth, aerial photography can be
used for identification (Cowardin and Myers, 1974) and for quantification
(Drake, 1976). Complex patterns not distinguishable from the ground can be
seen (Wrigler and Home, 1974). For submersed species penetration of the
water column is a serious problem on which progress is being made (Specht
et al., 1973). Though relatively inexpensive for evaluating large and/or
inaccessible areas, aerial photography is expensive when used for a few
small test plots. Aerial photography from a surface-towed ballon may
provide some of the advantages of an elevated point-of-view with a cost
appropriate for small scale evaluations (Edwards and Brown, 1960). When
supported by appropriate ground truth and/or other efficacy data, aerial
photography, as well as conventional photography, is an acceptable evalua-
tion technique.
References.
American Public Health Association, Inc. 1971. Standard Methods for the
Examination of Water and Wastewater. 13th Ed. 874 pp.
Comes, R.D. and L.A. Morrow. 1971. Control of waterlilies with dichlobenil.
Weed Sci. 19:402-405.
Cowardin, L.M., and V.I. Myers. 1974. Remote sensing for identification
and classification of wetland vegetation. J. Wildl. Manage. 38:308-314.
Drake, B.C. 1976. Seasonal changes in reflectance and standing crop bio-
mass in three salt marsh communities. Plant Physiol. 58:696-699.
Edwards, R.W., and M.W. Brown. 1960. An aerial photographic method for
studying the distribution of aquatic macrophytes in shallow waters. J^
Ecology. 48:161-163. •
Pager, E.W., A.O. Flechsig, R.F. Ford, R.I. Clutter, and R.J. Ghelardi. 1966.
Equipment for use in ecological studies using scuba. Limnol. Oceanog. 11:
503-509.
Gallagher, J.E., and W.F. Evans, and A.R. Cooke. 1968. A variation of
plastic enclosures for field-testing aquatic herbicides. Proc. Northeast
Weed Control Conf. 22:362.
Golterman, H.L. ed. 1971. Methods for Chemical Analysis of Fresh Waters.
IBP Handbook No. 8. Blackwell Scientific Publication, Oxford and Edinburg.
166 pp.
image:
121
Johnston, C.S., I.A. Morrison, and K. MacLachlan. 1969. A photographic
method for recording the underwater distribution of marine benthic
organisms. J. Ecology 57:453-459.
Kvet, J., and J.P. Ondek. 1973. Zonation of higher-plant shoot biomass
in the littoral of the Opatovicky fishpond. Pp. 87-92 in Ecosystem
Study on Wetland Biome in Czechoslavakia Czechosl. S. Henjny, ed.
IBP/PT-Report No. 3 Trebon.
Lawrence, J.M., and R.D. Blackburn. 1962. Evaluating herbicidal activity
of chemicals to aquatic plants and their toxicity to fish in the labora-
tory and in plastic pools. Auburn Oniv. Agric. Experiment Sta.: Auburn,
Ala. 23 pp. (mimeo.)
Livingston, R.J. R.S. Lloyd, and M.S. Zimmerman. 1976. Determination of
sampling strategy for benthic macrophytes in polluted and unpolluted
coastal areas. Bull. Mar. Sci. 26:569-575.
McGilvery, F.B., and J.H. Steenis. 1965. Control of alligatorweed in
South Carolina with granular silvex. Weeds 13:66-68.
Rejmankova, E. 1973. Seasonal changes in the growth rate of a duckweed
community. Folia Geobot. Phytotax. (Praha) 8:1-13.
Schwoerbel, J. 1970. Methods of Hydrobiology Fresh Water Biology. Pergamon
Press: Oxford, London. 200 pp. '
Shima, L.J. R.R. Anderson, and V.P. Carter. 1976. The use of aerial color
infrared photography in mapping the vegetation of a .freshwater
marsh. Chesapeake Sci. 17:74-85.
Spect, M.R., D. Needleer, and N.L. Fritz. 1973. New color film for water
penetration photography. Phytogram. Engin. 39:359-369.
Stanley, R.A., E. Shackelford, D. Wade, and C. Warren. 1976. Effects of
season and water depth on Eurasian watermilfoil. J. Aquatic Plant Manag.
14:32-36.
Tarnocai, C., and S.J. Kristof. 1976. Computer-aided classification of
land and water bodies using Landsat data, Mackenzie Delta area, N.W.T.,
Canada. Arctic Alpine Res. 8:151-159.
M.E.U. Taylor. 1971. Report on the Nelson Lakes Survey 1971 by Cawthron
Institute. Nelson Lakes National Park Board, Nelson, N.Z. 66 pp.
Timmons, F.L. 1970. Research on aquatic and bank weeds. Unique challenges
and techniques. Proc. West. Soc. Weed Sci. 23:6-10.
Walker; C.:R. 196.4; Dichlobenil as a herbicide, in..£ish habitats. .Weeds 12:
267-269.
. .1964. Simazine and other s-triazine compounds .as aquatic
herbicides in fish, habitats. Weeds 12:,134-139.
image:
122
Walker, C.R. 1965. Oiuron, Fenuron, Monuron, Neburon, and TCA used as
aquatic herbicides in fish habitats. Weeds 13:297-301.-- .
Weber, C.I., ed. 1973. Biological field and laboratory methods for measur-
ing the quality of surface waters and effluents. National Environmental
Research Center, U.S. EPA., Cincinnati, Ohio. EPA-670/4-73-001. xi +
146 pp. Appendices.
Wile, I. 1973. Use of remote sensing for mapping of aquatic vegetation
in the Kawartha Lakes. Remote Sens. Water Res. Manag. Proc. 17:331-336.
Wood, R.D. 1963. Adapting scuba to aquatic plant ecology. Ecology 44:
416-419.
Wrigley, R.C. and A.J. Home. 1974. Remote sensing and lake eutrophication.
Nature 250:213-214.
(e) Pure-culture technique for evaluating algicides. This procedure
is a modified version of a method first described by Fitzgerald and Faust
in 1963. (See also Fitzgerald, 1962, and 1964). This modified procedure
has been used at the Agency's Chemical and Biological Investigations Branch
Laboratories (Benefits and Field Studies Division) for several years to
help determine effectiveness of algicides as part of the Agency pesticide
enforcement activities. These modified procedures are published in the
Manual of Biological Testing Methods for Pesticides and Devices.
A method of test for evaluating algicides using pure-rculture techniques.
1. Scope 1.1 Products intended for the control of algae in swimming
pools, industrial cooling water systems, and other water systems should be
shown to be effective for the purpose claimed. This pure-culture test would
determine the effectiveness of a chemical formulation to control standard
algae species, and also show the algicidal vs. algistatic properties of the
formulation being tested.
2. Test Organisms
2.1 Chlorella pyrenoidosa. (Wis. 2005) or Chlorella pyrenoidosa
Chick No. 26 (Obtained from Dr. Richard C. Starr, Culture Collection of
Algae, Department of Botany, University of Texas, Austin, Texas. 78712.
2.2 Phormidium inundatum and Phonaidium retzii, should be included in
the test as representative algal species found in swimming pools. Other
algae may also be included if deemed necessary. For culture media for
Phormidium species, see Hughes, et al. (1958). The concentrations of stock
cultures must be adjusted in such a way that test inoculum will be reproduc-
ible and equivalent to a standard of Chlorella. This may be accomplished
by adjusting Phormidium inocula to a predetermined Chlorella inoculum
optical density. (See Fitzgerald, 1964).
image:
123
2.3 Modifications for use in developing presumptive efficacy data for
industrial cooling system products: Use the following algae: Chlorella
pyrenoidosa, Euglena gracilis, Scenedesmus obliquus. Obtain from the
addresses in section 2.1 as follows: C. pyrenoidosa - Fitzgerald or Starr;
E. gracilis and S_. obliquus Starr.
3. Culture Medium
3.1 Allen's Medium (Difco Algae Broth may be used) for Chlorella
pyrenoidosa.
Distilled water 1000 ml .
NH4C1 50 mg
NaN03 1000 mg
K2HPO4 250 mg
MgSO4 . 7H20 513 mg
CaCl2 • 2H2O 66 mg
FeCl3 3 mg
3.2 Stock cultures are maintained on Allen's medium (or Difco Bacto
"Algae Culture Agar") as 1.5 percent agar slant cultures in tubes.
3.3 Stock cultures stored at 22 to 24°C with 16 hrs of cool white
fluorescent light per day, 4306 Ix (400 ft. candles) initially and then
removed to low light conditions.
3.4 Media for algae used for industrial cooling system assays:
3.4.1 Hunter's Medium for Scenedesmus obliquus:
Amount per liter:
KH2PO4 0.8 g
MgS04 . 7H20 1.9 g
L-glutamic acid 10.0 g
DL-malic acid 4.0 g
CaC03 0.4 g
FeCl3 . 6H20 • 0.0167 g
Thiamine HCl 2.0 mg
(NH4)2HP04 0.4 g
Vitamin B12 0.4 ug
ZnS04 . 7H20 176.0 mg
MnS04 . 4H20 160.0 mg
Na2Mo04 . 2H20 • 40.0 mg
CoCl2 . 6H20 3.2 mg
CuS04 . 5H20 1.6 mg
H3B03 ' 1.2 mg
. -Nal ' . • ...;: .. . . ..-..•-' .47.0 ug ..
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124
3.4.2 Bristol's Medium for Euglena (or Difco Bacto Euglena Broth):
Bristol's medium, supplemented (per liter)
NaN03 505 mg
CaCl2 50 mg
MgS04 . 7H20 150 mg
K2HP04 150 mg
KH2P04 350 mg
Nad 50 mg
MnS04 . 4H20 300 ug
F.eCl3 1 ug
ZnS04 200 ug
H3B03 200 ug
CuS04 . 5H20 • 6 ug
yeast extract 1 g
proteose peptone 1 g
glucose 15 g
4. Apparatus
4.1 Glassware: 50-ml Erlenmeyer flasks with cotton plugs for each
species. Suitable pipettes for addition of chemical formulation to test
flasks.
4.2 Bacteriological loop (0.01 ml) for making subcultures.
4.3 Controlled-environment chamber for-holding temperature at 22-
24°C. Chamber equipped with cool white fluorescent lights giving 4306 Ix.
Lights are regulated with time clock to operate for 16 hours per day.
4.4 Automatic pipetter (facilitates preparation of large numbers of
test flasks).
4.5 Steam sterilizer.
4.6 Spencer Bright Line Hemacytometer (or equivalent) for counting
algae.
4.7 Bunsen burner for sterilizing bacteriological loops.
4.8 Laboratory microscope for use in making algae counts.
5. Procedure
5.1 Transfer algae from stock agar cultures (3.2) to media and hold
at 22-24°C under 16 hours fluorescent light, 4306 Ix in suitable containers
for up to 3 weeks to inoculate test flasks.
5.2 Place 30 ml of medium into each 50-ml Erlenmeyer flask. This may
be accomplished' quickly with the use of an-automatic pipetter. Plug flasks
with cotton and sterilized at 103 kpa (15 psi) for 20 min. After they have-
.cooled to room temperature the flasks are inoculated with C_. pyrenoidosa.
image:
125
Inoculate from an actively growing liquid culture (5.1) at a concentration
to give an initial cell count of 300,000 cells/ml in each test flask. No
more than 0.3 ml aliquot should be taken from the liquid stock culture for
addition to each test flask in order to give the desired algae concentration
of 300,000 cells per ml. Centrifugation of liquid stock culture or other
means may be necessary in order to obtain desired concentrations of algae
cells.
5.3 Add chemical formulation at each rate to be tested to 10 flasks.
Ten inoculated flasks are used as untreated controls for each species.
5.4 To determine algicidal vs. algistatic properties of the chemical
formulation, 0.01 ml aliquots, using a 0.01 ml bacteriological loop, are •
aseptically transferred from the primary cultures after 2 days of incubation,
to 10 additional 50 ml Erlenmeyer flasks, each containing 30 ml of sterile
growth medium. Subcultures are also made of controls. The subcultures are
kept under the same environmental conditions as the primary cultures for
three weeks.
5.5 Flasks should be continuously shaken during test (approximately
100 oscillations per minute).
6. Determination of results.
6.1 Evaluation: Record visual observations weekly for duration of the
test. Observations are made of both the primary cultures and subcultures.
A rating scale may be used to help determine actual percent control by the
chemical formulation such as, 0-5,-"0", being no visible growth, and "5"
very heavy growth; Other means, such as colorimetric methods, may be
employed to evaluate results of the test.
6.2 Interpretation: For a product to be satisfactory it must provide
at least 70% control of algae growth in the primary cultures for 3 weeks.
When using a rating system, percent control is obtained by subtracting the
average rating for the 10 treated flasks (RT) from the average rating of
the 10 untreated control flasks (RC), then dividing by the average rating
figure of the untreated flasks (RC), multiplied by 100. [Percent control =
100 (RC - RT)/RC.] In order for an algicidal product to claim "kill" or
"destroy" algae on the label, there must be no visible sign of algae growth
in subcultures after 3 weeks.
References.
Chemical and Biological Investigations Laboratories. 1978. Manual of
Biological Testing Methods for Pesticides and Devices. Schneider, 3.A.,
and A. J. Culver, Jr. ed. U.S. Environ. Prot. Agency, Benefits and
Field Studies Div., Beltsville, Md.
Fitzgerald, George P. 196-2. Bioassay for algicrdal chemicals in swimming
pools. Water and Sewage Works 109:361-363.
Eitzger.a-ld, -. George p.. 196-4.. Factors in the. testing, and application..of
.algicides.. Appl'. Microbiol. 12:247-253'. " , ; ' '. ;
image:
126
Fitzgerald, G.P., and L. Faust. 196'3. Bioassay for algicidal vs. algistatic
chemicals. Water and Sewage Works 11.0:296-298.
(f) Method for evaluating algicides in simulated swimming pools
1. Scope
1.1 Products intended for use to control, prevent or inhibit algae
growth in swimming pools should be effective. This test method is designed
to determine effectiveness of candidate products to control algae in simulated
swimming pools.
2. Inoculum
2.1 Test Algae - The genera of algae infesting swimming pools may
vary greatly from pool to pool. The species may differ geographically, in
response to climate, water hardness, pH, and other factors. Since this
method is not a pure culture technique, it would be very difficult to
maintain a predominant flora in a given pool situation. Therefore, the
choice of test organisms will be wide. However, representative isolates
of the following algae should be included in the inoculum for each test
pool:
Chlorella pyrenoidosa
Phormidium inundatum
Phormidium retzii
These genera have been found in swimming pool water. Pure cultures of
these algae can be obtained by isolation from sources in every location
where needed, or from commercial or institutional culture collections, such
as American Type Culture Collection, Rockville, Md. 20852, and the Culture
Collection of Algae, University of Texas, Austin, Tex. 78712. They should
be maintained in synthetic nutrient medium. (See 3. Synthetic algal nutrient
medium.)
2.2 Maintenance of Stock Cultures.
2.2.1 Medium - see 3.
2.2.2 Incubation Conditions - 24°C +_ 1, cultures exposed to 16 h photo-
period, 3229 Ix (300 ft candles), cool white fluorescent light.
2.2.3 Age of cultures - Two to three weeks with good growth evident.
2.3 Preparation of inoculum for test.
2.3.1 Culture units - Prepare one flask of medium (see 3) for each
algae times each pool in the test. Use 1000 ml culture flasks containing
500 ml of medium. Autoclave 20 min at 103 kpa (15 psi) and allow to cool
prior to inoculation.
image:
127
2.3.2 Inoculate sufficient culture units, using aseptic methods, to
supply the test pools.
2.3.3 Incubate as in 2.2.2 and 2.2.3.
3. Synthetic algal nutrient medium
3.1 Final concentration of nutrients (1).
3.1.1 Macronutrients - The following salts, Biological or Reagent
grade, in mg/1 of glass-distilled water:
Compound Concentration (mg/1)
NaN03
K2HP04
MgCl2
MgS04 .
CaCl2 .
NaHCOg
7H20
2H20
25.5
1.044
5.7
14.7
4.41
15.0
3.1.2 Micronutrients - The following salts. Biological or Reagent
grade, in ug/1 of glass-distilled water:
' Compound Concentration (ug/1)
H3B03 185.52
MnCl2 264.264
ZnCl2 32.709
CoCl2 0.78
CuCl2 0.009
Na2Mo04 . 2H20 7.26
FeCl3 96.0
Na2EDTA . 2H20 300.0
3.2 Stock solutions.
3.2.1 Macronutrients - Stock solutions of individual salts may be
made up in 1000 times the final concentration.
3.2.2 Micronutrients - The trace metals, FeCl3, and EDTA are combined
in a single stock mix at 1000 times the final concentration.
3.3 Preparation of medium
3...3.1 Combination of stock solutions - One ml of each- of the stock
solutions (.3,2..1, and 3.2.2) is. added to .glass-distilled water to give a
final volume of one liter. '••'.-:.. '
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123
4. Apparatus
4.1 Test pools, 1.22 by 0.61 m (4 by 4 by 2 ft) deep are.constructed
of 19 mm (3/4 in.) plywood, framed with 50.8 by 101.6 mm (2 by 4 in.)
lumber. Vinyl or polyethylene liners I/ are fitted into the supporting wood
box. The corners are either heat-sealed to make a clean joint or the excess
plastic is folded under, the watertight integrity being carefully preserved.
Pools of this size hold 908 liters (240 U.S. gal). Each pool is equipped
with a pump, in-line filter, and heater. The heater is controlled by
thermoregulator, adjusted to.24°C. The pump is a submersible Little Giant
Model 2E38N, 110V 60 cycles, or equivalent. The filter is a cellulose
cartridge type such as Teel (W.W.' Grainger) Stock No. 1P635 equipped with
filter cartridge Stock No. 1P753. Filter elements of different manufacture
but of similar composition may be substituted.
5. Procedure of test
5.1 Preparation of Pools - Fill with tap water to 25 mm (1 in.) from
top. Allow to equilibrate for 24 hr.
5.2 Replications - Each treatment should have a minimum of two
replicates. If this is not feasible because of space limitations, two
consecutive tests must be conducted.
5.3 Nutrients, temperature, and light - Add to each pool 10Og of Ra-
Pid-Gro Soluble Plant Food (Ra-Pid-Gro Corporation, Dansville, N.Y. 14437)
or equivalent, at the time of filling. After the 24 hr. equilibration
period adjust the temperature to 24°C + 1 with the circulating pump running
and the filter in place. Maintain 16 h photoperiod, 3229 Ix, cool white
fluorscent light over all pools, or daylight, if available.
5.4 Inoculation - inoculate each pool with one flask (2.3.1) of each
algae being used in the test. Remove all attached algae from flask with
a brush or other means and wash into pool with pool water.
5.5 Chlorine standard - Maintain at least one pool as a chlorine
standard with no additional algicidal treatment added. For pools stabilized
with isocyanuric acid (trihydroxytriazinetrione), maintain chlorine residual
at 1.0 to 1.5 ppm continuously. For non-stabilized pools, chlorine residual
should be incorporated in all tests at the rate of 0.6 to 1.0 ppm. Metering
devices may be used, provided they will accurately maintain the desired
chlorine level.
5.6 Water flow rate - Adjust flow rate to provide a complete filtration
cycle in about 6 h. For a 908 liter pool, this would require a pumping
rate of 151 1/hr (40 gal/hr), or 2.5 1/min (0.66 gal/min).
5.7 Adjust pH daily and maintain between 7.2 to 7.6 during the period
of the test. To raise pH, add sufficient soda ash (sodium carbonate) to
bring the pH to the above range in each pool. These chemicals should be
I/ Liners may be obtained from Plastimayd Corp., 2204 S.E., 7th Ave.,
Portland, Oregon 972.14 . . .
image:
129
placed in a plastic strainer and allowed to dissolve under the inlet water
flow. Algal growth will result in an increase in the pH. This increase
must be counteracted with the proper treatment above.
5.8 Treatment - A minimum of two replications per treatment should be
included in each test. Apply product at initial and maintenance doses.
Maintain 1 or more untreated control tanks and one chlorine standard tank.
5.9 Length of test period - Maintain conditions as in 5.3 above for a
minimum of four weeks.
6.0 Reporting Results - Examine tanks weekly. Rate amount of^algal
growth using a scale such as:
Numerical rating Status of algal growth
0 No apparent growth
1 Barely visible attached mat
2 Clearly visible mat, slight bloom
3 Well defined mat, moderate bloom,
bottom still visible
4 Walls and bottom covered by mat,
bottom barely visible
'5 Heavy bloom, loss of water trans-
parency, heavy attached or.free-
floating mats
6.1 Products which give a final average reading greater than that of
the chlorine standard should be considered unsatisfactory for use in swimming
pool service. In addition to estimation of algae by visual inspection,
swimming pool algicide effectiveness should be evaluated by determining the
remaining inoculated algal species in pool water samples taken at bi-weekly
intervals throughout the test period. Other valid means of evaluation may
be used when determining the results of the test.
References.
(1) Chemical and Biological Investigations Branch Laboratories. 1978.
Manual of Biological Testing Methods for Pesticides and Devices.
McCann, J., and A.J. Culver, Jr., eds. U.S. Environ. Prot. Agency,
ARC-East, Beltsyille, Md. 20705.
(.2) National Eutrophicatipn'Research program. Environmental Protection
Agency;-.August 1971, "Algal Assay Procedure., Bottle Test", U.S.
• tSoyernment Printing Office, 1972', Region No. 10. Pp... 11-12.
.Water and/Wastewa^er", .ABHA-.,:".13'th Ed-.., ..19'71.
"' '
image:
130
Series 93: EFFICACY OF FUNGICIDES AND NEMATICIDES
§ 93-1 General considerations.
For complete guidance on the performance of fungicides and nematicides,
the information contained in §§ 90-1 Overview and 90-2 General considerations
must be considered in conjunction with the information in §§ 93-2 through
-15.
§ 93-2 Definitions.
(a) The term "fungicide" means any substance or mixture of substances
intended for preventing or inhibiting the growth of, or destroying any
fungus (or any plant disease agent such as bacteria, mycoplasma, and virus)
declared to be a pest under § 162.14 of the FIFRA Sec. 3 regulations,
except those fungal control agents described in § 92-4 and -5 as "micro-
biocides", those defined in § 91-1 as "antimicrobial agents", and those
subject to the provisions of the Federal Food, Drug, and Cosmetic Act,
as amended (21 USC § 301 et seq.)
(b) The term "nematicide" means any substance or mixture of sub-
stances intended for preventing or inhibiting the multiplication or estab-
lishment of, preventing or mitigating-the adverse effects of, repelling,
or destroying any member of the Class Nematoda of the Phylum Nemathelminth.es
declared to be a pest under § 162.14 of the FIFRA Sec. 3 regulations.
§ 93-3 Suggested performance standards; acceptable levels of pest control.
(a) Due to the diverse factors involved in the control of plant
diseases or nematodes, the degree of control that is acceptable or attain-
able under certain use conditions for a particular pest/site combination
may not be acceptable under other conditions. The acceptable level of
pest control for fungicides and nematicides will vary for each pest/site
combination, user group, end use of treated site, and the degree of
control intended to be claimed on labeling (e.g., controls, aids in
control, prevents, suppresses). In general, the acceptable level of
control for a given use will be that which the ultimate user of the
pesticide would find acceptable. [See Subdivision H, §§ 103- for more
information on fungicide labeling].
(b) For claims that a product "controls" or "prevents" plant disease
or nematode pests, the product should generally provide, under moderate to
severe pest pressure, at least 70% control of the pest organisms or their
symptoms (compared with untreated controls) over the period in which
control is intended. For control of nonagricultural fungal pest problems.
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the product should generally provide complete control of the pest problem
for the expected storage period or service-life of the substance to be
protected.
(c) Under certain circumstances a level of effectiveness less than
that which is considered optimum or complete may be claimed and be appro-
priate. Lesser claims, such as "aids in control" or "suppresses", may
be made if less than 70% control of plant diseases or nematodes is obtained,
provided that a measurable benefit (e.g., increased quality or quantity of
crop yield) can be demonstrated. Such benefits are usually only attained
under certain circumstances which must be determined from the test results.
These use limitations must be stated on the product label. For example,
the label might state that the suppression of a given pest may only provide
a benefit when low pest populations exist.
§ 93-4 Products for use against above-ground plant pests.
(a) Control of plant diseases that attack above-ground plant parts
should be evaluated in tests conducted under in-use conditions which utilize
comparisons of untreated and treated plants. Separate tests should be con-
ducted using each intended type of application equipment, application rate
and schedule, type of spray (i.e./ full coverage/ low volume, or aerial),
type of control program (i.e., preventive or curative), and cultural
practice (e.g., greenhouse, field, irrigated, nonirrigated). Field tests
should be conducted at a sufficient number of geographic locations.so as to
include the expected range of rainfall, relative humidity, air temperature,
frequency and type of irrigation, planting dates, crop cultivars (varieties),
and other pertinent factors (appropriate for the pest/crop combination)
associated with the intended geographic area of use which may affect
product efficacy.
(b) The control of two or more concurrent diseases of the same crop
may be evaluated in the same test, provided that the disease symptoms are
readily distinguishable and allow for separate evaluations. In cases
where different stages of the same disease attack different plant parts,
the stage(s) intended to be controlled must be reported and supported by
the appropriate pest control data. A considerable number of disease
assessment criteria are available, e.g., number af lesions per leaf,
number of affected plants per specified row length. Since the precision
and reliability of each criterion will vary with the pest/crop combination
under study, the investigator should carefully select the best criteria
for detecting valid treatment differences in each test. In addition to
the criteria used, the determination of proper time and number of disease
evaluations is also important. Such observations are generally made at
the time of initial application and at periodic intervals, throughout the
.growing season. When repeat applications are .intended, it is advisable
to make disease observations on each application date to determine appro-
priateness of. the .time interval between applications.
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(c) Test reports, when applicable, should include detailed information
on the afore-mentioned test and evaluation variables, as well as the
following: the disease level at the time of initial application in curative
tests; the disease level and date when disease first appeared in test
plots, in preventive tests; a complete description of any rating system
used (i.e., criteria for each rating value); the nature and degree of
phytotoxicity which may result from use of the product at or above the
intended label dosage rates; and the nature and extent of any noticeable
spray deposits on plant foliage, fruit, or flowers.
§ 93-5 Products for use against soil-borne plant pests.
(a) Tests for fungicides and nematicides applied to soil should be
designed to determine safety to crops grown on the target site, and to
determine effectiveness against target soil-inhabiting organisms present
in soils of different textures, moisture and pH levels, organic and
mineral compositions, and under a range of climatic conditions common to
potential target sites.
(b) All specialized application equipment and techniques should be
adequately described.
(c) Evidence of pest control or repression of diagnostic symptoms
should be provided. Data which show beneficial effects such as plant
growth or yield increase are unacceptable as the sole proof of product
effectiveness; however, such data are necessary to demonstrate product
usefulness. Depending upon the plant pathogen or nematode and crop
involved, important factors to be measured in assessing pest control
include, but are not limited to, one or more of the- following:
(1) Crop emergence,
(2) Pest mortality or survival,
(3) Crop stand,
(4) Crop lodging,
(5) Crop root injury,
(6) Disease-free or marketable crop yield,
(7) Crop yield or growth response, and
(8) Pest and/or symptom expression ratings.
(d) When a general nematode claim on product labeling is intended,
effectiveness data should be developed to demonstrate control of at least
one species of root-knot, cyst, migratory endoparasitic nematode, and
ectoparastic nematode of importance on the crop(s) or crop planting
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site(s) to be treated. However, the test results should demonstrate that
the product will control all the important parasitic nematodes associated
with a given site; otherwise claims should be limited to the specific type(s)
that can be controlled. Nematode control may be effectively evaluated by
comparisons of treated and untreated plants, plant parts, or soil using
one or more of the following criteria:
(1) The degree of galling on roots, or root-knot index (rootknot
nematodes).
(2) Number of nematodes per unit volume or weight of soil (any
parasitic nematodes). •
(3) Number of nematodes per unit measure of roots or other plant
parts (lesion and other endoparasitic nematodes).
(4) Number of cysts containing viable eggs and/or larvae per unit
measure of soil and/or roots (cyst nematodes).
(e) The overall results should statistically demonstrate that nema-
tode control achieves a desired quality, quantity, or other measurable
crop benefit. Pest population comparisons should be made between treated
and untreated plots before or at treatment and at appropriate intervals
thereafter. Proper sampling times will vary depending upon the nature
of the chemical and type of crop and nematode. For example, final popu-
lation counts obtained at the end of'the growing season in treated areas
often exceed those in untreated areas, particularly when early season
control results in increased plant growth; therefore 'such counts are
unreliable measurements of control. See § 93-30 Item 15 for additional
guidance on conducting nematicide tests.
§ 93-6 Products for post harvest use on fruits and vegetables.
(a) The evaluation of pre harvest or post harvest fruit and vegetable
treatments for control of post harvest fungal or bacterial disease needs
replicated tests on lots which are of sufficient size to represent
commercial size storage containers.
(b) ' Measurement of disease levels should be taken following actual
or simulated packing, storage, and transportation procedures normally
encountered with the particular crop under study.
(c) Whether crops are treated before or during storage, disease
measurements should be obtained and recorded .periodically during storage.
5br each replicate, these measurements ordinarily should..be expressed, in
terms of the percentage of diseased produce. .'.-'• '
• (d.) • Usually., separate tests should..be conducted, on .each, .specific fruit
or •vegetable, and., on each-.organism, to be''cbn.trolied.« However.-,..if two. or
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irore readily distinguishable diseases are present on the same, host simul-
taneously, their control may be evaluated in the same test. Disease
control data on naturally- or artifically-inoculated produce is acceptable.
(e) The product should be tested with each type of equipment (bin-
type dumpers, tank washers, spray washers, brush cleaners, hydro-coolers,
and similar devices) named in the directions for use.
§ 93-7 Products for use as grain preservatives.
(a) Fungicides intended for use in the preservation of stored, high-
moisture grain should be evaluated in both laboratory tests and in-use
site tests. Tests should include treatments over a range of dosages and
grain moisture levels. High-moisture grain generally implies a moisture
content of at least 15 percent. Product effectiveness evaluations in both
laboratory tests and in-use site tests are necessary and should include
comparisons of treated and untreated grain samples. These evaluations are
based on visible mold growth or actual microorganism counts and auxiliary
criteria, such as temperature rise and respiration rate of stored grain.
(b) Laboratory tests should be designed to determine the presumptive
dosage rate(s) necessary to control fungi in or on naturally-infested or
artificially-infested grain (usually inoculated with species of Aspergillus,
Alternaria, Fusarium, and Penicillium). When high moisture .grain is not
available, naturally dried grain reconstituted to the desired moisture
level may be used. Test samples may consist of one quart (946 ml) to 25
gallon (94.6 1) volumes of grain in partially closed glass or plastic
containers. Insulated containers, such as thermos bottles, are necessary
for tests with small samples where ambient storage temperatures are not
controlled. Test variables should include, but are not limited to:
treatment rates, grain moisture levels, kinds of grain, and spoilage
organisms.
(c) Once the preliminary efficacy of the product is established in
the laboratory, additional tests must be conducted under actual in-use
storage conditions. If treated grain is to be stored in enclosed outdoor
structures (e.g., bins, sheds, or silos) such tests should be performed
in either a structure of this type having a capacity of at least 100
bushels (35.2 hi), or in small-scale unexposed outdoor structures (i.e.,
structures contained within a shed or similar enclosure) with a capacity
of at least 10 bushels (3.5 hi). If other types of storage are intended,
the product should be tested under those storage conditions. Tests should
contain a minimum of two (preferably more) replicates for each treatment.
The fungicide should be tested at the proposed application rate(s) in
several geographic locations where grain is commercially produced and
stored. Since stored high-moisture grain will spoil rapidly, testing of
the product on grains under in-use storage conditions should begin within
a few hours of harvest. The duration of testing must correspond with
claims to be made on the labeling (generally 6 to 12 months). Applicable
effectiveness evaluations should be recorded periodically during storage.
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(d) Depending upon the end-use of treated grain, additional studies
may be needed. If treated grain is to be used in animal feeds, appro-
priate feeding studies should be performed, preferably with poultry and
cattle, to demonstrate the acceptability of treated feeds to animals. If
grain is to be used for planting, phytotoxicity tests must be conducted
under field conditions. [Por guidance on phytotoxicity tests, see also
§ 93-8(b) of this subdivision and Subdivision J of these guidelines.] In
cases where treated grain may be used for human food or processed into
products for human consumption, an evaluation of the acceptability of
flavor, milling and baking qualities, fermentation properties, or other
important characteristics should be conducted.
§ 93-8 Products for use as seed treatments.
(a) Seed-treatment products intended for use in the control of post-
planting diseases of seed, seedlings, or mature plants should be evaluated
in laboratory, or greenhouse and field tests, utilizing seed lots and/or
soil known to contain high levels of specific disease-producing fungi.
Product performance under field conditions should be demonstrated utilizing
the intended types of application equipment. Since treated seed may be
stored for a number of months before planting, data should be developed
which demonstrate that the product will be effective and nonphytotoxic
after storing the treated seed for the maximum storage time generally
associated with the type of seed intended to be treated. If such storage
data are not obtained, the time of application on labeling should be limited
to the maximum time period between treatment and planting that is supported
by the test results.
(b) Seed decay and seedling blight disease control can be evaluated
in laboratory and greenhouse tests, provided that the results of such
tests are supported by a limited number of field tests. The field tests
need not have a wide geographical distribution to support claims for seed
decay and seedling blight control. In.laboratory and greenhouse tests,
moderate to severe disease incidences can be assured by techniques such
as initially slowing germination with reduced temperature or by increasing
inoculum levels of specific pathogens. Results are usually expressed as
the percent emergence and percent survival of seedlings on two or more
observation dates. Noticeable delays in seed germination or seedling
emergence observed under field conditions will require that such tests
be continued for the duration of the growing season to determine effects
on yield.
(c) Products intended for control of soilborne and/or seedborne
diseases such as blights, rusts., and smuts, which primarily affect mature
plants, must -be tested under different soil and climatic field .conditions
in'areas where the crop is likely to -be grown...Evaluations should be made
of emergence, stand, percent disease, and yields.' .. •
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(d) In any seed treatment field tests, well-replicated small plots
should be used with seeding rates and plant spacings comparable to general
cultural practices. The number of replications and the size of each plot
must be consistent with the known variability of soil, the character of
the disease, and the anticipated disease incidence. Tests in any one
location should contain a minimum of 4 to 6 replicates per treatment.
Replicates generally consist of one or two rows of 50 to 100 plants for
cotton, peanuts, and other large-seeded crops, or single rod-row plots
for grains and other small-seeded crops. larger plots will be needed if
samples are to be collected for residue analysis, or if grower planting
or harvesting equipment is to be used, or if low disease incidence is
expected. Tests should be designed to span several planting dates and
locations over a range of conditions, and for evaluation of several of
the principal' varieties of each crop for phytotoxic response. It is
desirable to have seed treatment materials evaluated in regional trials,
such as those conducted by the Regional Cotton Seed Treatment Committee of
the Cotton Disease Council. (For further information, contact: National
Cotton Council, P.O. Box 12285, Memphis, Tennessee 38112).
§ 93-9 Products for use on ornamental and flowering plants.
Final-stage testing of fungicides and nematicides for use on orna-
mental plants must be conducted in replicated field or greenhouse plots,
depending on the intended use of the product. Separate greenhouse and
field tests for effectiveness and phytotoxicity are required if both use
areas are intended. In the case of florist crops, acceptable levels of
phytotoxicity and visible pesticide spray deposits are generally lower
than those tolerated for other ornamental crop uses. The commercial
grower's assessment of the product under actual use conditions is there-
fore strongly recommended for uses on florist crops.
§ 93-10 Products for use on bulbs, corms, and tubers.
Tests to control storage rots in ornamental bulbs, corms, and tubers
should be conducted with 5 or more replications of 100 or more bulbs.
Apparently healthly bulbs selected from heavily diseased stock and/or
artificially inoculated healthy bulbs should be used for disease control
tests; bulbs for phytotoxicity tests (see also Subdivision J) should be
selected from stocks that are as disease-free as possible. Disease
measurements are usually obtained during and at the conclusion of the
storage season. lfcw_ever, testing should continue throughout a complete
growing cycle and an additional storage and growing cycle (or forcing
cycle) to determine the residual activity and phytotoxicity of the product.
Effectiveness evaluations are usually based on comparisons of the final
yield (weight, size, and number) of healthy and diseased bulbs within
and between treatments. Separate tests should be conducted for each
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plant species and each pathogen. However, if two or more readily-distin-
guishable diseases are present on the same host simultaneously, their
control may be evaluated in the same test.
§ 93-11 Products for use on trees.
(a) The number of mature trees of a given species to be included in a
test depends upon the nature of the disease and the method of treatment.
When specimens are the same size and vigor and are growing on similar
sites (e.g., similar as to stand density, cultural conditions, soil type,
and available water and drainage) or when transmission of disease is
dependent upon climatic factors, few trees (i.e., less than 100} per
treatment are needed. However, a minimum of 100 single-tree replicates
per treatment would ordinarily be required to produce statistically
sound data when any of the above conditions are variable, or when disease
transmission is dependent upon the presence of insect vectors or root
grafts. Since securing enough trees within a small geographic or climatic
area (e.g., one or more contiguous counties) is often impossible, the
tests must be carefully designed to yield valid test results. When such
testing conditions exist, pairing should be used for making.treatment
comparisons (i.e., each treated tree is paired with an untreated tree
which is in close proximity and is similar in size and degree of disease
development at the time of treatment). In cases where the disease is
widely distributed, separate tests from the major geographic and climatic
areas are necessary, or else labeling claims would be limited to the specific
area(s) of test.
(b) Depending on the nature and extent of the disease, replicated
blocks of 9 to 100 sq ft (0.84 to 9.3 sq m) each should be used for tree
seed and seedling bed treatments. Separate tests should be conducted for
each crop production practice (greenhouse, field, container) where treat-
ment is intended. Cue hundred single-tree replicates, or replicated
blocks of 4 to 20 saplings each, may be necessary to produce statistically
sound data for trees grown in nurseries.
§ 93-12 Products for use on turf.
(a) The size of plots and number of replicates for tests on lawns
and other fine turf areas depends on disease uniformity and severity in
the test area, and the type and size of application equipment. The
control of:,'two. .or .more diseases can be evaluated .in the same test when
,their symptoms.,ara readily distingiiishable.. . . ' ,
fto) Ifo support a general claim./for. use ••on turf, lawns, o-r grasses
.(nonrrgra;zing--ar*a§).>. t;.he ;data -should, demonstrate.ef:£ecti.venerss:.against .'.
-the sams.l-palinogen'oij .a.t'-'leiis-t two .cpoi, .seasoja grasses. ••(«;«-<3» ,. cbehtgr.ass.,
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bluegrass,-fescue) and at least two warm season grasses (e.g., Bermuda-
grass, Zoysia, St. Augustinegrass). Additional tests should be conducted
on other representative grasses likely to be treated to determine whether
or not the product is phytotoxic at dosages equal to and greater (generally
2-4x) than label rates. (See also Subdivision J.) Claims for use on speci-
fic types of grass will be acceptable for those types for which adequate
test data are developed. Products intented to contol diseases of Dichondra
and other non-grass lawn plants should be tested on the intended hosts under
their normal field conditions.
§ 93-13 Treatments for wood and wood products.
(a) Products which are used to control fungal rot and decay of raw
and finished wood, or to control surface molds and fungal staining of
fresh-cut lumber, require registration. These products are commonly
applied to logs, lumber, plywood, millworked products, poles, posts,
pilings, timber, wood chips, sawdust, and other wood-based material.
Note: Water repellents and sealers which bear claims for wood preservation
need not be registered, but the label must clearly indicate that the
product has no direct effect on pests and that effectiveness is attribu-
table only to the water repellent or sealant properties.
(b) Wood treatment products can be applied by several pressure and
nonpressure methods of application which provide a wide range of preser-
vative penetration and retention levels. Each application method is
intended to afford protection for various wood dimensions, periods of
time, extremes of exposure, and occasionally wood species. Farm and
home applications for treating fence posts and other wood include two or
more flowing brush or spray coats, 3-minute dips, hot and cold baths, or
12- to 48-hour soaks. The typical variety of commercial application
methods include:
(1) Applications to timber and lumber using pressure methods of
impregnation (e.g., "full-cell" or "empty-cell" processes); occasionally
double diffusion techniques are used.
(2) Treatment of sawmill lumber, poles, and pilings by passing the
freshly cut pieces through pressure tanks or spray hoods.
(3) Treatment of stored chips and sawdust by spray applications as
they move on a conveyor belt to the storage area.
(4) Treatment of window sash, frames, and other raillwork, either
before or after assembly, using diptank or vacuum process methods.
(5) Treatment of standing poles using diffusion processes, which
may involve grease, liquid, or impregnated bandage surface applications,
liquids injected into poles, or liquids placed in holes bored in poles.
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(c) Effectiveness data should generally be developed under field
conditions, utilizing wood products representative of those intended to
be treated, in several geographic areas representative of the exposure
extremes under which the treated wood will be used. The test program
and individual test procedures must be designed to consider the perti-
nent factors associated with the intended use pattern(s) such as:
wood species; physical dimensions of wood products; soil moisture and
temperature exposure; rain, humidity, solar radiation, and air tempera-
ture exposure; surface roughness of wood, wood quality; effect of water
repellents or sealants on efficacy of formulation; duration of pest
control; soil contact exposure; proportion of heartwood and sapwood;
wood moisture content at time of treatment; heartwood penetrability;
and protection afforded wood joints, bolt or nail holes, and other diffi-
cult to treat surfaces. See § 93-30 Items 9-11 for suggested procedures
for evaluating certain wood treatment uses. Note; Once adequate efficacy
data have been obtained to support one method of application for a given
wood treatment product, data which demonstrate comparable penetration
and retention levels may be used to support other methods of application
to the same wood products.
§ 93-14 Treatments for industrial materials and equipment.
(a) Industrial fungicides are used to prevent fungal deterioration,
disfigurement, or functional impairment of a great variety of products
and equipment such as broom corn, cellulose sponges, coatings, cordage,
drilling muds, dye baths, emulsions, fabrics, fuels, latex, leather,
metalworking fluids, paper, paperboard, plastics, resins, rubber, and
transportation and storage equipment. Test procedures for evaluating
pest control will vary with the material or equipment to be protected,
nature of the pest problem, fungicide formulation, and method of appli-
cation. Such tests must be conducted under actual or simulated use
conditions which reflect the most severe pest problem situation(s) likely
to be encountered. The test program/individual test procedures should be
designed to consider the pertinent factors associated with the intended
use patterns(s), such as: selection of an appropriate range of represen-
tative test substrates for each type of substrate intended to be treated;
presence of appropriate level(s) and type(s) of fungal pest organisms;
use of highly susceptible test substrates (when appropriate, some evalua-
tions should also include low and moderately susceptible substrates);
conditions conducive to maximum pest growth/damage; duration and level
of pest control needed by intended users; conditions most likely to
adversely affect fungicide efficacy (e.g., temperature, humidity, pH,
solar radiation, leaching); the most appropriate means of expressing the
dosage rate; and the most meaningful measurements to be used in expressing
pest numbers, (ox growth) and, when appropriate., pest damage. When labora-
tory, tests (other than those cited, in the § 93-^30) are being, considered
as the sole means of demonstrating product efficacy, it is recommended
that the proposed'methods;be;submitted to the Agency for review .prior to
initiating .such, tests.. Any Yrequests for review of-.test .methods' must be
. acrcompanied.'by.'documentatioh, which demonstrates. that the proposed .method .
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is reliable (e.g., data from studies designed to compare results from the
laboratory test with in-use tests). When new or unusual pest problems
are involved, background information on the nature and extent of the pest
problem should also be submitted.. See § 93-30 Items 7, 8, 12, 13, and 14
for suggested methods to evaluate some of these uses.
(b) Information should also be presented which demonstrates whether
or not undesirable changes in physical characteristics (e.g., unwanted
discoloration, tenderizing of fabrics) and interference with processes
or reactions will occur when fungicides are used as directed, under normal
use conditions. If the effectiveness tests are not adequate to make these
evaluations, then additional tests designed for this purpose must be con-
ducted.
§ 93-15 Products for control of mold and mildew on surfaces.
(a) The efficacy of products intended to prevent the growth of mold
and mildew is greatly affected by the type of surface to which the products
are applied. Test methods for representative surfaces are included in
§ 93-30 Items 1-4. If the surfaces to be treated, or the methods of appli-
cation, or the organisms to be controlled by the product, are not the same
as those indicated in the method, the method should be modified to reflect
these differences. Modifications should also be made so that the method
will more clearly reflect actual in-use conditions (including any special-
ized use situations). For example, tests for products with fungistatic
claims intended for use in shower stalls should include test data to
indicate whether leaching will alter the efficacy of the product. Any
modifications of test methods must be reported along with justification
for the change submitted.
(b) The efficacy of products that claim to kill mildew is depen-
dent upon concentration and length of time the active ingredient is
in contact with the organism. Methods for testing such products are
included in § 93-30 Items 5 and 6. The test method should be modified
for surfaces other than hard, nonporous surfaces. .
(c) Mildewcides and mildewstats should also be tested to determine
whether or not bleaching, staining, spotting or other undesirable
effects occur on the surfaces, articles, and materials to be protected.
§ 93-16 Products for control of organisms producing mycotoxins (Reserved)
(a) See also § 93-7 Products for use as grain preservatives.
(b) The registrant is advised to consult with the Agency on
their test protocol before initiating product performance testing.
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§ 93-30 Acceptable methods.
I. Surfaces (Mold and Mildew)
Item 1 - Fabric Mildew Fungistatic Test Method
1. Scope.
1.1 Products intended for use to control, prevent, or inhibit the
growth of fungi which cause mildew on various articles or surfaces
should be tested to demonstrate fungistatic effectiveness. . This method
is designed to determine effectiveness of products intended to control
mildew and nonpathogenic fungal growth on indoor articles or surfaces
composed of fabric. It also indicates the duration of protection
afforded, thereby providing a basis for recommending when applications
are to be repeated. This method is not applicable for evaluating
laundry additive products unless the appropriate modifications are
made to simulate actual use conditions.
2. Summary of Method.
2.1 Treated, dried strips.of cotton fabric are sprayed with a mixed
spore suspension of mildew causing organisms and incubated at high
humidity. Mildew growth on treated and untreated cotton strips is
rated at weekly intervals for up to four weeks.
3. Apparatus. . • .
3.1 Glassware: Flasks with cotton plugs suitable for preparation
of agar, diluent, and conidial suspensions. French square jars (500
ml) or equivalent screw cap containers. Caps modified by center
drilling and inserting an appropriate size stainless steel or brass
bolt to which a hook (formed from a 6-7 cm length of #22 nickel-chromium
wire or other non-corrosive wire) is attached. Hook position adjusted
so that the bottom ends of attached cotton fabric samples when in
jars are about 13 mm above water. (See 7.5.)
3.2 Tissue grinder (Homogenizer) No. 4288B, Arthur H. Thomas Co.,
Phila., Pa.
3.3 Atomizer, DeVilbiss #152 (or equivalent) operated at 69 kPa
(10 psi).
3.4 Counting chamber: Suitable for determining spore concentrations.
. . 4. Test Specimens.
- • / ,* - . .
4...1 Cut 2.5 by 75 mm strips from 136. to 203 g/m2 (4 to 6 az/yd2:)
cotton muslin^l/. .Fabric that will hang without curling excessively .: '
is preferable. NOTE; .-The above test fabric .i.s suitable for testing ..
products ..intended-, for. use on-;.general househo.id :f.abric. .Products:.. •
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intended solely for use on heavier fabrics, must be tested on cotton •
fabric weighing approximately the same as that'of the. lowest -fabric
weight to be treated under actual use conditions.
5. Test Fungi.
5.1 Aspergillus niger (ATCC 6275) 3/ and Penicillium variabile
(NKRL-3765 or ATCC 32333) 4/. Maintain stock cultures of each on neo-
peptone agar (lOg neopeptone, 20g dextrose, 20g agar, and 1 liter
distilled water) or Emmons Agar. Incubate new stock cultures 7 to 10
days at 25°C, then store at 2 to 10°C. '
6. Selection of Treatments.
6.1 Test Fungistat: Dosages of the test fungistat evaluated should
range from ineffective to effective levels so that the minimum effec-
tive dosage of test material can be determined. NOTE: Where both
wipe-on and pump spray (i.e., non-pressurized containers) application
methods are intended for the proposed formulation, only the wipe-on
applications method need to be tested.
6.2 Untreated Control: Ten untreated fabric strips are required
to establish the test validity and ascertain the degree of control
obtained with the test fungistat. '.
6.3 Standard Fungistat: A fungistat registered for use on fabric
may be included in the test as a comparative treatment. The product
selected must be used in accordance with label directions and should
involve a method of application comparable to that of the test fungi-
stat.
7. Procedures. NOTE; Aseptic procedures must be followed through-
out the course of the test.
7.1 Preparation of Conidial Suspensions: Conidial suspensions of
fungal organism are prepared by washing spores from the surface of
7 to 10 day agar cultures with sterile 0.85% saline solution contain-
ing- a surfactant such as 0.05% isboctylphenoxypolyethoxyethanol. 5_/
Spore chains may be broken up by transferring suspension to a heat
sterilized tissue grinder and reciprocating the piston several times.
Hyphal fragments should be removed by filtering the suspension through
a thin layer of sterile cotton or other suitable material. Conidial
suspension may be stored at 2 to 10°C for up to four weeks. Inoculum
for test should be adjusted to contain five million conidia per ml on
the day of use by appropriate dilution of stock suspension with saline
solution. 6/
7.2 Preparation of test specimen: Invalid tests due to failure of
mildew growth on the untreated test fabric can be reduced or eliminated
by applying a nutrient solution to the fabric. To insure luxuriant
growth on fabric strips saturate fabric with a sterilized glycerol
nutrient solution of the following composition: 98.7% distilled
water, 1.0% glycerol, 0.1% potassium phosphate, monobasic, -0.1%
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ammonium nitrate, 0.025% magnesium sulfate (heptahydrate), and 0.05%
yeast extract. TJ Adjust solution to pH 5.3. Sufficient nutrient
solution should be prepared to saturate all the fabric to be used
with a single test. Soak the fabric in nutrient for three minutes
or until saturated. Squeeze excess liquid with the fingers and allow
fabric strips to dry before proceeding with application of test pro-
ducts or standard fungistat.
7.3 Treatment: Treat both sides of ten fabric strips for each
formulation, dosage rate, and method of application being evaluated.
Ebr spray applications, the type of sprayer and distance from nozzle
to cloth surface, as well as the degree of wetness, must be control-
led and specified. If a standard fungistat is used, ten fabric strips
should be treated in accordance with label directions for use. Imme-
diately after treatment, fabric strips should be placed in a verti-
cal or near vertical position to permit excess liquid to drain. In-
clude ten untreated strips as controls. All samples are allowed to
dry before proceeding.
7.4 Inoculation: Place equal volumes of well-agitated conidial
suspensions of A. niger and £. variabile in a DeVilbiss atomizer
(or equivalent), maintain agitation and lightly spray both sides of
each fabric strip.
7.5 Incubation: The fabric samples are then suspended in individ-
ual 500 ml jars containing approximately 90 ml water, and incubated
at approximately 28°C. The caps ar.e tightened, then backed off 1/8
turn to allow for some ventilation.
8. Determination of Results.
8.1 Evaluation: Observations are made and recorded weekly for
four weeks, or until treatments fail and abundant growth occurs
on all treated strips. The presence or absence of observable mold
on the fabric strips is the criterion for determining the effective-
ness of the test product. Where no growth is visually evident at
the end of the test period, examination at approximately 15X magni-
fication must be conducted to confirm the absence or establish the
presence of subvisual growth. The untreated control strips must
have a minimum of 50% of their surface area covered with fungal
growth after 7 days to consider the test valid.
8.2 Interpretation: A product dosage is considered acceptable when
all ten treated replicates are free of fungal growth. The results of
this test must be correlated to intended label claims. The directions
for use must specify retreatment every 7, 14, or 21 days, as necessary
•depending on. the length of time that all of the test strips remain
free. of. mildew- growth.. Labeling, of .products which do not permit growth
after four weeks incubation must .specify a. retreatment schedule, such
as "repeat -as necessary when new growth appears", and. should .indicate
that treatments should be effective for at .least 28 days..
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144
9. Data Reporting. ,• . .. •. •
9.1 Test reports must include all pertinent details of the test con-
ditions and variables. Such information shall include at least the
following types of information:
9.1.1 Complete description of formulations tested (type of formula-
tion, name and percentage of active ingredients, and EPA Registration
Number of any standard f ungistat used).
9.1.2 Dosage rates (specify whether rate is in terms of product
or active ingredients, and whether on a weight and/or volume basis).
9.1.3 Complete description of all appropriate application proce-
dures and materials including details such as the type of sprayer
(pump vs. pressurized spray), spray application distance and duration,
applicator material used for wipe-on applications (damp cloth),
degree of wetness obtained on surfaces (dampen, thoroughly wet, etc.),
and time interval between application and rinsing.
9.1.4 Density (weight/unit area) of test fabric.
9.1.5 Test validity data—number of untreated controls wiith 50% or
more of the surface area covered with fungal growth after 7 days.
9.1.6 Effectiveness data—number of replicates with fungal growth
at each observation date for each treatment being evaluated (including
untreated controls). To demonstrate'differences among treatments, it
may be necessary to use additional criteria, such as the percentage of
surface area covered with fungal growth or the density of fungal growth.
9.1.7 Adverse effects data—describe the nature and extent of any
adverse effects noted on the fabric as a result of treatment.
9.1.8 Modifications—describe the nature of any changes made in the
test method and provide the rationale for each change.
Footnotes
V Cotton muslin and heavier types of cotton fabric are 'available from
Test-fabrics Inc., 200 Blackford Avenue, P.O. Box 53, Middlesex,
N.J. 08846.
2_/ DeVilbiss Atomizer available from DeVilbiss Co., Toledo, Ohio 43682.
3_/ Cultures of A. niger (ATCC 6275) are available from: American
Type Culture Collection, 12301 Parklawn Dr., Rockville, Md.
20852.
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145
4/ Cultures of P. variabile (NRRL 3765 or ATCC 32333) are available
from: ARS Culture Collection Investigations Fermentation Labora-
tory, USDA, 1815 North University St., Peoria, 111. 61604, or
American Type Culture Collection, 12301 Parklawn Dr., Rockville,
Md. 20852.
5_/ Triton X-100, Itohm & Haas Co., Phila., Pa. 19104, or other suitable
wetting agent such as dioctyl sodium sulfosuccinate, or Aerosol
OT solid A-349, Fisher Scientific Co.
j>/ Ptor detailed instructions: Tuite, John, Plant Pathological Methods,
Fungi and Bacteria, Burgess Publishing Co., Minneapolis, Minn.,
1969, pp. 183-184.
7/ The glycerol as a humectant provides a more equal.distribution of .
moisture to all areas of the substrate. The glycerol, yeast
extract and mineral salts also provide the nutrients necessary
for fungal growth.
Item 2 - Hard Surface Mildew Fungistatic Test Method
1. Scope.
1.1 Products which are intended for use to control, prevent, or
inhibit the growth of fungi which cause mildew on various articles
or surfaces should be tested to demonstrate fungistatic effectiveness.
This method is designed to determine effectiveness of products in-
tended to control mildew and non-pathogenic fungal growth on indoor
hard nonporous surfaces, such as painted walls, ceilings, floors,
metal,'glass, tile, porcelain, and plastic. This.method is not
satisfactory to "demonstrate whether or not the product will produce
adverse effects on plastic, painted, or metal articles and surfaces.
2. Summary of Method.
2.1 The test is conducted by treating the glazed side of square
sections of tile. After drying, the glazed side of tiles are sprayed
with an inoculum-nutrient solution, redried, and incubated individu-
ally in petri dishes. Mildew growth on treated and untreated tiles
is visually rated after 7 days of incubation.
3. Apparatus.
3.1 Glassware: Flasks with cotton plugs suitable for preparing
agar, solutions, and spore suspensions. Petri plates as containers
for drying and incubation of tiles.
3.2 Tissue grinder (Homogenizer): No. 4288B, Arthur H. Thomas Co.
3.3 Atomizer: DeVilbi-ss #152 (or, equivalent.) operated .at 69 k-Pa
; psi).; •• • • '•••' '•••'--'_: -•• ..•'..'- . '• . ••. .-..-•••.
,3;4 Counting chamber.:, . suitable for determining spp.re .concentration*
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146
4. Test Specimens..
4.1 Ceramic tiles: 25 mm square tiles with glazed surface, steri-
lized for 2 hours in hot air oven at 180°C.
5. Test Fungus and Materials.
5.1 Aspergillus niger (ATCC 6275). I/ Maintain stock culture on
neopeptone agar (10 neopeptone, 20g dextrose, 20g agar, and 1 liter
distilled water) or Emmons agar. Incubate new stock cultures 7 to 10
days at 258C, then store at 2 to 10°C.
5.22. Water agar: Two percent water agar (20g agar per liter of
distilled water).
5.3 Sterile Czapek solution: Distilled water 1000 ml; sodium ni-
trate 3g; potassium phosphate dibasic Ig; potassium chloride O.Sg;
magnesium sulfate O.Sg; ferrous sulfate O.Olg; sucrose 30g.
5.4 Sterile saline solution: 0.85% sodium chloride and 0.05% iso-
octylphenoxypolyethoxyethanol 2/ in distilled water.
6. Selection of Treatments.
6.1 Test Fungistat: Dosages of the test fungistat should range from
ineffective to effective levels so that the minimum effective dosage
of the test material can be determined. NOTE: If'both wipe-on and pump
spray (i.e., non-pressurized containers) methods of-applications are
intended for the proposed formulation, only the wiperon application
method needs to be tested.
6.2 Untreated Control: Ten untreated glazed tiles are included for
purposes of determining the validity of the test and the degree of
control obtained with the test fungistat.
6.3 Standard Fungistat: A fungistat registered for use on hard,
non-porous surfaces may be included in the test as a comparative
treatment. The product selected must be used in accordance with
label directions and should involve a method of application comparable
to that of the test fungistat.
7. Procedures. NOTE; Aseptic procedure must be followed through-
out the test.
7.1 Preparation of Conidial Suspension: Wash spores from the sur-
face of 7 to 10 day cultures of test fungus with sterile saline
solution. Pour resulting spore suspension into a sterilized tissue
grinder, reciprocate piston several times to break up spore .chains.
Filter suspension through a thin layer of sterile cotton or other
suitable material to remove spore chains and hyphal elements. Con-
idial suspensions may be stored at 2 to 10° for up to four weeks.
Standardize conidial suspensions to contain five million conida per
ml. Determine spore concentrations using a counting chamber 3j/' and•< .
image:
147
adjust to proper concentration with saline solution. Place 1 ml of
the standard conidial suspension in 20 ml sterile Czapek's liquid
medium and agitate.
7.2 Treatment: Treat the glazed side of ten tiles for each for-
mulation, dosage, and method of application being evaluated. For
spray applications the type of sprayer and distance from nozzle to
tile surface, as well as the degree of surface wetness, must be con-
trolled and specified. If a standard fungistat is used, ten tiles
should be treated in accordance with the label directions for use.
Immediately after treatment, tiles should be placed in a vertical
or near vertical position to permit excess liquid to drain. Place
five tiles in each of two sterile petri dishes and allow to dry at
37°C with lids ajar. Include ten untreated tiles, as controls.
7.3 Inoculation: Place well agitated &• niger conidial-nutrient
suspension in a #152 DeVilbiss atomizer (or equivalent), maintain
agitation and lightly spray the glazed surface of each tile. Tiles
contained in petri dishes (with lids ajar) are then returned to 37°C
oven and dried.
7.4 Incubation: Each tile, sprayed side up, is then placed in an
individual petri dish containing hardened sterile water agar. Plates
are incubated at 25°C and a minimum of 95% relative humidity (a wet-
type incubator has been found suitable for this purpose).
8. Determination of Results.
8.1 Evaluation: Observations are made and recorded after 7 days
of incubation. The presence or absence of observable fungal growth
on tiles is the criterion for determining the effectiveness of the
test product. When no visual growth is evident at the end of the
test period, examination at approximately 15X magnification must be
conducted to determine the presence or absence of growth. Untreated
control tiles must be at least 50% covered with fungal growth after
7 days in order to consider the test valid.
8.2 Interpretation: A product dosage is considered acceptable when
all ten treated replicates are free of fungal growth. The results of
this test must be correlated with the intended label claims. Products
which do. not permit growth after 7 days must specify a retreatment sched-
ule, such as, "repeat as necessary when new growth appears11. Product
labeling must state some of the appropriate hard, nonporous surfaces
(such as those listed in scope of method) on which the product is
intended to be used.
. 9. Data Reporting.
9.1 Test.;reports. must include .ail pertinent details of the'test .,
conditions and variables. Such info-rmation shall include at least
the following types of information: . '-'-•'.
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148
9.1.1; Complete description of formulatio.n.(s) tested (type of for-
mulation, name and percentage of active ingredient(s), and EPA'Regis-
tration Number of any standard fungistat used).
9.1.2 .Dosage rates (specify whether rate is in terms of product
or active ingredient, and whether on a weight and/or volume basis).
9.1.3 Complete description of all appropriate application proce-
dures and materials including details such as the type of sprayer
(pump vs. pressurized sprayer), spray application distance and dura-
tion, applicator material used for wipe-on applications (damp cloth),
degree of wetness to be obtained on surfaces (dampen, thoroughly wet,
etc.), and time interval between application and rinsing.
9.1.4 Test validity data—the number of untreated controls with
50% or more of the surface area covered with fungal growth after 7
days.
9.1.5 Effectiveness data—the number of replicates with fungal
growth on observation date for each treatment being evaluated
(including untreated controls). 1b demonstrate differences among
treatments it may be necessary to use additional criteria, such as
the percentage of surface area covered with fungal growth or the
density of fungal growth.
9.1.6 Adverse effects data—describe the nature and extent of any
adverse effects noted on the glazed tiles as a'result of treatment.
9.1.7 Modifications—describe the nature of any changes made in
the test method and provide the rationale for each change.
Footnotes
_!/ Cultures of A. niger (ATCC 6275) are available from: American
Type Culture Collection, 12301 Parklawn Dr., Hockville,
Md. 20852.
2_/ Triton X-100, Jtohm & Haas Co., Phila., Pa. 19104, or other suit-
able wetting agent.
3/ For detailed instructions: Tuite, John.j 1969. Plant Patholog-
ical Methods, Fungi and Bacteria. Burgess Publishing Co.,
Minneapolis, Minn. Pp. 183-184.
Item 3 - Leather Mildew Fungistatic -,Test Method
1. Scope.
• 1.1 Products intended for use to control, prevent or inhibit the
growth of fungi which cause mildew on'Various articles or surfaces
should be tested:to demonstrate fungistatic effectiveness. This method
is designed to determine effectiveness of..products intended, to control
mildew and non-pathogenic fungal growth.on indoor articles or surfaces
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149
of leather, such as book covers, luggage, shoes, and sporting goods.
It also indicates duration of protection afforded, thereby providing
a basis for recommending when to repeat applications.
2. Summary of Method.
2.1 This method simulates use conditions by utilizing sections of
vegetable tanned cowhide which, after treatment with the product, are
artificially inoculated with a spore suspension of mildew-causing or-
ganisms and incubated at high humidity. Mildew growth on treated and
untreated leather surfaces is rated during the four.week test.
3. Apparatus.
3.1 Glassware: Flasks with cotton plugs suitable for preparation of
agar, diluent, and conidial suspensions. French square jars (500 ml)
or equivalent screw cap containers. Screw caps adapted to allow
suspension of leather sections 30 to 40 mm below the jar caps. Caps
modified by center drilling cap and inserting appropriate size stain-
less steel or brass bolt to which a hook (formed from a 6-7 cm length
of #22 nickel-chromium wire or other non-corrosive wire) is attached.
3.2 Tissue grinder (homogenizer): No. 4288B, Arthur H. Thomas, Co.
3.3 Atomizer: DeVilbiss #152 (or equivalent) operated at 69 kPa
(10 psi).
3.4 'Counting chamber: Suitable for determining spore concentration.
4. Test Specimens.
4.1 Leather: Squares (25mm) of vegetable tanned cowhide (1.0 to
1.5 mm thick) j./ with a hole punched in the corner of each square to
permit it to be suspended from hook on modified French jar lid.
5. Test Fungi.
5.1 Aspergillus niger (ATCC 6275) 2_/ and Penicillium variabile (NRKL-
3765 or ATCC 32333). 3/ Maintain stock cultures of each organism on
neopeptone agar (lOg neopeptone, 20g dextrose, 20g agar, and 1 liter
distilled water) or Bmmons agar. Incubate new stock cultures 7 to 10
days at 25°C, then store at 2 to 10°C.
•. 6. Selection of Treatments:
6.1 Test Fungistat: Dosages of the test fungistat evaluated should
range from ineffective to effective levels so that the minimum effective.
• dosage, of test material can. be determined. . NOTE.; Where both wipe-on
and pump spray (i.e., non-pressurized containers) application methods
are intended for the proposed formulation, .only, the wipe-on .application
method .need to be tested. •••
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6.2 Untreated Control: Ten untreated leather squares are required
to establish the test validity and to ascertain the degree of control
obtained with the test fungistat.
6.3 Standard Fungistat: A fungistat registered for use on leather
may be included in the test as a comparative treatment. The product
selected must be used in accordance with label directions and should
involve a method of application comparable to that of the test fun-
gistat.
7. Procedures.
NOTE; Aseptic procedures must be followed throughout the course
of the test.
7.1 Preparation of conidial suspensions: Conidial suspensions
of each fungal organism are prepared by washing spores from the
surface of 7- to 10-day neopeptone agar cultures with sterile 0.85%
saline solution containing a surfactant such as 0.05% isooctylphenoxy-
polyethoxyethanol. 4/ Spore chains should be broken up by transfer-
ring the suspension to the heat-sterilized tissue grinder and recip-
rocating the piston several times. Hyphal fragments should be re-
moved by filtering the suspension through a thin layer of sterile
cotton or other suitable material. Conidial suspension may be stored
at 2 to 10°C for up to four weeks. Standardize test conidial suspen-
sions to contain five million conidia per ml (determine spore con-
centration with a counting chamber 5/) by adding sterile saline solu-
tion. ' .'.-'.; '.;.-.'•
7.2 Treatment of Leather Squares: Treat both sides of ten leather
test squares for each formulation, dosage and method of application
being evaluated. Wipe on/or spray application must simulate intended
method of use. For spray applications, the type of sprayer and dis-
tance from nozzle to leather surface, as well as the degree of sur-
face wetness, must be controlled and specified. Dip application of
product to leather test squares is not an acceptable substitute for
wipe-on or spray methods of application. If a standard fungistat is
used, the leather squares should be treated in accordance with label
directions for use. Immediately after treatment leather squares
should be placed in a vertical or near vertical position to permit
excess liquid to drain. All treated leather should be allowed to
dry before the mildew spore suspension is applied.
7.3 Inoculation: Place equal volumes of well agitated A. niger
and P_. variabile conidial suspensions in a #152 DeVilbiss atomizer
(or equivalent), maintain agitation and lightly spray both sides
of each leather square with the mixture.
7.4 Incubation: Suspend leather squares in individual modified
500 ml French square jars containing approximately 90 ml distilled
water and incubate at approximately 28°C. Tighten the caps, then
back off 1/8 turn -to allow for some ventilation.
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151
8. Determination of Results.
8.1 Evaluation: Observations are recorded weekly for four weeks
or until treatments fail and abundant growth occurs on all treated
squares. Hie presence or absence of observable mold on leather
squares is the criterion for determining the effectiveness of the
test product. Where no visual growth is evident at the end of the
test period, examination at approximately 15X magnification is re-
quired to confirm the absence or establish the presence of subvisual
fungal growth. The untreated control squares must have a minimum
of 50% of their surface area covered with fungal growth after 7
days for the test to be valid.
8.2 Interpretation: A product dosage is considered acceptable when
all 10 treated replicates are free of fungal growth. The results of
this test must be correlated to the intended label claims. The direc-
tions for use must be correlated to the intended label claims. The
directions for use must specify retreatment every 7, 14, and 21 days,
depending on the length of time all treated test squares remained free
of mildew growth. label directions for products which remain effec-
tive for the duration of the four week test must specify a retreatment
schedule, such as "repeat as necessary when new growth appears" and
should indicate that treatments be effective for at least 28 days.
9. Data Reporting.
9.1 Test reports must include all pertinent details of the test
conditions and variables. Such information shall include at least
the following types of information:
9.1.1 Complete description of formulation(s) tested (type of for-
mulation, name and percentage of active ingredient(s) ,• and EPA Regis-
tration Number of any standard fungistat used).
9.1.2 Dosage rates (specify whether rate is in terms of product or
active ingredient, and whether on a weight'and/or volume basis).
9.1.3 Complete description of all appropriate application pro-
cedures and materials including details such as the type of sprayer
(pump vs. pressurized spray), spray application distance and duration,
applicator material used for wipe-on applications (damp cloth), degree
of wetness to be obtained on surfaces (dampen, thoroughly wet, etc.),
and time interval between application and rinsing.
9.1.4 Test validity—the number of replicates with 50% or more of
the surface area covered with fungal growth after 7 days.
9.1.5 . .Effectiveness—the number of replicates..with fungal, growth
at.-each .observation date for each treatment .being evaluated (including.
untreated controls). To demonstrate the differences among-, treatments
it may be .necessary to use, .additional criteria, Such as the percen-
tage of surface area, covered with fungal.growth or the density of
fungal.-growth. •
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152
9.1.6 Adverse effects data—describe the nature and extent of any
adverse effects noted on leather as a result of treatment.
9.1.7 Modification—describe the nature of any changes made in the
test method and provide the rationale for each change.
Footnotes
I/ Vegetable tanned leather is available from Kberle Tanning Company,
360 Church Street, Westfield, Pennsylvania 16950.
2/ Cultures of A. niger (ATCC 6275) may be obtained from American
Type Culture Collection, 12301 Parklawn Dr., Rockville, Md.
20852.
V Cultures of P. variabile (NRRL 3765)(ATCC 32333) are available
from: ARS Culture Collection Investigations Fermentation Labora-
tory, USDA, 1815 North University St., Peoria, 111. 61604,
or American Type Culture Collection, 12301 Parklawn Dr., Rock-
ville, Md. 20852.
4/ Triton-X-100, Bohm & Haas Co., Phila., Pa. 19104, or other suitable
wetting agent such as dioctyl sodium sulfosuccinate, as Aerosol
OT solid A-349, Fisher Scientific Co.
5/ For detailed instructions see: Tuite, John, Plant Pathological
Methods, Fungi and Bacteria, Burgess Publishing Co., Minneapolis,
Minn., 1969, pp. 183-184.
Item 4 - Wood Block Mildew Fungistatic Test Method
. 1. Scope. - ,
1.1 Products intended for use to control, prevent or inhibit the
growth of fungi which cause mildew on various articles or on sur-
faces should be tested to demonstrate fungistatic effectiveness. This
method is designed to determine effectiveness of products intended
to control mildew and non-pathogenic, fungal growth on indoor articles
or surfaces composed of wood which have not been painted or coated.
It also indicates duration of protection afforded, thereby providing
a basis for recommending when to repeat applications. This test is
not satisfactory to support claims to control rot and decay of wood,
or mold and stain on fresh-cut lumber.
2. Summary of Method.
2.1 This test utilizes wooden blocks which are treated with the
test products and then sprayed with a mixed spore suspension of
test fungi. During incubation the mildew growth on treated and
untreated wood blocks are visually rated at weekly intervals for
four weeks.
3. Apparatus.
3.1 Glassware: Flasks with cotton plugs suitable for preparing
agar, solutions, and spore suspensions. French square jars (500 ml)
ojr. equivalent screw cap containers, with modified caps .('10 jars per
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153
treatment evaluated). Jar caps modified by center drilling the caps
and inserting stainless steel or brass bolts to which hooks (formed
from a 63 mm piece of #22 nickel chromium wire) are attached for
suspending the test samples. Length of the wire from cap to the
bottom of the hook approximately 5 cm.
3.2 Tissue grinder (Hbmogenizer) No. 4288B Arthur H. Thomas Co.
3.3 Atomizer, DeVilbiss #152 (or equivalent operated at 69 kPa
(10 psi).
3.4 Counting chamber: Suitable for determining spore concentra-
tion .
4. Test Specimens.
4.1 Pine sapwood blocks 25 by 25 x 15 mm. The wood should be free
of excessive resins, knots, growth rings, and other defects, and
contain no heartwood. Block should be smooth-surfaced on all six
sides, and kiln dried after sawing to avoid infestation by wsod rot-
ting fungi. Each block is corner drilled to allow hanging as des-
cribed in 3.1; other means of hanging can be used.
5. Fungi.
5.1 Aspergillus niger (ATCC 6275) I/ and Penicillium variabile
(NRRL-3765, or ATCC 32333) 2/. Maintain separate stock cultures on
neopeptone agar (lOg neopeptone, 2.0g dextrose, 20g agar, and 1 liter
distilled waiter) or Emmons agar. incubate new stock, cultures seven
to ten days at 25°C, then store at 2 to 10°C.
6. Selection of Treatments.
6.1 Test Fungistat: A fungistat registered for use on unpainted
wood surfaces may be included in the test as a comparative treat-
ment. The product selected must be used in accordance with label
directions and should involve a method of application comparable
to that of the test fungistat.
7. Procedures. NOTE; Aseptic procedures must be followed through-
out the test.
7.1 Preparation of Conidial Suspensions: Separate conidial suspen-
sions of each fungal organism are prepared. Prepare a'Sterile saline
solution which contains 0.85% sodium chloride and 0.5% isooctylphenoxy-
polyethoxyethanol' 3/. in distilled water. Wash spores from the surface
of 7- to 10-day neopeptone agar cultures with the sterile saline solu-
tion. Pour the spore suspension into, a-heat, sterilized tissue grinder,
and reciprocate' the piston several times to break up spore chains. .
Filter suspension through a•thin layer of sterile cotton or other
suitable,,mater,ial to remove spo.re chains and hypbal elements. - Gonidial
.suspensions may be stored at 2 to 10°C .for up .to four weeks:., Standard-
ize, test, conidial suspensions; to con-ta'in.-five;!niii"iipfri':-eoni<Ji:a:;,per ml
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154
(determine spore concentration with a counting chamber) 4/ by adding
sterile saline solution. ••••••.
7.2 Treatment: Treat all sides of ten wood blocks for each formu-
lation, dosage rate, and method of application being evaluated. For
spray applications, all sides should be sprayed at a specified dis-
tance to obtain the desired degree of wetness. If a standard fungi-
stat is used, wood blocks should be treated in accordance with the
label directions for use. Include ten untreated blocks as controls.
All samples are allowed to dry before proceeding.
7.3 Inoculation: Place equal volumes of well agitated A. niger
and P. variabile conidial suspensions in a #152 DeVilbiss atomizer
(or equivalent), maintain agitation, and lightly spray all surfaces
of the test blocks.
7.4 Incubation: The blocks are then suspended in individual jars
above approximately 90 ml of water, and incubated at approximately
28°C. The caps are tightened, then backed off 1/8 turn to allow
for some ventilation.
8.1 Evaluation: Observations are recorded weekly for four weeks
or until abundant growth occurs on treated blocks. The presence
or absence of observable mold on wood blocks is the criterion for
determining the effectiveness of the test product. Where no visual
growth is evident at the end of the test period, examination at
approximately 15x magnification must be conducted to determine the
presence or absence of subvisual growth. The untreated control
blocks must have a minimum of 50% of their .surface area covered with -
fungal growth after 7 days for the test to be considered valid.
8.2 Interpretation: A product dosage is considered acceptable when
all ten treated replicates are free of fungal growth. The results of
this test must be correlated to the intended label claims. The direc-
tions for use must specify retreatment every 7, 14, or 21 days, as
necessary, depending on the length of time that all of the test blocks
remain free of mildew growth. Products which do not permit growth
after four weeks incubation must specify a retreatment schedule, such
as "repeat as necessary when new growth appears" and should indicate
that treatments should be effective for at least 28 days.
9. Data Reporting.
9.1 Test reports must include all pertinent details of the test
conditions and variables. Such information shall include; at least
the following types of information: .
9.1.1 Complete description of.formulation(s) tested (type of
formulation, name and percentage of active ingredient(s), and EPA
Registration Number of any standard fungistat used).
9.1.2 Dosage rates (specify whether in terms of product or active
ingredient and whether on a weight and/or volume basis).
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155
9.1.3 Complete description of all appropriate application procedures
and materials including details such as the type of sprayer (pump vs.
pressurized spray), spray application distance and duration, applicator
materials used for wipe-on applications (damp cloth), degree of wetness
to be obtained on surfaces (dampen, thoroughly wet, etc.), and time
interval between application and rinsing.
9.1.4 Test validity data—the number of untreated controls with 50%
or more of the surface area covered with fungal growth after 7 days.
9.1.5 Effectiveness data—the number of replicates with fungal growth
at each observation date for each treatment being evaluated (including
untreated controls). Differences among treatments may be demonstrated
by use of additional criteria, such as the percentage of surface area
covered with fungal growth or the density of fungal growth.
9.1.6 Adverse effects data—describe the nature and extent of any
adverse effects noted on wood blocks as a result of treatment.
9.1.7 Modifications—describe the nature of any changes made in the
test method and provide the rationale for each change.
Footnotes
J./ Cultures of A. niger (ATCC 6275) are available from: American Type
Culture Collection, 12301 Parklawn Dr., Rockville, Md. 20852.
I 2/ Cultures of P. variabile (NRRL-3765, ATCC 32333) are available from:
I ARS Culture Collection Investigations Fermentation Laboratory,
r USDA, Northern Utilization Research and Development Division,
;, 1815 North University Street, Peoria, 111.; or American Type Cul-
ture Collection, 12301 Parklawn Dr., Itockville, Md. 20852.
;.' V Triton X-100, Rohm & Haas Co. Phila., Pa. 19104, or other suitable
: wetting agent such as dioctyl sodium sulfosuccinate (Aerosol
! OT solid A-349, Fisher Scientific Co.).
i, 4_/ For detailed instructions: Tuite, John, Plant Pathological Methods,
| Fungi and Bacteria, Burgess Publishing Co., Minneapolis, Minn.,
f 1969, pp. 183-184.
t; . • '
j. Item 5 - Glass Slide Mildew Fungicidal Test Method
[
t' 1. Scope.
[.' 1.1 Products, intended for use to kill fungi which cause mold and
^ mildew growth should be tested to demonstrate "cidal" effectiveness.
ji This method is designed to determine the effectiveness of products
(• intended to kill mildew organisms on hard, nonporous surfaces.
j. Residual effectiveness if intended or claimed must be demonstrated
r using other tests,, (i.e., Fabr.ic Mildew Fungistatic Test Method., Item
t 1.;. Hard Surface Mildew Fungistatic Test Method., item 2; .Leather Mildew
fj Fungistatic Test Method, Item 3; Hood Block Mildew Fungistatic Test
li -... Method, Item 4) depending on the natuf.e of the surfaces.or articles
| 'on which the product is intended-; to be used. .^.Thi.s/method is.-especially
['•; applicable for testing products applied as sprays-or formulated as
t •. pressurized sprays. -.-.-.
image:
156
2. Summary of Method. . :
2.1 This method is conducted using specially prepared sterile
square glass slides which are seeded with a standardized spore sus-
pension of the test organism. The slides are sprayed individually
with the -test fungicide and then placed into large test tubes contain-
ing culture medium plus a fungicide neutralizer for incubation and
subsequent evaluation for the presence or absence of fungal growth.
3. Apparatus.
3.1 Glassware: Lipless 32 x 200 mm test tubes with plugs. Petri
dishes 15 x 100 mm. Flasks with plugs. All glassware sterilized
two hours in hot air oven at 180°C.
3.2 Transfer loop (or equivalent device) which will deliver approx-
imately 0.01 ml of spore suspension.
3.3 Packs and baskets: Suitable for holding test tubes.
3.4 Microscope slides: Noncorrosive (25 x 25 mm) slides placed
individually in a petri dish matted with two. pieces of 90 mm dia-
meter filter paper (Whatman Nb. 1 or equivalent) and placed in a hot
air oven for sterilization.
3.5 Tissue grinder (Homogenizer) No., 4288B Arthur H. Thomas Co.
3.6 Counting chamber suitable for determining spore concentration.
4. Reagents and Materials.
4.1 Distilled water or water of equal purity.
4.2 Neopeptone—neutralizer broth: prepare by dissolving 0.7g
lecithin and 5.0g sorbitan monooleate I/ in 400 ml of hot water and
boiling until dissolved. Add l.Og sodium thiosulfate, lOg neopeptone
and 20g dextrose, and sufficient water to make one liter of culture
medium. The pH of the medium will be approximately 7.2 after auto-
claving.
4.4 Saline solution as 0.85% sodium chloride and 0.05% isooctyl-
phenoxypolyethoxyethanol 2/ in distilled water.
5. Test Fungus.
5.1 Aspergillus niger (ATCC 6275) 3/. Maintain stock cultures on
neopeptone agar (lOg neopeptone, 2Og dextrose, 20g agar and 1 liter
distilled water). Incubate stock culture for 7 to 10 days at 25°C,
then store at 2 to 10°C.
image:
157
6. Selection of Treatments.
6.1 Test Fungicide: A sufficient number of dosages of the test
fungicide should be evaluated in order to determine the minimum effec-
tive dosage.
6.2 Untreated Cbntrol: Two untreated slides are included for deter-
mining the validity of the test.
6.3 Standard Fungicide: A fungicide registered for use on similar
surfaces may be included in the test as a comparative treatment. The
product selected must be used in accordance with label directions and
should involve a method of application comparable to that of the test
fungicide.
7. Procedures. NOTE; Aseptic procedures must be followed through-
out the course of the test.
7.1 Preparation of Conidial Suspension: Conidial suspensions of
the fungus are prepared by washing the spores from the surface of 7-
to 10-day old neopeptone agar cultures with sterile saline solution.
The spore suspension is poured into a heat sterilized tissue grinder
and the piston reciprocated several times to break up the spore
chains. Filter suspension through a thin layer of sterile cotton
or other suitable material to remove spore chains and hyphal elements.
Qonidial suspensions may be stored at 2 to 10°C for up to four weeks.
Standardize test conidial suspensions to contain five million conidia
per ml by adding sterile diluent. Determine spore concentration with
a counting chamber 4.
7.2 Inoculation: Agitate spore suspension to disperse spores evenly
throughout, transfer approximately 0.01 ml of the spore suspension by
means of a transfer loop onto each 25 mm square sterile test slide
(contained in a petri dish) and spread evenly over the upper surface..
Cover the dish immediately and repeat the procedure until twelve slides
have been prepared (use two slides as controls). Allow all slides to
dry for 40 minutes at 37°C or let stand several hours at room tempera-
ture.
7.3 Treatment: Spray ten inoculated slides with the test product
concentration at a specified distance to obtain the desired degree of
wetness. Immediately after treatment, drain excess liquid from slides
and maintain in a petri dish for an exposure of one minute. NOTE;
Products which are capable of keeping surfaces totally wet for longer
than one minute under actual use conditions, should.be tested under a
longer exposure time. To determine the duration of such an increased
exposure time the following test procedure should .be employed. Tests
must utilize a hard, nonporous surface (e.g.,. glass,, mental, or porcelain.)
of at least one square foot .in area which are treated in accordance
with the proposed label directions for use. The test surface(s) must
be positioned vertically, -unless the product is intended solely for
'us^-on hori-zontal surfaces..(.e.g..,, .£I-ot>rs;)-..in'- which-.ca-se horizontal.'.-••'•..-,..
'•positions, mast'.'be used. The test must be conducted at a temperature ;
image:
158
of 20 to 25°C and a relative humidity of..5,0% or less. The length of
time (in seconds) from application to when any portion of the treated
surface, begins to appear dry should be recorded. The average length
of this drying time for a minimum of three replicates shall serve as
the basis for determining the increased treatment exposure time.
Products which keep surfaces totally wet for longer than 10 minutes
should utilize a 10 minute exposure time.
7.4 Incubation: Transfer each slide by means of flamed forceps
to separate 32 x 200 mm test tubes containing 20 ml of neopeptone
neutralizer broth. Shake culture medium thoroughly. Transfer two
unsprayed slides, as viability controls, to individual culture tubes
in the same manner. Incubate all tubes at 25°C for at least three days.
8. Determination of Results.
8.1 Evaluation: The presence or absence of fungal growth, after
3 days, is the criterion for determining "cidal" effectiveness of
the test product. For a valid test, fungal growth must be present
in both viability control replicates.
8.2 Interpretation: A product dosage is considered acceptable when
all ten treated replicates are free of fungal growth. The results of
this test must be correlated with the intended label claims. Products
which pass this test may be labeled as fungicides or mildewcides
which kill mold and mildew organisms. If the product is not tested
for residual effectiveness, the labeling must state "non-residual"
or "kills on contact." •;'..'. • ,
9. Data Reporting.
9.1 Test reports must include all pertinent details of the test
conditions and variables. Such information shall include:
9.1.1 Complete description of formulation(s) tested (type of
formulation, name and percentage of active ingredient(s), and EPA
Registration Number of any standard fungicide used).
9.1.2 Dosage rates (specify whether in terms of product or active
ingredient, and whether on a weight and/or volume basis).
9.1.3 Complete description of all appropriate application proce-
dures and materials including details such as the type of sprayer
(pump vs. pressurized spray), spray application distance and dura-
tion, degree of wetness to be obtained on surfaces (dampen, thorough-
ly wet, etc.), and time interval between application and rinsing.
9.1.4 Effectiveness data—the number of replicates with fungal
growth for each treatment being evaluated (including untreated
controls).
image:
159
9.1.5 Modifications—describe the nature of any changes made in
the test method and provide the rationale for each change. For
example: the duration of any exposure time which is longer than
1 minute exposure specified in section 7.3, plus the results of
the drying time test used to support the change in exposure time
should be reported.
Footnotes
_!/ Tween-80, ICI United States, Agricultural Division, Wilmington,
~ Del. 19898.
2/ Triton X-100, Rohrn & Haas Co., Phila., Pa. 19104, or other suit-
able agent such as dioctyl sodium sulfosuccinate (Aerosol
OT solid A-349, Fisher Scientific Co.).
V Cultures of A. niger (ATCC 6275) may be obtained from American
Type Culture Collection, 12301 Parklawn Dr., Rockville,
Md. 20852.
4/ For detailed instructions: Tuite, John. 1969. Plant Patholog-
ical Methods, Fungi and Bacteria. Burgess Publishing Co.,
Minneapolis, Minn. Pp. 183-184.
Item 6 - Use-Dilution Mildew Fungicidal Test Method
1. Scope.
1.1 Products intended for use to kill fungi which cause mold and
mildew growth should be tested to demonstrate "cidal" effectiveness.
This method is designed to determine the'effectiveness of products
intended to kill mildew organisms on hard non-porous surfaces.
Residual effectiveness, if intended, must be demonstrated using
other tests (i.e., Fabric Mildew Fungistatic Test Method, Item 1;
Hard Surface Mildew Fungistatic Test Method, Item 2; Leather Mildew
Fungistatic Test Method, Item 3; Wood Block Mildew Fungistatic Test
Method, Item 4) depending on the nature of the surfaces or articles
on which the product is intended to be used. This method is especially
applicable for testing products which are applied by non-spray methods
of application (wiping, mopping, etc.).
2. Summary of Method.
2.1 This method is conducted using polished cylinders (penicillin
cups) as carriers which are seeded with a standardized spore suspen-
sion of the test organism. After carriers are dried, they are immersed
in the use-dilution of the product, and then placed in test tubes con-
taining culture medium plus fungicide neutralizer. After incubation,
evaluation as the presence or 'absence of fungal growth is made.
..3. Apparatus. '•'-.'
3.1 Glassware: Lipiess 25 by 150 mm test, tubes-with cotton: plugs. ,
Petri plates 15 by 100 mm with filter paper. Erlenmeyer flasks (250 and
with ec-ttblv.plugs,. - .-: -'':•;.'=•. .. '.-',< ^-. '•'--.•
image:
160
3.2 Racks and Baskets: Suitable for holding test tubes.
3.3 Water Bath: Suitable for maintaining 20°C for the exposure period
of the product.
3.4 Carriers: Polished Cylinders (Penicillin Cups), 8 mm type 304,
stainless steel. I/
3.5 Tissue grinder (homogenizer): Ifo. 4288B, Arthur H. Thomas Co.
3.6 Counting chamber: Suitable for determining spore concentration.
4. Reagents and Materials.
4.1 Distilled water or water of equal purity, unless otherwise speci-
fied.
4.2 Neopeptone-neutralizer broth: Prepare by dissolving 0.7g leci-
thin and 5.0g sorbitan monooleate 2/ in 400 ml of hot water and boiling
until dissolved. Add l.Og sodium thiosulfate, lOg, neopeptone, and 20g
dextrose. Adjust pH 7.2 +_ 0.2 using tris buffer, bring volume to one
liter with water and mix thoroughly. Place 10 ml of the medium in cotton-
plugged 25 by 150 mm test tubes for sterilization.
4.3 Tris buffer stock solution: 0.1 M. Dissolve 12.Ig tris(hydroxy.-
methyDaminomethane in 500 ml water and bring to one liter. Approxi-
mately 70 ml of this buffer solution is used per liter of culture medium.
The pH of the medium is approximately 7.2 after autoclaving.
4.4 Sterile distilled water.
4.5 Asparagine solution: stock supply of sterile 0.1% asparagine water
solution.
4.6 Sodium hydroxide solution: approximately IN (4%).
4.7 Saline solution: 0.85% sodium chloride and 0.05% isooctylphenoxy-
polyethoxyethanol 3_/ in distilled water.
5. Test Fungus. '
5.1 Aspergillus niger (ATCC 6275) V. Maintain stock cultures on
neopeptone agar (lOg neopeptone, 20g dextrose, 20g agar and 1 liter
distilled water). Incubate stock culture 7 days at 25°C then store
at 2 to 10°C.
6. Selection of Treatments.
6.1 Test Fungicide: A sufficient number of dosages of the test
fungicide should be evaluated in order to determine the minimum
effective dosage. . .
6.2 Untreated Control: Two untreated carriers are included for
determining, the validity of the test. - •
image:
.161
6.3 Standard Fungicide: A fungicide registered for use on similar
surfaces may be included in the test as a comparative'treatment.
The product selected should involve a method of application compara-
ble to that of the test fungicide.
7. Procedures. NOTE; Aseptic procedures must be followed during
the course of the study.
7.1 Preparation of spore suspension: A spore suspension of fungus
is prepared by washing the spores from the surface of 7- to 10-day
neopeptone agar culture with sterile saline solution. Spore chains
may be broken up by transferring the suspension to a: heat-sterilized
tissue grinder and reciprocating the piston several times. Filter
suspension through a thin layer of sterile cotton or other suitable
material to remove spore chains and hyphal elements. Oonidial sus-
pensions may be stored at 2 to 10°C for no longer than four weeks.
Standardize test conidial suspensions to contain five million conidia
per ml by adding sterile diluent. Determine by spore concentration
with a counting chamber. 5/
7.2 Preparation of Carriers: Soak metal carriers overnight in 1N
sodium hydroxide solution, rinse with tap water several times,
then rinse twice with distilled water; place cleaned carriers in
multiples of ten in 25 by 150 mm test tubes with closures (or cotton
plugs) and cover with asparagine solution. Autoclave at 103 kPa
(121°C) for 20. min., cool/ and hold at room temperature.
7.3 Inoculation: When the carriers are cooled, pour off the aspar-
agine and cover with standard spore suspension of A. niger and allow
to stand 10 min. Pour off the spore suspension and remove the 10
carriers to a sterile petri plate matted with filter paper. Allow
the carriers to dry at room temperature or in an incubator at 37°C
with the petri plate lids ajar. (Note: Drying at room temperature
may take up to 12 hours; incubator, 1/2 to 2 hours).
.7.4 Treatment: Place 10 ml of the use-dilution of the product
in each of ten, 25 by 150 mm test tubes with closures. Place 10 ml
of sterile distilled water in each of two, 25 to 150 mm test tubes
with closures to serve as viability control (untreated controls).
All test tubes are placed in suitable rack and immersed in a water
bath to cover the lower 2 in. of the tubes. Allow the tubes to
come to temperature equilibrium at 20°C, then place the metal
carriers in the test tubes at convenient intervals. When the
carriers have been in contact with the product (or water for the
untreated controls) for 1 minute, remove them aseptically, allow
excess liquid to drain from the carriers and place them individually
in 25 to 150 mm test tubes containing 10 ml of neopeptone neutra-
.lizer brdth. Immediately after placing carriers in test tubes,
swirl tubes 3 times.. NOTE; Products -which are capable of keeping •
surfaces totally wet for longer than one minute under actual use co.nr-
ditions should..be-tested under .a longer treatment exposure .time.
To. determine, the. duration of s.uch. an '.increased.-.treatment..:time the .
following procedure should be empioye'dV '-'-Tests, must, utilise'- hard '
image:
162
nonporous surfaces (e.g., glass/ metal, or porcelain), of at least
one square foot in area and which are treated in accordance with
the proposed label directions for use. The test surface(s) must
be positioned vertically, unless the product is intended solely
for use on horizontal surfaces (e.g., floors) in which case horizon-
tal surfaces must be used. The test must be conducted at a tempera-
ture of 20 to 25°C and a relative humidity of 50% or less. The
length of time (in seconds) from application to when any portion
of the treated surface begins to appear dry should be recorded.
The average length of this drying time for a minimum of three
replicates, shall serve as the basis for determining the increased
treatment exposure time. Products which keep surfaces totally
wet for longer than 10 minutes should utilize a 10-minute exposure
time.
7.5 Incubation: Incubate all tubes at 25°C for at least three days.
8. Determination of Results.
8.1 Evaluation: The presence or absence of fungal growth in the
culture medium, after at least 3 days incubation, is the criterion
for determining the "cidal" effectiveness of the test product.
Fungal growth must be present in both viability control replicates if
the test is to be valid.
8.2 Interpretation: A product dosage is considered -acceptable when
all ten treated replicates are free of fungal growth. The results of
this test must be correlated to the intended label' claims. .Products
which pass this test may be labeled as fungicides or mildewcides
which kill mold and mildew organisms. If the product is not tested
for residual effectiveness, the labeling must state "non-residual"
or "kills on contact."
9. Data Reporting.
9.1 Test reports must include all pertinent details of the test
conditions and variables. Such information shall include:
9.1.1 Complete description of formulation(s) tested (type of
formulation, name and percentage of active ingredient(s), and EPA
Registration Number of any standard fungicide used).
9.1.2 Dosage rates (specify whether product or active ingredient,
and whether on a weight and/or volume basis).
9.1.3 Effectiveness data—the number of replicates with fungal
growth for each treatment being evaluated (including untreated con-
trols).
9.1.4 Modifications—describe the nature of any changes made in
the test method and provide the rationale for each change. For
example: the duration of any exposure time used which is longer
than the 1 minute exposure specified in section 7.4, plus the
image:
163
results of the drying time test.used to support the change in ex-
posure time should be reported.
Footnotes
I/ S and L Metal Products Corporation, 58-29 Fifty-seven Drive,
Maspeth, N.Y. 11378. '
2/ Tween-80, ICI United States, Agricultural Div., Wilmington,
Del. 19897.
3/ Triton X-100, Rohm & Haas Co., Phila., Pa. 19104, or other suitable
wetting, agent such as dioctyl sodium sulfosuccinate, as Aerosol
OT solid A-349, Fisher Scientific Co.
4/ Cultures of A. niger (ATCC 6275) are available from: American Type
Culture Collection, 12301 Parklawn Dr., .Hockville, Md. 20852.
V For detailed instructions: Tuite, John, 1969. Plant Pathologi-
cal Methods, Fungi and Bacteria, Burgess Publishing Co., Minneap-
olis, Minn., 1969, pp. 183-184.
II. Fabric, Cordage, and Fibers (Rot, Decay, Mold, and Mildew)
Item 7 - Mildew Resistance of Textile Materials: Soil Burial Method
Federal Test Method Standard. October 5, 1972. Mildew resistance
of textile materials; soil burial method. Method 5762.1
in Textile test methods No. 191, General- Services Admini-
stration , Washington , D.C. 20407. ••'.'
This test method may be used to support product claims for con-
trol of rot and decay as well as mold and mildew of textile materials
which are intended for soil contact uses. Products which pass this
test may also bear these same claims for uses on textile materials
which do not involve soil contact. Rot and decay evaluations should be
based on visible deterioration and breaking strength determinations,
whereas mold and mildew evaluations should be based on visible fungal
growth.
Item 8 - Mildew Resistance of Textile Materials: Mixed Culture Method
Federal Test Method Standard. December 31, 1968. Mildew resistance
of textile materials; mixed culture method. Method 5760 in
Textile test methods No. 191, General Services Administra-
tion, Washington, D.C. 20407.
.-. This .method may be used .to support claims for control of rot and
decay and mold1 and ..mildew of textile materials which are not intended
for soil contact-uses. Hot and decay evaluations should be based on.
visible deterioration and breaking strength determinations, whereas .'
mold and mildew evaluations, should be based "oh visible fungal growth:.
image:
164
III. Wood (Rot and Decay)
Item 9 - Standard Method for Field Tests with Stakes
American Hbod-Preservers' Association. 1969. Standard method for
field tests with stakes. AWPA Method M7-69 in AWPA Manual
of Recommended Practice.
Separate tests should be conducted for each method of applica-
tion (pressure, dip, brush, spray, etc.).
Item 10 - Standard Method for Evaluating Wood Preservatives by Field
Tests with Stakes
American Society for Testing and Materials. 1974. Standard method
of evaluating wood preservatives by field tests with stakes.
ASTM Designation: D1758-74.
This method should be used only for pressure methods of applica-
tion (or equivalent methods which provide the same retention and
penetration of preservative). ,
Item 11 - Standard Method for Field Tests with Posts
American Wood-Preservers' Association. 1950. Standard method for
field tests with posts. ' AWPA Method M8-56 in AWPA Manual '
of Recommended Practice.
Separate tests should be conducted for each method of application
(pressure, dip, brush, spray, etc.)
IV. Paper and Paperboard (Mold and Mildew)
Item 12 - Evaluating Antimycotic Properties of Paper and Paperboard
Vinson, L.J. 1953. The Lever spore cloud method for the evalua-
tion of antimycotic properties of paper and paperboard used
in the soap industry. TAPPI 36(5):234-236.
This method may be used for determining the effectiveness of
fungicides applied at the dry end of paper and paperboard manufacture.
If the paper or paperboard is intended for uses other than the soap
industry, the test may be modified by using other organisms.
image:
165
V. Polymeric Materials (Mold and Mildew)
Item 13 - Resistance of Synthetic Polymeric Materials to Fungi
American Society for Testing and Materials. 1970. Standard Recom-
mended practice for determining resistance of synthetic poly-
meric materials to fungi. ASTM Designation: G21-75. Amer.
Soc. for Testing and Materials, Phila., Pa. 19103
This procedure is limited to materials into which a pesticide
is incorporated during the manufacturing process.
VI. Coatings (Mold and Mildew)
Item 14 - Test for Resistance to Growth of Hold on Surfaces of Interior
Coatings
American Society for Testing and Materials. 1973. Tentative
method of test for resistance to growth of mold on the
surface of interior coatings in an environmental chamber.
ASTM Designation: D3272-73T. Amer. Soc. for Testing and
Materials, Phila., Pa. 19103.
VII. All Crops (Plant Parasitic Nematodes)
Item 15 - Nematicide Test Procedures
The following procedures are to be used for general guidance on plan-
ning, conducting, and evaluating nematicide field tests:
American Phytopathological Society and Society of Nematology.
1978. Methods for evaluating plant fungicides, nemati-
cides, and bactericides - Section IV Nematicide test pro-
cedures . The American Phytopathological Society, 3304
Pilot Knob Road, St. Paul, Minn. 55121. Pp. 99-134.
American Society for Testing and Materials (Phila., Pa. 19103; 1977)
- Standard guidelines for field evaluation of nematode
control agents - Site selection procedures. E 612-77.
- Standard guidelines for evaluation of nematode con-
trol agents - Side effects of nematicide applications
v :to.other organisms. E 613-77.
- Standard' guidelines'for; field-evaluation, of nematode ;
. . control .agents - Test .materials -and' env.iro.nmental: -.'.';' .
conditions. E 628-77. '•'•••'
':- Standard-guiaelo.nes for. field.evaluation of nematode .": . .-••
'••"•• . ...•• control agents - -Determination -of nematode population
.. • '•'' responses to. control agents. E 629-^77.. .-...- ...'..''
image:
166
Series 94: EFFICACY OF TERRESTRIAL HERBICIDES, PLANT REGULATORS,
DESICCANTS, AND DEFOLIANTS
§ 94-1 Overview.
(a) General. Terrestrial herbicides, plant regulators, defoliants,
and desiccants are numerous and extremely diverse in their physical and
chemical properties. Moreover, they are applied by various methods under
actual use situations and may interact variably with the environment once
introduced into the biosphere. These factors directly influence both bio-
logical performance (efficacy, and safety to crops and desirable nontarget
plants) and residual life (dissipation or persistence) of each pesticide
product. Therefore, tests should be conducted to determine the range of
conditions within which the product is useful without producing detrimental
effects on the crop, on other desirable plant species within the area in-
tended for treatment, or on nontarget plant species in adjoining sites.
Testing under actual use conditions is the acceptable means for evaluating
the ultimate- performance of herbicides, plant regulators, desiccants, and
defoliants. Each product should be able to demonstrate its usefulness
from applications made according to use directions and under conditions
recommended in labeling. Label claims include those expressed, such as
weed species controlled, and those implied, such as crop safety and safety
to desirable nontarget plant species. Data should encompass the variables
expressed on the label, those expected under actual use conditions, and
those describing experimental designs, procedures, conditions, and results
from supporting tests. -
(b) Definitions. The terms herbicide, plant regulator, desiccant,
and defoliant are defined in the Act and in § 162.3 of the FIFRA Sec. 3
regulations and are reiterated in §§ 104-2 through -5 of Subdivision H.
(c) Phytotoxicity. Product performance tests with herbicides, plant
regulators, defoliants, and desiccants would often also provide concurrent
information on phytotoxicity to nontarget plants. Information for determining
phytotoxicity are explained in Subdivision J of these guidelines. It is
likely that some of the field studies indicated in appropriate sections of
this subdivision can be easily combined with studies in by Subdivision J,
if necessary.
§ 94-2 General considerations.
(a) Data and test considerations. For complete guidance concerning
product performance, §§ 90-1 and 90-2 of this subdivision should also be
studied. The product performance recommendations in this section are
applicable only to herbicide, plant regulator, desiccant, and defoliant
products formulated and packaged for use as pesticides. Data requested
image:
167
for these pesticides should address test conditions which will vary according
to desired effects, crops (sites), use conditions, cultural practices,
application techniques, and biological activity of the product. Factors
to be evaluated and reported should include:
(1) Dosage rates. Dosage rates should be reported in units of active
ingredient or acid equivalent as appropriate. Bates may be expressed as
units of ingredient per unit of land area to be treated; units of concentra-
tion, such as parts per million, applied to obtain a specified degree of
foliage coverage, such as runoff; or units of ingredient per unit volume,
as in a ripening room. If a product is applied more than once within a
year or growing season, each rate and the interval between applications
should be indicated. If products are applied in a tank mixture or are applied
serially, rates and intervals, as appropriate, should be-reported with iden-
tification and formulation for each product. Where a range of rates is
recommended, labeling should indicate, and data should support, the conditions
for which each rate within the range is recommended.
(2) Test location. The state or geographical region where each test
is conducted should be reported. The name of the nearest town or city of
significant size should be reported.
(3) Statistical analysis. Where preliminary test results such as
efficacy, phytotoxicity, commodity quality, or yield, indicate poor or in-
consistent efficacy, or adverse effects on crops or commodities, statistical
analysis is needed in subsequent tests evaluating the same response(s).
As examples, analysis is needed when a plant regulator label claiming
yield increase produces inconsistent results (increases and decreases com-
pared to untreated control yields), and when a herbicide decreases crop
yields compared to check plots where commercially acceptable levels of
weed control are maintained. An additional situation where statistical
analysis is useful is in evaluation of interactions resulting from compara-
bility studies supporting tank mixes or serial applications.
(4) Product formulation. To support registration, data should demon-
strate usefulness of the exact formulation (product) for which registration
is sought.
(5) Application equipment. The application equipment used in testing ?«'.
products in small plots should be designed to simulate grower equipment. This
can be readily accomplished by using the basic components of commercial
application equipment in the design of the small plot equipment. For ex-
ample, nozzle types, sizes, and arrangements on small plot sprayers can be
identical to those used by growers on commercial ground sprayers; or single-
row commercial granular application equipment mounted on a garden tractor
for small plot trials should produce results comparable to a multiple of
such units on a large tractor. For large-scale trials, commercial applica-
tion equipment should be used. Specific detail as to descriptions of equipment
design, adjustment, and operation should .be provided in test reports involving
aerial applications and applications using conventional farm equipment (such
•as tillage or planting equipment), irrigation systems, mechanical incorpor-
ation, directed,sprays, subsurface placement, .or.band rather than broadcast
•distribution. " ...•••••. ,'.'•.
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(i) Aerial application. Aerial applications should include information on
dosage, spray volume, maximum height from nozzles to target, --type-of aircraft
(fixed wing or helicopter), ground speed, swath width, nozzle type, orifice
size, core, nozzle pressure, nozzle discharge orientation, number and arrange-
ment of nozzles, adjuvants used, carrier used, wind velocity and direction,
air stability, air temperature, and relative humidity. See § 126-1 through
-4 for additional data guidance. Since some of the label-recommended
measures, limitations and/or restrictions proposed to reduce drift can, by
altering droplet size and distribution (coverage), decrease efficacy and/or
increase crop injury in the target area, field efficacy and crop phytotoxicity
data (see Subdivision J) should be developed in support of using the application
equipment, equipment adjustment and operation, procedures and conditions
as allowed or recommended in labeling to reduce drift. Although it is
preferable to evaluate swath displacement and performance on the same or
an adjacent site, it is not mandatory. Regardless of the site for this
performance evaluation, the equipment, equipment operation and adjustment,
and procedures should be documented in detail. Aerial application performance
evaluation should be run in conjunction with the spray drift evaluation
study described in Subdivision R. Application conditions should be documented
and should be very similar to those found in the spray drift evaluation
study.
(ii) Irrigation system application. Data on efficacy and on crop phyto-
toxicity, yield, and quality should be developed as in conventional herbicide
or plant regulator applications. In the case of irrigation system applica-
tions, however, multiple plots within a treated'field should be examined for
efficacy, crop phytotoxicity, yield, and quality of harvestable commodity
as an indication of pesticide distribution. Data from such, plots should be
reported for each individual plot and not simply averaged, together. Further,
information on effects of the product(s) on injection pumps (metering
devices), gaskets, fittings, hoses, and nozzles, with special attention to
wear, clogging, and deterioration, should be provided if requested. It is
very important that, in addition to the standard data requirements for
conventional applications, data should cover soil texture, percent soil
organic matter, relative soil moisture condition (dry, medium, wet) at
application, acre-inches of water applied, and precipitation quantities
within one week after application.
(A) For sprinkler (overhead) irrigation systems, plots should be located
at several nozzle positions along the length of the lateral. Plots should be
placed at both extreme ends of the lateral as well as in areas where sprink-
ler patterns overlap. Comparisons should be made between adjacent plots
treated conventionally with ground application equipment and irrigated with
untreated water, and plots treated through the irrigation system.
(B) For surface irrigation systems (flood and furrow), plots should be
located at the points where the treated water enters the field and at the
lower end of the field. Where furrow irrigation is used, data should indicate
the spatial relationship between crop rows and furrows. If weed control in
furrow irrigation applications is intended only for the furrow itself and
not the bed between the furrows, the data should so indicate.
(iii) Directed sprays. When sprays are directed towards or away from
certain portions of the soil or plants, data should indicate nozzle arrangements,
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nozzle orientations, and the extent of spray contact with soil or plants.
(iv) Subsurface soil application. When herbicides or plant regulators
are applied directly beneath the soil surface, test reports should include
information on equipment. For example, for injection equipment, the follow-
ing must be specified: nozzle spacing, depth of operation, nozzle pressure,
speed of operation, and volume of liquid or gas applied per unit area.
(6) Timing of applications. Where crop plants are or will be involved
in the application of any herbicide, plant regulator, desiccant or defoliant,
their stage of growth or development at application should be described in
test reports and labeling. Test data supporting products intended for use
as herbicides should indicate the stage of weed growth at application. If
weeds have emerged, growth stage should be indicated in terms of the numbers
and/or types of leaves, weed height, or stage of development such as "prior
to flowering". .Reports involving herbicide applications to emerged weeds
should indicate those instances where weed growth rates detrimentally affect
product efficacy.
(7) Meteorological conditions. Where product efficacy and/or adverse
effects are dependent upon or affected by meteorological conditions, the
specific factor(s), such as temperature, wind conditions, precipitation,
or daylength, affecting product activity should be measured and reported.
Edaphic factors, such as relative soil moisture conditions (dry, medium,
wet) and temperature, which are directly affected by meteorological condi-
tions, should also be measured and reported when such factors affect product
efficacy and/or adverse effects.
(8) Spray.dilution. In certain foliage applications, the concentra-
tion or dilution of product in the finished spray and volume of finished
spray applied per leaf surface area or per plant are more closely related
to product activity levels than the quantity of product applied per unit
land area. As an example, a consistent plant response from a given concen-
tration of plant regulator applied to the point of foliage runoff in a
fruit orchard may require from 50 to 500 gallons (189.2 to 1892.5 liters)
of equally diluted finished spray per acre (hectare), depending upon popu-
lations and sizes of the trees involved. In cases such as this, data
should include volume of finished spray applied per unit land area,
plant population (number of trees per unit land area), age of the planting,
concentration of product in the spray solution, and the extent of foliage
coverage (such as volume of finished spray per tree or application to the
point of runoff). Regardless of the presence or absence of foliage at
application, performance data should support the range of spray
volumes and spray dilutions recommended in the label use directions.
(9) Adjuvants. Products with labeling which allows or recommends
the additions of separately packaged adjuvants to the spray tank should be
- supported., with data., indicating, their:-.benefits (if claimed} .and any detri-
mental effects. tsuch as increased, crop phytotoxicity} which may result ;
from tKeir addition to the herbicide,, plant .regulator, desiecant, or
defoliant. The only adjuvants actually permitted for use- with a "pesticide-
will be.thtfse adjuvant brand-.names or def,in6d..'adjuvant classes.Specifically •
•named on the pesticide label. The adjuvant, rate or. -range of rates'-should be..
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indicated on the pesticide label, and should be supported with data on effi-
cacy and any detrimental effects. If a range of adjuvant fates is recommend-
ed, the maximum and minimum rates within that range should be evaluated in
conjunction with the intended pesticide product.
(i) Adjuvants claiming increased pesticide product efficacy. If claims
are made or implied on pesticide product labels for increased efficacy from
the pesticide adjuvant combination over use of the pesticide alone, such
claims should be supported with efficacy comparability data from adjacent plots
where the pesticide is applied with and without the adjuvant and where the
comparison is analyzed statistically.
(ii) Adjuvants without pesticide efficacy claims. If adding an adju-
vant to the pesticide product or spray mixture is mentioned on the label
of the pesticide product, but no claims are made for increased performance,
then crop effects data should be developed to support maintained performance
and no increase in detrimental effects. Efficacy comparability data from
adjacent plots (with and without the adjuvant) should be developed as described
in paragraph (a)(9)(i) of this section.
(iii) Adjuvants essential to pesticide efficacy. If the label of a
pesticide product indicates that an adjuvant must be used in addition to
the pesticide, field efficacy data [as in paragraph (a)(9)(i) of this
section] should be presented which substantiates this essentiality, since
such a statement is an implied efficacy claim.
(10) Untreated control (check) plots. In efficacy evaluations, all
treated plots, plants, and/or commodities should be compared directly to
untreated control plots, plants, and/or commodities. All quality and/or
yield evaluations of plant regulator, desiccant, and/or defoliant treated
plants or commodities should be compared to control plants or commodities
receiving the same pesticides (herbicides, insecticides, fungicides) except
the one being evaluated. Detailed descriptions of plots and plants used as
control treatments for comparisons of efficacy and detrimental side effects
should be included for each test. Data should indicate if the control plot or
plants were untreated (received no herbicide, plant regulator, desiccant or
defoliant), received standard cultural practices (which should be described),
and received other pesticides (which should be listed). Data from untreated
plots or plants should include measurements of the same factors to be evaluated
in determining efficacy and detrimental side effects resulting from the pro-
posed herbicide, plant regulator, desiccant, or defoliant application. For
example, data from untreated weedy control plots for a herbicide should include
weed species present, relative weed populations (density), and weed vigor;
data from tests with a plant regulator such as a fruit ripener should include,
for example, fruit coloration, sugar/acid ratio, yield, quality, and dollar
value per unit for untreated fruit. If, in addition to the untreated control
.plots, plants, and/or commodities, a registered product is also applied (as
a standard) for comparison of efficacy and/or detrimental effects, data
should indicate the standard product's name, active ingredient, dosage rate,
and performance results.
(11) Tank mixture compatibility. Herbicides, plant regulators, desic-
cants, or defoliants along with other pesticides, recommended carriers, and
adjuvants which are to be mixed together in the same tank for simultaneous
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application should be physically compatible to allow uniform and useful appli-
cation. While tests conducted under actual use conditions should support
performance and compatibility of such mixes, laboratory tests should be
conducted to evaluate physical compatibility of maximum rates of all components
of the tank mix in the minimum carrier volume recommended per unit area.
[See also § 94-3(b)(6).] When adjuvants added to increase compatibility
are recommended or allowed, effects of the minimum recommended rates of
these adjuvants on tank mixture compatibility should be evaluated. Data
should indicate the order of addition of components to the test container,
field equivalency rates per unit area for each component, and the field
•equivalency spray volume per unit area. Data should also indicate the extent
of agitation used in the tests. It is important, where components are in
some degree incompatible in static tests, that effects of agitation and
sprayability be evaluated and reported.
(i) Laboratory tests for water carrier. If the carrier is limited to
water, tests should evaluate effects of water temperature, pH, and hardness
of compatibility, sprayability (using conventional screens, nozzles, and
pressures), and resuspendability. Water temperatures tested should include
ranges between 4°C and 32°C, pH levels tested should include 6, 7, 8, and
10, and hardness tested should include both soft water (less than 15 ppm
calcium carbonate) and hard water (greater than 200 ppm calcium carbonate).
Resuspendability should be evaluated after 24 hours without agitation. If
any of these variables are found to markedly reduce compatability, label
instructions should include appropriate limitations.
(ii) Laboratory tests for fluid fertilizer carrier. If fluid ferti-
lizers are used as partial or total carriers, laboratory tests should evaluate
compatibility, sprayability, and resuspendability using major classes of
fluid fertilizers, such as high nitrogen and complete analysis, and suspen-
sions. Laboratory tests are of value in that compatibility problems with
major classes of fluid fertilizers can be detected and addressed through
label cautions. Due to the larger number of possible fertilizer sources
and analyses, laboratory tests may involve only major classes of fluid
fertilizers, and emphasis should be directed, via the label, towards use of
a "jar test" by the applicator prior to large scale mixing. Employing the
exact fertilizer source and analysis intended for application in a jar test
with maximum rates of all other components is the only fully reliable method
for determining compatibility.
(12) Cultural practices. Cultural practices for a given crop vary with
production areas and. between growers within an area. The effects of cultural
practices on the product's usefulness should, therefore, be addressed.
(i) Irrigation. Irrigation and watering practices should be studied as
a variable if the product is to be used in irrigated areas or greenhouses,
respectively. The influence of different irrigation practices should be
studied in the use•area.; Irrigation data.should include a;description of
equipment -and techniques used in water application, the number and -timing
of irrigations, and quantity of water in acre-inches (.hectares-centimeters)
applied at each irrigation. Also, description of the chronological rela-
tionship between irrigation, applications'and.herbicide,. plant regulator,
: desiccant.. and/or defoliant application is needed. Where flood irrigation .
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is utilized (such as in rice production), depth, duration, and any "flushing"
should be described for each test. When irrigation is used to activate a
pesticide product in the absence of precipitation, the maximum application-
to-irrigation interval (producing the desired efficacy level) should be support-
ed by data. When irrigation is allowed or recommended (in labeling) just
after pesticide application, the minimum application-to-irrigation interval
(not detrimentally affecting pesticide performance) should be supported by
data. Since crop safety is often influenced by pesticide placement in the
soil profile, and irrigation may directly affect such placement, label-rec-
ommended or -allowed irrigation practices should be supported by crop safety
data (phytotoxicity and yield). When irrigation practices result in loss
of pesticide-contaminated water (as in runoff or drainage) from the target
area, data should address effects of such water on nontarget plants.
(ii) Mowing. Mowing operations may have a direct effect on efficacy
or detrimental effects of herbicides and plant regulators intended for
use on lawns, turf, golf courses, median strips, pastures, rangeland, and
hay and forage crops. Lush foliage may reduce efficacy by intercepting
products which are soil active, or maturing weed seedheads may constitute
a source of reinfestation even after herbicide application. Plant regulators
and herbicides which are dependent upon folia'ge contact for translocation
may require lush unraowed foliage for effectiveness. Mowing just prior to
or just after herbicide applications may, by mechanically injuring desirable
plants or by decreasing growth rates, increase injury to desirable plants
from the interaction of mechanical and chemical effects. Mowing just prior
to application may be a requirement for plant regulators intended to maintain
the neat appearance of grassy areas by retarding grass growth. In situations
where mowing is routinely a \part .of cultural practices, or may influence
efficacy or detrimental effects, such practices should .be reported in test
results, and, where appropriate, should be accounted for in use directions
and/or cautions on product labeling.
(b) Comparability tests. (1) In certain circumstances, the registrant
may have the option of establishing comparability of performance between pro-
posed and registered uses of formulations rather than conducting a large-scale
testing program in which all variables are again extensively reevaluated per-
tinent to the amended use or altered formulation. These circumstances may
apply when a registrant desires to amend a registered label by altering
directions for use, rates, or application techniques, or by adding tank mixes,
serial applications, adjuvants, or alternate carriers, or if a registrant
desires initial registration of an alternate formulation of a product which
has been previously registered. Use of this approach will generally decrease
the time and expense involved in generating performance data. Prior to
initiating this alternate approach, discussions with Agency personnel
should be held to determine the propriety of this approach and the performance
data encountered in the specific situation. Establishing comparability
should direct comparisons of results from plots (preferably adjacent)
subjected to identical test conditions.
(2) Since tests may be fewer in number and smaller in size than
those involved in full scale testing programs, more emphasis should be placed
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on selection of test conditions such that comparability will be evaluated
under the most rigorous test conditions. As an example of rigorous condi-
tions for a herbicide, plots should be selected which are likely to contain
the more resistant weed species of those claimed on the label.
(c) Data guidance for manufacturing-use products. Data are recommended
for certain of the "manufacturing-use" products, including those pesticides
intended for formulation of other pesticidal products, and those pesticides
intended for repackaging. In support of those pesticides intended for
reformulation, proof of pesticidal usefulness is needed, applicants may either
develop usefulness data [as to the type of pesticide(s) named] based upon
presumptive laboratory data and include a label statement notifying formtilators
of their responsibility for providing supporting data for their formulations,
or have usefulness data for each product formulation and include sample
labeling for each formulation. For those pesticides intended for repackaging,
in-use performance data which support the registered label of an identical
formulation may be referenced. If additional uses of formulations other
than those on the registered label of the identical formulation are sought,
then additional usefulness data should be developed.
(d) Usefulness. Usefulness of herbicide, plant regulator, desiccant,
or defoliant products includes both efficacy and any detrimental side effects
to crops or harvestable commodities, and adverse effects to nontarget plants.
For each of the above types of products, efficacy requirements and detrimental
effect evaluation considerations are discussed in §§ 94-3 in terms of specific
use patterns. '
§ 94-3 Terrestrial herbicides.
(a) General considerations for herbicides. Efficacy data should
support each weed species claimed on the label. See § 104-2(a)(3)(i)
for appropriate weed nomenclature. Only when data support consistent
broad spectrum control of weeds should the use of general claims such as
"existing vegetation", or "annual weeds" be used on labels. Efficacy data
should support control of each species at each minimum rate recommended
for a particular situation. As an example, if recommended rates vary with
soil textures (such as coarse, medium, and fine), then data should support
weed control by the minimum recommended rate for each of the three textural
groups. If rates are further defined for ranges of soil organic matter
content within a particular soil texture, weed control data should support
control of species at the minimum rate recommended for that specific situation.
•If rates depend on conditions defined in labeling use directions or in
limitations, such as soil texture, weed species, weed height, weed density,
• crop height., or other measurable- conditions, then supporting data shpuld
include aiv'assessment of these conditions so. that, use of recommended rates
can be verified.. Tests using excessive rates ...for' a" particular- situation
may be of value, in evaluating crop tolerance-if a-crojx.is involved; however,
use isf test rates" above those recoinmenaed,. by, labeling.for. .a., particular. -
situation are '..of 'SOQ.; value in- supporifcin-g;"' efficacy. elaims:; - -Use -:o-£' rates :.;
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proving to be efficacious, but lower than those recommended for a particular
situation, may be considered in evaluating efficacy. Weed control from
herbicide applications should be expressed as a comparison with an untreated
control. Except for the herbicide application, both treated and untreated
plots should receive similar cultural practices. Methods of measuring the
weed control achieved can take the form of either visual observations by
experienced personnel or actual weed counts per given area (stand) and/or
measurement of weed height, vigor, or weed weight. Control may. be indicated
as either percent control or as a rating. Percent control can be calculated
by the formula: weed growth in untreated control plot less weed growth in
treated plot, divided by weed growth in untreated control plot x 100 =
percent weed control. If a rating other than percent control is desired,
some other rating scale can be used, such as zero representing no weed
control (equal to untreated control) and ten equalling 100% weed control.
Where a rating scale other than percentage is used, an explanation of the
scale which indicates the levels representing no control, partial control,
commercially-acceptable control, and perfect control should be included in
the report.
(1) Suggested performance standard. In order to justify a label claim
for weed control, the degree of control in supporting data need not be
perfect (100%),-but should consistently reach a level which is considered
commercially acceptable. Commercially acceptable levels may vary slightly
from one use pattern and weed problem to the next; however, the acceptable
level should, in all cases, be expected to provide a meaningful benefit to
the ultimate user of the product. For the claim of "control", the product
usually must provide at least the equivalent of 70% reduction in weed
stand and/or vigor when compared to untreated check plots under moderate
to severe weed pressures. A lesser label claim for "suppression" or "partial
control" of certain weed species may be appropriate on labels containing
valid claims for "control" of other weed species. The terms "suppression"
and "partial control" may indicate either consistent weed stand and/or
vigor reductions at levels considered less than commercially acceptable,
or inconsistent levels of weed stand and/or vigor reduction. Even though
considered a lesser claim than "control", claims for "suppression" and
"partial control" should be supported by efficacy data which demonstrates
a benefit. A herbicide label bearing claims for only "suppression" and
"partial control" of weed species, without any accompanying claims for
"control" of other weed species, may be considered inappropriate for
registration depending upon the demonstrated level of benefit to the
user. Combined claims using wording such as "control or suppression" are
not acceptable. If certain conditions or rates allow only "suppression" or
"partial control" of weed species while other conditions or rates allow
"control" of the same species, these conditions or rates should be identified
in labeling and be associated with the appropriate level of response reflected
in the supporting data.
(2) Reporting of data. The degree of control of each weed species in
a test should be reported on a species-by-species basis. Since weed species
vary in their susceptibility to specific herbicides, and since label claims
are made on a species-by-species basis, reporting only broadleaf and grass
control or reporting a single percent control with a list of weeds present
is not considered sufficiently detailed to.siopport specific label claims.
In addition to individual weed control-ratings, information.is needed
which indicates weed pressure by. species in'the untreated check plots.
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Such data.should include both weed populations and vigor. In addition to weed
control data, secondary benefits derived from weed control may in certain
cases be supportive data. Depending upon the objective of weed control in
a particular use pattern, supportive data may include such evaluations as
decreased trash at harvest, increased harvest efficiency, increased water
retention from chemical fallow, or increased carrying capacity from renovated
rangelands. In no case should such supporting data be considered solely
as evidence of efficacy in lieu of data on herbicidal effectiveness.
(b) Specific data considerations. In addition to the general herbicide
considerations, the following specific use patterns should have additional
data as indicated.
(1) Soil incorporated products. For herbicides intended to be soil
incorporated, data and labeling should address application-to-incorporation
intervals, and intervals between incorporations (if repeated). Data should
include identification and/or description of incorporation equipment, depth
of equipment operation, and depth of incorporation of the pesticide.
(2) Systemic herbicides applied to foliage. The quantity of.weed
foliage (expressed in terms of weed height or development) provided for
herbicidal activity should be reported in performance data and indicated in
product labeling. Further, where systemic foliage applications are intended
to control roots or other underground plant parts, performance data should
address (and product labeling should indicate) a minimum application-to-tillage
interval which will allow sufficient time for translocation and herbicidal
effects to take place.
(3) Seedbed preparations for preplant and preemergence jtp crop)
applications, selected methods of seedbed preparation common to the test
crop should be studied and reported. . It is suggested that such methods cover
the extremes from poorly- to well-prepared seedbeds to study effects of clods
and plant debris on product efficacy and possible crop injury. These extremes
should be within the limits normally encountered by growers. If seedbed prep-
aration does not affect weed control or crop injury in preliminary trials, the
reporting of seedbed preparation in subsequent trials may not be critical.
(4) Cultivation practices. Cultivation practices should be those common
for the crop in the test site. Since many crops are being grown under minimum
tillage practices, it may be desirable to determine the effectiveness, both
initial and residual, of the herbicide without cultivation. If cultivation
is required to supplement the product's usefulness, subsequent trials should
be designed to show the effects of the product used without cultivation, the
effects of cultivation only, and their combined effects. Some cultivation
practices may reduce the effectiveness of the product through movement of
treated and/or untreated soil; these should be recognized and studied as
variables,. .When .cultivatipn is .allowed or recommended, or when it affects
product usefulness-, cultivation .practices should be addressed in .test reports
and•labeling.. .Weed control ratings recorded after cultivation should be so
'•indicated. ' "" '• •.' •..•,•••• -. ;.
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(5) Herbicide use with fertilizers... (i) Fertilizer practices should be
within the range recommended for the crop in the test location; however,
where herbicides are tank-mixed with fluid fertilizers or "are impregnated
onto dry fertilizers, .specific information is needed. As to information on
fluid fertilizer/herbicide tank mix applications, field test data should indi-
cate spray volume per unit area, fertilizer analysis, compatibility, efficacy,
and detrimental effects. For additional considerations on pesticide/fluid
fertilizer tank mixes, see §§ 90-2(b)(4) and 94-2(a)(11)(ii).
(ii) Reports of data supporting use of herbicide impregnated dry
fertilizers should include, in addition to efficacy and detrimental effects,
the. quantity of fertilizer applied per unit area, the fertilizer analysis,
the quantity of product impregnated onto a ton of fertilizer, and the tillage
(soil incorporation) operations. In addition, see § 104-2(c)(3) and (4).
(6) Tank mixtures, (i) Tank mixes containing herbicides are viewed
as new products, regardless of the history of individual components, for
which all label claims should be supported. Claims for a tank mix should be
indicated on the label of at least one of the registered components.
Refer also to § 164-4 of Subdivision N.
(ii) Claims may be made by: listing the specific weed species supported
by data from actual testing of the proposed mix; claiming all weeds from the
labels of each product involved in the mix and supporting these claims with
appropriately referenced efficacy data and performance comparability data
which indicate the extent of antagonism and synergism between the products;
or combining these' approaches when a label claims all weeds from the labels
of each product involved in the mix and claims additional weed species not
listed on any of the component product labels.
(iii) When tank mix labeling claims all weed species from labels of
each product involved in the mix, but supporting data for each species have
not been generated from use of the proposed mix, either the claims should be
restricted to those actually supported by tank mix data, or performance
comparability plots should be established and appropriate efficacy data
supporting the claims for each component should be referenced. Comparability
tests should demonstrate any change in efficacy or detrimental effects due to
antagonistic or synergistic effects of each product on the other(s). It is
not required that each weed species to be claimed for the tank mix be present
in comparability test plots, but rather such tests include those claimed
species considered most difficult to control. Antagonism and synergism are
best evaluated in adjacent replicated plots where possible interactions are
subjected to statistical analysis. •
(iv) For conventional tillage practices, adjacent plots should compare
usefulness of each product alone (at appropriate rates) with usefulness of
the proposed tank mix.
(v) When products which are tank-mixed have divergent modes of action,
such as a minimum tillage tank mix containing a contact herbicide for emerged
vegetation control combined with a residual herbicide for preemergent (to
weeds) weed control, observations of weed control-should be timed such that
contact and preemergent activities are reported separately. In situations
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where both, contact and preemergent products are tank-mixed or applied seri-
ally for minimum tillage weed control, it is relatively simple to claim only
those weeds directly, supported by tank mix data; however, when preemergence
weed control is to be supported by comparability data, evaluation of inter-
actions .between the two types of products may be more complex. Effects of
a preemergent (to weeds) herbicide on usefulness of a contact product can
be evaluated in adjacent minimum tillage plots comparing contact product
activity with and without the preemergent product. Effects of the contact
chemical on preemergence activity under minimum tillage conditions is
practically impossible to evaluate due to the existing vegetation cover.
Since effects of the contact chemical on preemergence activity should be
evaluated without interference from the existing vegetation, and herbicidal
control of existing vegetation would not resolve questions of antagonism
or synergism, possible interaction of the contact product on emergence
activity should be evaluated in adjacent conventionally-tilled plots. In
the conventionally-tilled plots, where existing vegetation has been mechan-
ically turned under the soil surface, the preemergence product should be
evaluated with and without the contact product.
(vi) Data in support of two or more herbicide products mixed partially
or completely in fluid fertilizers should either support all label usefulness
claims using the fluid fertilizer as carrier, or establish performance
comparability with water as the carrier in adjacent plots.
(7) Serial applications. Data considerations for serial applications)
of one or more herbicide(s) preceding or following another herbicide on the
same crop area in the same growing season are identical to those described
for tank mixes, except that the .times of application (or application interval)
should be indicated for each product or tank mix involved in the serial appli-
cation. Physical compatibility data as for components of tank mixes are not
required for serial applications. [See also § 90-2(a)(3) and §164-4 of Sub-
ivision N for criteria on serial applications.]
(8) Noncrop area applications. Use sites, such as rights-of-way,
lumberyards, or fence rows, should be indicated in test reports and labeling.
If claims are made for longevity of weed control, such claims should be
supported with efficacy data. Where bare ground is desired (for example,
for fire prevention in lumber yards), the following should be reported: weed
escapes, duration of weed control, the percent bare ground before and after
application, identification, dosages and dates of previous herbicide appli-
cations, and the extent of weed reinvasion from untreated areas.
(9) Chemical fallow applications. Labels and performance data support-
ing products for use in.chemical fallow programs should address the longevity
of weed control expected, the extent of mechanical tillage required to sup-
plement herbicide activity, the extent of mechanical tillage allowed which
.will not decrease, herbicide activity, and scheduling of harvest., application,
tillage/ fallow ,/arid replanting appropriate for 'the .-cultural practices follow-
ed:, in the geographical .area for which use of the. product .is intended.
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§ 94-4 Plant regulators.
(a) Scope. Plant regulators include but are not limited to products
that increase yields, increase flowering, thin flowers and fruits, increase
fruitset, decrease fruit drop, accelerate ripening, concentrate sugar content,
enhance abscission, chemically "pinch" .ornamentals, reduce internode elonga-
tion, inhibit tobacco sucker growth, and promote rooting of cuttings. Data
supporting efficacy claims and freedom from adverse effects on crop plants
from plant regulators are unique for each use pattern and crop. Often,
varying concentrations or varying applications of a product to a single
plant species at different growth stages will cause a wide variety of
responses. Many plant regulators eliciting a response in one variety or
cultivar of a species will not perform similarly at all on another variety
within the same species. Further, many of the plant response levels are
extremely subtle and at times may be inconsistent within the same closely
controlled tests.
(b) Suggested performance standard. Efficacy data in support of plant
regulator claims need not demonstrate perfect (100%) plant response, but
the response attained should be at a level considered commercially acceptable.
Commercially acceptable plant response levels vary from one use pattern,
plant species or variety, and environmental condition to another; however,
the response level should, in all cases, be expected to provide a meaningful
benefit to the user of the product.
(c) Suggested performance reliability. Due to the frequency of subtle
and inconsistent responses of plants or commodities to plant regulators, •'•
efficacy evaluations should be replicated and statistically analyzed where
greenhouse or small plot tests are conducted. When commercial application
equipment is utilized in large field plots where independently replicated
applications may not be practical or economical, efficacy evaluations
should be based upon multiple independent observations and/or samples
taken from within each treated area. As the response levels approach the
limits of usefulness, increased replication and sampling are needed to
support efficacy claims and absence of significant undesired effects.
(d) Efficacy and adverse effects data. The following data should
be considered in addition to the general considerations in § 94-2. The
untreated control crops or commodities should receive the same cultural
practices, post-harvest treatment, and pesticide applications (excluding
the product being evaluated) as the -treated plants .or commodities. Due to
the varied responses, sites, crops involved, use patterns, and application
techniques (all frequently involving the same product), each product is
evaluated by the Agency on a case-by-case basis and according to claims
made on the specific product label for the specified crop or commodity.
For example, a ripener for use on tomatoes, tobacco, and pineapples should
be evaluated using different criteria for each crop. As a result, a document
detailing the criteria for all plant regulator uses on all crops or commodities
would be enormously extensive. Therefore, the data considerations which
follow are not intended to serve as an exhaustive list nor to exclude
other' plant regulator uses, but instead to include only general guidance
which may be applied to certain categories, of plant regulator uses. See
Subdivision J for a full explanation on hazard evaluation to nontarget plants.
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(e) Specific data considerations. (1) Products increasing yields.
(i) Comparisons should be made between the treated crop (and/or commodity)
and the untreated crop (and/or commodity) with respect to:
(A) Crop or commodity yield per unit area;
(B) Crop stand and vigor;
(C) Crop or commodity quality (grade, dollar value, flavor, size,
shape, color); and
(D) Observations on plant growth, flowering, fruitset, yields, and
plant or commodity quality obtained during the second growing season after
initial application (if the product is intended for yearly or more frequent
use on perennial plants).
(ii) The following should also be reported: crop identification as to
species and variety, and (as applicable) cultivar, rootstock, or parentage;
stage of crop or commodity development at application and at harvest;
application-to-harvest interval; harvest technique employed; and any other
pesticides applied to the crop or commodity in the same growing season.
(2) Products increasing flowering, (i) Comparisons should be made
between treated plants (and/or commodities) and untreated plants (and/or
commodities) with respect to:
(A) Number of flowers per plant;
(B) Flower quality (size, shape, color);
(C) Timing of full bloom after treatment;
(D) Growth rate, and conformation of vegetative plant parts;
(E) Data as in paragraph (d)(1) of this section, if increased
flowering is intended ultimately to increase yields; and
(F) Observations on plant growth, flowering, fruitset, yields, and
plant or commodity quality obtained during the second growing season after
initial application (if the product is intended for yearly or more frequent
use on perennial plants).
(ii) The following should also be reported: detailed crop identification,
stage of plant growth at application, application-to-harvest interval, and
any other pesticides applied to the crop or commodity in the same growing
season.
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(3) Products thinning flowers and/or fruit, (i) Comparisons should
be made between treated plants (and/or commodities) and untreated plants
(and/or commodities) with respect to:
(A) Percent flower drop and/or fruit drop per plant:
(B) Effects on remaining flowers and/or fruit, conformation of
vegetative growth, and phytotoxicity;
(C) Data as in paragraph (d)(1) of this section, if thinning
of flowers and/or fruit is intended ultimately to increase crop or commodity
yields and/or quality; and
(D) Observations on plant growth, flowering, fruitset, yields, and
plant or commodity quality obtained during the second growing season after
initial application (if the product is intended for yearly or more frequent
use on perennial plants).
(ii) The following should also be reported: stage of plant growth
or development at application, detailed plant identification; and any
other pesticides applied to the crop or commodity in the same growing
season.
(4) Products increasing fruitset. (i) Comparisons should be made
between treated plants (and/or commodities) .and untreated plants (and/or
commodities) with respect to:
(A) Number of fruit set per plant; • •
(B) Fruit quality (grade, size, shape, color, flavor, dollar value;
(C) Data as in paragraph (d)(l) of this section if increased
fruitset is intended ultimately to increase crop or commodity yields and/or
quality;
(D) Number of successful crosses and number of seeds per cross, if
increased fruitset is intended ultimately to enhance plant breeding; and
(E) Observations on plant growth, flowering, fruitset, yields, and
plant or commodity quality obtained during the growing season after initial
application (if the product is intended for yearly or more frequent use
on perennial plants).
(ii) The following should also be reported: state of plant growth or
development at application, detailed plant identification, and any other
pesticides applied to the crop or commodity in the same growing season.
(5) Products decreasing preharvest fruit drop. (i) Comparisons
should be made between treated plants and/or commodities and the untreated
plants and/or commodities with respect to:.
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(A) Percent fruit drop or retention;
(B) Fruit quality (grade, size, shape, color, flavor, dollar value),
and storage and shelf life characteristics;
(C) Harvest date;
(D) Effects on plant growth, immature fruit, yield; and
(E) Observations on plant growth, flowering, fruitset, yield, and
plant or commodity quality obtained during the second growing season
after initial application (if the product is intended for yearly or more
frequent use on perennial plants).
(ii) The following should also be reported: application-to-harvest
interval, stage of fruit growth or maturity at application, detailed
plant identity, duration of effectiveness, harvest technique, harvest
efficiency, and any other pesticides applied to the crop or commodity in
the same growing season.
(6) Products accelerating ripening, (i) Comparisons should be made
between treated plants (and/or commodities) and untreated plants (and/or
commodities) with respect to:
(A) Sucrose content;
(B) Sugar/acid ratio;
(C) Internal and external coloration;
(D) Quality (grade, size, shape, firmness, texture, flavor, dollar
value);
(E) Effects on plant or commodity yield, rate of maturation, storage
and shelf life characteristics, and harvest efficiency; and
(F) Observations on plant growth, flowering, fruitset, yields, and
plant and commodity quality obtained during the second growing season
after initial application (if the product is intended for yearly or more
frequent use on perennial plants).
(ii) The following should also be reported: application-to-harvest
interval, detailed plant identification, plant or commodity stage of
growth or maturity at application, and any other pesticides applied to the
crop or commodity in the same growing season.
(iii) Refer to § 94-^30 paragraph II.G. for additional guidance.
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(7.) Products concentrating or preserving sugar content, '(i) Compari-
"sons should be'made between' treated plants • (.and/or comoditieis-) and untreated
plants (and/or commodities) with respect to:
(A) Soluble solid content of expressed juice;
(B) Sucrose purity;
(C) Sucrose yield per unit land area;
(D) Effects on plant and commodity yield, quality, rate of maturation,
harvest efficiency, and storage and shelf life characteristics; and
(E) Observations on plant growth, flowering, fruitset, yields and plant
and/or commodity quality obtained during the second growing season after
initial application (if the product is intended for yearly or more frequent
use on perennial plants).
(ii) The following should also be reported: stage of plant growth or
maturity at application, application-to-harvest interval, detailed plant
identification, duration of effectiveness, plant stand, age of planting,
and any other pesticides applied to the crop or commodity in the same growing
season.
(8) Products enhancing fruit abscission, (i) Comparisons should be
made between treated plants (and/or commodities) and untreated plants
(and/or commodities) with respect to:
(A) Pull force for fruit removal; . .
(B) Fruit quality (grade, size, shape, color, flavor, dollar value);
(C) Harvest efficiency;
(D) Effect on fruit and leaf drop, and yield; and
(E) Observations on plant growth, flowering, fruitset, yields, and
plant or commodity quality obtained during the second growing season
after initial application (if the product is intended for yearly or- more
frequent use on perennial plants).
(ii) The following should also be reported: application-to-harvest
interval, stage of plant growth or maturity at application, harvest
technique, ultimate fate of fruit (fresh market or processing), detailed
plant identification, and any other pesticides applied to the crop or
commodity in the same growing season.
(iii) Refer to § 94-30 paragraph II.H. for additional guidance.
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(9) Products chemically pinching ornamentals, (i) Comparisons should
be made between treated plants and untreated plants with respect to:
(A) Number and quality of buds, flowers, and lateral branches;
(B) Plant conformation;
(C) Effects on flowering cycle, usability for cuttings, and growth
rate; and
(D) Observations on plant growth, flowering, and plant quality ob-
tained during the second growing season after initial application (if
the product is intended for yearly or more frequent use on perennial
plants).
(ii) The following should also be reported: application-to-harvest
interval, plant stage of growth or development at application, detailed ....
plant identification, and any other pesticides applied to the crop in .... .
the same growing season.
(10) Products reducing plant internode elongation, (i) Comparisons
should be made between treated plants and untreated plants with respect to:
(A) Length of internodes;
(B) Plant size and conformation;
(C) Effects on growth rate, flowering, fruitset, yield, crop or com-
modity quality, and harvest efficiency; and
(D) Observations on plant growth, phytotoxicity, flowering, fruitset,
yield and plant or commodity quality obtained during the second growing
season after initial application (if the product is intended for yearly
or more frequent use on perennial plants).
(ii) The following should also be reported: stage of growth at appli-
cation, application-to-harvest interval, duration of effectiveness, detailed
plant identification, and any other pesticide applied to the crop in the
same growing season.
(11) Products decreasing tobacco sucker growth, (i) Comparisons should
be made between treated plants (and/or commodities) and handsuckered and
topped-but-not-suckered untreated plants (and/or commodities) with respect
to:
(A) Number and fresh weight of suckers at harvest;
(B).. Effects on leaf drop, yield, and cured leaf quality (.percent
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usable by tobacco companies, chemical and physical properties, and
smoke taste test); arid -.-•'•'' '• • '''••:•-
(ii) The following should also be reported: detailed plant identifi-
cation, stage of plant growth or sucker development at application, and
any other pesticides applied to the crop in the same growing season.
(iii) Refer to § 94-30 paragraph II.A. for additional guidance.
(12) Products promoting rooting of cuttings, (i) Comparisons should
be made between treated cuttings and untreated cuttings with respect to:
(A) Number or weight of roots per cutting and/or percent of rooted
cuttings per treatment?
(B) Effects on growth rate and conformation; and
(ii) The following should also be reported: type of cutting (stem,
root, leaf, softwood, herbaceous, greenwood, or hardwood), rooting medium,
detailed plant identification, application-to-observation interval, and
any other pesticides applied to the plant in the same growing season or to
the cuttings during the period of observation.
(13) Products inhibiting Irish potato tuber sprouting, (i) Comparisons
should be made between treated tubers (or tubers from treated plants) and
untreated tubers (or tubers from untreated plants) with respect to the
factors listed below:
(A) Sprouting (sprout number or weight per tuber), tuber.weight loss
and rot loss, tuber appearance (flattening, shriveling, discoloration, skin
texture), suberin and wound periderm formation, heat generated in piled
potatoes, quality (sugar content, mealiness, fry color, flavor, chipping)
both before and after storage, usefulness of tubers as propagation stock,
shelf life (at time and marketing); and
(B) Yield, and grades, if the plant regulator is applied preharvest.
(ii) The following should also be reported: storage conditions (temper-
atures, humidity, air circulation, and duration); stage of plant and/or
tuber growth or maturity at application; environmental factors affecting
usefulness if plant regulator is applied prior to harvest; rates and repeat
treatments applied; other pesticides applied to plants and tubers; duration
of exposure to sprout inhibitor; crop variety; length of growing season;
application equipment; finished spray volume per unit area or per unit
measure of tubers; or volume or weight of gas or liquid per unit volume of
storage space.
(iii) Refer to § 94-30 paragraph II.B. for additional guidance.
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(14) Products increasing branch angle in young nonbearing fruit
trees, (i) Comparisons should be made between treated plants and untreated
plants with respect to:
(A) Branch angle (angle less than 90° formed by the main truck and
the primary branches) prior to treatment, at the end of the growing season
(during which treatment(s) were applied), and at the end of the next growing
season (during which no treatments were applied). If treatments must be
or are allowed to be applied during more than one growing season, then branch
angles and all other data in paragraphs (d)(14)(i) and (ii) of this section
should be reported at the end of each season treated; and
(B) Effects on vegetative growth as to its appearance (color, size,
shape), and growth rates of shoot tips, and trunk. Observations made the ,.:.
growing season following treatment should include effects on winter hardiness,
time of leaf expansion in the spring, effects on appearance and growth of
vegetative parts, and branch angles.
(ii) The following should also be reported: tree age, variety, root-
stock, transplant-to-application interval, stage of vegetative growth
at application, other pesticides applied, effects of meteorological
conditions on product performance, rates, finished spray volumes applied
per unit land area and per tree, extent of spray coverage, number and
rates of repeated applications, application-to-observation interval(s),
and application equipment.
(iii) Refer to § 94r30 paragraph II.C. for additional guidance.
(15) Products controlling alternate bearing in apples, (i) Comparisons
should be made between treated plants and untreated plants with respect to:
(A) Data collected the year of treatment, including: fruitset, fruit
drop, yield, delay of maturity, fruit quality (size, shape, color, flavor),
and incidence of "bitter pit";
(B) Data collected the year after treatment, including: all factors
listed in paragraph (d)(15)(i) (A) of this section, as well as data on the
number of blossoms and time of blossoming;
(C) Data collected both in the treated year and the following year,
including: performance effects of cultural practices such as pruning and
use of plant regulators for fruit thinning and decreasing preharvest
fruit drop; effects of environmental conditions such as temperature,
frost, and rainfall; and effects on vegetative growth and appearance.
(ii) The following should.also be developed: application rates,
number of.applications, finished spray volume .per unit land area and per
tree., extent of ;spray coverage.,, apple variety, tree: age, stage of 'growth
at. application, other pesticides applied in the same season, and application
equipment... , .':.: • . • . . ••. . .. • ••• -: '."..""• . . '
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Ciii) If the product is intended for use-on the same trees every'year,.
then'data in paragraphs (d)(-15) ••(!')' and (ii) of this section should
be reported for applications repeated more than one growing season.
(iv) Refer to § 94-30 paragraph II.D. for additional guidance.
(16) Products altering sex expression in cucurbits, (i) Comparisons
should be made between treated and untreated plants with respect to:
(A) Sex of flowers at each node, number of flowers per plant,
vegetative growth rates and appearance, plant stand, time of flowering
and harvest, and duration of effectiveness;
(B) Effects of cultural practices and environmental conditions on
product performance;
(C) Data in support of hybrid seed production, including number of
seeds per fruit, seed yield per acre, percent seed germination, seedling
vigor, and seed storage life; and
(D) Data in support of increasing yields in once-over harvest programs,
including fruit yield per unit area by grades, market value by grade, and
fruit quality (shape, color, and flavor).
(ii) The following should also be reported: application rates, number
of applications, stage of growth at application, variety, plant population,
application equipment, and finished spray volume per unit area.
(iii) Refer to §"94-30 paragraph II.E. for additional, guidance. .
(17) Products increasing berry size of seedless table grapes. (i) Com-
parisons should be made between treated plants (and/or berries) and untreated
plants (and/or berries) with respect to:
(A) Berry size, yield (weight per vine), sugar/acid ratio, color,
flavor, number of berries per cluster, and effects on vegetative growth
(growth rate and formative effects), effect on harvest date, and shelf
life;
(B) Effects on flowering in addition to data in paragraph
(d)(17)(i)(A) of this section, to be collected and reported for the year
following application. (One-half the number of the same plants treated
the first growing season must be retreated during the following growing
season while the othet half will not be retreated); and
(C) Effects of cultural practices, such as girdling, application
of other plant regulators, irrigation, and effects of environmental
conditions, such as temperature and rainfall, on product performance.
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(ii) The following should also be reported: application rates, number
of applications, other pesticides applied, stage of growth or maturity at
application, application-to-harvest interval, variety, age of planting,
finished spray volume applied per unit land area or per vine, extent of
spray coverage, and application equipment.
(iii) Refer to § 94-30 paragraph II.H. for additional guidance.
(18) Products decreasing compactness of tightly clustered varieties
of grapes. (i) Comparisons should be made between treated plants (and/or
berries) and untreated plants (and/or berries) with respect to:
(A) Visual estimate of compactness or looseness, incidence of rot,
cluster length, fruit set (number of berries per cluster), number of shot
berries per cluster, berry yield (weight per vine), berry quality (size,
sugar/acid ratio, color, flavor), effects on vegetative growth (growth
rate, formative effects), and effect on rate of crop maturation;
(B) Effects on flowering in addition to data in paragraph
(d) (18)(i)(A) of this section, to be collected and reported for the year
following application. (One-half the number of the same plants treated
the first growing season must be retreated during the following growing
season while the remaining half will not be retreated); and
(C) Effects of cultural practices, such as girdling, application of
other plant regulators, irrigation, and environmental conditions (such as
temperature and rainfall), on product performance.
(ii) The following should also be reported: application rates, number
of applications, variety, age of planting, stage of growth or maturation
at application, other pesticides applied, finished spray volume per acre
or per vine, extent of spray coverage, application-to-harvest intervals,
and application equipment.
(iii) Refer to § 94-30 paragraphs II.I. for additional guidance.
(19) Products reducing acidity in citrus, (i) Comparisons should be
made between treated plants (and/or commodities) and untreated plants
(and/or .commodities) with respect to:
(A) Harvest dates when treated and untreated fruit attain their respec-
tive harvest maturities. A representative sample of untreated fruit should
also be harvested and evaluated, as appropriate for paragraph (d)(19)(i)(B)
of this section, at the same time as the treated fruit;
(B) Sugar/acid ratio, fruit yield per tree, juice content, fruit
quality (size, shape, color, flavor, blemishes), dollar value, effects
on vegetative growth (growth rate and formative effects), effects on
development-of flowers ;.and fruit present, on the: tree at application
;but not intended for treatment, effects on artificially-induced ripening
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response (coloration-), effects, on performance of abscission agents, and
effects-on frost resistance;
(C) Effects on flowering in addition to data in paragraph
(d)(19)(i)(A) of this section, to be collected and reported for the year
following application. (One-half the number of the same plants treated the
first growing season while the remaining half will not be retreated); and
(D) Effects of cultural practices (such as irrigation, pruning, and
application of other plant regulators) and effects of environmental conditions
(such as,temperature and rainfall) on product performance.
(ii) The following should also be reported: application rates, number of
applications, stage of plant growth or fruit maturity at application, appli-
cation-to-harvest interval, crop variety, rootstock, age of planting, other
pesticides applied, finished spray volume per unit land area or per tree,
extent of spray coverage, application equipment, and fate of fruit (fresh
market or processing).
(iii) Refer to § 94-30 paragraph II.J. for additional guidance.
(20) Products inhibiting apple scald. (i) Comparisons should be made
between treated fruit and untreated fruit with respect to:
(A) Scald symptoms, number of scalded and .unaffected fruit, percent of
fruit surface affected, and appearance (severity) of scald-infected areas;
(B) Fruit quality (flesh firmness, color, blemishes, flavor, .odor) upon
removal from storage, shelf life, and visible residues on fruit surface; and
(C) Effects of varied storage conditions, maturity at harvest, apple
varieties, other plant regulators (for ripening, abscission), and tank
mixes with other pesticides (such as fungicides) on product performance.
(ii) The following should also be reported:. concentration of finished
spray or dip, other pesticides applied, extreme weather conditions prior to
harvest, method of application, application equipment, storage conditions
(duration, temperatures, humidity, and air circulation), harvest-to-
application intervals, and duration of exposure (if dipped or fumigated).
(iii) Refer to § 94-30 paragraph U.K. for additional guidance.
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§ 94-5 Desiccants.
(a) General. Desiccants may be used for a variety of purposes including:
increasing harvest efficiency, reducing plant moisture content, modifying
the time of harvest by terminating growth and hastening maturity, or opening
up crop foliage to increase canopy penetration by light or air.
(b) Suggested performance standard. Efficacy data in support of
desiccant claims need not demonstrate perfect (100%) plant response, but
the response attained should be at a level considered commercially acceptable.
Commercially acceptable plant response levels- vary from one use pattern,
plant species .or variety, and environmental condition to another; however,
the response level must, in all cases, be expected to provide a meaningful
benefit to users of the product.
(c) Performance reliability. Efficacy evaluations used for supporting
data must be replicated and statistically analyzed where greenhouse or small
plot tests are conducted. When commercial application equipment is utilized
in large field plots where independently replicated applications may not
be practical or economical, efficacy evaluations must be based upon multiple
independent observations and/or samples taken from within each treated area.
As the response levels approach the limits of usefulness, increased replica-
tion and sampling is needed to support efficacy claims and absence of
undesired adverse effects.
(d) Specific considerations for plant desiccants. (1) Cotton.
(i) Purpose of treatment. The purposes of desiccating cotton plants are
to'reduce moisture and chlorophyll' content, and -.to desiccate regrowth .
(after defoliation) where stripper-type harvest equipment is to be used.
(ii) Efficacy data considerations. Data considerations will depend, to
some extent, on the intended use of the product. The following considerations
are the most, commonly appropriate ones for products claiming desiccation of
cotton plants:
(A) .Label claims for "desiccation" only. Label claims for "desiccation"
only should be supported by data which include:
(J_) Desiccation evaluation. For both treated plots and untreated
checks, the percent dried leaves, stems, and stalks'. should be determined,
using pre- and post-application green versus dried leaf, stem, and stalk
counts. Alternatively, visual observations and ratings of the same responses
may-be appropriate. .
(2^ Recording intervals. The application-to-maximum effect interval
and application-to-harvest interval should be reported.
- • (3;.). Percent ^defoliation.• The considerations of § 94-6(d-) (1.) of this
subdivision, apply ror all. cotton^defoliation .datta to be .'reported. .
>•„_•: .- <4.)\r Effects., on terntina-lsv'•••; .The, .perceittt-age o£ cotton, plants .with dead
terminal branches should'be rejHi'rte'd, . V. ' -.',- . ; ". ,
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OB) Label claims in addition to "desiccation". When other more specific
claims, such as "increased harvester efficiency", are made in. addition to
"desiccation", the product efficacy should be supported with data pertaining to
each specific claim made.
(iii) Concurrent effects. Data from tests should indicate the percent of
each stalk killed, extent of regrowth, effects on fiber quality, and effects
on premature boll opening. The quantity of regrowth per plant after desicca-
tion (or defoliation) should be determined by harvesting and weighing the
regrowth or by visual observations over the period from inception of regrowth
until cotton harvest time. The extent of detrimental effects from regrowth
should be evaluated and reported in terms of their effects on harvest and
quality of the cotton.
(iv) Additional instruction. Refer to § 94-30 paragraph III.A. for
additional guidance.
(2) Sorghum, (i) Purpose of treatment. The purposes for desiccating
sorghum plants are to decrease moisture in leaves and in immature seedheads
arising from tillers, decrease seed germination in heads, and decrease lodg-
ing by allowing earlier harvest.
(ii) Efficacy data considerations. (A) Moisture content. The moisture
content in seed or other plant parts (depending on purpose of desiccation)
should be determined from plants sampled and weighed at treatment and at har-
vest, and dried in a forced-draft oven until constant dry weight is attained.
Percent moisture must be presented as a percent of fresh weight. Desiccation
of some plant parts such as leaves may be' evaluated visually. .
(B) Recording interval. The application-to-maximum effect interval
and application-to-harvest interval should be reported.
(iiij Concurrent effects. Effects of the desiccant on the seed, in
terms of germination,. seedling vigor, color, texture, starch and protein
content, flavor, weight, milling properties, and yield should be evaluated in
comparison to untreated checks and reported. Untreated check plots should be
of sufficient size so that one-half can be harvested at the time of treated
plots and the remainder harvested at the usual time for untreated sorghum.
(iv) Additional instruction. Refer to § 94-30 paragraph III.B. for
additional guidance.
(3) Irish potato, (i) Purpose of treatment. The purpose for desiccat-
ing potato plants is to kill vines prior to harvest. Killing of vines may
facilitate both manual and mechanical harvesting; decrease the formation
.of oversize, misshapen, and second-growth tubers; terminate growth for earlier
harvest or to maintain tuber quality; and harden (set) tuber skin.
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(ii) Efficacy data considerations. (A) Vine desiccation. The degree
of vine desiccation should include evaluation of effects both on leaves and on
stems.
(B) Recording intervals. The application-to-maximum effect interval
and application-to-harvest interval should be reported.
(C) Secondary claims, Any specific claims other than desiccation,
such as maintenance of tuber quality, should be supported with appropriate
data.
(iii) Concurrent effects. Effects of the desiccant on tuber yield
and quality (grade), storage quality, cooking quality, seed piece quality,
flavor, skinning, vascular discoloration, and sprouting should be evaluated
in comparison to untreated checks and reported.
(iv) Additional instruction. Refer to § 94-30 paragraph III.C. for
additional guidance.
(4) Sunflower. (i) Purpose of treatment. The purposes of desiccating
sunflower plants are to reduce the quantity of green (fresh) plant material
passing through mechanical harvesters and to reduce moisture in the seed
heads.
(ii) Efficacy data considerations. (A) Harvester efficiency. Reduction
in bulk is indicated in increased harvester efficiency. Harvester efficiency
should be expressed in terms of length of time required for harvesting treated
and untreated plots of equal size and stand.
(B) Moisture content. Moisture content of seeds may be determined in
a manner similar to that for sorghum. See paragraph (d)(2)(ii)(A) of this
section.
(C) Recording intervals. The application-to-maximum effect interval
and application-to-harvest interval should be reported.
(iii) Concurrent effects. Effects of the desiccant on seed yields,
flavor, and quality should be evaluated by comparison with untreated checks
and reported.
(iv) Additional instruction. Refer to § 94-30 paragraph III.D. for
additional guidance.
(5) Seed crops, (i) Purpose of treatment. The purposes of applica-
tions of desiccants to crops such as alfalfa, clover, dry beans, and other
crops grown, for seed production are to reduce moisture content and increase
.harvester efficiency. . . . ..
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(iiV -Efficacy data considerations. The efficacy data considerations for
seed crops are similar to those for sunflowers. See paragraph-(,d)(4:){ii)
of this section.
(iii) Concurrent effects. Effects of the desiccant on seed yields,
pod dehiscence, seed weight, seedling vigor, and percent germination should
be evaluated by comparison to untreated controls and reported.
(6) Onion. (i) Purpose of treatment. The purposes for desiccating
onion plants are to dry tops, facilitate harvest, and allow flexibility in
scheduling of harvest.
(ii) Efficacy data considerations. (A) Moisture content. Moisture
content of tops should be reported and may be determined in a manner similar
to that for sorghum [see paragraph (d)(2)(ii)(A) of this section] or may
be evaluated visually.
(B) Harvest efficiency. Increased harvest efficiency, if claimed,
should be expressed in terms of length of time required for harvesting
treated and untreated plots of equal size and stand.
(C) Recording intervals. The application-to-maximum effect interval
and application-to-harvest interval should be reported.
(iii) Concurrent effects. Effects of the desiccant on onion yields,
bulb weight, sunburn of bulbs, and flavor should be evaluated by comparison
to untreated checks, and reported.
(iv) Additional instruction. Refer to § 94-30 paragraph III.E. for
additional guidance.
(7) Sugarcane. (i) Purpose of treatment. The purpose for desiccating
sugarcane plants is to dry leaves for improved burning which may increase
harvest and refining efficiency.
(ii) Efficacy data considerations. (A) Moisture content. Leaf desic-
cation should be evaluated by comparison to untreated check plots and reported.
Leaf desiccation may be determined by visual observation of browning or by
determining the percent moisture by drying leaves (minus the midribs) to a
constant dry weight in a forced-draft oven.
(B) Burning efficiency. If.improved burning is claimed, visual
estimates of percent foliage burned off compared to burned untreated check
plots, or weight of trash remaining on stalks after burning both treated
and untreated check plants, should be reported.
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(C) Recording intervals. The application-to-maximum effect interval
and application-to-harvest interval should be reported.
(iii) Concurrent effects. Effects of desiccant application on sugar-
cane sucrose yield per unit area, ratoon growth, and effects on growth of
the terminal buds of seed pieces should be evaluated by comparison to untreated
check plants, and reported.
(iv) Additional instruction. Refer to § 94-30 paragraph III.F. for
additional guidance.
§ 94-6 Defoliants.
(a) General. Defoliants may be used for a variety of purposes, many
of which are the same as indiated for desiccants. Since the purpose of
defoliation is the removal of plant leaves (or, occasionally, other above-
ground parts), the percent leaf drop should be determined by making pre- and
post-application leaf counts or by visual observation, for both treated and
untreated plots.
(b) Suggested performance standard. Efficacy data in support of
defoliant claims need not demonstrate perfect (100%) plant response, but
the response attained must be at a level considered commercially acceptable.
Commercially acceptable plant response levels vary from one use pattern,
plant species or variety, and environmental condition to another; however,
the response level should, in all'cases, by expected to provide a meaningful
benefit to the user of the product.
(c) Performance reliability. Efficacy evaluations should be replicated
and statistically analyzed where greenhouse or small plot tests are conducted.
When commercial application equipment is utilized in large field plots where
independently replicated applications may not be practical or economical,
efficacy evaluations should be based upon multiple independent observations
and/or samples-.taken from within each treated area. As the response levels
approach the limits of usefulness, increased replication and sampling is
necessary to support efficacy claims and absence of undesired adverse effects.
(d) Specific considerations for defoliants. (1) Cotton. (i) Purpose
of treatment. The primary purpose of defoliant application to cotton is
leaf removal which in turn may facilitate harvest by reducing trash and
improving harvester efficiency, may preserve seed and fiber quality, or
may cause mature green bolls to open sooner.
(ii) Efficacy data considerations. (A) Defoliation evaluation. The
:percentage of • leaf drop must be supported by comparison .with untreated
.Controls/and reported.. '....'.•'. . • .. . ' •
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(B) Harvest efficiency. If claimed, increased .harvest efficiency
should be supported by comparison of length of time required-to harvest
treated and untreated plots of equal size and stand. Harvest efficiency
may include comparisons of the amount of unpicked lint residues remaining
after harvest of treated and untreated plots.
(C) Secondary claims. If other secondary effects of defoliation are
claimed in labeling, specific data considerations will be applied, as appro-
priate, on a case-by-case basis.
(D) Recording intervals. The application-to-maximum effect interval
and the application-to-harvest interval should be reported.
(iii) Concurrent effects. Effects of defoliant applications on cotton
yields, boll abscission, immature boll opening, regrowth and cotton quality
(lint spotting and staining; fiber length, strength, and diameter; seed oil
content; seed germination; seed vigor; and trash content) should be evaluated
by comparison to untreated check plots, and reported.
(iv) Additional instruction. Refer to the § 94-30 paragraph IV.A.
for additional guidance.
(2) Okra. (i) Purpose of treatment. Defoliants are applied to
okra to remove leaves and facilitate harvest.
(ii) Efficacy data considerations. (A) Defoliation evaluation. The
percentage of leaf drop should be evaluated by comparison with untreated
controls, and reported. • . : '
(B) Harvest efficiency. If claimed, increased harvest efficency should
be supported by comparison with the length of time required to harvest
treated and untreated plots of equal size and stand.
(C) Recording intervals. The application-to-maximum effect interval
and applicator-to-harvest interval should be reported.
(iii) . Concurrent effects. Effects of the defoliant on okra yield,
flavor, and pod damage should be evaluated by comparison to untreated check
plants, and reported.
(3) Deciduous hardwoods and ornamentals in nurseries, (i) Purpose
of treatment. Application of defoliants to these plants is for leaf removal
prior to storage, harvest, or shipping.
(ii) Efficacy data considerations. (A) Defoliation evaluation. The
percentage of leaf drop should be evaluated by comparison with untreated con-
trols, and reported. If partial defoliation occurs, data should be provided
illustrating any differentiation between the extent of defoliation on new
and old wood.
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(B) Secondary claims. If secondary effects, such as increased survival,
are claimed in labeling, then appropriate supporting data, such as percent
survival, are needed.
(C) Recording intervals. The application-to-maximum effects interval
and application-to-harvest (storage or shipping) interval should be reported.
(ii) Concurrent effects. The following should be evaluated and reported:
effects of the defoliant on bark (desiccation, discoloration), on stem
growing points (tip dieback) and growth rates, and on dormant or expanding
buds by comparison to untreated check plants• Evaluations should be made
just prior to application, at the time of storage, harvest, or shipping,
and at the flush of spring growth following application.
(iv) Additional instruction. Refer to § 94-30 paragraph IV.B. for
additional guidance.
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§ 94-30 Acceptable methods.
In general, several test methods are necessary to prove the usefulness
of a given pesticide product. The methods selected should test the product
in the manner in which it is to be used and in a manner which will show the
effect of field variables on the product's usefulness. The data obtained
should support the product label claims and must be representative of the
results that the grower will obtain from commercial usage of the product.
While data considerations under specific situations may be identified, the
validity of test methods used to generate the data will be evaluat-
ed on a case-by-case basis in the scientific review process. Due to the
number, variety, specificity, and continuing evolution of valid test methods
for performance evaluation of terrestrial herbicides, plant regulators,
desiccants, and defoliants, the following test methods and references are
cited only as examples of test methods which may generate a portion of the
product performance data for registration purposes.
Terrestrial herbicides.
Methods reported in the literature for testing the usefulness of
herbicides vary with the physiological and biochemical characteristics of
the herbicide, the formulation, crop, geographical location, application
equipment, cultural practices, and other factors. Instead of citing a
multitude of very specific test methods having very limited usefulness,
the Agency has cited two published works which reflect several basic methods
common to many herbicide evaluations.
A. Herbicide application. Wiese (1977) discusses the importance of
precise and uniform application of herbicides, along with the equipment,
techniques, and procedures required to obtain such application. One prime
consideration in the application of herbicides, whether in small plot or
large-scale trials, is uniformity of compound distribution and coverage.
B. Measuring plant responses. Frans and Talbert (1977) discuss plot
designs and field evaluation procedures along with statistical treatment.
POT registration purposes, adequate counts and/or ratings must be taken to
show the effectiveness of the compound against individual weed species and
to show crop safety. The crop injury and weed control evaluations must be
interpreted in terms of "acceptability". This can be done for weed control
by reference to phytotoxicity, yield, and quality (grades) of the commodities
from those check plots where acceptable levels of weed control are maintained.
References. .
Frans, R.E., and R.E. Talbert. 1977. Design of. Field Experiments and the
Measurement and Analysis of Plant Responses. Pp. 15-24 in Research
Methods in Weed Science. B. Truelove, ed. 2nd Ed. S.- Weed Sc.i. Soc.
Auburn Printing, Inc.: Auburn, Ala. 221 pp. • •
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Wiese, A.P. 1977. Herbicide Application. Pp. 1-14 in Research Methods
in Weed Science. B. Truelove, ed. 2nd Ed. S. Weed Sci. Soc. Auburn
Printing, Inc.: Auburn, Ala. 221 pp. '
II. Plant regulators.
Many bioassay, laboratory, and greenhouse methods have been published
for the detection of chemicals which modify plant growth, development, and
response. Only a few published methods are available, however, for evaluat-
ing the potential commercial utility of these chemicals under field condit-
ions.
Testing under actual field conditions remains the only acceptable method
for evaluating the efficacy of potential plant regulators. Because responses
obtained with plant regulators cover such a wide range (for example, bud
inhibition, berry size increase, and fruit coloring), specific field test
methods are outlined. These are based to a large extent on methods employed
to evaluate the efficacy of plant regulators presently marketed. New methods
and protocols for evaluating plant regulators are continually being developed
because both new uses and new chemicals are being sought. In all cases,
however, data obtained from field tests should support the label claims for
the product and must be.representative of the results that the producer can
.expect when the product is used in accordance with label instructions under
commercial conditions. ..•'.• -•'...-..'
A. Control of axillary bud (sucker) growth on tobacco. Plant regulat-
ing chemicals have been developed which inhibit the growth of axillary
suckers. These growth-inhibiting chemicals may be divided into two types:
systemic and contact. The systemic regulators are applied as sprays to
leaves, are absorbed by the plant, and are translocated throughout the plant,
but especially to the meristematic areas of the axillary buds. The contact-
type sucker control chemicals are also applied as sprays but are directed
down the stalk so that they contact the axillary buds. They kill the small
suckers without damaging more mature leaf tissue. Test methods and factors
affecting the^efficacy of various tobacco sucker control agents have been
discussed in a number of publications (Collins et al., 1972; Seltmann and
Priest, 1972; Seltmann and Marshall, 1967; Smith et al., .1971; Steffens et
al., 1969a; Steffens et al., 1969b; and Spaulding et al., 1970).
1. Scope.
1.1 The Regional Tobacco Growth Regulator Committee, composed of State
and Federal researchers, extension workers, representatives of tobacco and
chemical industries, and..representatives of the .Qiv-ironmentai Protection
-Agency, have'worked together to evaluate .ftew plant regulating-chemicals.
.potentially-'useful in'tobacco production. The following-tests and procedures
describe, those, .used, by the State and USDA personnel on. the. Committee in
evaluation o-f--new/plant regulators for tobacco. .As such, the following
wording, procedures;,/-and., gud'dance-.; are. those .off. the.. Committee/and. ho-t . "'•'':•
frecessacily, tho-se e*f.. EPA-i --.While EPA is not bound-by Committee requirements
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or decisions, Committee personnel are the recognized experts in tobacco plant
regulators, and prospective registrants are encouraged by EPA to participate
in the Committee's program. EPA cannot specifically require comparisons
with "standard products" or public revelation of active ingredients as in-
dicated in Committee requirements; however, the detailed types of data re-
quired by the Committee do correspond to EPA specifications for efficacy,
phytotoxicity, yield, and chemical and physical properties, quality, and
flavor of cured leaves. Any such data generated for the Committee will
also, upon submission to EPA, be considered as partial support for product
registration. Consequently, the Committee's procedures and requirements
serve well in providing meaningful guidance toward product registration.
Two types of field tests are conducted annually, usually at 15 locations
in 10 states. Tobacco is evaluated with respect to efficacy of treatment,
agronomic characteristics, commercial utility, chemical and physical prop-
erties, and smoke taste. Leaf samples of known history are made available
for assessment of residues of residues of applied chemicals.
1.2 The flow chart (Figure 1) summarizes the progression of tests
established. After each test, the Committee will evaluate the data to
determine if any entry is eligible for the next test in the progression.
After company proprietary tests, the Committee makes recommendations to
the Environmental Protection Agency regarding label registration.
2. Test chemicals.
2.1 New chemicals; These include structures, mixtures, sequential treat-
ments of chemicals, or management-chemical regimes not previously tested.
Two or more chemicals .to be treated .in sequential applications or as manage-
ment-chemical regimes will be considered."one" treatment. If a management-
chemical regime involves a new chemical or a new management procedure, it
will be considered a new chemical treatment.
2.2 Modified chemicals; These include formulations of previously approved
chemicals which have been slightly altered (salt form, surfactant, etc.). The
company submitting the chemical must supply data to indicate if the entry is
to be considered a modified chemical.
3. Types of tests and test methods.
There are 4 sequential levels of testing: company preliminary tests:
regional preliminary tests; regional advanced tests; and company proprietary
tests.
.3.1 Company preliminary tests. These tests are field trials conducted
by chemical companies to provide data to justify entry of a chemical into
the regional preliminary testing program.
3.1.a. Test procedures.
3.1.a.1. Minimum of 2 flue-cured and 2 burley tobacco locations, if the
chemical is to be entered iri both flue-cured and air-cured tobacco classes.
Both locations for any one class are to be in the area where the class is
commercially produced and in different environmental areas.
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Figure 1. Sequence of evaluation for tobacco sucker control products.
Year 1 Company Preliminary Evaluations
•
?7
•
Data Evaluated^
•
•
•
•Modified . New
Chemical Chemical
Year 2 Company Proprietary2 Regional Preliminary Evaluation
Evaluation
•
Data Evaluated^
Determined to be a
Modified Chemical
New Chemical
Year 3 Company Proprietary^ Regional Advanced2
Evaluation Evaluation
Year 4 . Company Proprietary2
• Evaluation
^Evaluation by Committee to determine test level required.
2After Regional Advanced and Company Proprietary Evaluations,
the Committee makes recommendations to the Environmental
Protection Agency..regarding label registration. Experimental
use permits are optional. .
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200
3.1.a.2. Non-replicated fifteen-plant plots minimum.
3.1.a.3. Efficacy based on sucker weight; the treatment should be 90 per-
cent as effective as the performance of the appropriate standards included
in the same test. Data are to be obtained for flue-cured tobacco 2 weeks
after treatments (observational) and at final harvest (sucker number and
green weight). Etor burley tobacco, sucker number and green weight are to
be taken at harvest.
. 3.1.a.4. Leaf phytotoxicity data (including necrosis, malformation,
etc.) taken at the appropriate stage (scale O.to 5); leaf drop and stalk
girdling are not to exceed that of appropriate standards.
3.1.a.5. Records of cultural practices and environmental conditions.
3.2. Regional preliminary tests; These tests are conducted by State or
Federal agricultural experiment station scientists who are Committee members.
3.2.a. General testing procedures.
3.2.a.l. The formulation of the chemical (or very closely related for-
mulation) must have been evaluated on field-grown tobacco.
3.2.a.2. Each person conducting the test must know the identity of the
active ingredient on a confidential basis. 'The overall committee coordinator
may request the chemical composition of the complete formulation on a con-
fidential basis. . . - • . '
3.2.a.3. The company is to supply'all information available oh toxicity
to humans, animals, etc., and any information available on the breakdown,
persistence, action, etc., of the chemical.
3.2.a.4. The company is to supply sufficient quantities of the formu-
lated chemical to conduct the evaluations at all proposed locations.
The formulated chemical appropriately packaged is to be shipped by the
company to the individual locations by the specific dates as per instruct-
ions supplied by the Committee.
3.2.a.5. The company if submitting the chemical is to indicate if the
chemical is new or modified and suggest in writing the rate of application,
the procedure for application, and the timing for application. The company
is also to suggest the procedure for harvesting and the.sequence for curing
if different from the standard field practice.
3.2.a.6. After treatment, and during the course of the field evaluation,
the research worker will determine by visual observation the potential of
the treatment for achieving the desired results. This information will be
immediately forwarded to the coordinator for that tobacco class so information
on efficacy across a particular class can be summarized. After a decision
is made by the coordinator, based on the information available, complete
data will be collected from only those treatments showing the desired results.
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3.2.b. Field test procedures and data procedures.
3.2.b.l. Suggested distribution for flue-cured tobacco, 6 locations;
for burly tobacco, 4 locations; for cigar filler tobacco, 2 locations;.
I: for dark tobacco, 2 locations; and for Maryland tobacco, 1 location.
I 3.2.b.2. A minimum of thirty-plant plots (non-replicated).
(.' 3.2.b.3. Agronomic observations, including:
P - -
|: — Efficacy based on percent sucker control by weight, number of
|i suckers per plant, and weight per sucker;
i' " • .
I- — Leaf phytotoxicity (including necrosis, malformation, etc.) data
f taken at a. maximum manifestation (0 to 5 scale). Leaf drop and stalk-
s' girdling information is to be reported; and
I,
— Yield and quality based on U.S. standard grades.
' 3.2.b.4. Cured leaf data (on successful entries only), including:
f -— Percent useable by treatments and locations. Evaluations conducted by
\- tobacco companies;
[;'
[: — Chemical and physical properties (composite sample by treatment for
¥• each class) (see Table 1); and
I • • •
\: — Evaluation of smoke taste for the identification of unacceptable off-
£: taste due to treatment. Tobacco companies conducting smoke panel tests must
i know the identity of applied chemicals. Cigarettes will be made from a
s: composite sample by treatment for each class (flue-cured and burley).
|:
;>: 3.2.C. Procedures for passing the regional preliminary test.
£•
[• 3.2.c.l. Minimum sucker control based on sucker weight must be 90% as
•f effective as the performance of the appropriate standards included in the
£ same test.
g. 3.2.C.2.- Leaf phytotoxicity — (including necrosis, malformation, etc.).
f Average of 2.5 or less (0-5 scale). Leaf drop and stalk girdling are not
- to exceed appropriate standards.
K
l> 3.2.C.3. Yield, quality based on U.S. standard grades, tobacco company
jf evaluations, physical and chemical properties, and smoke taste tests must
•f compare favorably with appropriate standards.
M
k- - - . •
g . r 3.3. Regional advanced tests. After successfully passing the preliminary
| "tests.,.-chemicals'-may'be entered in advanced tests, also conducted by State
K . and Federal scientists-Committee member's. * ' i '.';."' '•'•••/'-
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Table 1. Chemical and Physical Property Analyses
Flue-Cured Tobacco
Burley Tobacco
Nicotine or total alkaloids
Reducing sugars
Total volatile bases
minus nicotine
Total ash
Equilibrium moisture content
Filling value
Nicotine or total alkaloids
Total nitrogen
Total volatile bases
minus nicotine
. Total ash
Equilibrium moisture content
Filling value
Alkalinity number of water-
soluble ash
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3.3.a. General considerations.
3.3.a.l. To enter the Regional Advanced Evaluations, an entry should
have passed Regional Preliminary Evaluations.
3.3.a.2. The identity of the active ingredient of an entry should be
revealed as public information. The overall coordinator may request the
chemical composition of the complete formulation on a confidential basis.
3.3.a.3. The company is to supply all information available on toxicity
to humans, animals, etc. Any information available on the breakdown, per-
sistence, action, etc., of the chemical should also be supplied.
3.3.a.4. The company is to supply sufficient quantities of the formulated
chemical to conduct the evaluation at all locations proposed. The formulated
chemical appropriately packaged is to be shipped by the company to the indi-
vidual locations by specific dates supplied by the Committee.
3.3.b. Field test and data procedures.
3.3.b.l. Suggested distribution for flue-cured tobacco, 6 locations;
for burley tobacco, 4 locations; for cigar filler tobacco, 2 locations;
for dark tobacco, 2 locations; and for Maryland tobacco, 1 location.
3.3.b.2. Plot size ~ randomized block design with 4 replicates, 30
plants/replicate.
3.3.b.3. Agronomic data, including: . . • •
— Efficacy based on percent sucker control by weight, sucker number
per plant, and weight per sucker;
— Leaf phytotoxicity data (including necrosis, malformation, etc.)
taken at maximum manifestation (0 to 5 scale). Leaf drop and stalk
girdling information is to be reported; and
— Yield and quality based on U.S. standard grades.
3.3.b.4. Cured leaf data, including:
— Usability, body, and,texture for flue-cured, and usability and
quality rating for burley (by location) is to be evaluated by tobacco
companies;
— Chemical and physical property determinations by locations (see
Table 1); and .
• • — .Evaluation of .smoke .taste, fo-r the identification of unacceptable
off-taste due to treatment. .Tobacco companies conducting smoke panels
should know, the identity-of the applied chemicals.. Cigarettes will be made
from a composite sample by treatment for each class .(fLue-cured .and burley).
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3. 3. c. Procedures for passing the regional advanced test.
3.3.c.l. Sucker control — based on sucker weight, control must be 90%
as effective as the performance of the appropriate standards included in
the evaluation.
3.3.C.2. Leaf phytotoxicity — (including necrosis, malformation, etc.).
Average of 2.5 or less (0 to 5 scale). Leaf drop and stalk girdling are not
to exceed appropriate standards.
3.3.C.3. Yield and quality based on U.S. standard grades must compare
favorably with appropriate standards.
3.3.C.4. Tobacco company evaluations — The cured leaf from treated
plants must compare favorably with appropriate standards.
3.3.C.5. Chemical and physical properties — When the data are statisti-
cally analyzed, the 0.5% level of significance will be used to determine if
the specified chemical and physical properties compare favorably with approp-
riate standards.
— Flue-cured — The specified chemical and physical properties should be
within the range of the standard hand-suckered check and the standard sequen-
tial treatment, or be better than the hand-suckered check.
— Burley — The specified chemical and physical properties should be
within the range of the standard hand-suckered check.and the standard
chemical treatment, or be better .than the hand-suckered check. .
3.3.C.6. Smoke taste — These tests are designed to ascertain if the
chemical treatment imparts an off-taste to the smoke of cigarettes manufac-
tured from treated tobacco. They are to be conducted as comparison tests
using a known check to compare a treated sample. A coded check sample will
be used as an internal standard. In addition to the simple evaluation of
"acceptable" or "not acceptable," the percentage and the number of panelists
will also be reported by each tobacco company making smoke panel tests.
Each company is to specify which treatment, if any, is considered objection-
able. If a majority of the companies reject the treatment, after the internal
standard is considered, the treatment will be dropped from further evaluations.
3.4. Company proprietary tests. The chemical company sponsoring a chemical
is responsible for conducting these final "on-fann"-type.tests. All data is
to be submitted to the Regional Committee for evaluation.
3.4.a. General considersations.
3.4.a.l. Using the same formulation evaluated in the advanced and/or
preliminary tests, the company is to conduct field tests at minimum of 3
flue-cured and/or 2 burley tobacco locations.
3.4.a.2. Applications should be made with mechanical sprayers similar
to those used in the commercial production of tobacco..
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205
3.4.b. Field test and data procedures.
3.4.b.l. Distribution of field locations (minimum number). For flue-
cured tobacco, 1 southern (Fla., Ga., or S.C.); 1 eastern (N.C.); and 1
old belt (N.C., Va.). For burley tobacco, 1 high elevation (N.C., Va.)
and 1 lower elevation (Tenn., Ky.).
3.4.b.2. Suggested plot size — 1/10 acre when as single or l/40th acre
small plots replicated three times. Locations within the same general area
can be considered as replicates. Data are to be taken on at least three
10-plant subsamples. One 30-plant topped-but-notsuckered plot is to be
included for calculation of percentage sucker control.
3.4.b.3. Agronomic data, including:
— Efficacy based on percent sucker control by weight, sucker number per
plant, and weight per sucker; and
— Phytotoxicity at maximum manifestation (0 to 5 scale). Leaf drop and
stalk girdling information is to be reported.
3.4.C. Modified chemicals and/or formulations. Modified chemicals and/or
formulations going through Company Proprietary Evaluations which have not
been subjected to smoke tests will require such tests in comparison to
appropriate standards. Approximately 25 Ibs. of cured tobacco are required
from each treatment for making cigarettes used for smoke taste evaluations.
. 3.4.d. Evaluation standards. -
3.4.d.1. Treatments (applied in accordance with label instruction):
— Contact — fatty alcohol (FA) normal primary octyl-decyl or decyl
alcohols.
— Systemic — diethanolamine salt of maleic hydrazide (MH).
— Sequential — fatty alcohol followed by maleic hydrazide.
— Untreated — Handsuckered (HS) (suckers are to be removed when they
are 4 to 6 in. long) and topped-and-not-suckered (TNS).
3.4.d.2. Treatments for each tobacco type:
. . — Flue-cured — The standard treatments should include the TNS, HS, and
the sequential treatments except that the HS is not required in Company
Preliminary and Company Proprietary Evaluations.
. — Burley — The standard,treatments should include the 'TNS, HS, .and the
MH treatments, expect that the.HS treatment is not required.in Company
Preliminary and Company Proprietary Evaluations..
3.4.d.3. The degree of sucker control, is meas.ured as percent -by which
treatment reduces sucker/green weight compared with. TNS-.check, - 90.% as ••''...
effective'as appropriate'standards .included in the evaluations. .
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206
3.4.d.4. Leaf phytotoxicity is rated either as:
— Leaf injury — on a 0 to 5 scale (0 = none; 1 = very slight; 2 = slight;
3 = slight to moderate; 4 = moderate; and 5 = severe).
Rating to include necrosis, malformation, etc.; or
— No leaf drop or stalk girdling compared to appropriate standards.
3.4..d.5. Tobacco company evaluations (visual): •
— The data should include percent usable cured tobacco by the evaluating
tobacco companies (average and total usable tobacco by at least one company).
— Regional Preliminary — report percent usable only.
— Regional Advanced — report flue-cured, percent usable, and leaf body
and texture evaluations; burley,. percent usable and quality rating.
— The cured leaf from treated plants must compare favorably with the
standard HS and sequential treatments for the flue-cured and with the
standard HS and the chemical treatment for the burley tobacco class.
3.4.d.6. Chemical and physical properties:
— When the data are statistically analyzed, the 0.05 level of significance
will be used to determine if the specified chemical and physical properties
compare favorably with the standards.
— Flue-cured — The specified chemical and physical properties should be
within the range of the standard HS check and the standard sequential treat-
ment, or be better than the HS check.
— Burley — The specified chemical and physical properties should be with-
in the range of the standard HS check and the standard chemical treatment,
or be better than the HS check.
References.
Collins, W.K., S.N. Hawks, Jr., and B.U. Kittrell. 1972. Effects of
contact and systemic sucker control agents applied in button and early
flower stages on yield and value of flue-cured tobacco. .lob. Sci.
16:134-135.
Seltmann, H. , and H.V. Marshall, Jr. 1967. • Pack sprayer for research
plots. Tob. Sci. 11:100-101.
Seltmann, H., and J.A. Priest. 1972. Simulation of machine application
with hand-held equipment. Tob. Sci. 16:100.
Smith, B.C., L.A. Link, G.L. Steffens, :and W.O. Atkinson. 1971. Regional
tests with contact, and systemic tobacco sucker control agents. III.
. Fire-cured tobacco. Tob. Sci. 15:87-89.
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207
Spaulding, D.W., G.L. Steffens, and J.H. Hoyert. 1970. Regional tests.
with contact and systemic tobacco sucker control agents. IV. Maryland
tobacco. Tob. Sci. 14:98-100.
Steffens., G.L., D.W. Spaulding, Fred Clark, Z.T. Ford, H.W. Lundy, J.D.
Miles, M.J. Rogers, H. Seltmann, and J.F. Chaplin. 1969a. Regional
tests with contact and systemic tobacco sucker control agents. I.
Flue-cured. Tob. Sci. 13:113-116.
Steffens, G.L., D.W. Spaulding, W.O. Atkinson, C.E. Bortner, L.A. Link,
B.C. Nichols, H.F. Ross, H. Seltmann, and Luther Shaw. 1969b. Regional
tests with contact and systemic tobacco sucker control agents. II.
Burley tobacco. Tob. Sci. 13:117-120.
B. Control of tobacco leaf ripening.
1. Scope.
1.1. Chemical ripening agents which hasten leaf yellowing may allow for
the more efficient utilization of harvesting and curing equipment by enabling
enabling the grower to harvest and cure tobacco when the equipment normally
would be idle. The producer may also be better able to program the harvesting
and curing of crops which tend to mature faster or slower than normal due to
weather conditions. It has been shown that tobacco leaves chemically ripened
in the field undergo chemical changes and loss of moisture which normally
occur in the yellowing stage of curing. Because of these changes, the length
of the curing cycle can be reduced by one or two days which results in increas-
ed barn availability and a slight reduction in usage of curing fuel and elec-
tricity in bulk barns.
1.2. Time and care are also involved in curing and handling the air-cured
tobacco classes. Chemical treatments which cause some loss of moisture prior
to harvest would allow more efficient utilization of barns because the tobacco
could be spaced closer together. Chemical treatments which shorten the curing
time would also reduce the risk of barn rots and other similar problems.
1.3. In an effort to hasten yellowing of tobacco that remained in an
immature state, plants have been treated with herbicides (Seltmann and
Peedin, 1968). It has long been known that ethylene gas can have dramatic
physiological effects on plants. Gases and mixtures of gases that include
ethylene have been introduced into curing barns in order to hasten the
uniform development of desired leaf color. The effects on plants of chemi-
cals which act through an ethylene evolution system have been reported
(deWilde, 1971). Such chemicals have been evaluated on tobacco and have
been found to have potential for yellowing leaves under field conditions
and thus have practical implications (Cutler and Gaines, 1971; Miles et al.,
19.72; Steffens et al., 1970). The concept of controlled yellowing or ripen- •
ing. of tobacco has gained considerable interest, and candidate chemicals
as well, as mahagemeftt-^chemical regimes are being actively sought arid eval-
uated. • V ' ; '•- ;.: ": ' • . :'-• : •- -„• : '
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1.4. As in the case of tobacco sucker control agents, the Regional Tobacco
Growth Regulator Committee has generated a set of guidelines for tobacco
ripening agents which parallels the sequential evaluation 'scheme for such
control agents. The previous section (II.A.) of this Appendix must be read,
since, with the exception of efficacy-related data, data considerations address-
ing tobacco classes, test locations and sequence, plot sizes, phytotoxicity,
yield and quality, and cured leaf data (percent usable, chemical and physical
properties, and smoke taste) are identical.
2. Data considerations are as follows:
2.1. Company preliminary evaluations. Data are to be,obtained on the
exhibition of the "ripening" response, speed of ripening, and color devel-
opment or reduction in curing time in the barn compared to appropriate
standards.
2.2. Regional preliminary evaluations. . •..-•"
2.2.a. Efficacy data — Record degree of ripening in relation to standards
by estimating percentage of yellowing and uniformity of color development
with respect to time. Record length of curing time compared to standards.
2.2.b. Requirements for passage of regional preliminary evaluations —
include the obvious increase in the rate of yellowing and/or curing.
2.3. Regional advanced evaluations.
2.3.a. Efficacy data —Record degree of ripening in relation to
standards by estimating percent of yellowing and uniformity of color
development with respect to time. Record length of curing time.
2.3.b. Requirements for passage of regional advanced evaluations:
2.3.b.1. Efficacy — Obvious increase in the rate of yellowing and/or
curing.
2.3.b.2. Tobacco company evaluatons — The cured leaf from treated
plants must compare favorably with the appropriate standards.
2.3.b.3. Chemical and physical properties — Specified chemical and
physical properties should compare favorably with those of the standard.
2.4. Company proprietary evaluations. Efficacy data recorded should
include the ripening of the treated leaf, the rate of color development,
and the uniformity of ripening of the average leaf. The effect on curing
time in the barn should be recorded.
2.5. Evaluation standards.
2.5.a. Ripening:
2.5:..a.1. Flue-cured — Conventional harvesting and curing procedures
are to serve as the standard for chemical ripening agents. An ethephon
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treatment should be included as a basis for comparison but is not to serve
as the standard.
2.5.a.2. Hurley and other air-cured classes — Conventional harvesting
and curing procedures will be the standard.
2.5.a.3. The method for the control of sucker growth on tobacco eval-
uated in ripening experiments should be uniform over all treatments in the
experiment.
2.5.b. Degree of ripening: The treatment should show an obvious improve-
ment in rate of yellowing and/or curing in comparison to conventional
practices, and the cured leaf from treated plants must compare favorably
with the appropriate standards.
2.5.C. Tobacco company evaluations (visual): The cured leaf from treated
plants must compare favorably with conventional harvesting and curing
practices.
2.5.d. Chemical and physical properties: The specified chemical and
physical properties should compare favorably with conventional harvesting
and curing procedures.
References.
Cutler, H.G., and T.P. Gaines. 1971. Some preliminary observations
on greenhouse-grown tobacco treated with 2-chloroethylphosphonic
acid at varying pH's.. Tob. Sci.. 15:100-102. . .
deWilde, R.C. 1971. Practical applications of (2-chloroethyl)phosphonic
acid in agricultural production. HortSci. 6:364-370.
Miles, J.D., G.L. Steffens, T.P. Gaines, and M.G. Stephenson. 1972.
Flue-cured tobacco yellowed with an ethylene releasing agent prior to
harvest. Tob. Sci. 16:71-74.
Seltmann, H., and G.F. Peedin. 1968. 2,4-D to ripen tobbaco may be a
detriment to the producer and the industry. Flue-Cured Tobacco Farmer:
June.
Steffens, G.L., J.G. Alphin, and Z.T. Ford. 1970. "Ripening" tobacco
with the ethylene-releasing agent 2-chloroethylphosphonic acid.
Beitrage zur Tobakforshung 5:262-265.
C. Control of white (Irish) potato tuber sprouting.
1. • Types of plant regulator applications to .induce dormancy.
In recent years, several types/of plant regulator applications have
been used to successfully-induce dormancy: "'
Ca) .Applying the. product as .preharves-t applications; •'-...
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210
(b) Dipping tubers in a solution of the.-product before storage;
(c) Dusting the tubers with the product before storage;
(d) Applying the regulating chemical as a volatile suspendable solid to
the tubers while in storage; or
(e) Applying the product as a spray or wax solution to the tubers as they
are packaged for the retail market. Factors (such as storage temperature)
affecting potato sprout inhibition and methods of testing have been discussed
in several publications (Sparks and Summers/ 1974; Zaehringer et al., 1966;
Rao and Wittwer, 1955; Hruschka et al.; 1965; Nylund and Ayres, 1964; Cun-
ningham et al., 1966; and Weaver, 1972).
2. Test conditions. The usual procedure is to randomly collect about
400 tubers for each variable to be tested. They should be field-run quality
but with no green, rot, or serious malformations. These are divided into
4 replicates of 100 tubers each. The method of applying the inhibitor depends
upon the time at which the material is to be applied. Several dosage levels
of the chemical under test are necessary.
In the case of preharvest applications of plant regulators to potato
plants, variables (such as time of application, dosage of chemical applied,
time of harvest, and effect of rainfall) must be considered in addition to
subsequent storage conditions. Replicated plots (usually 4 replicates)
sufficiently large to allow statistical analysis should be used.
Treated tubers or tubers from treated plants should be stored at var-
ious temperatures, subjected to various airflows and treated with air of
different humidity levels to determine length of control (Sparks, 1965
and Sparks, 1973). Retreatment with the inhibitor may be required if the
material is volatile and high rates of airflow are used. For comparison
purposes, untreated tubers and tubers treated with standard sprout inhibitors
are to be included. Standard chemicals may include CIPC (isopropyl-m-chloro-
carbanilate), TONE (1,2,4,5-tetrachloro-3-nitrobenzene), maleic hydrazide,
or other chemicals registered for this use.
3. Interpretation of test results.
a. At monthly intervals for 12 mo., data on weight loss, rot, and sprouting
should be taken.
b. At the end of the storage period, other data are also usually obtained:
i. Weight loss.
ii. Jtot loss.
iii. Sprouting.
iv. Flattening and shriveling.
.-• v. .Effect on suberin and wound per idem formation.
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vi. Heat generated in the potato pile (a gross measure of respiration rate.
vii. Quality change (sugars, mealiness, fry color, flavor, chipping).
viii. Appearance change (loss of net or russetting).
ix. Utilization as propagation stock.
References.
Cunningham, H.H., M.V. Zaehringer, and W.C. Sparks. 1966. Effect of
storage temperature and sprout inhibitors on mealiness, sloughing,
and specific gravity of Russet Burbank potatoes. Am. Potato J.
42:209-222.
Hruschka, H.W., P.G. Marth, and P.H. Heinze. 1965. External sprout
inhibition and internal sprouts in potatoes. Am. Potato J. 42:209-222.
Nylund, R.E., and L.C. Ayr.es.. 1964. Sprout inhibition of table stock
potatoes with CIPC-treated paper bags. Am. Potato J. 41:341-348.
Rao, S.N., and S.H. Wittwer. 1955. Further investigations on the use
of maleic hydrazide as a sprout inhibitor for potatoes. Am. Potato J.
32:51-59.
Sparks, W.C.' 1965. Effect of storage temperature on storage losses
of Russet Burbank potatoes. Am. Potato J. 42:241-246.
- Sparks,: W.C. 1973. Influence of Ventilation and humidity during
storage on weight and quality changes of Russet Burbank Potatoes.
Potato Res. 16:213-223.
Sparks, W.C., and L.V. Summers. 1974. Potato Weight losses. Quality
Changes and Cost Relationships During Storage. Idaho Agr. Expt. Sta.
Bull. 535.
Weaver, R.J. 1972. Plant Growth Substances in Agriculture. W.H. Freeman
S Company: San Francisco. Pp. 170-171.
Zaehringer, M.Z., H.H. Cunningham, and W.C. Sparks. 1966. Sugar content
and color of Russet Burbank potatoes as related to storage temperature
and sprout inhibitors. Am. Potato J. 43:305-314.
D. Control of branch angle in young trees. In many trees, especially
apples and pears, the primary-branches form narrow angles with the main
trunk which result in structurally weak crotches that often break under
moderate or heavy fruiting and seriously reduce the productiveness of
the tree. Also, such narrow crotches are very prone .to winter injury.
Within; a few years the entire tree.may .be lost from further weakening
and secondary.infection. The. development of wide branch angles avoids
the bark inclusion in the' tree crotches and trees can bear heavier crops
when they begin fruiting. The more open growth-:a-lso permits more light
into :the center-of of the tree, which i's particularly use-ful on spur type.
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Delicious apples .o.r regular Delicious on dwarfing, stock. • Fa.ctpr.s affecting
chemical 'treatments to modify crotch angle and methods of testing to demon-
strate the efficacy of a chemical treatment have been discussed in several
publications (Verner, 1938; Bukovac, 1963, 1968).
1. Test conditions. Young fruit trees, usually in the second or third
year after being set in the field, are sprayed with several concentrations
of the test chemical. The correct timing of application must be determined
[usually when the new shoot growth is 3 to 5 in. (7.6 to 12.7 cm) long].
The angle at which the branch leaves the main stem should be measured at
the end of the growing season. The effect on vegetative growth also should
be measured.
a. Some factors affecting product performance;
i. Volume of spray may be important. The trees should be sprayed to
runoff until sufficient data show that lower volumes can be used.
ii. Since response to chemical treatment can vary widely with variety,
the effect on all varieties should be studied.
iii. Tests should be made for each variety to determine the rate
[Ib. active ingredient/100 gal. (kg/1) of spray solution and volume of
spray solution applied per tree] and the stage of development of the
tree at the time of application that will give the best response under
the environmental conditions where the product will be used.
iv. The effect of seedling vigor and rate of growth should be'deter-
mined, especially in relation to inhibition of vegetative growth.
v. The effect of rainfall following application should be determined.
vi. The effect of a repeat application the following year should be
determined.
b. Interpretation of test results. To support label claims for modifying
crotch angle development in fruit trees, the treatment should increase the
angle at which the branch leaves the main stem. This is more important than
just causing the outer part of the branch to bend downward. In general, the
angle should be greater than 45° to insure wide angle laterals for a sounder
structural framework in trees.
References
Bukovac, M.J. 1963. Wide angle crotches are essential for structural
strength in apple trees. Ann. Rpt. Mich. State Hort. Soc. 93:63-67.
Bukovac, M.J. 1968. TIBA promotes flowering and wide branch angles.
Am. Fruit Grow. 88(5):18.
Verner, L. 1938. The effect of a plant growth substance on crotch
angles in young apple trees. Proc. Am. Soc. Hort. Sci. 36:415-422.
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213
E. Control of alternate bearing in apples. Many varieties of apples
bear heavily one year ("on" year) and very little the following year ("off"
year). Chemical treatment (return bloom treatment) applied in the "on"
year before blossom differentiation can increase yield during the "off"
year. In addition to leveling out crop production for the 2-year cycle,
such treatments often result in an increase in total yield for the period.
1. Test conditions. A series of test trees, in the "on" year, should
be paired on the basis of uniformity of rootstock, tree size, and the
intensity of blossoming in the spring of the year. One tree of each
pair is sprayed with the chemical treatment; the other should remain
untreated. Each treatment should be sprayed to runoff until data are
accumulated to show that lower volumes are equally effective. Various
rates need to be tested on each variety to determine the optimum rate.
Timing of application must also be determined. (The optimum timing
usually is 4 to 5 weeks after full bloom.) To maintain uniform crop
production, it is necessary to treat only during the "on" year. Because
the degree of blossoming in the spring will determine those years in which
treatments should be made, studies are needed on each variety on the degree
of spring blossoming and the amount of return bloom the following season.
Since climatic conditions can affect performance, it is absolutely
necessary to test the compound in each location where it will be used.
Growers should be cautioned to start with a small block of trees until
they gain experience in the performance of the compound under their
environmental conditions. Any effect of rainfall following application
should be determined.
The effect of a blossom-thinning treatment or the occurrence of frost
on blossom set should be determined before the application of the return
bloom treatment. Studies with various degrees of blossom thinning are
needed to determine the rate and effect of the return bloom treatment.
Tree vigor and size, as well as the mineral nutrition of the tree,
also can affect response.
2. Interpretation of test results. The effects of any treatment on the
control of biennial bearing will be noted in the year following treatment.
The yield should be taken at the end of the second season and compared
with untreated trees. However, since the objective is to even out the
productivity of the tree, yields should also be taken at the end of the
first season and the total yield for the 2 years should be compared.
Normally, yield alone will not give the total picture, so it is nec-
essary to grade the fruit and determine the market price for each grade.
This may show a greater cash return to the grower than if total yield
alone were the only criterion used. Return bloom treatments may also
.inhibit.vegetative 'growth, and an additional benefit may be ;a reduction
in the . a5s»unt..pf: pruning :hecessary> .There is also .a possibility of
delayed'bud growth in the spring which can., be helpful to the grower in
areas of late spring frosts. . .
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214
Some other effects which should be reported may include; that the
treatment can hasten or retard the maturity of the crop in the treatment
year; and/or the treatment may increase the severity of a physiological
disease known as "bitter pit." Factors affecting chemical treatments to
control alternate bearing in apples and methods of testing are discussed
in the references listed below.
References.
Bukovac, M.J. 1963. Chemical promotion of flowers. Am. Fruit Grow.
83(3):24.
Edgertbn, L.J., M.B. Hoffman, and C.G. Ebrschey. 1964. The effect
of some growth regulators on flowering and fruit set of apple trees.
Proc. Am. Soc. Hort. Sci. 84:1.
Greenhalgh, W.J. 1965. Alternate•cropping of the Delicious apple: The
effect of 2,3,5-triiodobenzoic acid spray on blossom bud initiation in
the "on" year. Aust. J. Exp. Agr. Anim... Husb. 5:482-486.
Stahly, E.A., and A.A. Piringer. 1962. Effects of photoperiod, light
quality, and two plant regulators on growth and flowering of Jonathan
apple trees. Proc. Am. Soc. Hort. Sci. 81:12.
F. Control of sex expression in cucumbers and squashes. The biochemical
control of sex expression in cucmbers and squashes aids in the production
of hybrid seed and also can be useful in increasing yield, especially
where once-over destruct harvest is .practiced. A chemical treatment is
made at the early seedling stage of growth to increase the number of pis-
tillate (female) flowers and to decrease the number of staminate (male)
flowers. These treatments bring about formation of female flowers at
lower nodes where normally only male flowers are formed in the standard
(monoecious) cucumber and squash varieties. Factors affecting this sex
response and methods of testing to demonstrate the efficacy of a chemical
treatment have been discussed in a number of publications (McMurray and
Miller, 1968 and 1969; Itobinson et al., 1969; Rudich et al., 1969; .Sims
and Gledhill, 1969; Weaver, 1972).
1. Test conditions. For sex ratio studies, field trials consisting
of single-row plots 8 ft. (2.4 m) long with 10 plants per plot are
adequate. Each treatment and control plot should be replicated 4 times.
For yield trials, however, single-row plots 40 ft (12.2m) long with
120 plants per plot should be used, with 6 replications per treatment.
The test plots should be located in the middle of a field containing
sufficient untreated monoecious plants to assure an adequate supply of
pollen. Application should be made to seedling plants to assure good
coverage (100 gal/acre) (1/ha).
To evaluate the effectiveness of a treatment on sex expression, the
sex of each flower at each node should b» determined on a weekly basis
during the blooming period. To determine the effect on yield, the plots
should be harvested twice a week, starting when the first fruit is at the
proper stage. For mechanical harvesting, -each plot should be harvested
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215
separately at the correct stage to take into account any delay or hastening
of maturity. In all cases, the yield should be separated by grade, and the
market price of each grade at each harvest date should should be determined.
2. Additional test conditions and variables that should be studied.
a. 5br hybrid seed production, the amount of seed per fruit should be
determined. Also, the percentage germination and the storage life should
be studied.
b. The response between varieties can differ significantly, and the
correct timing, rate, and number of applications should be studied on.each
variety under different environmental conditions.
c. The effect on delay or hastening of maturity should be determined.
d. Effects on fruit quality and fruit shape should be made.
e. Since the effect on sexual modification toward femaleness in the
treated plants may be temporary, studies should be made to determine the
length of effectiveness of each treatement.
f. The effect of rainfall following application should be determined.
g. The effect of the treatment on vine growth should be noted.
h. Any effect on time of flowering should be noted so that the pollina-
tion line can by planted at the proper time to. supply adequate pollen.
3. Interpretation of test results. To support claims as an aid to hybrid
seed production, the data should show an increase in the percentage of
female blossoms at the early stages of flowering. Seed production in the
treated fruit should not be decreased and the hybrid seed should have good
germination and storage life.
To support claims for increased yield, data are needed for yield and
grade for the period during which the crop is harvested and marketed.
Total yield during the growing season is often very misleading, especially
if a large percentage of the fruit falls in the low-priced grades. In
once-over destruct harvests, comparisons are needed between yield from
treated and untreated control plots harvested at the same stage of develop-
ment. If the chemical treatment hastens or delays the development of the
fruit/ it may not be necessary to harvest the treated and untreated control
plots at the same stage of development, but perhaps at different times.
The chemical treatment should not cause any adverse effect on fruit shape
or quality.
References.. ,'..... :,..._ ..-..'. .'-.-..•••.. ' •
McMurray,. A.L.,. !and, C.H, Miller. 1.968., Cucumber sex. expression modified
by 2-chloroethanephosphonic ac.id. Science 162:1397-1393.
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216
1969. The effect of 2-chlorethanephosphonic acid (Ethrel) on
the sex expression and yields of Cucumis sativus. J. Am. Soc. Hort.
Sci. 94:400-402.
Robinson, R.W., S. Shannon, and M. de la Gaurdia. 1969. Regulation of
sex expression in the cucumber. BioScience 19:141-142.
Rudich, J., A.H.'Halevy, and N. Ksdar. 1969. Increase in femaleness
of three cucurbits by treatment with Ethrel, an ethylene releasing
compound. Planta 86:69-76.
Sims, W.L., and B.L. Gledhill. 1969. Ethrel effects on sex expression
and growth development in pickling cucumbers. Calif. Agr. 23(2):4-6.
Sims. W.L., B.L. Gledhill, and B.J. Boyle. 1969. Effect of Ethrel on
Yield of Pickling Cucumbers, Field Experiments - 1969. Uhiv. of Calif.,
Davis, Ext. Release No. 129. 4 pp. (Nov. 1, 1969).
Weaver, R.J. 1972. Plant Growth Substances in Agriculture. W.H. Freeman
and Company: San Francisco. Pp. 212-221.
G. Control of fruit maturation and ripening. Plant regulators are
being tested experimentally and used commercially to achieve a number of
specific objectives related to fruit maturation and ripening. Included in
these objectives are: . :
(1) Enhancing pigmentation in cherries, cranberries and apples (Chaplin
and Kenworthy, 1970; Eck, 1969; Edgerton and Blanpied, 1968);
(2) Hastening or delaying maturity in peaches, tomatoes, and apples
(looney, 1972; Robinson et al., 1968; Unrath, 1971);
(3) The concentration of ripening in tomatoes, blueberries, and apples
(Iwahari and Lyons, 1970; Eck, 1970; Schomer et al., 1971);
(4) Improving uniformity of ripening and enhancing fruit quality of
.bananas (Russo et al., 1968); and
(5) Enhancing abscission and loosening fruits to facilitate mechanical
harvesting (Bukovac et al., 1969, 1971).
1. Test conditions. Test conditions that should be reported are plot
sizes, number of plants per plot, and number of replications and controls.
Information on product application should include: number of treatments;
rates of application; time of application; methods of application; a compar-
ison of dilute versus concentrated spray applications and low versus high
gallonage applications; and possible interaction with other chemicals applied.
Differential varietal responses are important and should be adequately inves-
tigated. Possible parameters in measuring growth regulator effects on the
maturation and ripening process incude such quality measurements as sugars,.„
acids, pigments, viscosity, firmness, dry weight, flavor, appearance, short-
and long-term storage quality., and size. Yield, also an important parameter,
might include measurement of total yield, marketable yield, grade, percent
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217
of crop mature at a single harvest, and yield distribution over the entire
harvest period. Residual effects should also be evaluated, particularly
as they may influence subsequent flowering and fruit set on perennial plants
and seeds of annuals*
2. Other conditions that should be studied.
a. Plant condition should be monitored, noting particularly any pos-
sible stress conditions.
b. Environmental conditions, particularly temperature, soil moisture,
and possible adverse weather conditions (before, during, and after treat-
ment) , should be monitored.
c. Soil residual effects on greenhouse and container-grown crops
should be measured.
d. Changes in insect and disease susceptibility should be noted.
e. Any chemical residue on the harvested fruit should be measured.
3. Interpretation of test results. Suggested interpretations in
terms of the quantitative level of response obtained from plant regulator
treatment might include:
a. A .15% improvement in yield, when yield is the primary objective;
. b..: Advance or delay of harvest by a week for a plant regulator
•treatment designed to modify the harvest season of particular crop;
c. A pigment enhancement of 50%, particularly in early harvested
fruit; and
d. Concentration of yield which results of 80% of the total harvest
occurring in a single picking for a plant regulator designed to concentrate
the harvest.
References.
Bukovac, M.J., F. Zucconi, R.P. Larson, and C.D. Kesner. 1969. Chemical
promotion of fruit abscission in cherries and plums with special reference
to 2-chloroethylphosphonic acid. J. Am. Soc. Hort. Sci. 94;226-230.
Bukovac, M.J., F. Zucconi, V.A. Wittenbach, J.A. Flore, and H. Inoue. 1971.
Effect of (2-chlorethyl) phosphonic acid on development and abscission of
maturing sweet cherry (Prunus avium) fruit. J. Am. Soc. Hort. Sci.
96:777-781.
Chaplin, M.H., and. A.L. Renwprthy.' 1970.- .The influence o:f sue cin-ic acid-
2,2-dimethylhydrazide on fruit ripening of the 'Windsor' sweet cherry. .
- J. Am. So c. Hort,.. Sci. 9St532-.536» . . .'.'. . ...
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218
Dostal, H.C., and G.E. Wilcox. 1971. Chemical regulation of fruit ripening
of field-grown tomatoes with (2-chloroethyl) phosphonic acid. J. Am. Soc.
Hort. Sci. 96:789-790.
Eck, P. 1969. Effect of pre-harvest sprays of Ethrel, Alar, and Malathion
on anthocyanin content of Early Black Cranberry (Vaccinium macrocarpon
Ait.). HortScience 4:224-226.
. 1970. Influence of Ethrel upon highbush blueberry fruit ripening.
HortScience 5:23-25. -
Edgerton, L.J., and G.D. Blanpied. 1968. Regulation of growth and fruit
maturation with 2-chloroethanephosphonic acid. Nature 219:1064-1065.
Iwahari, S., and J.M. Lyons. 1970. Maturation and quality of tomatoes
with pre-harvest treatment of 2-chloroethylphosphonic acid. J. Am.
Soc. Hort. Sci. 95:88-91.
Looney, N.E. 1972. Effect of succinic acid 2,2-dimethylhydrazide,
2-chlorethylphosphonic acid, and ethylene on respiration, ethylene ,
production and ripening of 'Redhaven1 peaches. Can. J. Plant Sci.
52:73-81.
Robinson, R.W., H. Wilczynski, F.G. Dennis, Jr., and H.H. Bryan. 1968.
Chemical promotion of tomato fruit ripening. Proc. Am. Soc. Hort.
Sci. 93:823-830.
Rom, R.C., -and K.R. Scott. .1971. - The effect of 2-chloroethylphosphonic •
acid (ethephon) on maturation of a processing.peach. HortScience
6:134-135.
Russo, L., Jr., H.C. Dostal, and A.C. Leopold. 1968. Chemical regulation
of fruit ripening. BioScience 18:109.
Schomer, H., M.M. Williams, and H.D. Billingsley. 1971. Effect of combin-
ations of growth regulators on maturity and quality of 'Tydeman's Red1
apples. HortScience 6:453-455.
Unrath, C.R. 1971. Effects of preharvest applications of ethephon on
maturity and quality of several apple cultivars. HortScienee 7:77-79.
H. Control of fruit abscission. Soon after mechanical harvesting of
fruits was tried, it became obvious that, in many cases, treatment of the
trees or vines with fruit abscission agents (chemical looseners) would be
beneficial, and that, with some types of mechanical harvesting equipment
such as air harvesters, it is absolutely necessary (Wilson and Coppock,
1968; 1969). Citrus, cherries, olives, apples, grapes, and pecans are
examples of crops whose harvest can be aided by the use of such compounds
(Weaver, 1972). Specific requirements for each crop may'vary somewhat,
but the overall principles involved appear to be similar. Although the
following test conditions relate primarily to citrus fruits, some examples
of problems encountered with.other crops area also included.. .
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1. Test conditions.
a. Method of application: The application method usually will be
by dilute or concentrated spray. Most of the current abscission chemicals
under investigation appear to function through contact rather than systemic .
action. A uniform spray coverage of trees and vines, therefore, is necessary.
Other methods of application should be evaluated as to their practicality
under field conditions (Wilson, 1966; 1967).
b. Timing and environmental conditions; Mast abscission agents produce
fruit loosening within 3 to 7 days following spray application, although
this may vary depending on the crop involved. With citrus, the abscission
agent can normally be applied at any time during the period when the fruit
is considered legally mature (Wilson, 1969).
Weather conditions may affect abscission chemicals (Wilson, 1969; 1972)
and should be studied. Although no loss of activity due to high temperatures
has been reported, abnormally wet and cold conditions may adversely affect
results either by washing off the material before it has time to act or
delaying or reducing its effects by slowing metabolic processes.
c. Consistency of performance; Failures caused by adverse environmental
conditions can occasionally be expected; unexplained failure occurring under
good environmental conditions, however, should be rare. Thus, inconsistency
in chemical performances should usually be interpreted unfavorably. The
effects of various root stocks should also be considered in evaluating results,
since, with citrus fruits, certain rootstocks produce fruit -which loosen more
readily when abscission chemicals are applied (Cooper and Wilson, 1971).
d. Physical measurements of chemical effectiveness; The most common
method used to determine degree of citrus fruit loosening is through use of
a pull-force measuring device (Hendershott, 1964). Most of these devices
measure straight pull-breaking force necessary to separate the stems from
the fruit. These pull forces may vary from a few grams for olives to 30 to
40 Ib (13.6 to 18.1 kg) for some citrus fruits. A measure of effectiveness
is a substantial reduction in the average pull-force measurements from that
of a control.
2. Interpretation of results. In evaluating abscission results, two
principal factors must be considered, depending on which of the methods of
removal discussed below is employed and the ultimate use of the fruit
(processing or fresh).
If fruit is to be shaken to the ground for mechanical or hand pickup,
maximum fruit loosening is desired. Under these conditions, there is a
high probability that substantial fruit drop will occur before harvest
(Wilson, 1973).., With citrus fruits, pull force will be reduced from 14 to
18 Ib (6.4 to 8>0 kg) for untreated fruit to. an average-of 2.5 Ib (1.1 kg.)
for treated fruit. Fruit drop of -20 to 50%. could be expected when fruit is
this loose. Under some .experimental conditions and sometimes under field
conditions, fruit'loosening will be so complete that the fruit will separate
from the stem.,when, touched .and prevent the-•fteraTstarement o-f pull force.
These results can.be .classed as ,"too loose-:to pull,,.11 ".a-H- fruit dropped;'" •
or some other appropriately descriptive .phrase.
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220
If fruit is to be shaken by a mechanical.device onto a catchframe,
fruit loosening should not be as complete as above. A chemical developed
for use with catchframes should loosen-, citrus fruit ideally to no less than
6 Ib (2.7 kg) average pull force.
Abscission agents are of two types: (1) chemicals which release ethyl-
ene or cause ethylene production by the fruit without peel injury; and
(2) chemicals which may cause superficial rind injury with resulting wound
ethylene formation. Although abscission not related to the action of ethyl-
ene may be possible, all present commercial abscission agents appear to
utilize the ethylene mechanisms. Many fruits, including citrus fruit intended
for fresh fruit utilization, apples, cherries, and (perhaps) olives and pecans
respond to chemicals with this mode of action.
Citrus fruits loosen readily when chemicals cause superficial peel injury.
Chemical damage to the rind is acceptable on citrus fruit for processing if it
does not cause fruit splitting and excess rotting under wet conditions (Wilson,
1972) or adversely affect juice flavor.
The amount of phytotoxicity which can be tolerated by trees and vines
before yields and/or fruit quality are adversely affected varies with the
crop and time of year. Ito specific percentages of leaf drop have been deter-
mined for citrus fruits which indicate the upper limits of phytotoxicity.
Experience has shown that a fairly large margin of safety is necessary.
With a crop such as apples, excessive leaf drop accompanying abscission sprays
applied in the fall occurs then anyway. With some other crops, economic
considerations may be such that some phytotoxicity can be- .tolerated.
Because mature fruits, leaves, immature fruits, and flowers are all
subject to abscission through basically the same physiological process, the
plant regulator(s) selected for abscission should, be specific to removal of
only mature fruits or nuts. The 'Valencia' orange, for example, poses a
distinct harvesting problem because its lengthy development period (11 to
18 mo) results in the presence of flowers of immature (green) fruits during
most of the harvest season. It is important, therefore, that any abscission
chemical selectively loosen mature fruit but cause minimum phytotoxicity
to flowers, immature fruits, or young growth which might be present.
References.
Cooper, W.C., and W.H. Henry. 1973. Chemical control of fruit abscission.
Pp. 475-524 in Shedding of Plant Parts. T.T. Kbzlowski, ed. Academic
Press: New York. 560 pp.
Cooper, W.C., and W.C. Wilson. 1971. Abscission chemicals in relation
to the harvest of 'Valencia' oranges. Proc. Fla. State Hort. Soe.
84:70-76.
Cooper, W.C., G.K. Rasmussen, B.J. Hagers, C.P. Reece, and W.H. Henry.
1968. Control of abscission-in agricultural crops and its physiological
basis. Plant Physio1. 43;1560-1576.
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221
Hendershott, C.H. 1964. The effect of various chemicals on the induction
of fruit abscission in 'Pineapple1 oranges. Proc. Am. Soc. Hort. Sci.
85:201-209.
Soule, J., W. Grierson, and J.G. Blair. 1967. Quality Tests for Citrus
Fruits. Fla. Agr. Exp. Sta. Circ. 315.
Wilson, W.C. 1966. The possibilities of using ethylene gas to produce
citrus fruit abscission under field conditions. Proc. Fla State Hort.
Soc. 79:301-304.
. 1967. Chemical abscission studies of citrus fruit. Proc.
Fla. State Hort. Soc. 80:227-231.
1969. Four years of abscission studies or oranges. Proc.
Fla. State Hort. Soc. 82:75-81.
. 1972. Field testing of weak acids for facilitating citrus
fruit harvest under Florida conditions. HortScience 7(1):21-33.
. 1973. A comparison of cycloheximide with a new abscission
chemical. Proc. Fla. State Hort. Soc. 86:56-60.
. 1973. Problems encountered using cycloheximide to produce
abscission of oranges. HortScience 8(4):323-324.
Wilson, W.C., and O.K. Ooppock. 1968. Chemical abscission studies of
oranges and trials with mechanical harvesters. Proc. Fla. State Hort.
Soc. 81:39-43.
1969. Chemical stimulation of fruit abscission. Proc. 1st
Int. Citrus Symp. 3:1125-1134.
Weaver, R. J. 1972. Plant Growth Substances in Agriculture. W.H. Freeman
& Company: San Francisco. Pp. 358-369.
I. Increased berry size of seedless varieties of table grapes. When
vines of Thompson or other seedless grape varieties are girdled at the fruit-
set stage, a large increase in berry size — sometimes as much as 100 percent
— usually results. Since about 1960, almost all Thompson Seedless grapes
intended for table use have been sprayed with 1 or 2 applications of the
plant regulator gibberellin. These treatments., along with girdling, increase
berry size in a uniform manner (Weaver, 1972).
1. Test conditions. The test chemicals are usually applied after
shatter (separation of calyptra from flower) following bloom (also the
normal girdling time). Four vines per replicate should be sprayed to wet
cluster area thoroughly. . -Use -5 .concentrations of. the test product and
compare results :w±th untreated contro-ls and with gibberellin at label rates.
2. Test results. At harvest the following measurements should be'made:
•/./a. ,Crop, weight,,pjer vine.. . • . . •"•'' .'":.' '• -. .'•""
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b. Average weight of 100 berries per vine to indicate berry size. (Take
berries from various cluster and from base, middle/ and apex of cluster.)
c. Brix value of juice taken with a refractometer.
References.
Stewart, W.S., D. Halsey, and F.T. Ching. 1958. Effect of the potassium
salt of gibberellic acid on fruit growth of Thompson Seedless grapes.
Proc. Am. Soc. Hort. Sci. 72:165-169.
Weaver, R.J. 1972. Plant Growth Substances in Agriculture. W. H. Freeman
& Company: San Francisco. Pp. 349-350.
J. Loosen compact-clustered varieties of Vitis vinifera (wine grapes).
The incidence of rot in seeded wine grapes is excessive in cultivars (varie-
ties) that produce compact clusters, for example, 'Zinfandel1 and 'Carignane'.
Berries are pushed off by other expanding berries, and the juice from the
fallen fruit fosters decay organisms. Tight clusters are also slow to dry
after rains. loosening of the cluster is a result of reduced set, cluster
elongation, and/or production of shot berries (Weaver, 1972); prebloom sprays
of the plant regulator gibberellin also loosen these clusters (Weaver and
McCune, 1959).
1. Test conditions. Test chemicals are usually sprayed onto the vines
2 to 3 weeks before bloom when the shoots are 15 to 20 in. (38.1 to 50.8 cm)
long; clusters should be 3 to 4 in. (7.6 to 10.2 cm) in length, but may range
from 2 to 5 in (5.1 to 12.7 cm) in length. Use 4 replicates of 4 vines per
treatment. Use 5 concentrations of the test product and compare results with
untreated controls and gibberellin at label rates for the variety. The clus-
ters must be thoroughly wet.
2. Test results. At harvest the following measurements should be made:
a. Estimate of looseness percentage. The compact control clusters are
considered to have 0 percent unoccupied space. Estimate that part of the total
volume of a cluster that is not occupied by berries.
b. Number of berries per cluster (for the determination of effect on
berry set).
c. Length of cluster from basal lateral to the apex of the cluster.
d. Berry size (average weight of 100 berries).
e. Degrees Brix value of juice taken with a refractometer.
f. Percentage of rot which has occurred in each treatment.
References.
Weaver, R.J. 1972. Plant Growth Substances in Agriculture. W.H. Freeman
& Company: San Francisco. Pp. 348-349.
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223
Weaver, R.J., and S.B. McCune. 1959. Response of certain varieties of
Vitis vinifera to gibberellin. Hilgardia 28:297-350.
K. Acidity reduction in citrus. The two main purposes for acidity re-
duction in citrus fruits are to produce early maturity of fruit and thereby
lengthen the marketing season, and to produce sweeter fruit. In analytical
chemical terms, "sweet" means "high ratio fruit" as measured by the standard
accepted Brix/acid determination. Maturity of citrus fruits generally follows
the pattern of gradual reduction of acid during the fruit season, and usually
there is a corresponding increase in soluble solids (sugars). Certain plant
regulators are used to induce acidity reduction in citrus fruits.
1. Test conditions. Field trials to reduce acidity may consist of
single-tree plots replicated 6 times. Materials are applied as sprays to
thoroughly cover fruit and foliage. With citrus fruits which maintain a
generally recognized low respiration rate, the time of application of the
sprays is usually 4 to 10 months before harvest. (The development of sprays
which would shorten this interval would be desirable.) At the present time,
maturity sprays are applied from postbloom until the end of the summer spray
period, although the most effective time is 1 to 6 weeks following bloom
— late March to early May.
Twenty fruit per tree, a total of 120 fruit per treatment picked at
random (inside and outside of the canopy), are taken from the treated,
control, and standard product trees at biweekly intervals 30 days before
and during the normal harvest period. The following measurements should
be taken:
a. Determine the % juice for each sample (total weight of squeezed
juice per unit weight of fruit).
b. Determine the % acid (total titratable acid) using phenolphthalein
as the indicator and 0.3125 N NaOH as the basic solution.
c. Determine Brix value for each sample.
d. Calculate ratio of Brix value to acid.
2. Interpretation of test results. At least 2 limitations to use of
an acidity-reducing compound should be considered:
a. Oranges, tangerines, grapefruit, tangelos, and tangelo hybrids can
be subject to excessive acidity reduction and produce a sweet fruit with an
undesirable "insipid" flavor.
b. "Flavor" of a fruit or fruit juice is made up of several components.
Although .a plant, regulator, may reduce juice acidity of immature fruit to what.
would be considered acceptable fox ^mature fruit, it. would not affect the raw,
immature flavors.also present .in the immature fruit. Normally these immature
,flavors diminish, at a Brix value, of 9.0 or higher. . '
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224
L. Apple scald inhibitors. Substantial information about scald, a
surface disfigurement particularly severe in apple fruits, is available, as
this problem has been widely studied for over 60 years. (Huelin, 1965;
Smock, 1961; Smock and Southwick, 1945).. The cause is still unknown or
indefinite. Because it is usually much worse on early pickings, it is asso-
ciated.with immaturity. Scald susceptibility varies from season to season,
so weather before harvest also is a factor. Many researchers believed that
volatile materials caused scald, since relatively high levels of esters in-
duce an artificial scald-like injury on apples; but this volatiles theory
is no longer widely accepted. Recently, Australian researchers (Pierson
et al., 1971) have suggested that oxidation products of alpha-famesene are
the direct cause of superficial scald. They further suggest that effects
of artificial antioxidants on the oxidation of alpha-farnesene could first
be measured in comparatively short tests before testing for control of scald
on apples•
For many years, use of tissue wraps containing mineral oil around each
fruit was the standard scald-control procedure. Plant regulating substances,
such as naphthaleneacetic acid (NAA) applied as a post-harvest dip, provided
considerable control but were not used commercially (Schomer and Marth, 1945).
Commercial scald control is now obtained by using 1 of 2 antioxidants (Smock,
1961): diphenylamine, and 1,2-dihydro-6-ethoxy-2,2,4-triinethylquinoline
(ethoxyquin). Both chemicals are registered for use on apples and provide
good scald control (Dewey and Dilley, 1964; Hardenburg and Anderson, 1962;
Huelin, 1964; Mattus and Rollins, 1968).
1. Test conditions.
a. Use freshly harvested applies, 50 to 100 replicate: More scald will
develop in control lots if fruit is picked 1 week before the best commercial
date. 'Stayman Winesap', 'York Imperial", "Rhode Island Greening", "Grimes
Golden", "Mclntosh", "Rome Beauty", and "Red Delicious" are scald-susceptible
cultivars.
b. Apply test product and appropriate standard products within the first
7 to 10 days after harvest. Dip or submerge apples for 10 sec. in test solu-
tions or suspensions. Keep test suspensions well-agitated.
c. Fruit temperature and treatment solution temperature should be mod-
erate (60° to 70°F) (15.6° to 21.1°C). Gold fruit will not take on a good
coating of test chemicals.
d. After treating fruit, tilt boxes or turn fruit to improve drainage
and prevent possible chemical burn in stem or calyx cavities. Examine for
initial visible residue.
e. To predict scald intensity, store treated and untreated fruit samples
in boxes with unsealed 1.5-mil polyethylene liners for 4 to 6 weeks at 70°F
(21.1°C (Smock, 1961).
f. To determine commercial scald control, store treated and untreated
fruit samples in unlined boxes for 5 to 6 months at 32°F (0°C)[38°F (3.3°C)
for 'Mclntosh'] with high relative humidity (90-95%-), then transfer to 70°F
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225
for 7 days to allow scald symptom development (Hardenburg and Anderson,
1972; Huelin, 1964; Mattus and ftjllins, 1963; Smock, 1961).
2. Interpretation of results.
a. Determine the number of fruit with no scald, slight scald, and
severe scald. Slight scald is that affecting 10% or less of the skin surface
and is light brown in intensity. Severe scald is that affecting more than
10% of the surface or is dark brown and markedly disfiguring.
b. Determine flesh firmness of fruit from treated and control fruit
samples using a fruit pressure tester to determine any adverse effect on
ripening or softening rate.
c. Examine fruit for "off odor," "off flavor," visible residue, lenticel
injury or chemical burn symptoms.
References.
Dewey, D.H., and D.R. Oilley. 1964. Control of Storage Scald of Apples.
Mich. State Univ. Ext. Bull. 470. 3 pp.
Hardenburg, R.E., and R.E. Anderson. 1962. Chemical Control of Scald on
Apples Grown in Eastern United States. U.S. Dept. Agr. Mktg. Res. Rpt.
538. 47 pp.
Heulin, F.E. 1964. Superficial scald, a functional disorder of stored
apples. II. Promoters and inhibitors. J. Sci. Food Agr. 15:227-236.
. 1968. Superficial scald, a functional disorder of stored
apples. III. Concentration of diphenylamine in the fruit after treatment!
J. Sci. Food Agr. 19:294-296.
Huelin, F.E., and I.M. Coggiola. 1970. Superficial scald, a functional
disorder of stored apples. VII. Effect of applied alpha-farnesene,
temperature and diphenylamine on scald and the concentration and oxida-
tion of alpha-farnesene in the fruit. J. Sci. Food Agr. 21:584-589.
Mattus, G.E., and H.A. Rollins, Jr. 1963. Apple Scald Control. Va.
Polytech. Inst. Circ. 940. 3 pp.
Pierson, C.F., M.J. Ceponis, and L.P. McColloch. 1971. Market Diseases
of Apples, Pears, and Quinces. O.S. Dept. Agr., Agr. Handb. 376. 112 pp.
Schomer, H.A., and P.C. Marth. 1945. Effects of growth-regulating sub-
stances on the development of apple scald. Bot. Gaz. 107:284-290.
Smock, R.M. 1961. Methods of Scald Control on the Apple. Cornell Univ.
Agr. Expt. Sta. Bull. 970. 55 pp.
Smock, R.M., and F.W. Southwick. 1945. Studies on Storage Scald of Apples.
Cornell. Univ. Agr. Expt. Sta.. Bull. 813. . 39 pp.
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226
III. Desiccants
Desiccants are intended to be applied to desirable plants or crop
plants for the purpose of altering plant parts by inducing or accelerating
the drying of plant tissues. The methods of evaluation of desiccation vary
with both the crops and investigators. Many investigators have developed
valid but varied techniques for evaluation of usefulness. Eor registration
purposes, testing under actual field conditions is currently the only accept-
able method for producing data for evaluating the usefulness of potential
desiccants. New protocols for evaluating desiccants are continually being
developed because both new uses and new chemicals are being sought. In all
cases, however, data obtained from field tests must support the label claims
for the product and must be representative of the results that the producer
can expect when the product is used in accordance with label instructions
under commercial conditions. The references presented are not intended as
an exhaustive list of methods, since the purpose of desiccation vary from
crop to crop, and many valid procedures exist. Referenced test methods are
presented for the crops on which desiccants are presently used.
References.
A. Use on cotton.
Miller, C.S., E.D. Cook, J.L. Hubbard, J.S. Newman, E.L. Thaxton, and
L.H. Wilkes. 1968. Cotton Desiccation Practices and Experimental
Results in Texas. Tex. Agr. Exp. Sta. MP 903.
Walhood, V.T. 1954. Effects of. defoliation and desiccation on some
fiber and boll properties. Proc. 8th Beltwide Defol. Conf., Nat.
Cotton Council, Memphis, Tenn. Pp. 67-69.
B. Use on sorghum. \
Bovey, R.W., F.R. Miller, and J.R. Baur. 1975. Preharvest desiccation
of grain sorghum with glyphosate. Agron. J. 67:618-621.
Bovey, R.W., and M.K. McCarty. 1965. -Effect of preharvest desiccation
on grain sorghum. Crop Sci. 5:523-526.
Bovey, R.W. 1969. Effects of foliarly applied desiccants on selected
species under tropical environment. Weed Sci. 17:79-83.
1968. Desiccation and defoliation of plants by different
herbicides and mixtures. Agron. J. 60:700-702.
Bovey, R.W., and P.R. Miller. 1968. Phytotoxicity of paraquat on white
and green hibiscus, sorghum, and alpinia leaves. Weed Res. 8:128-135.
Wilkins, R.J., and R.M. Tetlow. 1970. The effect of diquat and paraquat
used as desiccants on the moisture content of maize for silage. Weed
Res. 10:288-292.
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227
C. Pse on white (Irish) potatoes.
Ahrens, J.F. 1975. Vine killers on Katahdin potatoes in Connecticut.
Proc. NE. Weed Sci. Soc. 29:276-280.
Harrington, J.D. 1974. Effect of vine killers on top-kill and internal
quality of potatoes. Proc. HE. Weed Sci. Soc. 28:323-329.
Murphy, H.J. 1968. Potato vine killing, flmer. Potato J. 45:472-478.
Murphy, H.J. and M.J. Goven. 1976. Potato vine killing in Maine -1974.
Proc. NE. Weed Sci. Soc. 30:224-229.
Sieczka, J.B. 1976. The-use of UNI-N252 as a potato vine desiccant.
Proc. NE. Weed Sci. Soc. 30:230-234.
D. Use on sunflowers.
Gandy, E.E. 1976. Preliminary Sunflower Research Information. 25th
Oilseed Processing Clinic, New Orleans. La., 1975.
Shadden, R.C., J.A. Mullins, and T. McCutchen. 1970. Mechanical Harvesting
of Sunflowers in Tennessee. Intl. Sunflower Conf. Pp. 265-270.
Zimmer, D.E. 1972. Interesting Curiosity or Crop of the Future? Sun-
flowers. Crops and Soils Mag.; June-July.
E. Use on onions.
Isenberg, P.M., and M. Abdel-Rahman. 1972. Effectiveness of a new onion
top killer. HortScience 7:471-473.
Pendergrass, A., M.D. Gilbert, F.M.R. Isenberg, and K.J. Lisk. 1975.
Status of neo-decanoic acids applied to onion (Allium cepa L.) foliage
and phytotoxicity of neo-decanoic acids applied to organic soil. J.
Agri. Sci. Camb. 84:327-332.
F. Use on sugarcane.
Davidson, L.G. 1962. Effects of desiccants on sugarcane. Proc. 11th
Congress of the I.S.S.C.T., Mauritius.
Osenigo, J.R. 1976. Florida Sugarcane League, Inc. Personal communication.
IV. Defoliants.
Defoliants are intended to be applied to desirable plants or crop plants
for the purpose'of removing leaves by inducing or accelerating leaf abscission.
The methods of evaluation of defoliation vary with, both the crops and investi-
gators. Many-investigators have developed valid but varied techniques for
image:
228
evaluation of usefulness. For registration purposes, testing under actual
field conditions remains the only acceptable method for producing data
for evaluating the usefulness to potential defoliants. New methods and
protocols for evaluating defoliants are continually being developed because
both new uses and new chemicals are being sought. In all cases, however,
data obtained from field tests should support the label claims for the product
and must be representative of the results that the producer can expect when
the product is used in accordance with label instructions under commercial
conditions. The references presented are not intended as an exhaustive list
of methods, since the purposes of defoliation vary from crop to crop, and
many valid procedures exist. Reference test methods are presented for the
crops on which defoliants are presently used.
References.
A. Use on cotton.
Barker, G.L., J.A. Friessen, and R.O. Thomas. 1967. Effects of Chemical
Wilting Agents on Cotton Leaf Conditions and Mechanical Picking. Proc.
Beltwide Cotton Prod. Res. Conf. Pp. 71-77.
Cook, E.E.,- J.S. Newman, and E.L. Thaxton. 1964. Results of Cotton Harvest
-Aid Chemical Trials in the Blackland, El Paso and High Plains Area in
Texas. Proc. 18th Ann. Beltwide Cotton Defol. and Physiol. Conf. Pp. 1-8.
Hoover, M., and V.T. Walhood. 1974. Chemical harvest aids for cotton.
Calif. Agr. Ext. Serv. Pub. AXT-208.
Miller, C.S., and W.C. Hall. 1957. Effects of amino triazole salts and
derivatives on cotton defoliation, growth inhibition and respiration.
Weeds 5:218-226.
Miller, C.S., E.L. Thaxton, and C.A. Burleson. 1964. Harvest-Aid Chemical
Test Results in Texas, 1963. Proc. 18th Ann. Cotton Defol. and Physiol.
Conf. Pp. 14-16.
Miller. C.S., L.H. Wilkes, E.L. Thaxton, and J.L. Hubbard. 1971. Cotton
Wilt-Harvest and Wiltant Defoliant Effectiveness in Texas. Tex. Agr.
Exp. Sta. MP 1010.
Tharp, W.H., R.O. Thomas, V.T. Walhood, and H.R. Cams. 1961. Effects of
Cotton Defoliation on Yield and Quality. Agr. Res. Serv., U.S. Dept.
Agr. Prod. Res. Rpt. 46.
Thaxton, E.L., P.S. Canzono, A.J. Rutkowski, and L.H. Wilkes. 1967.
1966 Wilt-Harvest Test at Pecos, Texas. Proc. Beltwide Cotton Prod.
Res. Conf. Pp. 69-71.
Walhood, V.T. 1954. Effects of Defoliation and Desiccation on Some Fiber
and Boll Properties. Proc. 9th Beltwide Defol. Conf., Nat. Cotton Council,
Memphis, Term. Pp. 67-69.
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229
B. Use on deciduous hardwoods and ornamentals in nurseries.
Altken, J.B. 1973. Chemical defoliation of young budded pecan trees.
HortScience 8:50-51.
Larsen, P.E. 1966. Batassium iodide induced leaf abscission of deciduous
woody plants. Proc. ftmer. Soc. Hort. Sci. 88:690-697.
. 1967. Stimulation of leaf abscission of woody plants by po-
tassium iodide and alanine. HortScience 2:19-20.
1967. Five years results with pre-storage chemical defoliation
of deciduous nursery stock. Proc. Intl. Plant Prop. Soc. 17:157-172.
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Series 95: EFFICACY OF INVERTEBRATE CONTROL AGENTS
§ 95-1 General considerations.
(a)- Overview. A wide variety of pesticide products are used for
invertebrate control. These products are used in households, industry, and
agriculture, as well as in public health programs. They are applied against
a tremendous variety of pests by many different methods in numerous kinds
of formulations. Sections 95-1 through 14 provide guidance related to
efficacy test methods generally adequate to develop acceptable data on
invertebrate control pesticides.
(b) Applicable sections. (1) This section. The considerations contained
in this section apply to all studies in the series, from §§ 95-2 through -14,
unless one of these sections contains a specific consideration on the same
subject. In such a case, the specific consideration in the individual section
shall apply to the conduct of that particular study.
(2) Sections 95-2 through -14. Each of these sections contains
specific considerations related to the performance of studies for one group
of invertebrate control pesticides. These sections also provide suggested
performance standards, which are levels of pest control that should be
achieved through use of the pesticide product. These suggested performance
standards are to be used for guidance purposes only. [Also see paragraph
(d)(5) of this section.] .
(3) Section 90-2. Section 90-2 establishes general considerations
for evaluating the performance of invertebrate control .products. . .
(4) Hazards to non-target organisms. Subdivision E - Hazard Evaluation:
Wildlife and Aquatic Organisms, and Subdivision J - Hazard Evaluation:
Nontarget Plants, and Subdivision L - Hazard Evaluation: Nontarget Insects
contain requirements for data on the hazards to non-target species which
might result from use of a product. Sections in these two subdivisions
provide guidance on the kinds of information which must be reported if
adverse effects on non-target organisms are observed during an efficacy
.test.
(c) Definitions. The following definitions apply to all sections of
series 95 of this subdivision:
(1) An "invertebrate control pesticide" means any pesticide product
which is intended for preventing or inhibiting the establishment, repro-
duction, development or growth of, destroying, repelling or mitigating
invertebrate animals declared to be pests i/, including any member of the
i/ The pest names used in this unit on invertebrate control agents conform
to those listed in the "Common Names of Insects and Related Organisms" pub-
lished by the Entomological Society of America, 4603 Calvert Road, Box AJ,
College Park, Maryland 20740, and "The EPA List of Insects and Other Organ-
isms", Seventh Edition, published April 1982. Contact the Index Section,
ASIB-BFSD (TS-768), of the Agency for the latest update on invertebrate pest
names in the EPA listing.
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Class Insecta, other allied classes in phyla Arthropoda, Annelida, and
Mollusca, such as slugs and snails, but excluding nematodes. [Refer to
§ 162.3(ff)(10) and (11) of the FIFRA Sec. 3 regulations.]
(d) General considerations. (1) Efficacy data should be derived
from testing conducted under conditions typical of actual or proposed
use, or, where applicable, under controlled laboratory conditions which
simulate actual use.
(2) The test substance should be the formulated product evaluated at
various dosage levels including those dosage rates associated with its
proposed use. It should be tested under all techniques intended to be used
in applying the product.
(3) Data on the compatability of the test substance with other sub-
stances will be developed in accordance with § 90-2(a)(3)(i) through (vi),
.if the test substance will be used in sequence or with another substance.
(4) Data should demonstrate the effect of the test substance on various
life stages of pests, host plants or animals, fruit finish, yield, palata-
bility, marketability, carcass grade, or other significant factors. The
data should clearly establish the method of action of the test substance in
repelling, destroying, or mitigating pests.
(5) The efficacy of the test substance should be established with refer-
rence to the applicable suggested performance standard.
(i) The suggested performance standards contained in the following
sections are generally stated in terms of percent control, based on a
comparison of treated organisms and untreated control organisms. In certain
situations, the test substance may be evaluated in comparison to a product
of known efficacy. Under some other circumstances, the performance standards
are expressed as acceptable levels of damage.
(ii) The conditions under which the suggested performance standards
apply are listed generally in § 90-3(c) and the following sections. These
performance standards are not intended to be absolute or inflexible.
(iii) An analysis of variance and multiple range test or other appro-
priate statistical analysis should be conducted to determine the reliability
of data, when a question of relative effectiveness occurs.
(6) Dose response data should accompany applicable site/pest crop
combinations. The benefits such as increased yield, unblemished fruit,
reduction in nuisance pest levels, to be derived from each dosage rate to
be registered for control of a particular pest should be clearly defined and
reported. Dose response data for crops other than the pesticide site/pest
combination will be considered if submitted and referenced.
,(.e.) • Use of. §. 95-30 references. Section 95-30 (a) and...(b) and contains
references :.to.acceptable .test protocols.-for .evaluating t-he..efficacy of
invertebrate control agents when used in the'kind's of treatments described
:iii §'§95-2 through.-:14.. The reference-s contain criteria .covering .areas
.rsuch as.test.-design, site :selection., sample size., plot seleetio-n .and, manage--"':
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raent, application or treatment techniques and equipment, dosage selection,
levels of infestation (pest population), examination times, sampling and
evaluation procedures, statistical analysis information and reporting
procedures.
§ 95-2 Foliar treatments.
This section is concerned with efficacy test methods for invertebrate
control agents applied as foliar treatments to deciduous tree fruits, small
fruits, citrus and subtropical fruits, tree nuts, field crops, forage crops,
rangelands, and vegetable crops for protection from economic injury by
insects, mites, and certain other invertebrate pests.
(a) General considerations. The testing of a pesticide product for use
on food crops in home gardens should follow the same test procedures as are
appropriate for evaluating the product's commercial use, except that tests
with home use products should also include dosage levels higher than the
label-recommended rates.
(1) Site selection. Crop plantings should be selected which are
representative of cultivars (varieties), ages of plants, cultural practices,
and pest populations commonly encountered. For general considerations, refer
to § 90-2(a)(3)(iv). When selecting test sites, other factors such as
climate; texture, composition, and type of soil; and geographic location
should be considered. For general considerations, refer to § 90-2(b)(l) through
(3).
(2) Plot size. Plots of a size suitable for commercial applications
should be used. These should have dimensions large enough to avoid any
effects from drift onto untreated control and sampling areas. It may be
necessary to use several planting sites in the same immediate area to
deliver a form of replication where field dimensions preclude internal
replicate design.
(3) Number of trials. A minimum of 5 large-scale, geographically-
separated trials (e.g., when commercial application equipment is used) are
generally recommended, but the number of trials can vary somewhat due to the
accessibility of infestations, fluctuations in pest populations pressures,
behavior, insect resistance, and other important characteristics of the
target pest. The resistance problems may make it necessary to test in
certain geographic areas (e.g., greeribug on sorghum should be tested in
Trans-Pecos if a national label is requested).
(4) Application techniques and equipment. Data should be developed to
support conventional ground, aerial, or ultra-low volume applications and
crop/pest stage. -For general consideration, refer.to § 90-2(a)(3)(ii).
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(5) Evaluation and reporting procedures. The procedures used to
evaluate product performance should be specified. Reports should, include
for example, yield information, insect counts, percent damage, percent
marketable fruit, percent russeting, extent of catfacing, or other measures
for determining the effectiveness of the test substance. Results should
include the average infestation reductions throughout the sampled post-treatment
period compared with that in control plots or in preand post-treatment
periods. Raw data on pests counts, injury counts, or other measures of
control should be summarized. Any adverse effects such as phytotoxicity,
staining, discoloration, and other factors should be reported. For general
considerations, refer to § 90-2 (c)(l) through (3) and § 90-2 (d)(l) through
(4). • ,..-.--.- - ...... • -•••'•'•• - ....... , '• '. "'
-. '' f - • :
(b) Suggested performance standards. Unless otherwise specified,
these standards are presented on the basis of pre- and post-'treatment _ •••••
pest population counts from treated as well as untreated plots and standard
comparative teatments. All percentages of control refer to the performance
of the test substance (as determined by insect counts, yield data, and any
measures correlated to insect population pressures) against the vulnerable
stage(s) of the target pest, when evaluated according to a recognized
treatment program under actual field conditions.
(1) Fruit and nut crops. (i) Pome and stone fruits. (A) Apple •
maggot and other fruit flies. A minimum of 80% insect population reduction
(control), based on percent infested fruit, determined by counts of oviposi-
tional punctures and/or tunneling, made throughtout the growing season and
at harvest. Counts should include both dropped and harvested fruit.
(B) Aphids and leafhoppers. A minimum of 90% insect reduction.
Dormant treatments should give complete control of aphid eggs.
(C) Catfacing insects. A minimum of 90% control, based on evaluation
of catfacing injury of fruit prior to and at harvest.
(D) Major chewing insects. A minimum of 95% control of codling moth
and redbanded leafroller, and 80% control of tufted apple bud moth based
on percent fruit injury determined by counts made throughout the growing
season. Counts should include both dropped and harvested fruit.
(E) Foliage feeding mites. A minimum of 80% control where predatory
mites are present. A minimum of 99% control when no predatory mites are
present. Dormant treatments should give complete control of mite eggs.
(F) Scale insects. A minimum of 90% control, based on pretreatment
counts of live females before pesticide applications, and post-treatment
counts following the last pesticide application. Dormant treatments should.
give complete control based on pre- and post-application counts.
(G.). Twig borers on stone fruits. The twig borers include the oriental
fruit moth and.the peach twig borer. A minimum of 90% •con-'trol,. based on
counts of the number of. damaged terminals per tree -and .percent injured
fruit. Data...should include recorded percent injury from both dropped and
harvest fruit.". ' : -. •-.-.. , .'•' •-••.....'. ' ' •-..-""•
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•(H) Plum cruculio. A minimum of 90% control, based on percent infested
fruit, determined by counting egg-laying scars on fruit samples and number.
of larvae per 100 dropped fruit collected throughout the growing season and '
at harvest.
(I) Wood-boring insects. A minimum of 85% control determined from pre-
treatment poplulation estimates, and detailed examinations of the trunk and
larger limbs in the fall following the application of pesticide treatments.
(ii) Small fruits. (A) Grape insects. A minimum of 98% control of
grape berry moth, based on the comparison of injured berries for treated,
standard, and untreated plots. A minimum of 95% control of grape leafhopper
complex, based on pre- and post-treatment population counts on the number
of leafhoppers per leaf.
(B) Blueberry insects. Almost complete (99%) control of blueberry
maggot, based on larval counts obtained from post-treatment examinations.
(C) Strawberry insects. A minimum of 95% control of strawberry leaf-
roller, based on pre-treatment and post-treatment counts of the percentage
of leaves damaged.
(D) Cranberry insects. A minimum of 95% control of cranberry fruit
worm and cranberry tipworm, based on percentage of infested berries from
treated and untreated plots.
(E) Raspberry insects. A minimum of 95% control of raspberry fruit
worm and raspberry crown borer, based on comparison of injured fruits and
crowns in treated and untreated plots.
(F) Currant insects. A minimum of 90% control of currant borer based
on the number of injured shoots in treated and untreated plots. A minimum
of 95% control for currant fruit fly and currant aphid. Currant fruit fly
infestation is evaluated by examining berries to determine the number of
larvae and/or pupae in treated and untreated plots. Aphid control is
determined by pre- and post-treatment aphid counts.
(iii) Citrus and subtropical fruits. (A) Armored scale insects. A
reduction of 80% infestation over pre-treatment, based on 90-day post-treatment
counts, and 50% infestation reduction over pre-treatment based on 12-month
post-treatment counts.
(B) Soft scale insects. A reduction of 50% infestation over pre-treat-
ment, based on 12-month post-treatment counts.
(C) Other homopteran insects. A minimum of 95% control for whiteflies
and 80% control for mealybugs. Almost complete (99%) control for aphids,
based on an initial post-treatment count and 90% control based on a 14-day
post-treatment count.
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(D) Miscellaneous groups or species. No more than 10% scarred fruit
from thrips and less than one thrips per terminal, based on counts taken
within 4 weeks after treatment. Less than 15 orangeworm egg masses per
tree, based on per hour search basis counts. A minimum of 90% control for
fruitflies.
(E) Acari. A minimum of 80% control for all species of mites except
the citrus rust mite which should be almost complete 99% control of the
vulnerable stage based on 4-6 week post-treatment counts.
(iv) Tree nuts. (A) Armored scale insects. A minimum of 80% control
for all species, based on pre-treatment counts and post-treatment counts
taken not less than than 40 days after each treatment.
(B) Soft scale insects. A minimum of 80% control, based on pre-treat-
ment counts of the number of live scales on terminal growth or the number
of live scales on a circular area 15 mm in diameter randomly selected along
the midrib of each leaflet.
(C) Other homopteran insects. A minimum of 80% control of aphids,
based on pre- and post-treatment counts and expressed as aphids per leaflet.
A minimum of 90% control for spittlebugs, based on pre- and post-treatment
counts and expressed as the percent reduction in the number of terminals
infested with live spittlebug nymphs.
(D) Lepidopteran insects. Less that 5% infested nuts at harvest for
codling moth and filbertworm. Less than 30 infested fruit spurs per 300
fruit spurs per 6 tree subplot, based on post bloom counts for fruittree
leafroller. Harvest count should be less than 10% injured fruit for fruittree
leafroller. Less that 10% infested shucks or a 90% increase in the number
of nuts over number in untreated controls for hickory shuckworm. Less than
20% damaged kernels for naval orangeworm. A minimum of 80% control for
peachtree borer. Less than 15 worm-damaged terminals per count tree and
less that 10% worm-damaged harvested fruit for peach twig borer. Over 80%
retention of nuts on the tree indicates adequate control of pecan nut case-
bearer. A minimum of 80% control for pecan serpentine leafminer, and 90%
control for western tent caterpillar.
(E) Miscellaneous insects. Less than 15% damaged nuts, based on
harvest counts for the walnut husk fly and pecan weevil.
(F) Acari. A minimum of 80% control of European red mite and other
tetranychid mites. Less than one mite per leaf during 45 day post-treatment
counts for Pacific spider mite and twospotted spider mite.
(2) Field crops. For the following insect pests of corn, the data
should indicate that a product demonstrates effective control which is equal
to a better, than, other product, of known efficacy used as reference, standards.
.Efficacy is based upon:
(ij Corn. (A) Southwestern corn borer. Percent sta-Lk-. girdling., damage
to harvested ears and yield* . .......
'(.B) European corn borer. For' leaf-feeding f irst-genef atiori' European
corn, borers ,.va..visual;,.plant rating.-'damage scale of :lr.9j • for .second-generation
European ..corn borers, .number, of feeding tunnels determined by stalk evaluat-.
ions., borer damage-to harvested ears, and-yield. ; •.. . ...
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(C) Corn earwonn. United States standards for U.S. Fancy Grade fresh
.sweet corn permit no more than 10% damaged ears from all sources. Efficacy
is based on percent of injury-free ears, percent of worm-free ears, or
percent of worm-infested ears.
(D) Fall armyworm. Percent of injury-free or worm-infested plants
from observations made 1-7 days after the last insecticide applications.
(E) Corn rootworm adults. Adult counts are made pre-treatment and
1, 3, 7 and 14 days following applications. Soil core samples are taken
after the first killing frost, washed through custom-built corn rootworm
egg extractors, and the number of corn rootworm eggs counted. Egg counts
are used to estimate insecticide effectiveness in reduction the infestation.
(ii) Cotton. (A) Boll weevil. Based on percent damaged squares or
bolls per acre (hectare) correlated with subsequent harvest yield data.
(B) Bollworm and tobacco budworm. Based on percent damaged squares
or bolls per acre (hectare) correlated with subsequent harvest yield data.
(C) Lygus bugs.
harvest yield data.
A minimum of 85% control correlated with subsequent
(D) Mites. A minimum of 80% control, based on pre- and post-treatment
population counts.
(E) Pink bollworm. The data should indicate that a product demonstrates
effective control which is equal to or better than other products of known
efficacy used as reference standard which are determined by adult moth
counts from pheromone traps or weekly cutting and examination of bolls.
(iii) Peanuts. (A) Cabbage looper, green cloverworm, bollworm. A
mimimum of 80% control.
(B) Lesser cornstalk borer. A minimum of 90% control based upon a
reduction in the number of infested vines and/or pegs; or a 90% reduction
in the number of infested sample sites for whole-field applications where
pre-treatment data indicate a minimum of 30% of the sample sites as infested.
Those reductions should further correlate to yield increases as compared to
untreated sites to confirm the benefits of pesticide application.
(C) Granulate cutworm. The data should indicate a minimum of 90% control
based upon population counts (usually expressed as larvae per row ft.) and
reductions in damaged vines.
(iv) Sorghum. (A) Greenbug. A minimum of 90% control, based on pre-
and post-treatment insect counts, correlated with yield data.
(B) Sorghum midge. The data should indicate that a product demonstrates
effective control which is equal to or better than other products of known
efficacy used as reference standards. Population infestation is determined
by estimating the number of sorghum midges for a number of sorghum heads.
Level of sorghum midge infestation are determined by sorghum midge damage
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to individual florets. Yield data is considered to be the most appropriate
method of evaluating insecticide efficacy against the sorghum midge.
(C) Banks grass mite. Control is determined by pre- and post-treatment
counts of all life stages of this pest. Plant lodging data is considered to
be indicative of product efficacy of this part. Plant product-damage and/or
lodging data are also considered indicative of product efficacy when correla-
ted to mite counts.
(v) Grain crops; greenbug. A minimum of 90% control, based on pre-
and post-treatment insect counts, correlated with yield data.
(vi) Soybeans. (A) Soybean podworm, stink bugs. A minimum of 80%
control for soybean podworm and stink bugs, based on post-treatment counts
of live insects, usually 48 hours after treatment. Yield data aids in
supporting claims involving timing of applications according to economic
thresholds.
(B) Mexican bean beetle, bean leaf beetle. A minimum of 85% control,
based on pre- and post-treatment insect population counts. An estimation
of insect damage on a scale of 0 - 100% is appropriate for evaluating single
applications, but not multiple applications.
(C) Southern green stink bug. A minimum of 80% control based on post-
treatment insect counts of number of dead and living fourth instar southern
green stink bugs in artificially-infested caged tests.
(vii) Sugarbeets: beet armyworm, fall armyworm. A minimum of 85%
control, based on pre- and post-treatment insect counts.
(viii) Sugarcane: sugarcane borer. A minimum of 80% control, based
on the number of live and dead larvae in post-treatment counts, number of
bored points, and number feeding in the leaf sheath.
fix) Sunflowers: sunflower moth. A minimum of 80% control based on
the evaluation of insect numbers and damage on a minimum of 100 heads
collected after the last insecticide applicaion.
(x) Tobacco. (A) Tobacco budworm, tobacco hornworm, cabbage looper.
A minimum of 90% control for all of these insects, based on pre- and post-
treatment insect counts and leaf surface area damage.
(B) Green peach aphid, tobacco flea beetle. A minimum of 85% control.
The performance evaluation against the green peach aphid is usually based
on pre- and post-treatment counts of insects per leaf. The performance
evaluation for tobacco flea beetle is usually based on the number of holes
in treated plants compared to the holes in a like amount of leaves from
untreated check plants.
(.xi) Wheat. (A) Greenbug. A minimum of 90% control, .based..on pr.e-
.and post-treatment insect counts coupled with yield data.
"." .. ..(B) Pale western, cutworm-. A.minimum of .80% .contiol.^ based; on. population
r eduction ."determined' 'from soil samples evaluated at post-treatment intervals.
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(3) Forage crops: alfalfa, red clover , birdsfoot trefoil. (i)
Alfalfa weevil, Egyptian alfalfa weevil. A minimum of 80% control, based
on pre- and post-treatment counts of adults and larvae from treated and
untreated plots.
(ii) Spittlebugs. A minimum of 90% control, based on pre-treatment
nymphal stage counts and post-treatment counts before the new adults appear.
(iii) Potato leafhopper, aphids. A minimum of 80% control, based on
pre- and post-treatment counts correlated with a minimum of 90% control of
hopper burn.
(iv) Seed chalcids, plant bugs. A minimum of 80% control. For
seed chalcids, efficacy is evaluated by spliting seed pods at harvest to
determine shriveled versus plump seeds. Efficacy against plant bugs is
evaluated by counting nymphs in samples collected post-treatment in compara-
tive treated plots.
(4) Rangelands. (i) Grasshoppers. A minimum of 90% control, based
on population counts taken immediately prior to treatment (including dominant
species and life cycle stage) and post-treatment counts at appropriate inter-
vals, and compared to untreated control population fluctuations.
(ii) Range caterpillars. A minimum of 90% control, based on counts
immediately prior to treatment (include all larval stages) and at appropriate
post-treatment intervals estimated to embrace partial and maximum kill.
(iii) Harvester ants. A minimum of 90% control. Control evaluations
should be made post-treatment at appropriate intervals and expecially in
the spring of the year following application. Untreated check plots should
be included.
(iv) Imported fire ants. A minimum of 90% control based on counts
made for a minimum of 30 days for mound applications and 60 days for
broadcast treatments. Percent control is based upon the percent reduction
in both old and new active mounds in the treatment area as compared to
precounts and untreated controls.
(v) Black grass bug complex. 'A minimum of 90% control, based on pre-
and post-treatment population counts.
(vi) Chiggers. A minimum of 90% control, based on numbers of lesions
per bird (turkey) pre- and post-treatment throughout the test period. Refer
to § 95-8 (b)(vi). Grade of turkey as determined by Federal meat inspection
also used as a criterion for determining efficacy.
(5) Vegetables (field grown). (i) Crucifers. (A) Cabbage looper,
imported cabbageworm, diamondback moth, fall armyworm, beet armyworm,
garden webworm, Hawaiian beet webworm, corn earworm. A minimum of 95% con-
trol, based on pre- and post-treatment larval counts supplemented with rat-
ings of plant injury, yield .records, counts supplemented with ratings of
plant injury, yield records, and where appropriate, U.S. Grade crop evalua-
tion ratings. .
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•a
(B) Harelequin -bug-. A minimum of 95% control, based on pre- and post-
treatment adult and larval counts, supplemented with counts of characteristic
keg-shaped egg masses, plant conditions throughout the season either through
ratings or actual counts of the harlequin bugs or their feeding scars, yield
records, or unmarketable heads and their weights.
(C) Striped flea beetle, western striped flea beetle, western black
flea beetle, sinuate striped flea beetle. A minimum of 95% control, based
on pre- and post-treatment feeding scars on leaves. On seedlings, count
the number of injured plants per foot of row.
(D) Aphids and thrips. A minimum of 90% control, based on pre- and
post-treatment population counts of apterous aphids and thrips.
(ii) Cucurbits . (A) striped cucumber beetle, spotted cucumber
beetle, western spotted cucumber beetle, western striped cucumber beetle.
A minimum of 99% control, based on pre- treatment counts of adult beetles,
and post-treatment counts of adult beetles and fruit scars, correlated
with yield data (weight of fruit) format least 3 harvests.
(B) Squash bug;-' A minimum of 90% control, based on evaluation of
pre- and post-treatment adult feeding injury and actual counts of nymphs
per plot, coupled with yield data of marketable and non-marketable fruit
(including number, weight, and injured and malformed fruit).
(C) Squash vine borer. A minimum of 90% control, based on counts
of larvae, wilted plants, and borer tunnels, coupled with yield records.
(D) Pickleworm and melonworm. A minimum of 95% control based on
counts of percent infested fruit throughout the season.
(E) Melon aphid. A minimum of 90% control, based on pre- and post-
treatment melon aphid population counts coupled with foliage or tip
injury and yield data including marketable and unmarketable fruit.
(F) Cabbage looper. A minimum of 85% control, based on pre- and
post-treatment counts of the cabbage looper population coupled with
yield data.
(G) Mites . A minimum of 95% control, based on pre- and post-
treatment population counts.
(iii) Irish potatoes . (A) Colorado potato beetle. A minimum of
85% control, based on pre- and post- treatment population counts, coupled
with yield data, including evaluation of potatoes according to U.S. Grades.
(B) Potato flea beetle. A minimum of 95% control, based on counts
of pre-treatment feeding scars on leaves, post- treatment feeding scars on
leaves, and larval, injury to tubers, coupled with yield data and potatoes
evaluated according, to U.S. grades.
(C!) ; . £btafo. .Leafhopp'er. , A: minimum osf.'9S* ««ifeirol, .ba*sed on. .pre~-- -and
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post-treatment population counts of nymphs, number of injured plants andj ',
diseased tubers, coupled with.yield-data and potatoes evaluated according-
to U.S. grades. ' '
(D) European corn borer. A minimum of 75% control, based on number
of plants injured by the European corn borer, number of borers per plant,
coupled with yield data and potatoes evaluated according to U.S. grades.
(E) Potato psyllid. The test product should compare favorably with
other products of known efficacy used as reference standards. Control is
based on actual pre- and post-treatment counts of potato psyllid nymphs and
infested plants, coupled with yield data pertaining to U.S. marketability
grades.
(F) Potato aphid. A minimum of 80% control, based on pre- and post-
treatment population counts of apterous potato aphids.
(G) Potato tuberworm. The test product should compare favorably with
other products of known efficacy used as reference standards. The percent
control achieved is based on counts of the number of tubers damaged at
harvest time.
(iv) Lettuce: lettuce root aphid. The test product should demonstrate
efficacy equal to or better than products of known efficacy used as refer-
ence standards. Percent insect control is based on comparative evaluation
of root systems at harvest time to determine the presence of lettuce root
aphid damage.
(v) Lima beans, snapbeans, southern peas. (A) Lygus bugs. The test
product should demonstrate efficacy equal to or better than products of known
efficacy used as reference standards. Percent insect control is derived
from pre- and post-treatment population counts and percent Lygus-bug damaged
beans at harvest.
(B) Bean leaf beetle. A minimum of 95% control, based on pre- and
post-treatment population counts of the adult bean leaf beetle, at appro-
priate intervals supplemented by feeding hole counts, and inspection of
roots for larval injury.
(C) Bean aphid. A minimum of 90% control, based on pre- and post-
treatment population counts of all stages of the bean aphid.
(D) Cowpea curculio. The test product should demonstrate efficacy
equal to or better than other products of known efficacy used as reference
standards, based on counts of number of stings, feeding punctures, and
damaged peas at harvest coupled with yield data estimating loss due to
cowpea curculio injury.
(E) Potato leafhopper, beet leafhopper. A minimum of 90% control,
based on pre- and post-treatment population counts, coupled with an evalu-
ation of damage to foliage (potato leafhopper) and curly top damage (beet
leafhopper).
(vi) Peas. (A.)-. -Pea aphid. A-minimum of 90% control, based on pre-
• . • ' 'o-e-1 ' : v--'
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and post-treatment insect population counts.
(B) Pea weevil. (Reserved.)
(vii) Peppers. (A) European corn borer. A minimum of 95% control,
based on European corn borer injury determined by pepper dissection and
examination at harvest, coupled with yield data including percent marketable
fruit, percent loss due to the European corn borer, and number of "rots."
(B) Green peach aphid^ A minimum of 90% control, based on pre- and
post-emergence treatment and green peach aphid population counts.
(C) Pepper maggot. The test product should demonstrate efficacy equal
to or better than products of known efficacy used as. reference standards,
based on examination of fruit at harvest to determine the percent fruit :
infested with pepper maggots, pepper coupled with yield data indicating the
loss due to pepper maggot.
(viii) Sweet corn. (A) Corn earworm. A maximum of 5% damaged ears
for fresh market corn and 25% damaged ears for all other corn.
(B) Fall armywona. The product should demonstrate efficacy equal to or
better than other products of known efficacy used as reference standards,
based on post-treatment counts of the fall armyworm and fall armyworm injury.
(C) Corn flea beetle. A minimum of 95% control, based on number of
the corn flea beetle per specified number of sweet corn plants per plot,
the incidence of bacterial wilt coupled with yield data (noting numbers,
weight, size, and general conformation of the ears) from examinations at
appropriate intervals.
(D) European corn borer. A minimum of 95% control, based on post-
treatment counts at appropriate intervals of the European corn borer and
their injury (including the number of larvae, total number of tunnels, and
number of infested plants).
(ix) Poled tomatoes: tomato fruitworm, tomato pinwonn. A minimum
of 90% control, based on examinations at appropriate intervals to determine
percent damaged fruit. Extreme care should be exercised to differentiate
between tomato fruitworm and tomato pinworm damage.
(x) Tomatoes. (A) Tomato fruitworm. A minimum of 90% control.
Refer to poled tomatoes, paragraph (b)(5)(ix) of this section.
(B) Colorado potato beetle. A minimum of 95% control in direct-
seeded tomatoes. A minimum of 80% control for summer applications.
(C) Potato aphid. A minimum of 90% control, based on .pre- and post-
treatment potato aphid population counts and number of injured plants.
(6) Vegetables (greenhouse) . . (i.) Tomatoes , lettuce, cucumber, radish,
watercress., endive. (A) Aphids. A minimum of 90% control, based, on pr-e- :
-and post-treatment aphid population counts. .-..'.-
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(B) Leaf-eating beetles. A minimum of 90% control, based on pre- and
post-treatment beetle population counts. • .... ..-•.. ..
(C) Cutworms. A minimum of 90% control, based on post-treatment
counts of dead larvae found in soil depressions around the bases of
injured plants, and percent cutworm-injured plants.
(D) Garden symphylan. A minimum of 90% control, based on pre- and
post-treatment population counts at appropriate intervals, of living garden
symphylans, as determined from soil samples including part of the root
zone of living plants.
(E) Greenhouse whitefly. A minimum of 90% control, based on pre-
and post-treatment population counts and leaf examinations to determine
percent; mortality of immature and adult stages.
(F) Leafminers. A minimum of 90% control, based on pre- and post-
treatment examinations of leaves from treated and untreated plants to
determine cessation of feeding, shriveling or killed larvae, and
examination of mines, correlated to indicate percent dead or living larvae.
(G) Leaf-eating caterpillars. A minimum of 90% control, based on
pre- and post-treatment insect population counts and plant injury ratings.
(H) Slugs and snails. A minimum of 90% control, based on post-
treatment counts of living and dead slugs in treated areas. Correlate
with number of damaged fruit and foliage injury pre- and post-treatment
to determine percent control.
(I) Spider mites. A minimum of 90% control, based oh pre- and
post-treatment mite population counts.
§ 95-3 General soil treatments.
This section is concerned with efficacy testing of invertebrate control
pesticides used as soil applications for control of major insect pests
of corn, sugar beets, peanuts, tobacco, and vegetables as well as a
separate discussion on control of imported fire ant (See § 95-3(c)).
(a) General considerations. The testing of pesticide products for
use on food crops in home gardens should follow the same procedures as
are appropriate for evaluating their commercial use.
(1) Site selection. Crop plantings should be selected which are repre-
sentative of cultivars (varieties), ages of plants cultural practices,
and pesticide populations commonly encountered. When selecting a test site,
the soil texture, soil composition, and geographic location must be consider-
ed. For general considerations, refer to § 90-2 (a)(3)(iv), (b)(l) through (3).
(2) Plot 'size. Plots of size suitable for .commercial applications
should be used. These should have dimensions large enough to avoid any effects
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from movement into untreated control and sampling areas. Conditions may
dictate that large plots should not be replicated in one planting site which
may necessitate additional tests in different plantings.
(3) Number of trials. A minimum of 5 large-scale, geographically-
separated trials are generally needed, but the number of trials can
vary somewhat due to the accessibility of infestations, fluctuations in
pest population pressures, behavior, and other important considerations in
the biology of the target pest.
(4) Application techniques and equipment. In-furrow, broadcast, or
band applications should be evaluated if these methods are. specified on the
label. For general considerations, refer to § 90-2 (a)(3)(ii).
(5) Evaluation and reporting procedures. The procedures used to
evaluate product performance should be specified. Reports should include
yield information, insect counts, percent damage, or other measures for
determining the effectiveness of the test product. Results should include
the average infestation reduction throughout the sampled post-treatment
period compared with that in control plots or in pre- and post-treatment
periods. Raw data on pest counts, injury counts, or other measures of
control should be summarized. Any adverse effects such as phytotoxicity
should be reported. For general considerations, refer to § 90-2 (c)(l)
and (3).
(b) Suggested performance standards. Unless otherwise specified,
these standards are presented on the basis of pre- and post-treatment
pest population counts from the treated as well as the untreated lots
and standard comparative treatments. All percentages of control refer
to the performance of the test substance (as determined by insect counts,
yield data and other measures correlated to insect population pressures)
against the vulnerable stage(s) of the target pest, when evaluated according
to a recognized treatment program under actual field conditions.
(1) Field crops. (i) Corn. (A) Northern corn rootworm, southern
corn rootworm, western corn rootworm. The test substance should compare
favorably with other products of known efficacy used as reference standards,
when such comparisions are based upon both larval mortality and root damage
evaluations. It should show no adverse effects on yield. Use of a root rating
scale ranging from 1 (no damage) to 6 (severe damage), a rating no greater
that 3 is acceptable.
(B) Cutworms. A minimum of 85% control. In terms of retention, 85%
of the stand must be retained.
(ii) Peanuts. (A) Southern corn rootworm. A minimum of 90% control,
evaluated by examining random samples of pegs and pods at harvest, and
determining southern corn rootworm, injury.
(.B.) Lesser, cornstalk borer. A minimum of 90% control., based on pre-
.and post—treatment lesser cornstalk borer counts.
-.L; (C.) . Gr-anulate cutvoraU A minimum of '9iQ-%: control., based''ofa/pre*-.
and post-treatment living and dead larval population counts.
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(D) Burrowing bug. A minimum of 90% control, based on population
counts of adults and nymphs, obtained from soil samples taken at 10-14
day intervals after insecticide application, and from burrowing bug-
damaged kernels at harvest.
(E) Potato leafhopper. A minimum of 90% control, based on pre-
treatment and post-treatment adult and nymph potato leafhopper population
counts, and counts of the number of leaves with "hopperburn." Post-
treatment counts should be taken 10 to 15 weeks after treatment.
(iii) Sugar beets: sugarbeet root maggot. A minimum of 90% control,
based on damage ratings ranging from 1 (no damage) to 5 (severe damage). The
product must demonstrate a rating less than 2 (light damage) to be acceptable.
(iv) Tobacco. (A) Tobacco wireworm. A minimum of 90% control, based
on percent tobacco wireworm damaged plants 10-14 days after transplanting,
and root and stem damage within 4 weeks of transplanting.
(B) Darksided cutworm. A minimum of 90% control, based on percent
reduction of larval population from counts made as weekly intervals starting
one week after transplanting, and continuing until larvae pupate.
(C) Green June beetle. A minimum of 90% control, based on counts of
dead larvae on the soil surface approximately 6 weeks after seed germination,
when insecticides are applied just prior to seeding. For spring treatments
when larvae are active, count dead larvae 2 days post-treatment and continue
to determine mortaltiy for 1 week. Two weeks after treatment excavate the
treated area to a depth of 6 to 12 inches (15.2 to 30.5 cm) and count all
living and dead larvae.
(D) Tobacco flea beetle. A minimum of 90% control, based on pre-
and post-treatment tobacco flea beetle population counts, and feeding
punctures on damaged leaves throughout the season.
(2) Vegetable crops (field grown), (i) Cabbage, cauliflower,
broccoli, brussels sprouts, rutabaga, turnip, onion, beans, peas, potatoes.
For the following insect pests of vegetables, the data should indicate that
a product demonstrates effective control which is equal to or better than
other similar products of known efficacy used as reference standards.
(A) Cabbage maggot (cabbage, cauliflower, broccoli, brussels sprouts).
Control is determined from post-treatment counts of cabbage maggot-damaged
roots. The actual numbers of cabbage maggot tunnels or infested plants
are counted throughout the test period.
(3) Turnip maggot (rutabaga and turnips). Control is determined from
post-treatment counts of turnip maggot tunnels are counted throughout the
test period.
(C) Onion maggot (onion). Control is determined throughout the test
period by counting and removing injured plants until plant mortality cases,
and computing treatment effectiveness from the total plant stand.
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(D) Seedcorn maggot (beans and peas). Control is determined by
counting seedcorn maggot-injured plants per given area, and computing
treatment effectiveness from the total plant stand.
(E) Tobacco wireworm (potatoes). Control is determined by counting
the number of tobacco wireworm-injured tubers, coupled with yield data at
harvest.
(F) Southern potato wireworm (potatoes). Control is determined by
counting the number of southern potato wireworm-injured tubers, coupled
with yield data at harvest.
(G) Great Basin wireworm and Pacific Coast wireworm on potatoes.
Control is determined by counting the number of Great Basin wireworm
and/or Pacific Coast wireworm-injured tubers, coupled with yield data at
harvest.
(c) Imported fire ant. (1) Site selection. Sites should be selected
from several states within the range of the imported fire ant. Usually,
three such sites are necessary to provide information in the data derived.
(2) Plot size, (i) Mound application. In general, use a minimum of 10
mounds per plot. Usually, 3 to 4 plots are treated with the candidate
compound and 1 plot is used as a control.
(ii) Broadcast treatments. Plots should be of sufficient size to permit
the use of the application equipment specified on the label. Replication
may be difficult when very large plots are necessary.
(3) Suggested performance standards. (i) Control claims. A
minimum of 90% control based on counts made for a minimum of 30 days
for mound applications and 60 days for broadcast treatments. Percent
control is based upon the percent reduction in both old and new active
mounds in the treatment area as compared to precounts and untreated
controls.
(ii) Suppression claims. Broadcast treatments only A minimum of
80% control based on counts made for a minimum of 60 days and based upon
the adjusted percentage of reduction of the total number of old and new
active mounds in the treatment area and untreated controls. A lower
percentage' of control may be acceptable if testing was done under adverse
conditions, but in no case would less than 70% control be acceptable.
§ 95-4 Lawn and turf treatments.
This section concerns efficacy testing of invertebrate control
pesticides applied to lawns and turf for protecting these sites from
economic injury by insects and certain other invertebrate pests.
(a) General considerations. The testing of a pesticide, product for.
use on home lawns should follow the same procedures as are appropriate for
evaluating its commercial use.
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(1) Site selecion. Outdoor application sites should be selected from
areas where known infestations exist, and must reflect variations in climate
or other environmental conditions, edaphic conditions, and geographic dis-
tribution. Report test results from at least four different geograph-
ical areas where the pest is found. For general considerations, refer to
§ 90-1 (b)(l) through (3).
(2) Plot size. ' Plot sizes should be sufficiently large to accomodate
commercial application equipment. They usually range from 10" x 10' to a
size suitable for commercial aplications. The plots should have dimensions
large enough to avoid any movement of the test products into untreated
control or sampling areas. Conditions may dictate that large plots should
not be replicated in one planting site which may necessitate additional
tests in different plantings.
(3) Number of trials. A minimum of 5 large-scale geographically-
separated trials are generally necessary, but the number of trials can
vary somewhat due to the accessibility of infestations, fluctuations in
pest population pressures, behavior, and other important considerations
in the biology of the target pest.
(4) Experimental test design. A suitable experimental test design
should be used to reduce variability in the test data. Random observations
or pre-treatment counts should be used to determine population distribution
and to provide guidance in establishing plot location. Examples of experi-
mental designs generally used are randomized complete block design, complete
randomized design, and latin square design.
(5) Application techniques and equipment. Dusts may be applied with
a hand duster or lawn fertilizer spreader; granules and dry baits, with
a lawn fertilizer spreader; wettable powders and emulsifiable concentrates
(after dilution with water) with a compressed air or knapsack sprayer, a
hose proportioner or a watering can. For general considerations, refer to
§ 90-2 (a)(3)(i).
(6) Evaluation and reporting procedures. The procedures used to
evaluate product performance should be specified in the presentation of the
data Reports must include insect counts, percent damage or other measures
for determining the effectiveness of the test product. Results should include
the average infestation reduction throughout the sampled post-treatment
period compared with that in control plots in pre- and post-treatment periods.
Raw data on pest counts, injury counts, or other measures of control should be
summarized and submitted. Since grasses or other lawn plants differ signif-
icantly in their sensitivity to chemicals, and some high quality lawns and
golf greens represent a sizeable monetary investment, it is important to
observe any adverse effects such a phytotoxicity which might occur after
treatment. For general considerations, refer to §§ 90-2 (c)(l), (2) and
(d)(l) through (4).
(b) Suggested performance standards. These standards apply to control
of insect pests on all types of lawn turf grasses. Unless otherwise specified,
these standards are presented on the basis of pre- and post-treatment pest
population counts from the treated as well as the untreated plots and
image:
24.7
standard comparative treatments. All percentages of control refer to
performance of the test substance (as determined by insect counts, yield
data and other measures correlated to insect population pressures) against
the vulnerable stage(s) of the target pest, when evaluated according to a
recognized treatment program under actual field conditions. For (4), (5), •
(6), and (7) (below), the poduct should demonstrate effective control which
is equal to or better than other similar products of known efficacy used
as reference standards.
(1) Chinch bugs. A minimum of 95% control, expressed as knockdown
ability of the pesticide when counts are taken within 1 week of the data of
application. A minimum of 90% control is needed for additional residual
claims of greater than the 1 week period.
(2) White grubs. A minimum of 90% control based upon population
reductions of the most damaging larval instars when expressed as a factor
of the numbers of white grubs per unit area (sq. ft., sq. meter, etc.)
and compared to untreated controls.
(3) Sod webworms. A minimum of 90% control based on population
reduction counts obtained by counting larvae on a specified area (e.g.,
a 0.6m x 0.6m section of each plot) that emerge in 10 minutes as a result
of sprinkling 0.02-0.05% pyrethrum on the test area.
(4) Mole crickets. Different methods may be used to determine control.
The most common method is to count dead and moribund mole crickets on the
turf in each plot. Counts are made for four to seven days after treatment.
A second method is to drench a 1 m^ area with 21% pyrethum and count the
mole crickets that emerge within 15 minutes. A third method has been to
count surface burrows in open spaces after irrigation or rain.
(5) Spittlebugs. Control is based on the number of spittlebugs
determined in a specified size area of each plot; counts are made one,
three, and seven days after treatment.
(6) Lepidopterous larvae. Grass loopers of the genus Mocis and other
turf-feeding Lepidoptera (Spodoptera spp.). Based on pre-treatment and post-
treatment counts at 0, 24, and 72 hours.
(7) Margarodid scales (ground pearls). Based on counts made from
soil core samples taken during pre-treatment and at intervals of up to
one year after treatment.
§ 95-5 Outdoor woody ornamental plant treatments.
This section concerns efficacy testing of invertebrate control pesti-
cides used on outdoor woody ornamental plants for protection from economic
injury by insects and certain other invertebrate pests.. Such plants include
a large and constantly increasing number of cultivars, embracing more than
1000 species, 150 genera, and 60 families. The most common of these include
.arborvitae,. azalea., boxwood, camellia, chamaecxparis , cotoneaster., crabapple,
dogwood, .euonymus-, forsythia, holly, honey locust., juniper, laurel., lilac., -
magnolia, oleander, palm, privet, pyracantha, redbud, rhododendron, ...
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248
spirea, viburnum, and yew. ' . -
-Ca') General considerations. The -testing of. a pesticide product for "
use on woody ornamentals and shrubs in home gardens must follow the same
procedures as are appropriate for evaluating its commercial use.
(1) Site selection. Plantings for experimentation should be selected
which are representative of cultivars (varieties), ages of plants, cultural
practices, and pest populations commonly encountered. When selecting a
test site, other factors such as climate, soil texture, other substrates,
and geographic distribution must be considered. For general considerations,
refer to § 90-2 (a)(3)(iv), (b)(l) through (3).
(2) Plot size. Plot size should be sufficiently large to accomodats
commercial application equipment. Plots should be sufficiently separated
so that there is essentially no opportunity for drift on untreated controls
and sampling areas. Conditions may dictate that large plots not be
replicated in one planting; therefore, additionals test may be needed
at different locations.
(3) Number of trials. A minimum of 3 large-scale, geographically-
separated trials are generally necessary, but the number of trials can
vary somewhat due to the accessibility of infestations, fluctuations in
pest population pressures, behavior, and other important considerations
in the biology of the target pest.
(4) Application techniques and equipment. Application techniques
should be appropriate for the use contemplated and the size of the test
plots. Compressed air sprayers are frequently used and results are
comparable to those obtained from power equipment, provided that the same
dilution is used and the same amount of toxicant is applied to each plant.
Other application techniques such as aerial or ultra-low volume should be
evaluated if these methods are specified on the label. For general
considerations, refer to § 90-2 (a)(3)(i), (ii).
(5) Evaluation and reporting procedures. The procedures used to
evaluate product performance data should be specified. Reports should include
yield information, insect counts, percent damage, or other measures of
determining the effectiveness of the test products. Results should include
the average infestation reduction throughout the sampled post-treatment
period compared with that in control plots or in pre- and post-treatment
periods. Data on pest counts, injury counts, or other measures of control
should be summarized. Due to the complex nature of pesticide usage on
woody ornamentals and the sizable number of host plants involved, it is
necessary that plant tolerance to such applications be determined. This
is an integral part of effectiveness testing of pesticide products since
plants differ in their responses to chemicals, and a product registered
for use against a pest on one host may be registered for use against the
same pest on another host, provided phytotoxicity data indicates plant
tolerance. For general considerations, refer to § 90-2 (c)(l), (2), and
(d)(l) through (4).
(•b) Suggested performance standards. These standards apply to control
of insect pests on outdoor woody ornamental plants. Unless otherwise specified,
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these standards are presented on the basis of pre- and post-treatment pest
population counts from the treated as well as the untreated plots and stand-
ard comparative treatments. All percentages of control refer to the perfor-
mance of the test product (as determined by insect counts, yield and/or
damage data and other measures correlated to insect population pressures)
against the vulnerable stage(s) of the target pest, when evaluated according
to a recognized treatment program under actual field conditions.
(1) Adelgids. A minimum of 90% control, when measured as a factor of
a reduction in new galls as compared to pre-treatment and post-treatment
counts.
(2) Aphids. A minimum of 90% control based upon counts of living
aphids made during pre-treatment and at least 48 hours during post-treatment,
considered as adjusted mortality when compared to water-treated controls.
(3) Wood boring insects. Almost complete (99%) control in specimen
plantings and 85% control in production plantings, when based upon a measure-
ment of adult emergence and frass counts, as appropriate.
(4) Bud destroying insects and tip destroying insects. A minimum of
90% control, evaluated as a factor of insect counts, adult emergence, or
pupal counts and correlated to appropriate damage indices.
(5) True bugs. A minimum of 90% control, as based upon adjusted
mortality and correlated to damage reduction.
(6) Foliage feeding insects (defoliators). A minimum of 90% control,
based upon pre- and post-treatment adjusted mortality correlated to damage
reduction.
(7) Eriophyid mites. A minimum of 90% control, based upon a reduction
in new galls or damaged plant parts.
(8) Gall wasps and gall aphids. A minimum of 90% control, when measured
as a factor of a reduction in new galls, or the percentage of galls contain-
ing living insects.
(9) Leafminers. A minimum of 90% control, based upon the numbers of
damaged leaves on treated and untreated plants coupled with demonstration
of the ability of the material to inhibit the development of new mines
on previously infested leaves.
(10) Mealybugs. A minimum of 90% control, based upon population
reductions and correlated to a reduction of damage.
(11.) Root feeding insects. A mini mum of 90% control, based upon, popu-
.lation reductions and adjusted mortalities as compared to damage reduction.
(12) Scale insects. For scale .crawlers, a-minimum of 90%. control is
required when applications are compared to water-treated ..controls. For scale
-adults.,, a.minimum, of 85% control is required based upon adjusted mortality
as] compared to untireateei'controls 'ah'd - relative plant vigor. . :
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(13) Tetranychid mites. A minimum of 90% control, based upon population
reduction as compared to untreated check plots, when counts are made'with a
mite-brushing machine, ketone extraction, or other accepted sampling method.
(14) Thrips. A minimum of 90% control, based upon population reductions
as compared to untreated samples.
§ 95-6 Greenhouse floricultural treatments.
This section is concerned with efficacy testing of invertebrate control
pesticides used on greenhouse floricultural crops for protection from econ-
omic injury by insects and certain other invertebrate pests such as aphids,
beetles, cutworms, garden symphylan, greenhouse whitefly, leafminers, leaf-
eating caterpillars, slugs, snails, and spider mites.
(a) General considerations. (1) Site selection. Uses of insecticide
products in greenhouses are similar, regardless of location, due to controlled
environments. Data from several individual greenhouses are adequate as long
as such locations cover the range of variations in temperature, relative
humidity, soil or other substrates, or growing practices likely to be encoun-
tered. For general considerations, refer to § 90-2 (b)(l), and (2).
(2) Plot size. Plots size should be sufficiently large to accomodate
commercial application equipment. Conditions may dictate that large plots
not be replicated in one planting. In this event, additional tests may be
necessary at different plantings . -. :
(3) Number of trials. At least 5 individual trials, divided among 3
locations, each utilizing a single greenhouse, will provide adequate
data.
(4) Application techniques and equipment. Ultra-low volume spray
applications should be evaluated if these methods are specified on the label.
For general considerations, refer to § 90-2 (a)(3)(ii).
(5) Evaluation and reporting procedures. The procedures used to
evaluate product performance data should be specified. Reports should include
yield information, insect counts, percent crop damage, ot other detrimental
effects (also see Subdivision J), or other measures of determining the
effectiveness of the test product. Results should include the average infes-
tation reduction throughout the sampled post-treatment period compared
with that in control flat or in pre- and post-treatment periods. Data on
pest counts, amount of injury, or other measures of control should be summa-
rized. For general considerations, refer to §§ 90-2 (c)(l), (2), and
(d)(1) through (4).
(b) Suggested performance standards. These suggested standards apply to
control of insect pests on greenhouse floricultural crops. Unless otherwise,
specified, these standards are presented on the basis of pre- and post-treatment
pest population counts from the treated as well as the untreated plots and
standard comparative treatments. All percentages of control refer to the
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performance of the test substance (as determined by insect counts, yield
data, and other measures correlated to insect population pressures)
aginst the vulnerable stage(s) of the target pest, when evaluated according
to a recognized treatment program under actual use conditions.
(1) Twospotted spider mite. A minimum of 95% control, expressed as
population reductions from samples taken from leaves or flowers, as
appropriate.
(2) Aphids. A minimum of 99% control based upon population reductions
in both foliar and floral plant parts, as appropriate, or based upon the
percentage of infested plants or flowers; or a minimum of 90% control when
expressed as adjusted mortality based upon water-treated (water spray only)
controls.
(3) Flower thrips. Almost complete (99%) control of exposed flower
thrips, of 90% control of all flower thrips, as sampled by the wash method,
mechanical method, or irritation method, and compared to untreated controls.
(4) Iris borer. A minimum of 85% control, based upon a reduction of
infested rhizomes as derived from dissecting sample material from both treat-
ed and untreated plots.
(5) Mealybugs - Almost complete (99%) control, based upon comparison
with untreated checks reported as the mean number of individuals per plant.
(6) Greenhouse whitefly. Almost complete '(99%) control of the adult
stage, based upon comparison with untreated checks: or for immature stages,
a minimum of 95% control of either larvae or "puparia" when measured as a
factor of pre- and post-treatment counts of living individuals in the pres-
ence of acceptable populations recorded in untreated checks.
§ 95-7 Shade trees and forest land treatments.
This section is concerned with efficacy testing of invertebrate control
pesticides used on shade trees and forest lands for protection against direct
and indirect economic injury by insects and certain other invertebrate pests.
Forest pest control or suppression programs are usually conducted under the
supervision of state or Federal agencies. These programs are generally
directed at a single species of pest often of regional significance. As
such, extensive areas are often treated. The treatment of extensive areas
requires the careful development of data concerning effects on non-target
organisms. Data on directions for use also should be obtained since these
treatments usually involve application procedures which are different from
typical agricultural techniques. The objectives of the program may be for
suppression or management of a vector of forest diseases, for protection
of foliage, or control of a forest insect.
(a,) Definitions. The following, definitions! apply to this section.:
(.1) "Foliage protection" is defined 'as that level of pest population
•reduction. ;necessary to pr.eserve_ the tree fpl-iage..to ,the .point ..-that; mortality"
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252
of the tree(s) does not occur.
(2) "Control" is defined as'that level of pest poplulation reduction • .
necessary to preclude the need to treat the succeeding generation of the pest.
(b) General considerations. CD Site selection. Experimental plots
must be located in areas where a readily measurable population of pest insect
exists, where a predominance of preferred host sites (trees) suitable for
population sampling are present, and where the insect pest population is in
a healthy condition. For general considerations, refer to § 90-2 (a)(3)(iv).
The insects evaluated must be minimally affected by natural epizootic factors.
For defoliating insects, plots should be located within pest populations that
are increasing in severity and with no more than one year's noticeable defol-
iation of damage prior to the test season. Plots are to be located in geo-
graphic areas representative of the distribution of the pest. For general
considerations, refer to § 90-2 (b)(3).
(2) Plot size. Plot size should be adequate to evaluate the proposed
application techniques. Minimum plot size is generally 50 acres (20.3
hectares) for small single-engine fixed-wing aircraft and 20 (8.1 hectares)
acres for helicopter application. The acreage necessary to evaluate products
intended for- large scale control programs utilizing larger aircraft may in-
crease to several hundred acres (hectares) per plot depending on the equipment
used.
(3) Number of trials. A minimum of 3 large-scale, geographically-
separated trials are generally necessary, but the number of trials can vary
somewhat due to the accessibility of infestations, fluctuations in pest
population pressures, behavior, and other important considerations in the
biology of the target pest. "
(4) Application techniques and equipment. Data should be developed
from large scale plots utilizing application equipment and techniques that
can be expected to be used in control programs. If the product is likely
to be applied by multi-engine aircraft, then test results from experiments
using multi-engine aircraft should be developed. Forest applications
generally involve low volume or ultra-low volume spray rates because of
economics and logistics. The problem of delivery of this small amount of
pesticide to a target site becomes paramount. Information on droplet size
should be reported for each forest pesticide . For general considerations,
refer to § 90-2 (a)(3).
(5) Evaluation and reporting considerations. Depending upon the objec-
tives of the program and proposed pesticide usage, product evaluation may
involve sampling of the pest poplulation or determination of the level
of defoliation. Sampling may involve pre- and post-treatment determination
population levels, comparison with an untreated control plot, or with levels
of known economic damage. The development of insect life histories in
relation to effects upon host tree mortality of injury is encouraged. Where
the objective of the program is primarily foliage protection, methods for
estimating the loss of foliage must be employed. Each method should also
be accompanied by data substantiating that the defoliation is attributable
to the specified pest under evaluation. The use of infrared aerial photo-
graphy can be use to great advantage in determining degree of defoliation
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25.3.
over extensive areas. Potential nontarget effects include not only hazards
to wildlife and aquatic organisms (Subdivision E), but also effects such
as potential damage to automobile finishes and similar structures subjected
to pesticide deposition, and phytotoxic effects upon desirable trees and
understory shrubs. An evaluation of any non-target effects should be sub-
mitted or referenced with each application for registration. For general
considerations, refer to § 90-3 (f)(4)(ii).
(6) Untreated controls and comparative treatments. The proposed
product should be tested against a registered comparative treatment applied
to an adjacent area of planting, or compared to an untreated control plot.
Acceptable levels of damage may be used as a comparison, provided they
are clearly defined.
(c) Specific considerations. These considerations apply to control of
insect pests on shade trees and forest land. Forest applications directed
against foliage-feeding insects (defoliators) generally are used for either
foliage protection, or control of the pest. Gypsy moth, spruce budworm,
and Douglas-fir tussock moth programs are discussed in detail.
(1) Gypsy moth. Generally foliage protection and control are evalu-
ated in programs with the gypsy moth as the target pest. Experimental areas
must have a minimum of 200 egg masses per acre (hectare), and preferably
not more than 1000. The population should be on the increase, with a minimum
of virus incidence. Plots should contain a predominance of preferred host
trees (oaks). Pilot plot size should be no smaller than 50 acres (20.3 hec-
tares) and be replicated at least 4 times. Operational tests of at least
300 acres (121.5 hectares) should be included. The relationship of larval
development to stage of host leaf expansion should be specified. Usually
applications are directed against second instar larvae when the oak foliage
is 50 to 75% expanded. The methodology for sampling of the pest population
or estimation of defoliation should be given. Foliage protection should be
estimated in 20% or smaller increments on all tree species in subplots on
both test and control plots. Usually 20 one-fortieth acre (hectare) sub-
plots per plot are adequate. Population reduction or control should be
determined by measuring the pre- and post-treatment egg mass counts.
Additional evidence of mortality due to treatment must be determined by
measuring residual larval populations counted on 24-inch (61.0 cm) terminal
branches, by timed counts of larvae, counts on subplots, or larval counts
under burlap bands.
(2) Spruce budworm and western spruce budworm. The following conditions
apply to pesticide products intended for both the spruce budworm and western
spruce budworm. The population density in plots should be of sufficient
intensity to cause economic injury in the absence of treatment. This
number is usually 15 larvae per 15-inch (38.1 cm) branch taken from mid-crown
of the tree. The population should be healthy with minimum indication of
natural epizootic factors present. There should be a predominance of the
preferred host species (spruce/fir). Plot.size should be appropriate for
the application of the equipment employed. Since spruce budworm programs
usually employ large aircraft, plots of 500 acres. (202.5 hectare.s) or
greater are usually necessary. Information on the stage of larval development
in relationship to tree development should be provided.. Most spruce .budworm
programs are directed towards, the., late fourth instar larVae .at the time of
bud break, of the host tree. The sampling method should be described in
detail. Adequate.sampling for both-.spfuce budworms inv<3lLves the ed'l'ec'tlon
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of branch samples from the mid-crown of 30- to 50-foot-high (9.1- to
15.2-meter) trees. At least 15 different trees should be randomly selected
per plot with two to four 15-inch (38.1 cm) branches taken per tree.
Counts should be expressed as the number of larvae per 15-inch (38.1 cm)
branch or number per 100 shoots or buds. The degree of tree top-kail
and amount of regrowth after treatment should also be given.
(3) Douglas-fir tussock moth. Population density within plots should
be of sufficient intensity to cause economic injury in the absence of treat-
ment. This level is 20 or more egg masses per 1000 square inches of foliage.
The larval population should be essentially free of the influence of virus
infection. Preferred host trees (Douglas-fir or True firs) should predominate
in the experimental plots. Plot size should be adequate for the type of
application equipment employed. Plots should be 500 acres (202.5 hectares)
or larger. Information on the timing of application should be developed.
Application timing in relation to percent egg hatch and the details of
sampling methodology should be provided. Sampling must involve counts
of the number of larvae per 18-inch (45.7 cm) branch taken from the
mid-crown of 30- to 50-foot-high (9.1- to 15.2-meter) trees. A minimum
of 15 trees must be randomly selected per plot. The population level
should be expressed as the number of larvae per 1000 square inches of
foliage or number per 18-inch (45.7 cm) branch. Foliage protection
should be measured by optical examination of the volume of feeding per 100
buds per tree expressed as percent defoliation in increments of 10%.
(4) Other pest species. Specific data considerations and test standards
have not been developed for forest insect pests other than those listed
above. Registration guidance for other pests will be handled on a case-
by-case basis. Registrants are urged to contact the Agency as early in the
experimental program as possible- to develop data needs.
(d) Suggested performance standards. (1) Gypsy moth. Products
will be acceptable for foliage protection if 50% of the foliage is preserved
as a result of larval feeding reduction due to the treatment. Products will
be acceptable for control of the gypsy moth larvae if reduction of the popu-
lation to less than 2000 egg masses per acre (hectare) is achieved as a
result of the treatment, compared to significant population increases in
untreated plots.
(2) Spruce budworm and western spruce budworm. Products will be
acceptable for foliage protection if the results of treatment show protection
of 50% of the current year's foliage. Products demonstrating control
must reduce the population to levels where retreatment in succeeding
seasons will not be necessary.
(3) Douglas-fir tussock moth. Products will be considered acceptable
for foliage protection if the results of treatment show protection of 75%
of the current year's foliage. Products will be acceptable for control of
Douglas-fir tussock moth larvae if reduction of the succeeding year's popu-
lation is reduced to less than 20 eggs (0.1 egg masses) per 1000 square
inches of foliage as a result of the treatment compared to significant
population increases in untreated plots.
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§ 95-8 Livestock, poultry, fur, and wool-bearing animal treatments.
This section concerns efficacy testing of invertebrate control pesti-
cides used on cattle, horses, sheep, goats, swine, chickens, turkeys, other
domestic fowls, and fur-bearing animals such as mink and rabbits, for control
of the major arthropod pests that parasitize these animals.
(a) General considerations. (1) Site selection. Pests of livestock,
poultry, fur, and wool-bearing animals include, but are not limited to,
cattle grubs, nose bot, sheep ked, various biting flies -{horn fly and
stable fly), lice, ticks, housefly, face fly, mosquitoes, and sarcoptic
mange mites. Evaluating an insecticide that is to be applied directly to
such animals to control these pests should generally be based on adequate
tests on representative animals from herds or flocks in at least 5 widely-
separated regions where the insecticide is to be marketed, unless applica-
bility so limits use of the pesticide in special representative regions.
Tests on dairy cattle may not be necessary if comparable to efficacy data are
reported from tests using beef cattle. For general considerations, refer to
§ 90-3(b)(3).
(2) Sample size. Sample size should be representative of the number of
animals in a particular treatment group, v*iich can range from one to a herd
or flock of thousands.
(3) Number of trials. A minimum of 5 large-scale geographically-
separated trials are generally necessary, but the number of trials can vary
somewhat due to the accessibility of infestations, fluctuations in pest
population pressures, behavior, and other important considerations in the
biology of the target pest.
(4) Dosage selection. Dosage levels and concentrations should be
identified in the laboratory or small-scale tests before field testing.
It is usually necessary to repeat an application only once or twice for
lice, ticks and sarcoptic mange mites. The data should include support of
the schedule of repeated applications normally required against flies and
their continuous population pressure. For general considerations, refer to
§ 90-2-(a)(2).
(5) Application techniques and equipment. (i) Single oral dose.
For insecticides administered orally as drenches, boluses, or in capsules,
care should be taken that animals receive the entire dose. Record the
formulation, final concentration of active ingredient, total amount adminis-
tered, and dosage in terms of mg of active ingredient per kg of body weight
of animal. The regulation of many internal applications comes under the
authority of the Food and Drug Administration, Bureau of Veterinary Medicine.
Some of these pesticides may fall into this category.
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(ii-) Feed treatment. Insecticides administered to animals as part of
a feeding regimen should be mixed into the entire feed ration or fed in a
small amount of feed which, when consumed, is followed by untreated feed.
Record the formulation, final percent content of active ingredient (ppm
in feed), or total mg of active ingredient per kg body weight of animal,
amount of feed or treatment consumed, and length of treatment period.
(iii) Water treatment. Some insecticides may be administered to
animals through drinking water. Animals may be treated individually or in
groups, which, when having consumed the treated water, are given untreated
water. Regardless of the size of the group, the following should be recorded:
the formulation, final concentration of active ingredient in terms of ppm
in water or mg of active ingredient per kg of body weight of animal, average
consumption of water per animal, and duration of treatment.
(iv) Mineral, salt, or protein supplement. Insecticides of this
type are generally formulated at low concentrations in mineral, salt, or
protein supplements are offered free choice to animals. Because consumption
of salt, mineral, and protein supplement varies considerably from animal
to animal, it is important to determine whether or not all animals consumed
treated materials. Record the formulation, final concentration of active
ingredient in terms of percentage or ppm of treated supplement, average
consumption per animal per day, dosage in terms.of mg active ingredient
per kg body weight per day, and length of treatment period.
(v) Injections. For insecticides given to animals in the form of
intramuscular, intraperitoneal, or subcutaneous injections, record the
formulation, amount of material injected per animal,.location of injection,
and dosage in terms of mg of active ingredient per kg of body weight.
(vi) Whole-body sprays. For insecticides applied to animals as
whole-body sprays, care should be taken that animals are treated thoroughly
and that enough pressure is used to penetrate hair coat and assure wetting
of the skin. A variation of the whole-body spray is the use of spray-dip
machine to apply spray to animals. With either method, record the formu-
lation, final concentration of active ingredient, equipment used and
application techniques, and average volume of spray applied per animal.
(vii) Dip. For insecticides used to charge dipping vats, animals
should be immersed thoroughly in the dip fluids. Record the formulation,
final concentration of active ingredient, volume of liquid in the vat,
• age of charge at time of dipping, number of animals dipped, and data on
recharging (if necessary). Chemical analyses of active ingredient in vat
fluids before and after dipping are necessary to determine the actual amount
of active ingredient in the vat fluid.
(viii) Pour-on treatment. Insecticides applied to animals by the
pour-on technique, ready-to-use formulations, or emulsifiable concentrates
diluted with water or oil, are poured down the backline of animals in
ounce (milliliter) rates. In an extension of this technique, ready-to-
use formulations are applied to a spot on the backline a-t milliliter rates.
Record formulation, diluent, final concentration of active ingredient,
amount applied per animal, area treated, and dosage bajsed on mg active
ingredient per kg body'weight of animal.- ' . ' ; •
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(ix) Dust treatment (livestock). Insecticides are applied as dusts
by power duster or by hand or contained in dust bags and placed in the
pasture for free-choice use or placed in openings to feed, mineral, and/or
water sources so that animals are forced to use them on a daily basis.
With cattle grub control, it is important that dust bags be located so that
animals are forced to treat themselves on a daily basis to insure that
sufficient insecticide is applied for cattle grub control. In tests with
dust bags for control of ectoparasites, such daily treatment is not essential.
Record formulation, final concentration of active ingredient, average amount
of dust per animal, location of dust bags, and length of treatment period.
(x) Back spray. In tests to control cattle grubs, the animal's backs
are sprayed thoroughly with the contact insecticide. Care is taken to force
insecticide into the warble openings in the animal's backs. Record formula-
tion, final concentration of active ingredient, equipment used, application
techniques, and average volume of spray applied per back.
(xi) Floor or litter treatment. Insecticides are applied as mists or
fogs, dust, or granules directly to floor areas, litter, or nest areas.
Record formulation, final concentration of active ingredient, equipment
used, amount of insecticide per square meter of surface treated, and total
surface area treated.
(xii) Dust box treatment (poultry). Insecticides formulated as dusts
or granules are placed into dust box containers and fowl allowed to treat
themselves. Record formulation, diluent, final concentration of active
ingredient, amount of material per dust box, length of treatment period,
number of birds, and average amount of material used per bird during treat-
ment period.
(xiii) Vapor treatment. Strands, cords, or other devices impregnated
with insecticides are attached underneath or around cages containing infested
birds. Insecticides volatilize from the impregnated surfaces and kill ecto-
parasites on birds. Record the formulation, impregnated material, final
concentration of active ingredient, length or weight of impregnated material
per bird or per cage containing a specific number of birds, and length of
treatment period. Low volume or ultra low volume application must be evaluated
if these methods are to be specified on the label. For general considerations,
refer to § 90-3 (a) and (b).
(6) Record of toxicity and other adverse effects. Adjusted average
daily gains (ADG) on the test and control groups should be recorded and
reported. In testing petroleum oil formulations, the possible toxic effects
on the animal from the petroleum oil must be evaluated. For example, small
amounts of certain solvents cause tolerable minor itching and burning for
short periods after application. Higher concentrations may cause death if
not diluted before application. The recommended method of treatment for
pour-on and other ready-to-use products containing oils should be tested to
ensure that' the. recommended method of treatment will not result in. excessive
dosages of oil viiich may evoke adverse reactions in treated animals. Bie
final use dilution of emulsifiable concentrations should be tested to ensure
.that i-t does not contain dangerous amounts of oil. .Insecticides wh.ich are
tested for use on livestock, poultry, of fur or wool-bearing animals should
not injure the an-imal- even uhen application is repeated over .a long^period
of time.; the margin of safety to the. treated animal is a vital consideration
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in determining usefulness. Neither should the insecticides appear illegally
in or on meat, meat by-products, milk, or eggs. If the treated animal is
less than 3 months of age (excluding poultry), the effects of stress caused
by a particular operation such as castration, dehorning, or other similar
procedures, should be evaluated in conjunction with toxicity data. This is
especially important for feed-through insecticides. For general considerations,
refer to §§ 90-3 (d)(l) through (4), and for other requirements on toxicity,
refer to Subdivision F.
(7) Evaluation and reporting procedures. The evaluation procedures
used should be specified in the presentation of the data. Reports should include
formulations, final concentrations of active -ingredient in terms of ppm in
water or percent active ingredient in dusts, dosage in terms of mg of active
ingredient per kg of body weight of animal, length of treatment period,
equipment used, and other similar factors. The average daily gain in weight
should be reported for any tests of 7 to 14 days duration.
(8) Untreated controls and comparative treatments. When it is imprac-
tical to maintain untreated control animals, the effectiveness of the test
product may be measured by comparative treatments with products of well-known
efficacy used as reference standards. To evaluate the effect of treatments,
records should be kept on the comparative changes in meat, milk, or
egg production or some similar measurement; and insect counts following
each treatment. The number of animals in the control or comparative treatment
groups must be equal to the numbers of animals in a treatment group in small
scale tests; in large-scale tests, only a small portion of the animals need
to be untreated or given a standard treatment. Control groups should contain
animals of the same general size, age, condition, and origin as those in
the treatment groups and have similar infestations of parasites or be exposed
to similar populations of arthropod parasites.
(b) Suggested performance standards. Unless otherwise specified,
these standards are presented on the basis of pest population counts from
treated compared to untreated animals. All percentages of control refer
to the performance of the test product (as determined by insect counts,
yield of meat and milk, and any other measures correlated to insect population
pressures) against the vulnerable stage(s) of the target pest, when evaluated
according to a recognized treatment program under actual field conditions.
(1) Cattle (beef and dairy). (i) Cattle grubs. All of the following
percentages are based upon the reductions of larvae (warbles) in the backs
of animals, as derived from monthly observation and compared to untreated
control groups.
(A) Systemic treatments; single oral dose,' feed treatment, drinking
water treatment, mineral salt or protein supplements, injections, whole-
body sprays, dips, pour-ons. A minimum of 90% reduction in infestation.
(B) Contact treatments; dusts and sprays. A minimum of 75% reduction
in infestation.
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(ii) Horn fly. The percentages of control for the horn fly are based
upon pre- and post-treatment counts coupled with a comparison to separate
untreated control groups. Such percentages are derived from actual counts
of the number of adult flies per side per animal, and may additionally be
correlated average daily weight or milk production.
(A) Feed treatment, drinking-water treatment, and mineral salt or
protein supplements. A minimum of 90% control of emerging adults along
with a 70% reduction of adults on the cattle as measured by side or whole-
body counts.
(B) Whole-body sprays, dips, pour-ons. A minimum of 90% reduction in
infestation for 3 weeks.
(C) Dusts. A minimum of 95% reduction in infestation for 2 weeks.
(D) Backrubbers. A mimimum of 90% reduction in infestation, under
continued use for one month.
(E) LV, ULV and waxed-bar applications. A minimum of 90% reduction
in infestation under continued use.
(iii) Other biting flies; (stable fly, deer flies, and horse flies).
The percentages of control given for these pests are based upon the feeding
rates of flies before treatment compared to the feeding rates of flies on
the same cattle after treatment as correlated to the feeding rates on
untreated animals taken at the same time. Such feeding rates should be
expressed as populations of each separate pest species observed consistently
at a particular time of day associated with maximum fly activity.
(A) Whole-body sprays, dips, pour-on, and dusts. A minimum of 90%
reduction in infestation one day after application and 75% reduction in
infestation one week after application.
(B) Backnibber, LV, and ULV applications. A minimum of 90% reduction
in infestation under continued use.
(iv) Face fly: whole-body sprays, dips, pour-ons, dusts, back-
rubbers, LV, ULV, applications, smears, baits, ointments, and face wipes.
Reduction in infestation should range from 20-60% for 2-3 days from a single
application, based upon a comparison of the numbers of flies per animal pre-
and post-treatment correlated with populations of the pest occurring on
adjacent untreated herds observed at the same time.
(v) Lice. The percentage of control for lice are expressed as factors
of population reductions derived from skin examinations on those areas of
the body most susceptible to the particular species of louse. Such percen-
tages are additionally based upon pre- and post-treatment population counts
of both treated and untreated animals.
(A.) Whole-body sprays, dips, and pour-ons. A. ml minium-, of 90% reduction
in infestation for. one month .after trea-tmen-t
.-,..(B) Pusts. A,.mimimum of 90% reduction in infestation .for a single'-'•
.application for one'mon-th after treatment. '
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(C) Back rubbers, LV, and ULV applications. A. minimum of 90% reduction
in infestation under continued use.
(vi) Ticks. The percentage of control listed as suggested performance
standards for ticks are based upon pre- and post-treatment counts on the
same animals or as the average number counted on untreated controls during
the same post-treatment intervals. If pre- and post-treatment counts are
utilized as a basis for computing the percentage, then untreated control
counts should still be reported to permit correlation with natural tick
population dynamics for the site/pest complex.
(A) Whole-body sprays, dips, pour-ons, and dusts. A mimimum of 90%
reduction in infestation one day after application and 75% reduction in
infestation for one week after application.
(B) Back rubber, LV, and ULV applications. A minimum of 90%
reduction in infestation under continued use.
(vii) Scab mite and various mange mites; dips and whole-body sprays.
Performances of candidate materials for scab and mange mite i/ control is
based upon a determination of pre- and post-treatment population counts
obtained from skin scrapings and subsequent microscopic examination. Such
treatments should be compared to control groups in separate areas. The
standard is complete control from two treatments at two week intervals, or
as the product is used according to label directions.
(viii) Screwworm: dips, whole-body sprays, dusts, and smears. All
larvae in pre-infested wounds must be killed, and reinfestations must be
prevented for a two-week period.
(2) Horses. (i) Horse bots: stomach tube, pour-on, intramuscular
injection, and feed treatment. A minimum of 90% reduction as determined
by counts of expelled larvae compared with counts of those larvae remaining
in the animal after treatment (to be determined either by necropsy or by
trichlorfon drench).
(ii) Ticks, lice and mange mites 2/. (A) Whole-body sprays, dips.
A minimum of 75% reduction in tick infestation at the end of one week,
and 90% reduction in louse and mange mite infestations at the end of 2
weeks, based upon pre- and post-treatment counts in comparison with untreat-
ed controls.
The mange mites include Psorergetes mange mites, demodectic mange
mites, psoroptic mange mites, and sarcoptic mange mites.
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(B) Ear treatment for ticks only. A minimum of 90% reduction in
infestation for one week and 75% reduction in infestation for one month,
based upon pre- and post-treatment counts in comparison with untreated
controls.
(iii) Flies; dips, whole-body sprays, fine-mist sprays, toxicants
and/or repellents in aerosols, sponge-ons, wipe-ons, smears, baits, and
treated halters. The percentage given as a suggested performance standard
for fly control are based upon a reduction in infestation numbers for a
minimum period of 3 hours or that period of protection to be claimed on
the label, whichever is shorter. A minimum of 90% reduction in infestation
under continued use.
(3) Sheep and goats. (i) Sheep lice and goat lice: dips, sprays,
pour-ons, dusts, and treated collars. A minimum of 90% reduction in
infestation for one month; based upon average numbers of lice on treated
and untreated animals at specific intervals after treatment.
(ii) Sheep ked; dips, sprays, pour-ons, dusts, and treated collars.
A minimum of 90% reduction in reinfestation for one month, based upon pre-
and post-treatment counts as correlated to control populations.
(iii) Scab mite and various mange mites .i/: dips. A minimum of 90%
reduction in infestation based upon the number of infested animals post-
treatment as compared to the total number of infested animals pre-treatment
and correlated to control population fluctuations, as determined pre- and
post-treatment skin scrapings.
(iv) Fleeceworms and screwworm; sprays, dips, dusts, and smears.
Must initially kill all existing larvae and prevent subsequent reinfestation
for a period of 2 weeks.
(y) Sheep hot fly; single oral treatments, feed or water additives,
injection, and nasal treatments. A minimum of 90% reduction in infestation
following treatment, based upon the pre- and post-treatment infestation
rates and the infestation rate of similar untreated animals examined at the
same time.
(4) Swine. (i) Hog louse. The suggested performance standards for
the hog louse are expressed as percentages based upon the average numbers
of lice on the animals, pre- and post-treatment, as compared to the average
numbers of the hog louse on untreated swine. This percentage of control
should be demonstrated for the period of time claimed on the label unless
otherwise stated.
=f Th-e mange mites include Psorergetes mange mites, demodectic mange
. mites, psoroptic mange mites, and sarcoptic mange mites.
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(A) Whole-body sprays, dips, dus'bs/ and pour-ons. A minimum of 90%
reduction in infestation for one month following treatment.
(B) Back-rubbers and oilers. A minimum of 90% reduction in infestation
under continued use.
(C) Litter treatments. A minimum of 90% reduction in infestation for
a period of one month after one or two treatments.
(ii) Hog mange mite; dips and sprays. A minimum of 90% reduction in
infestation for a period of one month after one or two treatments. Percent-
ages are derived from a comparison between skin scrapings taken both pre-
and post-treatment from treated and untreated animals.
(5) Chickens, turkeys, and other domestic fowl. *(i) Lice; dips,
sprays, mist sprays, dusts, floor or litter treatments,, dust box treatments,
and vapor treatments. A minimum of 90% reduction in infestation, as
determined by comparing the average number of lice per bird in treated
groups with the average number of lice per bird in untreated groups, both
pre- and post-treatment, for a period of one month.
(ii) Northern fowl mite and tropical fowl mite; dips, sprays, mist
sprays, dusts, floor or litter treatments, dust box treatments, and vapor
treatments. A minimum of 90% reduction in infestation' for a period of
one month after treatment, based upon reductions in the number of mites
and the relative degree of feather discoloration in the vent area.
(iii) Turkey chigger: dips, mist sprays, dusts, floor or litter
treatments, dust box treatments, and vapor treatments. ' A minimum of 90%
reduction in infestation for a period of one month after treatment, based
upon the numbers of lesions pre- and post-treatment on both treated and
control groups of birds.
(iv) Manure-inhabiting fly larvae. (A) Feed treatment. A minimum
of 90% reduction in infestation under continued use, based upon both adult
fly reductions determined either by manure emergence or manure bioassay
testing, comparing both treated and control groups.
(B) Fly larvicides to manure. A minimum of 90% reductions in infes-
tation for a period of 2 weeks after treatment, based upon the same criteria
used for feed treatment in paragraph (b)(6)(iv)(A) of this section.
§ 95-9 Treatments to control pests of humans and pets.
This section is concerned with efficacy testing of invertebrate
control pesticides used on humans and pets. Humans may be infested by
three types of lice: the body louse, the head louse, and crab louse.
The demonstration of effectiveness against any species should therefore
be based on tests against lice of that particular type.. Dogs and cats
are generally infested by lice, fleas, ticks, and the species used must
be fully identified in the report. There are many factors involved in
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263
actual use conditions that testing cannot always adequately evaluate.
The habits of the host animals, infestation pressures of the parasite,
exposure to rain, dew, sunlight, or even pH of water and geographical
considerations can result in a change in the degree or duration of efficacy
for a particular product. In order to make label claims more meaningful,
an accurate description of product performance under a broad spectrum of
use conditions should be recorded.
(a) General considerations. (1) Number of trials. A minimum of 5
large-scale, geographically-separated trials are generally necessary, but
the number of trials can vary somewhat due to the accessibility of infes-
tations, fluctuations in pest population pressures, behavior, and other
important considerations in the biology of the target pest.
(2) Treatment techniques and equipment. Product treatments to control
pests of humans are applied as sprays, dusts, lotions, towels, ointments,
creams, sticks, soaps, and shampoos. Product treatments to control pests
of pets are applied as sprays, dusts, foams, flea collars, flea tags, soaps,
and shampoos. For general considerations, refer to § 90-3(a) and (b). For
guidance on testing flea and tick collars on dogs, see § 95-30(b) item No. 11.
(3) Evaluation and reporting considerations. Reports should distinguish
between killing and repelling effects, since repellent insecticides may
preclude any lethal effects. Insect repellency test data should demonstrate
minimal contact between the insect and the test chemical. Any adverse
effects should be reported. Breed, age, size, and hair length influence the
effectiveness of invertebrate control agents applied to dogs and cats,
and such factors must be evaluated and reported in testing pet treatments.
For general considerations, refer to § 90-3 (d)(l) through (4).
(4) Toxicology tests. Any product intended to be applied directly
to humans or pets, or used so as to provide frequent or constant product
exposure to humans or pets, must be tested under the full range of toxi-
cology testing required in Subdivision F. After submittal of such toxicol-
ogy data, consultation with the Agency is required before extensive
product performance testing dealing with human pest control products is
undertaken.
(b) Suggested performance standards. These standards are presented
on the basis of pest population counts from treated compared to untreated
subjects unless otherwise specified. (1) Pests of humans. (i) Body
louse. Demonstrate 100% control of the stage(s) claimed to be controlled
by the test product. Efficacy is based on numbers of the body louse before
treatment and ,at intervals after treatment, in tests conducted where
clothing, activities, and environments are typical of those of louse-infested
populations. Ovicidal activity must be demonstrated unless repeat applications
are intended.
(ii.) Head louse and crab louse. .Demonstrate 100% control of ,. . .
the stage(s)' claimed to .be controlled by the test product* .Efficacy/is .'
based.on-the numbers of eggs, .nymphs and'adults of the head louse or: craB
louse before and after treatment in tests conducted.where .activities-'and ._.<>.,•••"•'
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264
environments are typical of those of louse-infested populations. Ovicidal
activity should be demonstrated unless repeat applications are indicated.
(iii) Ticks, fleas, and mites. Should provide 100% in pest infestation
through a killing or repelling action when tested under simulated or actual
conditions. Or, the product should provide the protection time (in hours)
which is justified by the supporting data and appears on the label. The
minimum acceptable protection time is one hour, when using first confirmed
bite methodology, or sock testing.
(iv) Mosquitoes. Should generally provide a minimum of 2-3 hours pro-
tection time based upon first confirmed bite field tests, depending upon
the biting pressure evidenced in the testing. If the product provides long-
er protection times, then this may be stated on the label. Performance
standards for killing mosquitoes are listed under § 95-10 Mosquito, Black
Fly, Nonbiting Midge, and Biting Midge (Sand fly) Treatments.
(v) Biting flies. The data should indicate a minimum protection time
of 3 hours based upon first confirmed bite field tests. A product may be
registered for repelling biting flies if it provides one to three hours of
protection, but the direction should.be stated on the label.
(2) Pests of pets (dogs, cats, birds). (i) Fleas and ticks.
Provide 90% reduction in pest infestation through a killing action when
tested under simulated or actual field conditions.
(ii) Lice. Demonstrate 100% reduction in pest infestation
through killing or repelling action when tested under simulated or actual
field conditions.
(iii) Mites. Provide 100% reduction in pest infestation
through killing or repelling action when tested under simulated or actual
field conditions.
§ 95-10 Mosquito, black fly, nonbiting midge, and biting midge (sand
fly) treatments.
This section is concerned with efficacy data requirements for evaluation
of invertebrate control pesticides as outdoor and breeding ground treatments
against several groups of important insect pests which pose economic and
public health threats to man in urban, rural, agricultural, and wilderness
habitats. The insects for which data considerations are outlined here belong
to five families: Culicidae (mosquitoes), Simuliidae (black flies),
Ceratopogonidae (biting midges), and nonbiting midges in the families
Chironomidae • (midges) and Chaoboridae (phantom midges). These insects
inhabit a variety of ecological niches and show diverse biological, physio- •
logical, and behavioral patterns. In the evaluation of biologically active
compounds, the unique features of each pest species must be taken into
consideration. Members of the 5 families for which -these guidelines are
developed are mostly aquatic or semi-aquatic in their immature stages.
The mature stages are terres/tria^,.' dispersing, beyond the 'immediate vicinity
of breeding sources. .. :
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(a) General considerations. (1) Species, stage, age, and sex. The
test insect should be identified as to genus and species. Additional infor-
mation as to subspecies or strains should be recorded. Methods and proce-
dures utilized for assessment are dictated by the stage and habitat of
the insect. Pesticides are generally evaluated against the larval and/or
adult stages. In certain situations, however, registrants may have to
gather data on the egg and pupal stages. In larval evaluation, the ins tar
should be standardized and specified. The age or age range (if precise age
is not known) of the adult test insects should be reported. Reporting of
the calendar age of larvae is not essential if the larval instars are
specified. The sex of the adult test insects should be specified. Deter-
mination of the sex of the immature stages is difficult, and generally this
information is not necessary.
(2) Plot size. Plots which are suitable in size for commercial
applications should be used. Plot size can vary from a few square feet to
hundreds of acres (hectares), depending on the objectives of the test,
species, and type of control desired. Test plot dimensions should be large
enough to avoid drift onto sampling areas of untreated control plots.
(3) Number of trials. A minimum of 5 large-scale geographically-
separated trials are generally necessary, but the number of trials can
vary somewhat due to the accessibility of infestations, fluctuations in
pest population pressures, behavior, and other important considerations
in the biology of the target pest.
(4) Application techniques and equipment. Aerial or ultra-low
volume applications should be evaluated if these methods are specified on
the label. For general considerations, refer to § 90-3(a)(3)(ii).
(5) Evaluation and reporting procedures. The evaluation procedures
should be specified in the presentation of the data. Reports should include
larval counts, bite counts, percent mortality, knock-down time, or other
appropriate measures of determining the effectiveness of the test product.
Results should include the infestation or nuisance reduction, or preferably
both, compared with the control plots or areas. Raw data on pest counts or
other measures of control should be summarized. Insect resistance can be
a major problem when testing a product for mosquito control, and any evidence
of such should be reported when tests are run in areas where resistance
is a known problem. Other factors of special concern in determining a prod-
uct's usefulness are wildlife and fish hazards (see Subdivision E), possible
water or air pollution, effects on house paints, and spotting of automobiles.
Any adverse effects should be reported. Repellency from mosquito coils, can-
dles, and torches is dependent upon the concentrations and duration of smoke
or fumes which permeate a limited area. These types of products should demon-
strate repellency of mosquitoes and other small flying insects on patios,
torches., or other confined outdoor areas where there is little or no breeze.
For general considerations, refer to §§ 90-3(c)(l), (2), and (d)(l) through (4),
(.6) Sampling, techniques. Various, species and the, different stages of
the same species require specific sampling techniques. .Applicable techniques •
.for assessment of populations of test species or groups-.should be employed. '..
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(b) Suggested performance standards. Unless otherwise specified,
these standards are presented on the basis of pre- and post-treatment pest
population counts (including landing, bite, and trap counts) from treated
as well as the untreated pest plots and standard comparative treatments.
All percentages of control refer to the performance of the test product
[as determined by pest insect counts (landing and bite) and other measures
correlated to insect population pressures] against the vulnerable stage(s)
of the target pest, when evaluated according to a recognized aerial or
ground application treatment program under actual field conditions.
(1) Culicidae (mosquitoes). (i) Larvae. A minimum of 95% population
reduction, based on pre- and post-treatment infestation counts from tests
conducted under actual field conditions.
(ii) Adults. A minimum of 95% population reduction, based on pre-
and post-treatment infestation counts. When appropriate, laboratory colony
or caged wild mosquitoes can be used. The tests should be conducted under
actual field conditions.
(2) Simuliidae (black flies). (i) Larvae. A minimum of 80% population
reduction, based on laboratory evaluation techniques (jar, cloth trap, flush-
ing and draining, and trough) and the single stream technique to determine
effectiveness by comparing larval populations above and below the stream
treatment point, not more than one day before and one day after treatment.
(ii) Adults. Large scale control operations are evaluated by pre-
and post-treatment population estimates. Landing rate counts (a one-
minute count beginning after a 5-minute waiting period is satisfactory),
and standardized-sweeps about the head of observer inside and outside the
treated areas provide a useful index to the annoyance rate.
(3) Chironomidae (midges). (i) Larvae. A minimum of 95% population
reduction, based on samples taken prior to treatment, and 4, 7, and 15 days
longer after treatment.
(ii) Adults. A minimum of 95% population reduction.
(4) Chaeoboridae (phantom' midges). (i) Larvae. A minimum of 95%
population reduction, based on larval samples taken prior to and after
treatment.
(ii) Adults. A minimum of 95% population reduction.
. (5) Ceratopogonidae (biting midges). (i) Larvae. A minimum of 95%
population reduction, based on pre- and post-treatment counts. Populations
of larvae can be sampled by laboratory inspection of suspected breeding
media collected in the field, or by an emergence trapping method to determine
the level of control.
(ii) Adults. A minimum of 95% population reduction, based on pre-
and post-treatment population estimates determined by landing counts or
light trap collections.
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§ 95-11 Premises treatments.
This section is concerned with the efficacy data guidance for
invertebrate control pesticides used against pests of premises. Commercial
and industrial pesticide formulations used for premises treatments include,
but are not limited to, liquid or pressurized products for spray treatments
and pastes, powders, and granules for baits.
(a) Definitions. The following definitions are of special importance
in understanding this section:
(1) The term "premises" refers to the spaces within structures, their
walls (both inside and outside), and the immediate adjacent surrounding
grounds. Such structures include households (e.g., houses, apartments);
commercial, industrial and institutional buildings; agricultural structures
(e.g., barns); and food-handling establishments.
(2) The term "food handling establishment" refers to any place, other
than a private residence, in which food is held, processed, prepared,
and/or served. Food areas include those used for receiving, serving,
storing (dry, cold, frozen, raw), packaging (canning, bottling, wrapping,
boxing) and preparing (cleaning, slicing, cooking, grinding) food; for
edible waste storage; and for enclosed processing systems (mills, dairies,
edible oil extractors, and evaporators).
(3) The term "non-food areas" refers to premise areas such as garbage
rooms, lavatories, floor drains (to sewers), entries and vestibules, offices,
locker rooms, machine rooms, boiler rooms, garages, mop closets, and storage
(after packaging.)
(4) The term "crack and crevice treatment" means the application of
small amounts of insecticide into cracks and crevices in which insects
hide or through which they may enter the building. Such openings commonly
occur at expansion joints, between different elements of construction,
and between equipment and floors. These openings may lead to voids such
as hollow walls, equipment legs and bases, conduits, motor housing,
junction or switch boxes
(5) The term "spot treatment" is application to limited areas on
which insects are likely to occur, but which will not be in contact with
food or untensils and will not ordinarily be contacted by workers. These
areas may occur on floors, walls, and bases or undersides of equipment.
For this purpose, a "spot" will not exceed 2 square feet. Spot treatments
must be limited to 20% of the lower wall and floor surfaces. The 2 square
foot "spots" cannot be contiguous.
(6.) .The term "gener-al treatment" is application to broad expanses of
surfaces such as walls,, floors, and ceilings or as. an outside treatment.
(b) General considerations. The tes ting of . .a pes-ticide produc t for
..use in households should follow, the same procedures as are appropriate, for
its commercial uSeV- --"•'•• ''"-'• ...... : '
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('!) Site selection. The uniqueness of each application site or
structure, and the fact that identical sites or structures will usually
not be available, should be recognized. For general considerations, refer
to § 90-3(a) and (e)(3)(vi).
(2) Number of trials. Under controlled conditions in the laboratory,
at least 5 trials are usually necessary. A minmum of 5 large-scale field
trials are generally needed, but the number of trials can vary somewhat
due to the accessibility of infestations, fluctuations in pest population
pressures, behavior, and other important considerations in the biology
of the target pest.
(3) Test species. Test invertebrates should be representative of flying
and/or crawling species against which the test product is to be used. A
pest population may be difficult to estimate or to sample, and it will often
be necessary to limit testing to the laboratory, or to develop a system of
estimating the extent of an infestation in the field. Ihe pests to be
controlled often cannot be distinguished as separate entities and will
frequently need to be grouped according to control methods, geographical
locations, infested premises, life stage or form, type of damage and other
factors. Therefore, testing may need to be directed at the most important
pest or pests, life stage or pest complex, or at the species most resistant
to a particular pesticide.
(4) Exposure period. The duration of exposure to the test product is
important in order to achieve maximum control, and data must be reported in
terms of time length, such as 12-, 24-, or 48-hour exposure periods.
(5) Temperature and humidity. The temperature and humidity at the
time the product is tested should be recorded, since these factors are
important relative to the activity of the pest population, and the degradation
of the active ingredient(s).
. (6) Residual considerations. The amount of pesticide residue deposited
on treated surfaces is critical to the effectiveness of many treatments
against crawling pests. The amount of residue deposited should be determined
under actual or simulated use conditions, and the method(s) of determination
must be submitted with the test data. The types of surfaces to which resid-
dual pesticides are applied must be reported since surface .type has a pro-
nounced effect on the amount of active residue available to pests. In
general, the following absorptive and non-absorptive surfaces should be test-
ed for crawling pests: vinyl tile or linoleum, stainless steel, painted and
unpainted wood and ceramic tile.
(7) Application techniques and equipment. The application technique
should reflect the claims proposed on the label, whether crack and crevice,
spot, general, space spray, contact spray or total release. Insecticides
to be evaluated in or around dairy barns,-horse barns, stables, poultry
houses and yards, and other agricultrual structures may be applied as
sprays, dusts, and baits to stanchions, walls, floors, manure, bedding,
and other areas where the target insects- may rest or bceed. For general
considerations, refer to § 90-3(a). •
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(c) Test methods and suggested performance standards. These suggested
standards are determined on the basis of results obtained from laboratory
and simulated or actual field tests in which the efficacy of the test
product is compared with that of official test pesticides such as the
official test pressurized spray (OTPS), from the Chemical Specialties
Manufacturers Association, untreated controls, or a registered standard.
The performance standards are the same for household, commercial and industrial,
agricultural, structural and food handling establishment treatments.
(1) Non-residual aerosols and space sprays. (i) Flying insects.
The product should demonstrate efficacy equal to, greater than, or within 5
percentage points of the OTPS when tested according to the CSMA Aerosol
and Pressurized Spray Insecticide Test Method for flying insects or the
ANSI-ASTM E653-78 Standard Method for Testing Effectiveness of Aerosol and
Pressurized Space Spray Insecticides Against Flying Insects.
(ii) Crawling insect. (A) Contact spray. The product should be
tested against the proposed target pest and demonstrate performance equal
to, greater than, or within 10 percentage points of the OTPS when tested
according to the CSMA Cockroach Aerosol Method or the ANSI-ASTM E654-78
Standard Method for Testing the Effectiveness of Aerosol and Pressurized
Spray Insecticides Against Cockroaches.
(B) Space spray. There are no official test methods for space sprays.
However, the following general considerations apply to pesticides applied as
space sprays for crawling insects, as most of these insects are not exposed
at the time of application.
(a) The room size and dosage must be consistent with label claims
and directions.
(b) Replication is necessary to provide reliability.
(c) Controls should be utilized for comparison.
(d) As some products may knockdown but not kill a high percentage of
insects, mortality must be recorded at 1 and 24 hrs. post treatment.
(e) Usually German cockroaches are used in testing. When dosage rates
are very small, a larger species, such as American or Oriental cockroaches,
may also be necessary. Resistant strains should be utilized if the pesticide
has a history of resistance problems. Last ins tar nymphs and adults are
recommended for all species.
(f) A product registered for the use pattern should be used as a
positive control.
(g) The containers or enclosures for holding the roaches should be
representative of these areas which commonly shelter roacb/es (underneath
refrigerators, stoves, sinks, cabinets and in. certa-in wall voids.
• (ft.)'v The "number of. specimens "should-'be~"'su"f fieien-t Ho 'provl'de--accurate
statistical evaluation of the results. Perhaps the .greatest problems have
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arisen with respect to item (g) above. Note that open containers or those
with unobstructed holes in the top area are not representative of covered'
or enclosed situations.
•.v
(2) Residual sprays. (i) General; Efficacy data for residual sprays
should indicate the appropriate dosage and the utility of the formulation when
used as directed. Usually, laboratory testing is performed to establish
the effective dosage range, determine if the formulation is repellent to
the point of adversely effecting performance of the product, and to evaluate
the effects of the various substrates upon which deposition is to occur.
Field studies are then necessary to confirm the performance of the compound
under actual use conditions. See § 95-30 items 5 and 12.
(ii) Sprays. (A) Crawling insects. (a) Laboratory testing.
Laboratory testing should evaluate the proper dosage range utilizing treated
panels representative of field substrates. Usually these include such
items as painted and unpainted wood, glass, formica, chipboard or particle
board, stainless steel, concrete, and vinyl tile. Such treated panels
should be aged under conditions similar to field situations and challenged
with the target insect as regular intervals to determine the duration of
efficacy. Additionally, choice box testing or some other method of evalua-
tion must be performed to determine if a high degree of repellence is
likely to affect field performance. All laboratory testing should utilize
both untreated and positive controls to determine the relative vigor of
the laboratory populations.
(b) Field testing. Field testing for crawling insects involves
utilizing sites and methods of application essentially the same as those
to appear on the proposed label. Evaluation is based-upon pre- and post-
application counts of living insects. For example, cockroaches are normally
counted by flashlight, trapping, or flushing agents. Untreated areas or
sites should be used as controls if possible, and a positive control should
also be used in the testing.
(B) Flying insects, (a) Lab tests. Laboratory tests for residual
applications to control flying insects are usually done in screen cages with
one side replaced by the treated panel. Such testing should evaluate the
appropriate dosage range (usually mg. active ingredient per square foot),
and the degree of repellency exhibited by the formulation.
(b) Field testing. Field tests should utilize the proposed sites of
application. Counts may be made by visual observation, fly speck cards,
black light traps, or other suitable methods.
(3) Baits. (a) General. The important factors relating to testing
bait products are to:
(1) Establish the proper dosage and intrinsic attractancy of the
formulation in "free-choice" laboratory tests.
(2) Evaluate the utility of the product under actual-use conditions.
See § 95-30 items No. 5 and 12.
(b) Laboratory testing. The most important factor involved in lab
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testing is to provide a free-choice alternative food source to the test
insects. This may be laboratory dog chow for insects like cockroaches,
or sugar based materials for houseflies. Ihe formulation should demonstrate
acceptable toxicity in competition with the alternative food source.
(c) Field testing. Testing under actual use conditions should demonstrate
the utility of the product when used in situations vAiere a continuous source
of pest insects are available. The product should demonstrate both mortality
and a reduction in the overall population over time. Measurement techniques
are essentially the same as for residual sprays.
§ 95-12 Structural treatments.
This section concerns the guidance on efficacy data for evaluation
of invertebrate control pesticides used to control pests such as wood-
destroying beetles, wharf borer, carpenter ant, and carpenter bee in
structures such as buildings, structural members, and stored lumber.
(a) General considerations. (1) Site selection. When selecting an
application site for testing invertebrate control agents against structural
pests, such factors as temperature, humidity, moisture, soil texture, and
freezing-thawing conditions should be considered and reported. For general
considerations, refer to §§ 90-3 (a)(3)(iv) and (b)(l) through (3).
(2) Number of trials. A minimum of 3 large-scale geographically-
separated trials are generally necessary, but the number of trials can
vary somewhat due to the accessibility of infestations, fluctuations in
pest population pressures, behavior, and other important considerations
in the biology of the target pest.
(3) Residual considerations. The soil residual potential for termite
control products is important in determining the length of time that such
pesticides will be effective, and is measured by the time over which the
toxic barrier resists penetration. Soil treatments require that special
care be exercised to assure that water supplies are not contaminated with
the pesticide and that the treatment does not cause intolerable injury to
building foundations, plants, shrubs, and other ornamental plants.
(4) application techniques and equipment. Data should be developed to
support the use of pressurized, impregnation, or other specialized types of
application equipment: For general considerations, refer to § 90-3 (a)(3)(i),
(5) Evaluation and reporting procedures, (i) The procedures used
to evaluate product performance should be specified in the data. Reports
should include insect counts, amount of damage, or to the measures of deter-
mining the effectiveness of the test product. Results should include the
average infestation reduction throughout the sampled post-treatment period
compared with that in untreated controls. Data on pest counts or other
measures of control should be summarized. The test data should indicate
that there are no adverse effects such as warping or staining to •
wallpaper, paint, tile, varnish, or other.surfaces if they are likely.to
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be contacted by the product when applied according to label directions for
use. The data should also indicate that the material does not impart long-
lasting objectionable odors to furniture, cabinets, or other indoor treatment
sites which may be specified on the label.
(ii) wood-destroying beetles include species of Anobiidae, Lyctidae,
Bostrichidae, and one species of Cerambycidea; Hylotrupes bajulus (the
old house borer), all which may reinfest structural members of lumber in
storage. Tests for use of wood preservatives to control adults of powder-
post beetles, old house borer, carpenter ants, and certain other wood-
destroying insects should follow conventional test method procedures by
treatments designed for the control of flying insects; and these could be
evaluated by tests paralleling those for other household pests.
(iii) Wharf borer adults emerging into buildings or in areas around
the sites of former buildings are controlled by treatments designed for
the control of flying insects, and these are evaluated by tests paralleling
those for other household pests. [Refer to § 95-11].
(iv) Subterranean termite attacks in a structure are prevented or
controlled through impregnation of soil with a termite toxicant beneath
and adjacent to the structure. Protection of wood from attack by subter-
ranean termites may also be provided by impregnation of the wood by a
termite toxicant.
(v) Dampwood termites are evaluated by the same procedures that are
practical for the control of subterranean termites. [Refer to paragraphs
(b)(l)(i)(B) and (b)(1)(ii)(B) of this section].
/
(vi) Drywood termites are controlled by fumigation or treatment of
channels with dusts, liquids, or spot fumigants for the elimination of dry-
wood termites should be evaluated by observing the effect of such treatments
under field conditions. Absorptive dusts have been used as a protective
barrier treatment and should be evaluated on this basis in field treatments.
For general considerations, refer to §§ 90-3 (c)(2) and (d)(l) through (4).
(b) Specific considerations and suggested performance standards.
(1) Subterranean termites are by far the most important structural pests.
These insects damage wood and other cellulosic building materials causing
major property damage particularly in the southern areas of the United
States. Control procedures usually are reflective of the fact that colonies
of these termites require contact with the soil as opposed to drywood or
dampwood termites which may exist independently of soil moisture.
(i) Preventive treatments - soil barriers. (A) Data considerations.
Subterranean termite attacks in a structure are prevented or controlled
through toxicant impregnation of the soil beneath and adjacent to the
structure. Data should indicate that the treatment does not cause intolerable
injury to building foundations, plants, shrubs, or to other ornamentals.
For general considerations, refer to § 90-3 (c)(l).- The effectiveness of
prophylactic termite treatments is measured by the time over which the
toxic barrier remains effective in resisting penetration by the termites.
Therefore, the efficacy data should indicate the establishment and maintenance
of this toxic barrier.
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(B) Test methods. Published laboratory methods are not currently
recognized by the Agency as giving a reliable evaluation of soil toxicants
for termite control, but three field tests are recognized as giving a
reliable evaluation of soil toxicants: the Stake method, the Ground board
method, and the Modified ground board method.
(C) Suggested performance standards. Data derived from such testing
should provide complete resistance to termite attack for a period of 5
years, based upon annual reinspection. The tests should be in geographic
areas which provide year-around pest pressure (usually in the southern
0.S.).
(ii) Preventive treatments - wood impregnation. (A) Data considerations.
Subterranean termite damage in" a structure is also prevented by treatment
of susceptible materials. Ihese treatments are applied as dips, brush-on
applications, sprays, or pressure treatments. The activity of the materials
is such that damage to the treated cellulesic substance is precluded or
reduced either because the treated material is toxic upon ingestion or
sufficiently repellent that ingestion does not occur. The effectiveness
of such treatments is determined by a measurement of the duration for
which the treated material resists termite attack.
(B) Test methods. Modifications of the Stake method, Ground board
method, and Modified ground board method are acceptable in evaluating
impregnated wood products.
(C) Suggested performance standards. When acceptable data derived
from testing for at least 2 years but less than 5 years shows a complete
resistance to termite attack, the product may be registered contingent
upon a restriction which specifies annual reinspection. The tests should
be conducted in geographic areas which provide year round pest pressure
(usually in southern U.S.).
(iii) Infested sites. (A) Data considerations. Data should be
derived from experiments utilizing field infestations. These experiments
must address the considerations contained in § 90-3 and evaluate the effects
of the treatment as related to the infested site.
(B) Test methods. (Reserved).
(C) Suggested performance standards. Data should indicate not only
that the insects are killed, but that those insects in egg and nymphal
instars at the time of treatment will be controlled either by immediate or
residual toxicity. The data should clearly indicate that the entire colony
is destroyed by isolation from the essential water source by the establishment
of a pesticidal barrier, through the penetration of all wood galleries by
the pesticide, or by both routes.
(2) Dampwood termites and drywood termites-.. Dampwood termites may
exist in structural members .where a water source is constantly available
to the colony due to faulty plumbing., roof leaks, or other factors. Drywood
;termites, can. colonize in. wood.or othefCelluloaic gubs'tances .regardless . ,...
of.._ariy apparent water'source, since'they" are capable' -of' metabolizing suffi-
cient moisture from the materials they ingest. For/-tn'is reason., applications
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of pesticides for the control of these pests are usually preventative or
eradicant treatments of actual infested material. These wood treatments
generally parallel those for subterranean termites, and paragraphs (b)(l)(ii)
and (b)(l)(iii) of this section address the data considerations, testing pro-
cedures, and performance standards which are also applicable to compounds
used against dampwood and drywood termites.
(3) Other terrestrial pests of structures. These include insects
such as powder post beetles, the old house borer, carpenter ants, carpenter
bees, longhorned borers, the furniture beetle, and the wharf borer. The
biological characteristics, sites of infestation, types of damage and treat-
ments used for control of these pests are complex and often complicated in
nature. Data to support use patterns for these pests will be considered
on a case-by-case basis when applicable, until specific data considerations
are made available.
(4) Structural pests in aquatic sites. (Reserved).
§ 95-13 Stored product treatments.
This section concerns the data considerations for the determination of
the effectiveness of invertebrate control pesticides against pests of
stored products. These include the confused flour beetle, rice weevil,
lesser grain borer, Indian meal moth, cowpea weevil, Angoumois grain moth,
cadelle, granary weevil, sawtoothed grain beetle, and other stored product
insects. These pests should be controlled not only in the stored product
commodities but also in facilities where the commodities are stored,
processed, manufactured, packaged, and transported. Some species cause
damage in both the larval and adult stages. See § 95-30 reference no. 13
for examples of methods for evaluating stored-grain insect pests.
(a) General considerations. The testing of a pesticide product for
household use should follow the same procedures as are appropriate for the
product's commercial use.
(1) Site selection. Commodities to be protected include grain,
seeds, cereals and other dry foods, tobacco, woolen and feather goods/
furs, and furniture stored in such sites as grain elevators, boxcars,
ship's holds, and warehouses. The types of commodities and packaging
around which the test product is to be tested must be representative of
the types of commodities and packaging in areas in which the product is
intended to be used, such as pantries, storage closets, warehouses, and
other areas. Test sites should be selected which are representative of
pest populations commonly encountered. Other factors such as climate and
geographic location should also be considered and reported. For general
considerations, refer to § 90-3 (a)(3)(iv) and (b)(l), and (3).
(2) Number of trials. A minimum of 5 large-scale, geographically-
separated trials are generally necessary, but the number of trials can
vary somewhat due to the accessibility of infestations, fluctuations in
pest population pressures, behavior, and other important considerations
in the biology of the;target pest. , .
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(3) Exposure time. The length of the treatment exposure time should
be stated in terms of time length such as 12/ 24, or 48 hours, depending on
the formulation of the test product and the pest species involved.
(4) Temperature and humidity. Temperature and humidity should be
reported in the data since they can affect the activity of the pest
species and the effectiveness of the test product. This is particularly
true when testing fumigants.
(5) Residual considerations. The residual activity of the pesticide
product should be evaluated to validate control over periods of days and
weeks. Pesticides to be registered for use on stored food products are
required to have a residue tolerance level set or they mus.t have an exemption
from the requirement for a tolerance, as stipulated in the Federal Food,
Drug and Cosmetic Act (21 U.S.C. 346, 346a, and 348).
(6) Application techniques and equipment. Ultra-low volume application
equipment or fumigation techniques should be evaluated if these methods are
specified on the label.
(7) Evaluation and reporting procedures. The procedures for eval-
uating product performance should be specified in the presentation of the
data. Reports should include insect counts, amounts of damage, or other
measures of determining the effectiveness of the test product. Results
should include the average pest infestation reduction compared with with
that in the controls, and an assessment of any possible adverse effects
on the grade or quality of the treated commodity. Raw data on pest counts
or other measures of control should be summarized and submitted. Tests should
be conducted on palatability in all cases where food or feed germination
or viability should be provided. Space sprays and residual treatments should
be evaluated under conditions which minimize the contamination of stored
feed, food, or tobacco. Tolerances fcSr stored food and feed should not be
exceeded. For general considerations; refer to § 90-3 (c}(2) and (d)(l)
through (4). >
(b) Suggested performance standards. These suggested standards are
determined on the basis of results obtained from laboratory and simulated
or actual field tests in which the efficacy of the test product is evaluated
against products of known efficacy used as reference standards, or against
untreated controls. The following stored products pests are covered:
confused flour beetle, rice weevil, lesser grain borer, Indian meal moth,
cowpea weevil, Angoumois grain moth, cadelle, granary weevil, sawtoothed
grain beetle and other stored product insects.
(1). Aerosols. Aerosol products should demonstrate efficacy equal to or
better than the official test aerosol (OTA).
(2.) Fumigants. Fumigant products should demonstrate .efficacy compa-rable
to a registered standard such as methyl bromide.
(.3) Insect resistant- packaging. Insect resistan-t ^packaging should demon-
strate complete protection, .agadnst s.tored product pests. •-Control- is based
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276
on representative packages which should be examined for insect penetration, and
for insects in the commodity, at frequent intervals throughout the tests.
(4) Residual protectants. Residual protectants should demonstrate
efficacy comparable to a registered standard such as malathion.
(5) Vapors. (Reserved).
§ 95-14 Fabric treatments.
This section concerns the data considerations for efficacy evaluation of
pesticide products used for control of invertebrate pests of fabrics. The
principle pests of fabrics include the webbing clothes moth and black carpet
beetle.
(a) Definitions. The following definitions and explanations are of
special importance in understanding this section:
(1) The term "fabrics" refer to all animal, plant, and synthetic
fibers or blends to such materials.
(2) The term "temporary fabric treatments" refers to non-residual
spray applications which kill fabric pests by contact action. Such
treatments may require repeated application.
(3) The term "semipermanent fabric treatments" refers to residual
chemical applications to wool, fur,.-or hair'rugs, garments and blankets
as coarse sprays or, in some cases, in dry cleaning solutions. To prepare
for semipermanent mothproofing tests, the articles must be sprayed lightly
but uniformly until the surfaces are moist but never soaked or saturated,
and the sprayed articles thoroughly dried before testing.
(4) The term "permanent fabric treatments" refers to residual chemical
impregnation in the fabric during the dye process. Test to determine effec-
tiveness of these treatments require that the impregnated fabrics be tested
before and after repreated washing, drycleaning, and other tests such as the
performance tests of the American Association of Textile Chemists and
Colorists.
(b) General considerations. The testing of a pesticide for household use
should follow the same procedures as are appropriate for its commercial use.
(1) Test species. Although several species of insects attack woolen
fabric and other animal fibers, the webbing clothes moth and black carpet
beetle should be used in conducting efficacy tests for fabric protectants.
(2) Number of test insects. The number of test insects used is impor-
tant in obtaining an accurate evaluation of these types of treatments, and
at least 120 insects of each pest s-tage (larvae and adults') should be used
for each test when evaluating temporary fabric protectants (direct contact
sprays). When evaluating semipermanent and permanent, fabric protecfcahts,
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use the number of insects set forth in the official CSMA "Textile Resistance
Test", and the American Association of Textile Chemists and Colorists testr
method 24-1977.
(3) Number of trials. Four large-scale trials are generally preferred,
and the number of trials can vary somewhat due to the accessibility of infes-
tations, fluctuations in the pest population pressures, behavior, and other
important considerations in the biology of the target pest. Because of the
nature of fabric-insect problems, large scale trials are often impossible
to conduct. However, for permanent fabric treatments, laboratory application
data should be supplemented by pilot plant or mill scale application data.
(4) Observations. For agents providing temporary mothproofing, the .
test insects should be observed 24 and 48 hours after treatment and the
percentage of knocked-down (KD) and dead plus moribund (D+M) insects >
recorded. Data should be presented in tabular form. When testing semi-
permanent mothproofing products, the appropriate excrement-weight or
fabric-weight loss data, mortality data, and the analytical results should
be submitted in tabular form. Visual feeding damage ratings are useful.
(5) Application techniques and equipment. The data obtained must
reflect the effectiveness of the test product when applied in commercial
dry-cleaning and dying equipment. For general considerations, refer to
§ 90-5 (a)(3)(i).
(6) Evaluation and reporting procedures. The procedures used to
evaluate product performance should be specified in the data. Reports should
include mortality, fabric weight loss, excrement weight, analytical results,
or other measures of determining the effectiveness of the test product.
Data obtained should be based on the effect of the test product in protecting
fabrics from damage by the larval stage of the pests. All observed adverse
effects of the test product should be reported. For general considerations,
refer to §§ 90-3 (c)(2) and (d)(l) through (4).
(c) Suggested performance standards. These suggested standards are
determined on the basis of results obtained from laboratory tests following
procedures set forth in the official CSMA "Textile Resistance Test," and
the American Association of Textile Chemist and Colorists standard test
methods 24-1977 and 28-1977. The performance standards cover the following
fabric pests: webbing clothes moth and black carpet beetle.
(1) Temporary fabric treatments. Non-residual products that should pro-
vide almost complete (99%) control under repeated treatments every 30 days.
(2) Semi-permanent fabric treatments. Residual products should
provide protection of the fabric as specified by CSMA and the American
Association of Textile Chemists and Colorists test methods for 6 months
to one year..
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(3) Permanent fabric treatments. Residual products which are impreg-
nated into the fabric during the dyeing process should provide protection of
the fabric as specified by CSMA and the American Association of Textile
Chemists and Colorists before and after the performance tests listed below:
Manipulation Minimum requirements Maximum requirements
Washing 5 times
Drycleaning - 5 times
Stoddard solvent or
perchloroethylene
Hot pressing 5 times
Sea water 5 immersions
Perspiration:
acid - 5 immersions
alkaline - 5 immersions
Ligh t: Fade-Ome ter 4 0 S tandard Fading
10-15 times
10-15 times
10-15 times
100 Standard Fading
Abrasion
hours
2000 revolutions -
wear test machine
2000 revolutions for
6 minutes -
Accelerotor
hours
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2.79
§ 95-30 Acceptable methods
The following thirteen items contain acceptable protocols for the
testing of invertebrate control agents.
(a) The following 10 items are booklets prepared for the Agency by
eminent scientists in each discipline, under contract through the American
Institute of Biological Sciences (AIBS), and all booklets are available
from the National Technical Information Service (NTIS), 4285 Port Royal
Road, Springfield, VA 22151. . . -••.••••••.... • '
(1) Anonymous. 1977. Analysis of specialized pesticide problems. Inverte-
brate control agents. Efficacy test methods. Vol. I. Foliar treat- ••
ments I. Deciduous fruit trees, small fruits, citrus and subtropical'*
fruits, tree nuts. EPA contract number 68-01-2457, AIBS, Arlington,
VA. 80 pp.
(2) Anonymous. 1977. Analysis of specialized pesticide problems. Inverte-
brate control agents. Efficacy test methods. Vol. II. Foliar treat-
ments II. Field crops, forage crops, rangeland, vegetables—field
and greenhouse. EPA contract number 68-01-2457, AIBS, Arlington,
VA. 137 pp.
(3) Anonymous. 1977. Analysis of specialized pesticide problems. Inverte-
brate control agents. Efficacy test methods. Vol. III. General soil
treatments. EPA contract number 68-01-2457, AIBS, Arlington, VA. 99 pp.
(4) Anonymous. 1977. Analysis of specialized pesticide problems. Inverte-
brate control agents. Efficacy test methods. Vol. IV. Livestock,
poultry, fur, and wool-bearing animals. EPA contract number 68-01-2457,
AIBS, Arlington, VA. 68 pp.
(5) Anonymous. 1977. Analysis of specialized problems. Invertebrate control
agents. Efficacy test methods. Vol. V. Stored products and premise
treatments. EPA contract number 68-01-2457, AIBS, Arlington, VA. 58 pp.
(6) Anonymous. 1977. Analysis of specialized problems. Invertebrate control
agents. Efficacy test methods. Vol. VI. Lawns, ornamentals, forest
lands. EPA contract number 68-01-2457, AIBS, Arlington, VA. 55 pp.
(7) Anonymous. 1977. Analysis of specialized problems. Invertebrate control
agents. Efficacy test methods. Vol. VII. Human and pet treatments.
EPA contract number 68-01-2457, AIBS, Arlington, VA. 24 pp.
(8) Anonymous. 1977. Analysis of specialized pesticide problems. Inverte-
brate, control agents.. Efficacy test methods. Vol. VIII. Mosquitoes,
black, flies, midges and sand flies. EPA contract number 68-01-2457,
AIBS, Arlington, VA. 52 pp. ' . -. " : "-,
(9) .Anonymous.. 1978. Analysis of specialized problems. Invertebrate control
agents. IX.. Baculoviruses and en.tomogenotos-...bacter.iaf.. •;EPA..eontr.ax:'f " i-
,•.....-.._ number 68-0:1-2457, AIBS, Arlington., VA. L15 pp...
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280
(10) 'Anonymous. 1978. Analysis of specialized pesticide problems. Inverte-
brate control agents. Efficacy test methods. Vol. X. Turf, ornamen-
tals, forest lands. EPA contract number 68-012457, AIBS, Arlington,
VA. 81 pp.
(b) Items (11) and (12) are adapted from guidance documents prepared by
scientists in the Agency's Registration Division of the Office of Pesticide
Programs.
(11) Guidance for the testing of flea and tick collars for control of ticks
on doos.
1. Scope. There are many commercially produced pesticidal matrices
(as collars and tags) which may now or in the future bear label claims for
the control of fleas or ticks. Such claims are acceptable only upon a demon-
stration of the efficacy of the product, as claimed, when used according to
label directions. The strength and kind of acceptable label claims are
generally determined by four factors which will be discussed later in greater
detail. These factors are:
1.1 The degree of control against fleas and ticks.
1.2 The anatomical coverage of the product.
1.3 The species of parasites controlled.
1.4 The duration of the control afforded. ; -'•'..''
This protocol is designed to aid the investigator or registrant in the
development of data as it applies to the registration of various claims for
impregnated products, in such a manner as to include all of the above mention-
ed considerations.
2. Definitions:
2.1 Control animals - Those animals not treated with any pesticide
product.
2.2 Test collar animals - Those animals wearing the collar which is
under evaluation.
2.3 Placebo - A collar formulated from the same carrier matrix as
the collar to be tested, but lacking any active pesticide ingredients.
2.4 Qualified personnel - Individuals who count or apply the reinfes-
tations and are trained in finding and recognizing the parasites on the dog.
They should also be familiar with the biology of the parasite and the record-
ing techniques necessary in development of meaningful data.
2.5 Positive control - A collar registered for the proposed label claims.
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3. Definition of control. The magnitude of the infestation on the
control animals should be representative of the population as established
by the screening procedures. Test collar dogs will then be compared to the
controls by parasite count and body areas infested. The acceptance of a
collar for registration and ultimate control claims allowed will be dependent
on a number of factors. These may include the overall number of fleas or
ticks, the number of live attached ticks and live fleas on the test collar
dogs in comparison to the control animals; and the positive control animals.
4. Size of the test. The test should be large enough to permit good
statistical evaluation. The exact number of animals bo be evaluated cannot
be specifically stated. However, the investigator is reminded that the
validity of the resulsts of the testing program is directlyrrelated "to the
degree of variability within the test. Increasing the number of test animals
increases the reliability of the test results. In general, a minimum of six
animals per group is required, with 10 preferred in any single test.
5. Selection of test arthropods.
a. Ticks. Limited or single species testing may be utilized to support
restricted claims. For general tick control claims, however, testing should
be designed to show sufficient biological activity to control those species
of ticks common to dogs. Therefore, a minimum amount of testing should be
conducted to ascertain sufficient activity against representative tick
species. In order to support a general tick claim, data must be derived
on the brown dog tick (Rhipicephalus sanguineus) and at least one other
species of tick common to dogs. In addition to the brown dog tick, other
tick species common to dogs include the lone star tick (Amblyoma americana),
American dog tick (Dermacentor variabilis) and the Rocky Mountain wood tick
(Dermacentor andersoni). D. variabilis is preferred.
b. Fleas. The only species required to be tested is the cat flea,
Ctenocephalides felis. This flea is the predominant species on both dogs
and cats. Mixed flea populations may be used providing that an adequate
infestation of C. felis is established on the test animals and the fleas
of different species are counted and recorded separately.
6. Testing specifications.
6.1 All testing shall be conducted on male and female adult dogs of
various sizes and hair lengths.
6.2 Placebo collars shall be used on the control animals.
6.3 The animals should have a clean bill of health substantiated by
a veterinarian and should undergo a preconditioning period of at least
seven days before use. All animals should be washed with a non-residual
insecticidal shampoo before initiation of the testing.
6.4 All control and test collar dogs shall be prescreened for tick
attachment before initiation of the test. .Only, animals showing acceptable
•tick attachment shall be used in the study. .......
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6.5 Any other insecticides used on or near the animals during the
course of any testing period must be fully justified, including any effects
of such application on tick populations.
6.6 While not required, a positive control test using a collar regis-
tered for tick control would serve as a standard reference and aid in the
final evaluation of the test collars.
6.7 A minimum of 5 large dogs (40 Ibs.) must be represented in the
sum of the entire series of tests submitted for registration. It is recom-
mended, that at least 3 large dogs be included in each group in each test.
7. Exposure.
7.1 Adult parasites are recommended for infestation of the control
and test collar animals. All ticks should be of uniform age and feeding
status (unfed) since the last moult.
7.2 There are several basic methods for infesting dogs with fleas and
ticks. Any of these may be acceptable in developing data for registration.
Regardless of the infestation procedure, the primary consideration is to
achive adequate numbers of attached parasites with sufficient anatomical
distribution to allow evaluation of collar effectiveness. The infestation
procedure should be refined to the extent that equal infestation pressures
are applied to both control and test collar dogs. In general, a minimum
of 3 ticks and/or 5 fleas per dog (controls) are necessary for a valid test.
7.3 All animals should be housed individually in holding enclosures.
The holding environment should provide both indoor and outdoor exposure.
7.4 If the proposed label claims refer to the ability of the collar
such that performance is unaffected by washing, then the test collared
animals should be bathed biweekly with a non-insecticidal shampoo.
8. Reinfestation.
8.1 Since fleas and ticks will leave a dog in certain situations
regardless of the presence of a collar, a reinfestation of unfed parasites
must be made at specific intervals during the testing. All animals should
be reinfested periodically following the initial infestation for the entire
period of control proposed for the product. Frequent observations as
described below for the initial infestation should be recorded for each
subsequent reinfestation. The same period of observation (1-7 days) should
be utilized for these reinfestations, and all remaining parasites should
be removed at the end of each counting period. The duration of testing
must be consistent with label claims; therefore, infestation pressure must
be maintained throughout the entire period of control. It is suggested
.that the actual testing be continued in excess of the label claim being
sought to provide more confidence in the data derived.
8.2 The reinfestation pressure should be of sufficient degree to induce
an acceptable quantity of parasites to attach to the control animals.
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8.3 Hie same collar shall be used on an animal for the duration of a
study, and control and test collar animals shall not be interchanged prior
to a reinfestation challenge.
9. Observations.
9.1 All observations should be made and records maintained by qualified
personnel.
9.2 A pretreatment inspection of all animals should be made to ascertain
the number of parasites on the dogs prior to the installation of the collars.
9.3 The initial observations should be made soon after inoculation to
ascertain the knockdown potential of the collar. This observation (usually
at 2 hours, once again at 8 hours) is essential for a "fast acting" claim
if so desired by the registrant. It is realized that observations made this
soon after inoculation may affect the degree of attachment for the first
counting period.
9.4 Frequent counts of parasites are then recorded (e.g., every 2-3
days) after infestation for a period of 5-7 days or until substantial de-
tachment occurs after engorgement. These inspections should indicate the
number, sex, attachment and viability (living or dead) of the ticks along
with the specific locations on the animals (head, neck, torso, rump, legs,
feet). This information should be recorded in conjunction with the age of
the collar for the particular day on which a dog was inspected. For fleas;
only the number of parasites need be recorded.
9.5 Records should be kept for each individual animal, and the data
combined into tables which compare the various test groups (test collars,
controls, positive control animals).
9.6 All parasites remaining on animal (both test collar and control)
at the end of the counting period should be removed as well as possible.
9.7 All abnormal signs and symptoms during the course of the study
shall be noted and recorded for both control and test collar animals.
10. Evaluation and interpretation of data derived from testing. The
strength and kind of acceptable level claims are generally determined by
four factors:
10.1 The degree of control.
10-2 Anatomical sites of tick attachment.
10.3 The species of parasites tested.
10.4-..The duration of effectiveness. ;. . . . ; •
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Ticks. First is the degree of control afforded by the collar.
Such claims as "control ticks" must be supported by data which indicate
that the collar will effectively reduce substantial populations of ticks
to a level significantly below that of the control. For all practical
purposes, this would be control to the extent that the user would not
usually be required to rely on other pesticides or methods of control
during the time period specified on the labeling. It is realized that
100% tick control will rarely be achieved in tests where the outdoor
exposure may provide for a source of additional reinfestation. In general,
90% control is a desirable level of reduction, but even 80% control may
support label claims under certain circumstances.
It is also realized that some benefit is derived from collars which do
not give control of ticks, yet induce a consistent recognizable reduction
in the number of ticks on the test animals as compared to the control
animals. Label claims for "aids in the^control of ticks" are appropriate
for such products. A recognizable reduction is a level of protection
which is evident not only upon statistical analysis but which would be
apparent to the purchaser of the product in actual use situations. A
minimum of 50% reduction must be apparent for an "aids in control" claim.
The second area of consideration is the use of anatomical restriction
in those cases where the concentrations of pesticide generated from the
collar matrix do not control.ticks over the entire body of the dog. If
anatomical restrictions are necessary, they will be added to the tick
claim. A familiar example could be to specify control "in the head and
neck area only."
As stated in section 5.a., the species of ticks tested will determine
the label claims to be supported.
The final factor involved in the interpretation of the data is the
duration of testing as compared with efficacy. Label claims must be
justified by data which are derived from testing the collar for that period
of time which is to appear on product labeling. Such data must at least
show the collar to be efficacious over the control period proposed for the
product.
Fleas. For fleas, a minimum of 90% control, as compared to the counts
on the placebo animals, is required for the duration of testing. There are
no "aids in control" claims or anatomical restrictions in regard to claims
for the control of fleas.
11. Other data.
Note that other kinds of data are also considered when evaluating the
efficacy of a product. Such information as pesticide release rates, when
accurately measured and correlated with the effective dosage of the active
ingredient to ticks, may also be used. Such data should be derived from
in vivo tests and reported as mg. active ingredient released per day.
Additionally, consumer or veterinary testing is often utilized to further
indicate the performance of a collar. •
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This provisional protocol is not designed to be the only acceptable
test methodolgy for the generation of applicable data. It is merely one
acceptable method which attempts to resolve many of those items viiich have
resulted in data which were unacceptable to support registration in the
past.
(12) Cockroach bait test method.
I. In troduc tion.
This is a suggested test procedure for cockroach baits to be used to
control cockroaches in homes, warehouses, food establishments, etc. 'The
following outline is a suggested series of laboratory and field tests.
The important factors are to:
1. Establish the proper dosage and intrinsic attractancy of the
formulation in "free-choice" laboratory tests.
2. Evaluate the utility of the product under actual-use conditions.
II. Test protocol.
A. Choice box laboratory test.
1. Enclosures (4) constructed out of plywood. Dimensions approximately
2'x4'x4". Tops are either made of plexiglass or the sides are greased or
teflon coated to prevent escape. Each enclosure is divided into two halves
by a line drawn or painted down the center giving two 2'x2' areas. In each
side are placed a cardboard hide (12"xl2"xl2" ht. is suggested) and water.
In three of the enclosures, one side is treated with the proposed dosage
of bait (gm. per unit area) and on the other side an equal amount of Purina
Dog. Chow (R) is placed. Some of the bait and dog chow may be placed in the
hides. In the remaining box only Purina Dog Chow is used as a control.
Each box receives 50 last instar nymph or adult German cockroaches, Blatella
germanica placed either along the center line or 25 in each side. Boxes
are kept in areas providing a normal day/night photoperiod or a minimum of
8 hours darkness each day. Roaches are counted at 1, 3, 5, 7, 10, 14 and
21 days and the percent mortality can be derived from Abbott's formula. A
table of results and the counts for the individual boxes should be submitted.
Consumption may indicate that additional bait is necessary to give acceptable
mortality. If all the bait is consumed, then additional bait or dog chow
may be added as necessary. Such additions must be recorded and reported.
Testing may need to be repeated in situations where definitive results are
not obtained.
2. Ovicidal and chemosterilant action-trials may be designed to indicate
either a chemosterilant effect or toxicity to the eggs carried by the female.
For chemosterilants, the laboratory choice box. tes.t should be continued for
as many days as necessary to collect significant mimbers of oothecae. Upon
deposition, these should be collected from the boxes, and kept in separate
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conbainers. Percent eclosion should be recorded for treated boxes and
compared to the controls. To enhance deposition, a high number of non-gravid
adult females should be used at the beginning of the test. No gravid females
should be used at the initiation of a test for chemosterilant effects. For
oothecal toxicity, only by bearing females should be used. The oothecae are
again collected and percent eclosion compared to the control group.
B. Field trials (Experimental use permit may be required).
1. Once the appropriate dosage has been discovered in the laboratory
testing, the product must be field tested to be registered. Field testing
usually involves apartment houses, but food establishments or other areas
with heavy infestations may also be used. The most popular methods of
infestation assessment are via flashlight inspection or trapping. After
the pretreatment counts are made, and application has been completed,
additional counts are usually initiated on a weekly basis. Along with
cockroach numbers, the degree of sanitation and any other pertinent details
should be reported. Comparison treatments (i.e., a registered standard
bait material) should be used as a point of reference.
III. Modifications.
1. Most testing laboratories make modifications in protocol specifi-
cations as facilities dictate. For this reason we suggest that protocols
be prepared and submitted for EPA review prior to the initiation of testing.
Such protocols should be submitted to the product manager for the proposed
product.
(13) LaHue, D.W., and L. Bulla. 1977. Guidelines for Evaluating
Efficacy of Chemical Insecticides as Protectants of Stored Grain Against
Insect Pests. Bull. Ent. Soc. Amer. 23: 117-118.
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287'
Series 96: EFFICACY OF VERTEBRATE CONTROL AGENTS
§ 96-1 General considerations.
(a) Scope of vertebrate control agents. (1) A vertebrate pesticide
is defined as any substance or mixture of substances intended for preventing,
destroying, repelling, or mitigating any vertebrate animal which the Admin-
istrator declares to be a pest. Vertebrates include fish, amphibians, rep-
tiles, birds, and all wild and domestic mammals. Vertebrate pesticides
include oral, dermal, and inhalation toxicants; irritants; repellents (odor,
taste, or tactile reaction); chemical frightening agents; anesthetizing . . -
chemicals; and reproductive inhibitors.
(b) Scope of data considerations. Toxicity data may include oral, dermal
and inhalation toxicity tests for terrestrial vertebrates, or static and flow-
through toxicity tests for aquatic vertebrates. These data are used to assess
the appropriateness of dosage levels in subsequent efficacy testing. When
conducting toxicity tests, consult references 1, 9, 10, 11, and 16 in § 96-25
for acceptable protocols. For a dose-response curve consult reference 5.
Laboratory acceptance data provide information to establish bait carriers
without toxicants that will be acceptable to the target animals. Laooratory
efficacy data establish the preliminary effectiveness of the product under
cage or pen conditions. Field efficacy data aid in determining the
effectiveness of the product under actual use conditions.
(c) Pattern of considerations. (1) Specific product performance data
considerations are outlined below for vertebrate pesticides in §§ 96-2 through
96-19. For each type of product (e.g., commensal rodenticides), the data
considerations are addressed in the same order in which tests may be conducted:
toxicity data, laboratory acceptance data, laboratory efficacy data, and
field efficacy data.
(2) Under each of the kinds of data considerations stated above (toxicity,
laboratory acceptance, laboratory efficacy, and field efficacy data) are the
following major headings: test standards, test information, and acceptable
protocols. "Test standards" prescribe the standards of an acceptable
protocol. "Test information" suggests what kinds of test information
should be submitted. "Acceptable protocols" cite examples of adequate
protocols (when they exist) or refer the reader to those sections in § 96-30
that may be helpful in developing an acceptable protocol.
(d) Field studies. (1) Censusing methods. When conducting field
studies, some means should be used in determining the number of animals before
and after treatment to establish efficacy. For guidance on censusing, con-
sult the data - considerations .in the specific..product sections of. this subdivision
and in the Field Census Techniques section in § 96-30. •
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288
(2) Test and control areas. The use of test and'control areas when
appropriate in vertebrate control is necessary to insure that the reduction
of the target pest or its damage in test areas is due to the treatment and
not to other factors. Generally, both test and control areas should be of
equal size"and subjected to the same conditions including target animal
pressures and abundance. The exact site and location of these areas will
depend upon the target pests distribution, behavior and mobility. Exper-
imental areas should be similar and include those sites when actual use is
anticipated.
(3) Regional studies. Unless specified in particular sections, the
number of advanced field studies necessary for each species and use pattern
is five studies for each region claimed or implied on the label. These
regions are the northeast, northwest, southeast, southwest, and midwest
continental United States.
(e) Detoxification studies. Certain toxicants are applied in water
to kill the target animals (e.g., certain pest fish). Subsequently, these
aquatic areas may be restocked with more desirable species that may also
be susceptible to the toxicant. Therefore, detoxification studies aid in
determining how much time must elapse before restocking.
(f) Relation to other guidelines. (1) Besides the product performance
data discussed in the guidelines of this subdivision, additional data may be
needed when conditions such as pattern and location of use, claims made
or implied, method of application, water quality, exposure period, detoxifi-
cation, and species and subspecies differences indicate that the data are
insufficient to .answer specific questions. Registration applicants should
become especially familiar with § 90-3 General considerations of this subdi-
vision, as well as Subdivisions D - Product Chemistry, E - Hazard Evaluation:
Wildlife and Aquatic Organisms, and F - Hazard Evaluation: Humans and
Domestic Animals. Applicants should note that fish and wildlife toxicity
data (of Subdivision E) are required for all vertebrate pesticides.
Obtaining these data may require three kinds of tests: studies to determine
their toxicity to nontarget organisms through primary (direct) poisoning
and secondary (indirect) poisoning; reproductive studies; and field studies.
(2) In developing product performance data, testing personnel should
be thoroughly familiar with the life habits and behavior of the target species
and with Subdivision H - Labeling Guidelines for Pesticide Use Directions
particularly §§ 100-5 and § 106-1 through 19 dealing with labeling of
vertebrate control products. When designing efficacy tests for use under
captive conditions, corroborative methods of field application should be
kept in mind.
(3) When vertebrate pesticides are applied on or near plants or seeds,
data should insure that any phytotoxicity of the formulation does not
significantly reduce plant yield, plant quality, or seed germination.
Such studies should be conducted on representative plants or seeds and should
reflect the effects of application on plant growth or seed development through
a growing cycle. The data should also reflect effects of repeat applications
where such applications are recommended. The type and percentage of phyto-
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toxicity should be reported. Common types of injury are leaf burning, chlor-
osis, and stunting. Consult Subdivision J for further data recommendations on
phytotoxicity.
§ 96-2 Fish control agents.
(a) General. This section deals with efficacy data and test procedures
for toxicants and repellents that claim to control fish. Target animals
include lamprey, squawfish, and carp. Submission of efficacy data is waived
for this section.
(b) Acute toxicity studies. Submit the results of static jar or flow-
through LC50 tests on the active ingredient for each species and life stage' "•
(egg, larva, adult) claimed to be controlled by a fish toxicant or fish
repellent. The purpose of these studies is to assess the toxicity of the
active ingredients to the target species.
(1) Test standards. Refer to reference 1 of § 96-25 for test standards.
To evaluate the effect of water quality on toxicity of active ingredients,
for each major species to be controlled, LC5Q tests should be conducted at:
4 water hardnesses (e.g., 11, 44, 170, 300 ppm); 4 temperatures (e.g., 7, 12,
17, 22°C); and 4 pH levels (e.g., 6.5, 7.5, 8.5, 9.5). Refer to reference 2
of § 96-25 for a discussion of selected water quality factors.
(2) Test information. Indicate the species and origin of test
fish. Describe acclimation of fish, laboratory conditions (e.g., temperature,
light, water quality, pH, hardness, salinity), and changes in behavior during
the test that might affect efficacy. For example, were the fish repelled by
a toxicant or killed by a repellent? Submit a dose response curve with the
computation of the slope and LC50 with 95% confidence limits for each environ-
mental factor evaluated (e.g., ranges on temperatures, water hardness, pH
levels). For a complete list of test information, refer to page 47
of reference 1 in § 96-25.
(c) Laboratory efficacy studies. Submit the results of static jar or
flow-through LC99 toxicity tests for each species and life stage (egg, larva,
adult) to be controlled by the formulation. The purpose of these studies is
to assess the laboratory efficacy of the formulation and to interpret
the results of subsequent field tests.
(1) Test standards; Conduct LC99 tests, evaluating the same water
qualities required under the LC50 test described in (b)(2) of this section.
(2) Test information. Submit the same test information requested
under UCc,Q toxicity test described in (b)(3) of this section on the
formulations. Indicate the chemical composition of the formulation tested.
(d) Detoxification studies. Submit the results of LC50.detoxification
"tests for.each species claimed to- be controlled. The purpose of these studies
is to determine the detoxification rate of formulation. .
'• "(T) Test sTtandards': ''Consult ref e'rehees; 1 •-," 3., . a-ftd *4 *of §' 96-25 f or
deto'xi-f ication test.:.s:tandards.. Terminate- tests ,wh-e.n.;thev.'biologicaJL;-'ac-tl'vr.ty
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of the product has been reduced by .50% (half-life) for each major species
to be controlled. Conduct tests at: 2 water hardnesses (e.g., 11 and 300
ppm); 2 temperatures (e.g., 7 and 22°C); and two pH levels (e.g., 6.5 and
9.5).
(2) .Test information. Submit the same test information
as under the LCso toxicity test described in paragraph (b)(4) of this
section, indicate the chemical composition of the formulations, the
environmental conditions, and the time interval (half-life) when the
formulation has degraded to the point that a concentration of chemical
that would kill 50% of the test organisms initially will now kill only
25% of the target organisms.
(3) Acceptable protocols. For conducting LC50 (static jar or flow-
through tests) and for a complete list of information, refer to
references 1, 3, and 4 in § 96-25. For presenting a dose response curve,
consult reference 5 in § 96-25.
(e) Preliminary field efficacy studies. Submit the results of pre-
liminary field studies conducted in small aquatic areas under natural
conditions for animals claimed to be controlled. The purpose of these
studies is to assess the field efficacy of formulations under semi-labora-
tory and natural field conditions.
(1) Test standards. Use the target species. Conduct studies in
small water areas (plastic swimming pool, raceway, pond, or small stream).
Natural breeding populations of target and non-target organisms are
desirable. Hatchery or wild strains are acceptable. Test each formulation
and dosage level that is to be tested in the field. Monitor each group's
behavior frequently during the test to evaluate any unusual animal responses
that might affect efficacy. See Exhibit 1 in § 96-30 for acceptable test
standards, test information, and acceptable protocols.
(f) Advanced field efficacy studies. Submit the results of field
efficacy tests for each species and life stage (egg, larva, adult) claimed
to be controlled by a fish toxicant or fish repellent. The purpose of these
studies is to assess the field efficacy of the product under actual use
conditions.
(1) Test standards, (i) Number of regional studies. For each region,
water type (lake, pond, stream, marsh), and species claimed or implied, sub-
mit the results from five study areas. All ranges of pH, water hardness,
organic matter, temperature, and depth claimed or implied on the label should
be supported by adequate field data under those environmental conditions.
Minimum data considerations and the methodology to be used are similar to those
in Exhibit 1 in § 96-30.
(ii) Pre-treatment and post-treatment population censusing. The
density and distribution of the target fish before and after treatment
should be determined by appropriate methods. Adequate statistical anal-
ysis of the data should be provided. In the case of repellents the censusing
should be complete enough in the area surrounding the test area to show
the movement of fish from the treated area. ' •
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(iii) Site location and selection. The test and control areas
selected in each region should be representative of each of the major
environmental areas in the region where the toxicant or repellent may
be used (e.g., pond, lake, stream, marsh).
(2) Test information. For each test, indicate the species
controlled, formulation, location of the test and control areas, and
method, rate, and frequency of application. Provide a map showing
location and dimensions (area and depth) of test areas. Provide all
raw and summary data used to evaluate effectiveness. Include data on
target and nontarget organisms and the method and rationale of select-
ing experimental areas. The applicant is advised to refer to Subdivision
E of this part for details on evaluating .nontarget effects on wildlife
and aquatic organisms. Provide climatological data for study areas
during the test period. Describe the statistical methods used.
(3) Acceptable protocols. Refer to Exhibit 1 of § 96-30 and the
literature cited there to develop an acceptable protocol incorporating
the test standards above. Prior consultation with the Agency on such
protocols to be employed is strongly recommended.
§ 96-3 Aquatic amphibian control agents.
(a) General. This section gives information on efficacy data and
test procedures for toxicants and repellents that claim to control
aquatic stages of amphibians. Target animals include frogs, toads,
salamanders, and mudpuppies. Submission of efficacy data is waived
for this section.
(b) Acute and subacute toxicity studies. Submit the results of LC50
and LC99 toxicity studies for each species claimed to be controlled by an
amphibian toxicant. The purpose of these studies is to assess the appro-
priateness of dosage levels to be used in subsequent efficacy tests.
(1) Test standards. Use the target species. Cultured animals of the
target species are acceptable. Use at least 10 animals for each dosage
level and formulation. Cage animals individually if fighting or cannibalism
occurs. Observe any changes in behavior that may affect efficacy and gross
pathological changes found at post mortem. To evaluate the effect of water
quality on efficacy, LC50 tests should be conducted at 4 .water hardnesses,
4 temperatures, and 4 pH levels. (Consult reference 1 in § 96-25.) Also,
see § 96-2(b)-(l) of this subdivision.
(2.:) Test information. For test information, see § 96-2(b)(2) of
this subdivision.- .,-•-•-.-.:• -....••' •
(c) Laboratory efficacy studies for amphibian toxioants... Submit the
results' of the following laboratory e:£ficacy tes-t f-oc each .species claimed.
..to- be Gtwitrbiled w.ith,.a: toxicant. The purpose'of • these studies.'.is to- ass-es-s
Mveis-.-to:.-be.-rased:xn.-s-oJbsexjHeit-t -eff-icacy:'tests..
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(1) Test standards. Use the target species. Laboratory or cultured
strains are acceptable. Use at least 10 animals for each species and dosage
level. Group animals in lots of 10 if fighting or cannibalism does not occur
and if group caging of animals has been shown not to affect laboratory eval-
uations. If fighting or cannibalism occurs, cage and test animals separately.
Test each formulation that may be tested in the field. Monitor each group's
behavior frequently to evaluate any unusual animal responses that might
affect efficacy.
(2) Test information. For a complete list of test information,
see § 96-2(b)(2) of this subdivision.
(3) Acceptable protocols. For acceptable protocols, refer to § 96-2
(b)(3) of this subdivision.
(d) Laboratory efficacy studies for amphibian repellents. Submit the
results of laboratory efficacy tests for each species claimed to be control-
led with a repellent. The purpose of these studies is to assess the labora-
tory efficacy of formulations and to interpret results of subsequent field
tests.
(1) Test standards. Use the target species. Laboratory or cultured
strains are acceptable. Use at least 10 animals for each species and dosage
level. Group animals in lots of 10 if fighting or cannibalism does not
occur and if group caging of animals has been shown not to affect laboratory
evaluations. If fighting or cannibalism occurs, cage and test animals
separately. Test each formulation that may be tested in the field. Observe
any changes in behavior that may affect efficacy. Replicate tests three
times for all species and environmental combinations. Test the target organ-
isms in an environment (e.g., pond, artificial or natural stream) where the
organism is exposed to various concentrations of the repellent. Consult
references 6 and 7 of § 96-25 for additional information.
(2) Test information. Indicate the species and origin of
test animals. Describe the test apparatus and introduction of test animals.
Describe the acclimation of the target species to the test situation prior
to the introduction of the repellent. Provide a detailed description of the
changes in animal behavior and a summary of the results of the test, present
an EC50 dose response curve for each test.
(3) Acceptable protocols. For information on recommended test envir-
onments, refer to references 6 and 7 in § 96-25. References cited in
§§ 95-25 and 30 may be helpful in developing an acceptable protocol incorpor-
ating the test standards described in paragraph (d)(1) of this section. At
this time, no standard protocols for evaluating amphibian repellents are
available.
(e) Detoxification studies. Submit the results of LC50 detoxifica-
tion tests for each species claimed to be controlled. The purpose of these
studies is to determine the detoxification rate of'formulations. For test
standards, test information, and acceptable protocols,, refer to § 93-2(d)
of this subdivision. •• .
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(f) Field efficacy studies. Submit the results of field efficacy
tests for each species claimed to be controlled with an amphibian toxicant
or repellent. The purpose of these studies is to assess the field efficacy
of the product under actual use conditions.
(1) Test standards. For test standards, see § 96-2(f)(l) of this
subdivision.
(2) Test information. For information, see § 96-2 (f)(2) of this
subdivision.
(3) Acceptable protocols. Refer to the literature cited in § 96-30
to develop acceptable protocol incorporating the test standards described
in paragraph (f)(1) of this section. Those methods and references referring
to fish toxicants may, with minor revisions, be used for amphibian toxicants
and repellents.
§ 96-4 Terrestrial amphibian and reptilian control agents.
(a) General. This section provides efficacy data and test procedures
for toxicants and repellents that claim to control terrestrial
amphibians and reptiles. Target animals include frogs, toads, salmanders,
mudpuppies, snakes, lizards, and turtles. Submission of efficacy data is
waived for this section.
(b) Acute and subacute toxicity studies. Submit the results of tox-
icity studies for each species claimed to be controlled by an amphibian or
reptilian toxicant. Acute oral LD50 and subacute LD50 tests are needed
for oral toxicants. Dermal LD50 tests are needed for contact toxicants.
The purpose of these studies is to assess the appropriateness of dosage
levels used in subsequent testing.
(1) Test standards. Use the target species. Cultured animals
of the target species are acceptable. Cage test animals individually.
Use at least 10 animals for each dosage level. Observe changes in
behavior that may affect efficacy. Consult standard references on holding
conditions for amphibians and reptiles (see reference 8 of § 96-25) and on
conducting toxicity tests (See references 8, 9, and 10).
(2) Test information. Indicate the species and origin of
test animals. Describe acclimation of animals, laboratory and test condi-
tions (e.g., light, temperature, humidity, cage size, cage type), changes
in behavior during the test that may affect efficacy, and gross pathological
changes found at post mortem. Provide basic toxicity data for all target
animals .and "dose levels used. Submit a dose response curve wi.th computation
of the slope,:and ;LD50 within 95% confidence.limits for all, species tested.
tSee reference. 5 o£ 96-25).
(c) Laboratory efficacy studies.. Submit- the results of .laboratory
efficacy.-tests for each:;.species' -Claimed.. The piirpos-e-of tltese studies is . :
to assess the laboratory. efficacy of the product formulations. ...-.•"'•
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(1) Test standards. Use th« target species. Cultured animals of the
target species are acceptable. Use at least 10 animals for each species
and dosage level. Use at least 10 animals for controls. The exposure period
for an acute toxicant should be three days. The exposure period for a multiple-
dose toxicant or repellent should be fifteen days. Evaluate the pesticide
under a variety of environmental conditions.
(2) Test information. Indicate the species and origin of
test animals used. Describe acclimation of animals and laboratory test
conditions (e.g., temperature, humidity, light, cage size, and cage type).
Submit raw and summary data showing the response of the animals to the
pesticides. Submit dose response curve with computation of the slope and
appropriate end points (i.e., LDSO's, LDlOO's, ECSO's, and EClOO's) with
95% confidence limits for all species tested. (See reference 5 of § 96-25.)
(d) Field efficacy studies. Submit the results of field efficacy
studies for each species claimed to be controlled. The purpose of these
studies is to assess the field efficacy of the product under actual use
conditions.
(1) Test standards, (i) Number of regional studies. The density and
distribution of the target animals before and after treatment should be deter-
mined by appropriate methods. Adequate statistical analysis of the data
should be provided.
(ii) Pre-treatment and post-treatment population censusing. The
density and distribution of the target animals before and after treatment
should be determined by appropriate methods. Adequate statistical analysis
of the data should be provided. . ..
(iii) Site selection. Test sites should be selected that permit
evaluation of efficacy without significant movement of animals in or out
of the census area. For example, tadpoles cannot be evaluated adequately
during the metamorphosis into frogs or toads. Also, snakes cannot be
evaluated when they are moving in or out of denning areas.
(iv) Environmental factors. Conduct tests under those environmental
conditions (e.g., humidity, precipitation, temperature, wind) and application
rates, frequencies, and methods anticipated under actual use conditions.
(2) Test information. Indicate the formulation for each
study, location of sites, application method, and number of applications
used. Provide a map with dimensions locating all control and test sites.
Indicate the method used to select each site. Provide all raw and summary
data used to evaluate efficacy. Include data on target and nontarget organ-
isms. Provide climatological data for the study period.
(3) Acceptable protocols. References cited in § 96-30 may be helpful
in developing an acceptable protocol. incorporating the test standards describ-
ed in paragraph (d)(l) of this section.
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§ 96-5 Avian toxicants.
(a) General. This section gives efficacy and test procedures
for avian toxicants. These products are applied as baits in and around
buildings and in feed lots, and are applied as liquids or gels in or on
perches and around buildings. Target species include the herring gull,
English sparrow, house finch, pigeon, starling, brown-headed cowbird, red-
winged blackbird, rusty blackbird, and common grackle. Submission of efficacy
data is waived for this section.
(b) Acute and subacute toxicity studies. For a single-dose toxicant,
submit the results of an acute oral LD50 test and a subacute (5-day) oral
LD50 test on the target species. For a single-dose liquid or gel, also sub-
mit an acute dermal LD50 test. For a multiple-dose liquid or gel, also sub-
mit a subacute dermal LD50 test. The purpose of these studies is to assess
the appropriateness of dosage levels used in subsequent testing.
(1) Test standards, use the target species. Laboratory strains of
the target species are acceptable. Use at least 10 animals for each dosage
level for each sex. Cage test animals individually. Observe any changes in
behavior that may affect efficacy (e.g., subject quickly becomes agitated).
Observe gross pathological changes found at post mortem (e.g., hemorrhaging,
edema, ulcers, discoloring). For detailed test standards, consult standard
references on the subject. (See references 9, 10, and 11 of § 96-25.)
(2) Test information, indicate the species and origin of
test birds. Describe acclimation of animals, laboratory conditions (e.g.,
temperature, humidity, light, cage size), changes in behavior during the
test that may affect efficacy, and gross pathological changes found at post
mortem. Separate the mortality data by sex for each dosage level. Submit a
dose response curve for each sex with the computation of the slope and LD50
with 95% confidence limits.
(c) Laboratory acceptance studies. Submit the results of laboratory
acceptance studies for each species claimed to be controlled with an avian
toxic bait. The purpose of these studies is to assess the appropriateness
of bait carriers used in subsequent testing.
(1) Test standards. Use the target species. Laboratory strains of
the target species are acceptable. Use six adult males, six adult females,
and six juveniles of either sex for each species. Cage test birds individ-
ually. Provide a free choice between the habituated ration and the candidate
carrier. Continue recording daily food consumption until the percentage of
each food eaten stablizes. Conduct laboratory acceptance studies with all
candidate carriers. Do not fast animals prior to any feeding tests. Conduct
tests during regular feeding hours.
(2) Test information. Indicate the species and origin of the
test animals. • Describe .acclimation of animals and laboratory, conditions.
Provide daily:''food consumption data 'for each .animal. . Provide .summary data :
by sex and age, including the quantity and percentage of each diet consumed.
(3.) Acceptable protocols.: For conducting.laboratory acceptance studies,
refer to Section 8 of. reference T2 in.'§ ..96-25. References-• cited in §:96—30
may be. helpful in-:developing, an. .acceptable: protocol, incprpora:ting- 'th«-.-S:taiidards
described in paragraph.(c)(2) of this section. " •!"
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(d) Laboratory efficacy studies. Submit the results--of .laboratory
efficacy studies for each formulation and species claimed to be controlled
with an avian toxicant. The purpose of these studies is to assess the labor-
atory efficacy of formulations that will be tested in the field.
(1) Test standards. Use the target species. Laboratory strains of
the target species are acceptable. Use six adult males, six adult females,
and six juveniles of either sex for each species and formulation. Birds
should be tested individually in pens where they can fly freely. Provide a
free choice between the habituated ration and the candidate carrier for
toxic baits, and between treated and untreated perches for liquids and gels.
Test each formulation that may be evaluated in the field. Monitor each
bird's behavior periodically after it is exposed to the toxicant.
(2) Test information required. Indicate the species and origin of
test animals. Describe the introduction of test animals into pens, and
describe pen conditions. Indicate the time and date of introduction, a
description and time of all relevant behavior related to product efficacy,
and the time of death for each animal. Indicate the statistical methods
used to evaluate data. Provide summary data by sex, age, and species.
Submit a chemical analysis of the test ration for the percentage of active
ingredient. (Consult references 13 and 14 in § 96-25.
(3) Suggested performance standard. The level of efficacy that should
be attained for an acceptable test has not been established. However, a 90%
or greater mortality is suggested.
(4) Acceptable protocols. For conducting laboratory efficacy studies,
refer to Section 5 of reference 12 of § 96-25. For existing methods of
chemical analysis, see references 13 and 14 of § 96-25.
(e) Field efficacy studies. Submit the results of field efficacy tests
for each species claimed to be controlled with an avian toxicant. The purpose
of these studies is to assess ,the field efficacy of the product under actual
use conditions.
(1) Test standards. (i) Preliminary field Resting. Monitor prelim-
inary field testing very closely in a well-delineated area to uncover any
unforseen problems with product efficacy or nontarget hazard before extensive
regional testing is initiated.
(ii) Number of regional studies. For each site, formulation, species,
method of application, major region, and species claimed or implied on the
label, submit five field efficacy studies.
(iii) Pre-treatment and post-treatment population censusing. Direct
censusing is always required and involves visual counts of birds are made
at periodic intervals (e.g., early morning) for several days in order to
obtain a relative index of bird pressure. Both target and non-target bird
species should be censused. In indirect censusing,. random samples of crops
are taken and.evaluated for percentage of damage. Indirect censoising is
always needed when the chemical is intended to protect a crop. For
these situations, yield comparisons (projected or actual) and cost/benefit
analyses can. alsp be made. Make periodic counts of dead otarget ajid-non-
target, animals. Indicate the species :.and sex. .••.-. ° '-. . ,• '
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(iv) Site selection, it is important that all experimental groups
have comparable and adequate damage or bird pressure. Otherwise, the study
will be biased. An experimental area should not be included either as a test
or control unless it is experiencing a predetermined amount of damage or
pressure. Equal numbers of test and control areas can be selected in two
ways: at random, or by assigning areas with comparable damage to the test
and control groups.
(v) Rate, frequency, and method of application. In order to provide
adequate use directions on the label, efficacy testing should include
trials at different rates (e.g., 1 Ib/acre or 4 oz. per placement); frequen-
cies (e.g., 1 application per week or month); and method of application •
(e.g., ground, hand or aerial). If a claim is made that a product is
effective for a particular time period, then the test design should reflect
studies covering that period of time.
(vi) Effect of environmental factors and managerial practices.
Studies should be conducted under the environmental factors anticipated
under actual use conditions. Environmental factors (such as precipita-
tion, temperature, light, humidity, and wind), as well as managerial
practices (such as sanitation, timing of planting, and location of crops),
may have an impact on product effectiveness. These variables should be taken
into account in the experimental test design.
(vii) Suggested performance standard. Although the level of efficacy
that should be attained has not been established, a 70% reduction in the
target pest numbers, activity, or damage is suggested.
(2) Test information. Indicate the locations, formulations,
site, method, frequency, and number of applications for each study. Pro-
vide a map of test and control areas, including their dimensions. Provide
all raw and summary data used to evaluate effectiveness. Include information
on target and non-target species. Indicate method and rationale of selecting
experimental areas and of assigning them to test and control groups. Provide
climatological data for the study period. Include all information on the
statistical methods used.
(3) Acceptable protocols. For conducting field efficacy studies,
consult Section 9 of reference 12 in § 96-25. References cited in § 96-30
may be helpful in developing an acceptable protocol incorporating the test
standards above.
§ 96-6 Avian repellents.
(a) -General. This section furnishes information on efficacy.data
and test procedures for avian repellents applied to seed. Target s.pecies
include the starling, red-winged blackbird, .common grackle, pheasant, Oregon
junco, and Canada jay. Submission of efficacy data is waived for this
section, :'.'-.-. • . • ...''-
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(b) Preliminary acute and subacute'toxicity studies. Submit the results.
of toxicity studies for each species claimed to be controlled with an avian
repellent applied to seed. These studies shall include an acute oral LD50
test and a subacute (5-day) oral LD50 test for the bobwhite quail. The pur-
pose of these studies is to assess the possibility of hazard to the target
species.
(1) Test standards. Use the bobwhite quail. Laboratory strains are
acceptable. Use at least 10 birds for each dosage level for each sex. Cage
test birds individually. Observe any changes in behavior that may affect
efficacy. Consult standard references on the subject. (See references 9
and 11 in § 96-25.)
(2) Test information. Indicate species and origin of test
birds. Describe acclimation of birds, laboratory conditions (e.g., temper-
ature, humidity, light, cage size), changes in behavior during the test that
may affect efficacy, and gross pathological changes found at post mortem.
Separate the mortality data by sex for each dosage level. Submit a dose
response curve for each sex with the computation of the slope and LD50 with
95% confidence limits. (See reference 5 in § 96-25.)
(c) Advanced acute and subacute toxicity studies. If either test list-
ed above yields an LD50 of 1000 mg/kg or less, conduct tests using the target
bird claimed to be controlled by an avian repellent. The purpose of these
studies is to assess the toxicity and possibility of hazard to target species.
(1) Test standards. Use the target species or genus. Laboratory
strains are acceptable. Use at least 10 birds for each dosage level for each
each sex. Cage birds individually. Observe any changes in behavior that
may affect efficacy. Observe gross pathological changes found at post mortem
(e.g., hemorrhaging, edema, ulcers, discoloring). Consult standard references
on the subject. For detailed test standards, consult references 9 and 11 in
§ 96-25.
(2) Test information, indicate species and origin of the test birds.
Describe acclimation of birds, laboratory conditions (e.g., temperature,
humidity, light, cage size), changes in behavior during the test that may
affect efficacy, and gross pathological changes found at post mortem. Separate
the mortality data by sex for each dosage level. Submit a dose response
curve for each sex with the computation of slope and LD50 with 95% confidence
limits.
(d) Laboratory efficacy studies. Submit the results of a laboratory
efficacy test for each species claimed to be controlled. The purpose of
these studies is to assess the laboratory efficacy of the formulation.
(1) Test standards. Use the target species. Use animals that are
laboratory-reared or captured from wild environments. Maintain wild-
caught birds destined for testing in communal cages for a minimum of 2
weeks to acclimate them to captivity. Maintain laboratory-reared and
acclimated wild-caught birds for 3 to 7 days in the type of pen or cage
used for the test. Use test cages or pens that allow free movement of
the test bird and free access to food and water. Use a single, readily
available, -normal or preferred food of the test species, ^as - the habituated
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diet. At least 20% of the diet should comprise the food item to be tested.
Insure that the diet and general condition have stabilized for one week
prior to the test. Maintain stable environmental conditions in the labor-
atory. Do not fast test birds prior to testing. For at least two days
prior to the test, birds are to be individually caged and offered a weighed
quantity of food in excess of daily requirements in two identical food con-
tainers. Their locations should be switched daily. Birds consuming less than
60% of the mean food consumption of all pretest birds shall not be included
in further testing. Use a minimum of 5 adult males, 5 adult females, and
5 juveniles of either sex for each treatment and species. Transfer birds
to clean individual cages. Provide each bird with two clean food containers,
one with treated food and the other untreated food. Continue the test for
at least four days. Record daily food consumption for each diet and for
each bird. Reverse position of food containers daily. Use 3 to 5 treat-'"
ments to establish the ED50. Record death, sickness, and unusual behavioral
changes in test birds according to age, sex, and species.
(2) Suggested performance standard. A bird is considered to be
repelled if more than 75% of the total amount of food eaten during the 4
days of the test is untreated.
(3) Test information. Report date test was commenced. Identify the
species and origin of test birds. Describe acclimation of the birds and
laboratory conditions (e.g., light, temperature, humidity, cage size).
Submit the following information for each bird: sex, age, initial and
final weight. Indicate the composition and concentration of each formulation
and how the repellent was applied to the food. Submit the following
information for the food particles: the type, source, size, and average
weight. Report daily and summary treated and untreated food consumption
data by sex, age, and species. Provide information according to sex,
age, and species on deaths, sickness, or unusual behavioral changes.
Submit a dose response curve for each sex and age with the computation of
the slope and ED50 with 95% confidence limits. (See reference 5 in § 96-25).
(4) Acceptable protocols. For presenting a dose response curve, refer
to reference 5 in § 96-25. For conducting laboratory efficacy studies, con-
sult reference 15 in § 96-25.
(e) Field efficacy studies. Submit the results of field efficacy tests
for each species claimed to be controlled. The purpose of these studies is
to assess the field efficacy of the product under actual use conditions.
(ii) Pre-treatinent and post-treatment censusing. Direct and indirect
censusing in treated and untreated areas should be undertaken. In direct cen-
susing, the species (target and non-target) are counted with optical aids
for 3 or more days at specific times during the day (e.g., 7:00 to 7:30 AM).
In indirect censusing, the number of seeds removed per unit area is counted
until seeds are no longer subject to bird pressure.
(iii). Rate, frequency, and- method of application. In order to provide
adequate .use directions on- the label, efficacy testing s-hould include trials
at different rates (e..g., 10 oz. product per IOC Ibs. of seed), frequencies
(e.g., 1 application per week or month):, and methods..of application (e.g.,
.ground, hand.,, or aerial.). If a claim, is made ,that a product Is..effective.
for-a-particuia'r.-time, period-, "then the tast design: should reflect studies '*
covering that period,.of time;. .. ,..-,:., - ... . •. •'
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(iv) Effect of environmental factors and managerial practices. Studies
should be conducted with the environmental factors anticipated under actual use
conditions. Environmental factors (such as precipitation, temperature, light,-
humidity, and wind), as well as managerial practices (such as sanitation,
and timing and location of planting), may have an impact on product effective-
ness. These variables should be taken into account in the experimental design.
(2) Suggested performance standard. The level of efficacy that
should be attained for an acceptable test has not been established. However,
if 70% fewer seeds treated with the product are removed by the target
population, in comparison to untreated seeds, such a level of efficacy
would be is suggested.
(3) Test information. Indicate the target species. Include a map
with dimensions of all treated and untreated study areas. Indicate how
experimental areas were selected and how they were assigned to treatments
and controls. Report the method and frequency of application, the formulation,
dosage rates, temperature, precipitation, and the soil moisture. Include
all raw and summary data upon which direct and indirect censusing were made.
Record the species and sex of all dead animals (target and non-target) found
in treated and control areas. Describe all statistical methods used to
evaluate efficacy.
(4) Acceptable protocols. References cited in § 96-30 may be helpful
in developing an acceptable protocol incorporating the test standards describ-
ed in paragraph (e)(2) of this section.
§ 96-7 Avian frightening agents.
(a) General. This section deals with efficacy data and test procedures
for avian frightening agents. This guidance refers to those agents applied
as baits in and around buildings, in crops, in dumps, and at airports.
Target species include the herring gull, English sparrow, pigeon, starling,
brown-headed cowbird, red-winged blackbird, and common grackle. Submission
of efficacy data is waived for this section.
(b) Acute and subacute toxicity studies. Submit the results of an
acute oral LD50 test and subacute (5-day) oral LD50 test for each species
claimed to be controlled with an avian frightening agent. The purpose
of these studies is to assess the appropriateness of dosage levels used in
• subs.eque.nt testing.
(1) Test standards. Use the target species. Laboratory strains of
the target species are acceptable. Use at least 10 animals for each dosage
level for each sex. Cage test animals individually. Observe any changes
in behavior that may affect efficacy. Observe gross pathological changes
found at post mortem (e.g., hemorrhaging, edema, ulcers, discoloring).
Cheek standard references on the subject for detailed test standards.
(Consult references 9 and 11 in § 96-25.)
(2) Test information. Indicate the species and-origin of
test animals. Describe acclimation of animals, laboratory conditions
(e.g., temperature, humidity, light, cage size), changes in'behavior
during the test that may affect efficacy, and gross pathological changes
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found at post mortem. Separate the mortality data by sex for each dosage
level. Indicate the oral LD50 of the product for each sex. Submit a
dose response curve with the computation of the slope and LD50 with 95%
confidence limits. (See reference 5 in § 96-25.)
(c) Laboratory acceptance studies. Submit the results of laboratory
acceptance studies for each species claimed to be controlled with an
avian frightening agent. The purpose of these studies is to assess the
appropriateness of bait carriers used in subsequent testing.
(1) Test standards. Use the target species. Laboratory strains of
the target species are acceptable. Use six adult males, six adult females, *
and six juveniles of either sex for each species. Cage test animals indi-
vidually. Provide a free choice between the standard ration the animals
are accustomed to eating and the candidate carrier. Continue recording
daily food consumption until the percentage of each food eaten stabilizes.
Conduct laboratory acceptance studies with all candidate carriers. Do
not fast the animals prior to any feeding hours. Conduct tests during
regular feeding hours. Consult reference 12 in § 96-25 for additional
information.
(2) Test information. Indicate the species and origin of
test animals. Describe acclimation of animals and laboratory conditions.
Provide daily food consumption data for each animal. Report summary data
by sex and age, including the quantity and percentage of each diet consumed.
(3) Acceptable protocols. For information on laboratory acceptance
studies, review Section 8 of reference 12 in § 96-25. References cited in
§ 96-25 on avian frightening agents may be helpful in developing an accepta-
able protocol incorporating the test standards described in paragraph
(c)(2) of this section.
(d) Laboratory efficacy studies. Submit the results of laboratory
efficacy studies for each species claimed to be controlled with an avian
frightening agent. The purpose of these studies is to assess the labora-
tory efficacy of formulations that will be tested in the field.
(1) Test standards. Use the target species. Laboratory strains
of the target species are acceptable. Use six adult males, six adult
females, and six juveniles of either sex for each species and formula-
ation. Test animals individually in pens where the birds can fly from
from perch to perch. Test each formulation that may be tested in the
field. Monitor each bird's behavior continuously after it is introduced
into the pen with the candidate formulation. Consult reference 12 in
§ 96-25 for additional information.
(2) Test information. Indicate the species and origin of
test animals. Describe the introduction .of test animals into .pens, and
pen conditions. Provide the time of introduction, a description and time
of all relevant .behavior related to product efficacy., and time of death
for each anima-1. Report summary data by sex, age., and species,. .Submit a
chemical analysis of the;test ration for the percentage of active, ingredient.
(See references 13 and 14 in § 96-2'5.)
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(3) Acceptable protocols. For information on laboratory efficacy
studies, refer to Section 5 of reference 12 in § 96-25. For methods of
chemical analysis, consult references 13 and 14 in § 96-25.
(e) Field efficacy studies. Submit the results of field efficacy
tests for each species claimed.to be controlled with an avian frightening
agent. The purpose of these studies is to assess the field efficacy of
the product under actual use conditions.
(1) Test standards. (i) Number of regional studies. Submit 5 field
efficacy studies for each site (e.g., in and around buildings, in sunflowers,
in field corn, in sweet corn, in dumps, at airports), formulation, method of
application, major region, and species claimed or implied to be controlled.
(ii) Pre-treatment and post-treatment population censusing. Two basic
methods of censusing are used, one of which is direct and the other is in-
direct. In direct censusing, visual counts of birds are made at periodic
intervals, (e.g., early morning) for several days in order to obtain a
relative index of bird pressure. Both target and non-target bird species
should be censused. Direct censusing is always recommended. In indirect census-
ing, random samples of crops are made and evaluated for percentage of damage.
For some sites such as sunflowers, sweet corn, and field corn, the U.S. Depart-
ment of the Interior has developed standard methods of direct and indirect
censusing. Indirect censusing is always recommended when the chemical is
intended to protect a crop. For these situations, yield comparisons
(projected or actual) and cost/benefit analyses can also be made.
(iii) Site selection. All experimental groups should have comparable
and adequate damage or bird pressure. Otherwise, the study will be biased.
A field should not be included either as a test or control area unless it is
experiencing a predetermined amount of damage or pressure. Equal numbers
of test and control areas can be selected in two ways: at random, or by
assigning areas with comparable damage to the test and control areas.
(iv) Rate, frequency, and method of application. To provide adequate
use directions on the label, the tester should design efficacy testing to
include trials at different rates (e.g., 1 Ib. per acre or 4 oz. per place-
ment), frequencies (e.g., 1 application per week or month), and methods of
application (e.g., ground, hand, or aerial). .If a claim is made that a
product is effective for a particular time period, then the test design
should reflect studies covering that period of time.
(v) Effect of environmental factors and managerial practices. Studies
should be conducted under environmental factors anticipated under actual use
conditions. Environmental factors (such as precipitation, temperature,
light, humidity, and wind), as well as managerial practices (such as sani-
tation, timing of planting, and location of crops), may have an impact on
product effectiveness. These variables should be taken into account in the
experimental test design.
(vi) Initial field testing. Monitor initial field testing very closely
in a well delineated area to uncover any unforeseen problems with product
efficacy or nontarget hazard before extensive regional testing is initiated.
Consult reference 12 in •§ 96-25 for additional informatipn.,
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(yii) Suggested performance standard. Although the level of efficacy
that should be maintained has not been established, a 70% reduction in
activity or damages due to use of the product is suggested.
(2) Test information. For each study, indicate the formulation,
location and size of test area(s), and the method, number, rate, and frequency
of applications. Provide a map of test and control areas, including their
dimensions. Report all raw and summary data used to evaluate effectiveness.
Include information on target and non-target species. Indicate the method
and rationale of selecting experimental areas and of assigning them to
test or control groups. Report type and degree of damage being experienced ,
by test and control areas. Provide climatological data for the study
period. Describe all statistical methods used.
(3) Acceptable protocols. For information on field efficacy studies,
see Section 9 of reference 12 in § 96-25. References cited in § 96-30 may
be helpful in developing an acceptable protocol incorporating the test
standards described in paragraph (e)(2) of this section.
§ 96-8 Mole toxicants.
(a) General. This section presents efficacy data and test procedures
for mole toxicants applied as baits and fumigants to underground tunnels.
Target species include the eastern mole, Townsend mole, starnose mole, and
California mole. Submission of efficacy data is waived for this section.
(b) Acute and subacute toxicity studies. Submit the results of tox-
icity studies for each genus of mole claimed to be controlled by a toxicant
applied as bait or toxicant. For a single-dose bait, submit an acute oral
LD50 test. For a multiple-dose bait, submit an acute oral LD50 test and a
subacute (5-day) oral LD50 test. For a fumigant, submit an acute inhalation
LC50 test. The purpose of these studies is to assess the appropriateness
of dosage levels used in subsequent testing.
(1) Test standards. Use the target genera. Use animals that are labor-
atory-reared or captured from wild environments. Use at least 10 animals for
each dosage level. If external sexing is not possible, determine sex at post
mortem. Cage test animals individually. Observe any changes in behavior
that may affect efficacy. Observe gross pathological changes found at post
mortem (e.g., hemorrhaging, edema, ulcers, discoloring). For detailed test
standards, consult references 9 and 16 in § 96-25.
(2) Test information, indicate the species and origin of test
animals. Describe acclimation of animals, laboratory conditions (e.g., temp-
erature, humidity, light, cage size), and gross pathological changes found
at post mortem. Separate, mortality data for each dosage level.. Submit a
dose response curve for each species claimed.or implied with computation
of the slope and LD50 or LC50 with 95% confidence limits.- (See reference
.5 in § 96-25..) . .
"(c')"" Laboratory acceptance Studies <'" Su'b'mi't the 'results of laboratory
acceptance studies fdr.each genus claimed to be controlled with a toxic-'
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bait. ..The purpose of these studies is to assess the appropriateness of
bait carriers used in subsequent testing. .-• „. • .• ....-•••..
(1) Test standards. Use the wild target genus. Use at least 10
adult molestIf external sexing is not possible, determine sex at post
mortem. Cage test animals individually. Provide a free choice between
the candidate bait carrier and the habituated diet. Continue recording
daily food consumption until the percentage of food eaten stablizes.
Conduct laboratory acceptance studies with all candidate carriers that may
be tested in the field.
(2) Test information. Indicate the species and origin of
test animals. Describe acclimation of animals and laboratory conditions.
Submit individual and summary data on all moles, including the quantity
and percentage of each diet consumed.
(d) Laboratory efficacy studies. Submit the results of a laboratory
efficacy test for each genus claimed to be controlled. The purpose of
these studies is to assess the laboratory efficacy of the formulation.
(1) Test standards. Use the wild target genus. Use at least 10 adult
moles for each test. If external sexing is not possible, determine sex at
post mortem. Use at least 10 adult moles for controls. Greater than 10%
mortality in controls negates the test. For toxic baits, provide a free
choice between the habituated ration (e.g., fish-flavored cat food) and the
toxic bait. For fumigants, test animals individually in an artificial mole
burrow. (See reference 17 in § 96-25.) The exposure period for a single-
dose bait is 3 days. The exposure period for a multiple-dose bait is 15 days.
The exposure period for a fumigant is one hour. Test all formulations that
may be used in the field.
(2) Suggested performance standard. The level of efficacy that
should be attained for an acceptable test has not been established. However,
90% mortality in the test group and no more than 10% mortality in the
control group is suggested.
(3) Test information. Identify the species and origin of the target
animals. Describe acclimation of the animals and laboratory conditions.
For a fumigant, describe the method of application. For a toxic bait, provide
daily treated and untreated food consumption data for each test animal and
•species. For each animal, indicate the time of death. Provide summary data
by sex and species. Submit a chemical analysis of the test ration for the
percentage of active ingredient. (See references 13 and 14 in § 96-25.)
(4) Acceptable protocols. References 17 and 18 in § 96-25 and others
cited in the § 96-30 may be helpful in developing an acceptable protocol
incorporating the test standards above. For methods of chemical analysis,
consult references 13 and 14 in § 96-25.
(e) Field efficacy studies. Submit the results of field efficacy tests
for each species claimed to be controlled. The purpose of these studies is
to assess the field efficacy of the product under actual use conditions.
(1) Test standards. (i) Number of regional studies. Submit the
results of three acceptable field efficacy studies for each formulation,
method of application, major region,- and.species claimed or implied to be
controlled. • ::....
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(ii) Pre-treatment and post-treatment population censusing.. Locate
mole-infested fields, lawns, and other areas where interference by cats,
dogs, or other animals is not anticipated. Using surface burrows, identify
and mark burrow systems individually. A single burrow system would include
all interconnecting surface burrows. In a period of five days, identify 20
active burrow systems. Punch one-inch holes in the roof of each burrow of a
burrow system. Burrow systems are defined as "active" if holes have been
opened and plugged at least three times within five days. Mark the locations
where burrows are punched so that the same tunnels can be punched during the
post-treatment phase. As each of the 20 active burrow systems is located,
number them so that the closest active burrow systems have consecutive numbers.
(iii) Treatment phase. Treat the odd-numbered burrow systems, but not
the eve'n numbered burrow systems. In this way, only .alternate systems are
treated. Treat in a consistent manner, according to the directions to be
included on the product label. Maintain records on the methods of application, •
location and quantity of bait or fumigant, and the frequency of application.
(iv) Treated and untreated burrows. The use of treated and untreated
burrows insures that the reduction in the number of the target pests in treat-
ed burrows is due to the treatment and not to other factors.
(v) Verification of the target species. Because the method of censusing
is an indirect one, some live trapping before censusing should be done to verify
the species of mole. Record the species of mole captured.
(vi) Suggested performance standard. The level of efficacy that should
be attained for an acceptable test has not been established. However, a 70%
reduction in burrowing activity of the target population due to the use of
the product is suggested.
(2) Test information. Report the results of all studies, including
raw and summary data. Submit information for each treated burrow system on
the method and frequency of application, and the location, composition, and
quantity of bait or fumigant. Provide a general map showing the numbers and
approximate locations of punched tunnels, dimensions of the burrow systems,
distance to the closest active burrow system, and the location of treatments.
For all phases of the study, include climatological data (daily maximum and
minimum temperature, and daily precipitation) from a local weather station.
Submit information on mortality by species and sex for target and non-target
kills in study areas. Include all figures used to calculate the percentage
of efficacy.
(3) Acceptable protocols. Reference 17 in § 96-25 and others cited in
§ 96-30 may be helpful in developing an acceptable protocol incorporating the
test standards described in paragraph (e)(2) of this section. Prior consulta-
tion with the Agency on such protocols to be employed is strongly recommended.
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§ 96-9 Bat toxicants and repellents.
(a) General. This section gives ef-ficacy guidance for bat toxicants
and repellents applied indoors. Target species include the little brown bat
and the big brown bat. Submission of efficacy data is waived for this section.
(b) Acute and subacute toxicity studies. Conduct acute and subacute
(5-day) oral LD50 studies with target species. Inhalation and/or dermal LD50
studies may be required depending upon the nature of the active ingredient
and its apparent mode of action. The purpose of these studies is to assess
the appropriateness of dosage levels used in subsequent testing.
(1) Test standards. Use the target species. Use 10 adult animals of
each sex for each dosage level. Cage animals individually. Describe accli-
mation of animals, laboratory conditions (e.g., temperature, humidity, light-
ing, cage size), symptoms of poisoning, and gross pathological changes.
(2) Test information required. Indicate the species tested, the loca-
tion of capture, the length of time in captivity, and holding conditions.
provide weights of animals at capture, before dosing, and after death or
recovery. Ten males and 10 females should be tested at each dosage level.
Submit a dose response curve with the computation of the slope and LD50 with
95% confidence limits. (See reference 5 of § 96-25.) The time and date of
symptom onset and the date of death (if any) should be noted for all subjects.
(c) Laboratory efficacy studies. Submit the results of laboratory
efficacy studies for each target species. The purpose of these studies is
to assess the laboratory efficacy of formulations that will be tested in the
field. •
(1) Test standards. Use the target species. Use at least ten adult
males and ten adult females for each target species and each formulation.
Use a control group maintained under the same conditions as test subjects but
without exposure to control agent. Record weights of bats at capture and be-
fore and after test. Acclimate bats for two weeks prior to test. For main-
taining'bats in captivity, consult reference 19 in § 96-25. For claims of
repellency, quantitative definitions are needed. Repellency may be defined as
an apparent reduction of activity in a certain (treated) portion of a test en-
closure. Protocols should be reviewed by the Agency before tests are conducted.
(2) Suggested performance standard. No performance standards have
been established for bat products. However, 90% mortality would be adequate
for toxicants and 80% reduction in activity would be adequate for repellents.
(3) Test information. For each study, indicate the formulation,
type of claim, and details of capture of animals. Describe laboratory and
test enclosure acclimation procedures. Report weights of animals at capture,
at the onset of testing, and at the conclusion of testing. Submit a diagram
of the test enclosure, details of procedures and rates of application, records
of behavioral responses relevant to the claims being tested, records of symp-
toms of toxicity, and dates of death (if any). Provide all raw and summary
data for test and control animals. For all products, the length of time be-
tween the onset of symptoms and death should be noted. Also, note the length
of time that symptoms were present in any subjects that recovered.from apparent
poisoning.. • • '
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(4) Acceptable protocols. No specific complete test protocols exist for
laboratory evaluation of bat toxicants and repellents. However, references
cited in § 96-30 may be helpful in developing acceptable protocols incorpora-
ting the test standards indicated in paragraph (c)(2) of this section.
(d) Field efficacy studies. Submit the results of all field efficacy
tests for each species and each use pattern claimed. The purpose of these
studies is to assess the field efficacy of the product under actual use
conditions.
(1) Test standards. (i) Number of regional studies. For each formu-
lation, method of application, major region, type of site, and bat species-
claimed to be controlled, submit the results of at least five acceptable
field efficacy studies. Every field efficacy study site must use a corres-
ponding control site.
(ii) Site. Treatments should be made in indoor sites only.
(iii) Pre-treatment and post-treatment population censusing. Control
is measured in terms of estimates of population reduction (for toxicant
claims) or in percent bat activity reduction (for repellency claims). Esti-
mation procedures should be reviewed by the Agency prior to the conducting
of tests. Extensive pre-treatment data should be collected to determine normal
levels of occupancy of sites by bats. Procedures for censusing bats are
referenced in § 96-30. Under certain conditions, direct counts may be used.
Two sampling methods should be used unless it is possible for direct counts
to be absolute.
(iv) Suggested performance standard. No performance standards have
been established. However, 90% control due to use of the product is
suggested.
(2) Test information, indicate target species, methods of
censusing, pre-and post-treatment census results, application procedures,
and application rates. Report numbers of dead bats found and any non-target
deaths. Describe significant changes in bat behavior, observable symptoms
of toxicity, weather conditions (temperature, humidity, precipitation—espec-
ially for enclosures with leaky roofs), and any other data relevant to the
assessment of efficacy.
(3) Acceptable protocols. No standard protocols exist for field evalu-
ation of the efficacy of bat control products. However, references cited in
Section 96-30 may be helpful in developing acceptable protocols incorporating
the test standards described in paragraph (d)(2) of this section.
•§••96-10 Commensal, rodenticides .
(a) General . This section concerns guidance regarding efficacy and,
test procedures for commensal rodenticides used in. or around buildings.
• Target species. . are -commensal rodents s.uc-h- as the Norway .r.a.t,. . roof • rat,, Poly.T-
nesian"fa't, and house •ffl6us'e." Commensal' rodents are those that live at 'man's
expense, usually living in his home or buildings, and sharing with man! their
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diseases and many other undesirable traits, without contributing anything
beneficial to the; .relationship. Submission-of ef-ficacy data is waited in this
section.
(b) Acute and subacute toxicity studies. Submit the results of toxicity
studies for each species claimed to be controlled with a commensal rodenticide.
For single-dose rodenticides, submit an acute oral LD50 test. For multiple-
dose rodenticides, submit an acute oral LD50 test and a subacute (5-day) oral
LD50 test. The purpose of these studies is to test the appropriateness of
dosage levels used in subsequent testing.
(1) Test standards, use the target species. Laboratory strains of
target species are acceptable. Cage test animals individually. Test both
juvenile animals (within 21 days after weaning) and adult animals (over 35
days after weaning). Tests are not required for juvenile roof rats. For
each sex and age group, use at least 10 animals for each dosage level.
Observe changes in behavior that may affect efficacy. Observe gross patho-
logical changes found at post mortem (e.g., hemorrhaging, edema, ulcers,
discoloring). For detailed test standards, consult a standard reference
on the subject. (see reference 9 in § 96-25.)
(2) Test information. Indicate species and origin of test
animals. Describe acclimation of animals, laboratory conditions (e.g.,
temperature, humidity, light, cage size), changes in behavior during test,
and gross pathological changes found at post mortem. Separate the mortality
data by age and sex for each dosage level. Indicate the number dying at
each dosage level for each sex and age group. Submit a dose response curve
with the computation of the slope and LD50 with 95% confidence limits. (See
reference 5 in § 96-25.) . •
(3) When the applicant or registrant is notified by the Agency that
samples are necessary for confirmatory toxicity testing on rats and mice, such
samples should be sent to the EPA Animal Biology Laboratories at the following
address: Building 402 Agr. Res. Center-East, Beltsville, Md. 20705.
(c) Laboratory efficacy studies. Submit the results of a laboratory
efficacy test for each species claimed to be controlled with a commensal
rodenticide. The purpose of these studies is to assess the laboratory
efficacy of the formulation.
(1) Test standards. For complete test standards and protocols, refer
to Exhibits 2 through 20 in § 96-30. Use the wild target species for a new
claim. Otherwise, use either the wild target species or laboratory strains
of the target species. In the latter case, a roof rat claim may be supported
by Norway rat data, use at least 10 males and 10 females for each test. Use
at least 10 males and 10 females for controls. A standard challenge diet is
provided whenever the animals are exposed to a toxic bait. Refer to Exhibits
2 through 20 in § 96-30 for appropriate challenge diets. For a tracking
powder, the animal is always provided with a non-toxic route to all sources
of food and water. For a liquid bait, an alternate, nontoxic water source
must be provided. The exposure period for a single-dose toxicant is 3 days.
For a multiple-dose toxicant, the exposure period is 15 days. Some very
toxic anticoagulant baits may be tested for only 3 days. Rodenticide baits
presented to a species in an unopened place pack must haxe a test .on the
•formulation itself and with the formula-tion in the plage .•pack.
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(2) Suggested performance standards. In an acceptable laboratory
efficacy test, the product should pass the following levels of mortality
and acceptance. Anticoagulant dry baits should achieve 90% mortality and
33% acceptance (the toxic bait comprises 33% of the total food consumed).
Anticoagulant dry baits that are offered to the test rodent for a maximum
of 3 days should achieve at least 90% mortality. Acute dry baits, liquid
baits, and tracking powders should achieve 90% mortality. Red squill
baits and tracking powders should achieve 75% mortality. Wax blocks and
wax pellets should achieve 80% mortality and 25% acceptance. Greater than
10% mortality in controls negates the test.
(3) Test information. Indicate the species and strain of animal
used, the origin of test animals, and the weights of the animals at the
beginning and end of the test. Describe the laboratory conditions (e.g.,
temperature, humidity, light, and cage size) and acclimation of animals.
Submit the raw data for daily consumption of toxic bait and challenge diet.
Indicate the day of death for each animal. Provide summary data showing the
mortality and the total amount and percentage of toxic bait consumed. Submit
a chemical analysis of the test ration for the percentage of active ingredient.
(See references 13 and 14 in § 96-25.)
(4) Acceptable protocols. Refer to Exhibits 2 through 20 in § 96-30
for acceptable protocols. For existing methods of chemical analysis, consult
references 13 and 14 in § 96-25.
(5) Data submitted to support laboratory efficacy claims which are made
for products recommended for use on rats and mice may be confirmed by tests
conducted by the Agency's Animal Biology Laboratory when requested before
registration.
(d) Field efficacy studies. Submit the results of field efficacy stud-
ies for each species claimed to be controlled with a commensal rodenticide.
Data should be submitted for a new claim. If a dry bait will be marketed in
more than one form (meal, pellet, or wax block), field tests should be conducted
with at least one of these formulations. The purpose of these studies is to
assess the field efficacy of the product under actual use situations.
(1) Test standards. (i) Indoor and outdoor studies. If an indoor use
use is claimed, only the indoor studies are needed. For outdoor studies,
the following types of sites are acceptable: sheds (covered, non-enclosed
structures); small dumps that are not being continually reinfested; and small
isolated stockyards (all placements in tamper-proof bait boxes or in rat
burrows).
(ii) Number of regional studies. (A) Dry bait studies. Norway rat:
5 indoor studies (one in each of the five major regions of the country) and
2 outdoor studies (.each in a different region). Roof rat: 2 indoor studies
(in different regions) and 1 outdoor study. House mouse: 5 indoor studies
(1 in each region.) and .1 .outdoor study. .'••:•.••.• • ' •
(B.) Tracking powder or liquid bait s-tudies: Norway rat: 5 indoor
studies (any region),, and 2 outdoor studies, (any region.). Roof ra't: 2
indoor-studies, (any. region) and. .1 outdoor study*.. House .mous-e: 5-indoor .,--•
studies (any region), and 1 outdoor study. . .
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(iii) Pre-treatment and post-treatment population censusing. For
commensal rodenticides, at least two methods of pre-treatment and post-
treatment censusing are needed, one of which cannot be snap -trapping-. •••
However, the post-treatment census should be followed by 3 days of snap
trapping. The use of snap traps is necessary to complement the two census
methods as to the number of rodents present and/or to confirm the existence
of the target species, some acceptable censusing methods are live trapping,
food consumption, non-toxic tracking dust, and smoked milk cartons. If live
trapping is used, animals should be marked individually. If live trapping is
not one of the census methods, some live trapping prior to pre-treatment
censusing may be necessary to confirm the presence of the test species. If
food consumption is used, there should be at least one placement inaccessible
to animals but open to the air, to determine moisture uptake. Otherwise,
all baits should be dried to a constant weight prior to placement and after
removal. If non-toxic tracking dust or smoked milk cartons are used, some
preliminary work in the laboratory will be needed to determine how long
the dust or cartons have to be exposed before rats or mice begin leaving
tracks. A suggested rating system is: 0 = no activity, 1 = little activity
(1-3 tracks), 2 = moderate activity (4-6 tracks), and 3 = heavy activity
(7 or more tracks).
(iv) Site selection. Select sites that are isolated as much as possi-
ble from areas that may serve as reservoirs of reinfestation. Treat all
buildings at test sites. Every field efficacy study site should use a corres-
ponding control site.
(3) Suggested performance standard. Acceptable studies are those that
show 70% or greater reduction in the target population activity (due to use
of the product) in at least two of the censusing techniques outlined above,
and' that show no more than one target animal snap-trapped per 10 traps set
per night.
(4) Test information. Report the results of all studies.
Submit a sketch of the test sites, indicating the location and approximate
dimensions of all buildings. Also indicate where the baits, powder, and
traps were placed. Provide all raw data used for computing population
levels. Include all data on amount of toxic bait applied and consumed.
Weigh, identify, and sex all animals, including non-target species found
dead. Perform a post mortem on these animals to determine cause of death.
Indicate the method used for marking animals.
(5) Acceptable protocols. No specific complete test protocols exist
for field evaluation of commensal rodenticides. However, protocols cited
in § 96-30 may be helpful in developing an acceptable protocol incorporating
the test standards indicated in paragraph (d)(2) of this section.
§ 96-11 Rodenticides in orchards.
(a) General. This section furnishes information on efficacy data and
test procedures for rodenticides applied as baits or ground sprays in
orchards. Target species include the meadow vole and pine vole. Submission
of efficacy data is waived for this section.
Cb) Acute and subacute toxicity s-tudies. Submit •.the results of tox-
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icity studies for each species claimed to be controlled with a toxic bait
or ground spray in an orchard. For a single-dose bait or ground spray,
submit an acute oral LD50 test. For a multiple-dose bait or ground spray,
also submit an acute dermal LD50 test. The purpose of these studies is to
assess the appropriateness of dosage levels used in subsequent testing.
(1) Test standards. Use the wild target species. Use at least 10
animals for each dosage level for each sex. Cage test animals individually.
Observe any changes in behavior that may affect efficacy. Observe gross
pathological changes found at post mortem (e.g., hemorrhaging, edema,
ulcers, discoloring). Consult standard references on the subject for more
detailed test standards. (See references 9 and 10 in § 96-25.)
(2) Test information, indicate the species and origin of test animals.
Describe acclimation of animals, laboratory conditions (e.g., temperature,
humidity, light, cage size), changes in behavior that may affect efficacy,
and gross pathological changes found at post mortem. Separate the mortality
data by sex for each dosage level. Submit a dose response curve for each
sex with the computation of the slope and LD50 with 95% confidence limits.
(See reference 5 in § 96-25.)
(c) Laboratory acceptance studies. Submit the results of laboratory
acceptance studies for each species claimed to be controlled with a toxic
bait. The purpose of these studies is to assess the appropriateness of
bait carriers used in subsequent testing.
(1) Test standards. Use the wild target species. Use six adult
males, six adult females, and six juveniles of either sex for each target
species. Cage test animals individually. Provide a free choice between
the rat and mouse challenge diet and the candidate carrier. Continue
recording daily food consumption until the percentage of each food eaten
stabilizes. Conduct laboratory acceptance studies with all candidate
carriers. Do not fast the animals prior to any feeding tests. Conduct
tests during regular feeding hours.
(2) Test information. Indicate the species and origin of test
animals. Describe acclimation of animals, and laboratory conditions.
Submit daily food consumption data for each animal. Provide summary data
by sex and age, including the quantity and percentage of each diet consumed.
(3) Acceptable protocols. References cited in § 96-30 may be helpful
in developing an acceptable protocol incorporating the test standards describ-
ed in paragraph (c)(2) of this section.
(d) Laboratory efficacy studies. Submit the results of laboratory
efficacy studies for each species claimed to be controlled with a toxic
bait or ground spray. The purpose of these studies is to assess the labora-
tory efficacy of the formulations that will be tested in the .field.
• (.1) Test standards. Use the wild target species. Use at least 10
males aind 10 females .for each formulation. .Use at least 10 .males and 10
f-emales for controls.. Greater than 10% mortality in. the -con.tarois ..negates .-.•
the test. Provide a. free choice between the rat and mouse challenge diet ;
and -the toxic bait or sprayed vegetation. The exposure period for single'—
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dose toxicants is 3 days. For multiple-dose toxicants,, the. exposure period
Is 15 days. ' •"*,". " " *'
(2) Suggested performance standard. Under laboratory conditions, a
mortality of 100% is desirable while 90% is suggested.
(3) Test information. Indicate the species and origin of
test animals. Describe acclimation of animals and laboratory conditions.
provide daily food consumption data for each animal. Indicate the day of
death for each animal. Report summary data, including the mortality and the
total amount and percentage of toxic material and challenge diet consumed.
Submit a chemical analysis of the test ration for the percentage of active
ingredient. (See references 13 and 14 of § 96-25.)
(4) Acceptable protocols. References cited or exhibits included in
§ 96-30 may be helpful in developing an acceptable protocol incorporating the
test standard above. For methods of chemical analysis consult references 13
and 14 of § 96-25.
(e) Field efficacy studies. Submit the results of field efficacy tests
for each species claimed to be controlled with a toxic bait or ground spray
in an orchard. The purpose of these studies is to assess the field efficacy
of the product under actual use' conditions.
(1) Test standards, (i) Number of regional studies. For each formu-
lation, site, method of application, major region, and species claimed or
implied to be controlled, submit results of five field efficacy studies.
(ii) Pre-treatment and post-treatment population censusing. Two basic
methods of censusing may be used, one of which is direct and the other indi-
rect. In direct censusing, animals are livetrapped, individually marked,
released, and recaptured. (See reference 20 in § 96-25.) In indirect cen-
susing, sections of apples are used to obtain a relative index of orchard
mouse activity. (See reference 21 in § 96-25.) If this method is used,
some live trapping will be necessary to substantiate the identity of the
target species.
(iii) Rate, frequency and method of application. In order to provide
adequate use directions on the label, efficacy testing should include trials
at different rates (e.g., 1 Ib. per acre or 4 oz. per placement), frequencies
(e.g., 1 application per week or month), and methods of application (e.g.,
ground, hand, or aerial). If a claim is made that a product is effective
for a particular time period, then the test design should reflect studies
covering that period of time.
(iv) Effect of environmental factors and managerial practices. Studies
should be conducted under the environmental factors anticipated under actual
use conditions. Environmental factors such as precipation, temperature,
light, humidity, and wind may have an impact on product effectiveness as well
as managerial practices such as sanitation, timing of planting, and location
of crops. These variables should be taken into account in the experimental
test design.
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(2) Test information. Indicate the formulation, and location,
site, method, number, and frequency of applications for each study. Provide
a map of test and control areas, including their dimensions. Report all
raw and summary data used to evaluate effectiveness. Submit mortality infor-
mation by species and sex on target and non-target animals in experimental
areas. Indicate the method and rationale for selecting experimental areas and
assigning them to test or control groups. Provide climatological data for the
study period. Include all information on the statistical methods used.
(3) Acceptable protocols. For direct censusing of orchard mice, see
reference 20 in § 96-25. For indirect censusing of orchard mice with apple
slices, consult reference 21 in § 96-25. References 22 and 23.in § 96-25
also may be helpful in developing an acceptable protocol incorporating the
test standards described in paragraph (e)(2) of this section.
§ 96-12 Rodenticides on farm and rangelands.
(a) General. This section supplies information on efficacy data and
test procedures for rodenticides applied as baits on farms and rangelands.
Target animals include gophers, prairie dogs, marmots, ground squirrels, and
field mice. Registration guidance for feral commensal rodenticides are
provided in § 96-10. Submission of efficacy data is waived for this section.
(b) Acute and subacute toxicity studies. Submit the results of toxicity
studies for each species claimed to be controlled with a rodenticide applied
as a bait on farm and range lands. For a single-dose bait, submit an acute
oral LD50 test. For a multiple-dose bait, submit an acute oral LD50 test
and a subacute (5-day) oral LD50 test. The purpose of these studies is to
assess the appropriateness of dosage levels used in subsequent testing.
(1) Test standards. Use the wild target species. Use at least 10
animals for each dosage level for each sex. Cage test animals individually.
Observe changes in behavior that may affect efficacy, and gross pathological
changes at post mortem (e.g., hemorrhaging, edema, ulcers, discoloring).
Consult a standard reference for detailed test standards. (See reference 9
of § 96-25.)
(2) Test information required. Indicate the species and origin of test
animals. Describe acclimation of animals, laboratory conditions (cage speci-
fications, temperature, humidity, light), and changes in animals' behavior
that may affect efficacy. Indicate the the sex and weight of each animal.
Describe gross pathological changes found at post mortem. Submit a dose-
response curve for each sex with the computation of the slope and LD50 with
95% confidence limi'ts. (See reference 5 in § 96-25.)
(c) Laboratory acceptance studies. Submit the results of laboratory
acceptance studies for each, species claimed to be. controlled with a rodenti-
cide applied as a. bait on farms and range3ands.. The purpose of these studies
is to assess the appropriateness of bait .carriers used on subsequent testing.
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(1) Test standards. Use the wild target species. Use 6 adult males,
6 adult females, and 6 juveniles of either sex for each target species.
Cage test animals individually. Provide a free choice, between each candidate
carrier and the habituated diet. Replace the habituated diet with the stand-
ard challenge diet when it has been established. For example, see Exhibits
2 through 20 in § 96-30. Continue recording daily food consumption data
until the percentage of each food eaten stabilizes. Conduct laboratory
bait acceptance studies with all candidate carriers.
(2) Test information. Indicate the species and origin of test animals.
Describe acclimation of animals and laboratory conditions. Report food
consumption data for each animal. Provide summary data by sex and age,
including the quantity and percentage of each diet consumed.
(3) Acceptable protocols. References cited in § 96-30 may be helpful
in developing an acceptable protocol incorporating the test standards
described in paragraph (c){2) of this section.
(d) Laboratory efficacy studies. Submit the results of a laboratory
efficacy test for each species claimed to be controlled. The purpose of
these studies is to assess the laboratory efficacy of the formulation.
(1) Test standards, use the wild target species. Use at least 10
males and 10 females for each test. Use at least 10 males and 10 females
for controls. Greater then 10% mortality in the controls negates the test.
Provide a free choice between the habituated ration or the standard challenge
diet and the toxic bait. The exposure period for a single-dose toxicant is
3 days. For a multiple-dose toxicant, the exposure period is 15 days. Test
all formulations that may be used in the field.
(2) Suggested performance standard. In an acceptable laboratory
efficacy test, the product should kill at least 90% of the test animals.
(3) Test information. Indicate the species and origin of test animals.
Describe acclimation of animals and laboratory conditions. Provide daily
food consumption data for each test animal. Indicate the day of death of
each animal. Provide summary data by sex and species. Submit a chemical
analysis of the bait for the percentage of active ingredient. (See
references 13 and 14 of § 96-25.)
(4) Acceptable protocols. For methods of chemical analysis, consult
references 13 and 14 in § 96-25. References cited in § 96-30 may be helpful
in developing an acceptable protocol incorporating the test standards de-
scribed in paragraph (d)(2) of this section.
(e) Field efficacy studies. Submit the results of field efficacy
tests for each species claimed to be controlled with a rodenticide applied
as a bait on farms and rangelands. The purpose of these studies is to
assess the field efficacy of the product under actual use conditions.
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(1) Test standards. (i) Number of regional studies. Submit 5 efficacy
studies for each formulation, site, method of application, major region, and
species claimed or implied to be controlled on the label.
(ii) Pre-treatment and post-treatment population censusing. Two basic
methods of censusing may be used, one of which is direct and the other indi-
rect. In direct censusing, animals may be live-trapped, individually marked,
released, and recaptured. (see reference 20 in § 96-25.) Also, visual counts
of rodents may be made at periodic intervals (e.g., early morning) for several
days in order to obtain a relative index of rodent pressure. In indirect cen-
susing, mark mounds or burrows individually. Depending on the-species, these•'••
would be either opened or closed. After one or two days, determine which
burrows still show activity. For gophers, use at least 25 active mounds in
treated and control areas. (See reference 24 in § 96-25.)
(iii) Control site. A control site is needed for every study.
(iv) Rate, frequency, and method of application. Efficacy testing
should include trials at different rates (e.g., 1 Ib per acre or 4 oz per
placement), frequencies (e.g., 1 application per week or month), and methods
of application (e.g., ground, hand, or aerial). If a claim is made that a
product is effective for a perticular time period, then the test design should
reflect studies covering that period of time.
(v) Effect of environmental factors and managerial practices. Studies
should be conducted with the environmental factors anticipated under actual
use conditions. Environmental factors (such as precipitation, temperature,
light, humidity, and wind), as well as managerial practices (such as sanitation
and timing of planting), may have an impact on product effectiveness. These
variables should be taken into account in the experimental test design.
(2) Suggested performance standards. An acceptable level of efficacy
should result in at least a 70% reduction of rodent activity.
(3) Test information. Report the results of all studies.
Submit a sketch of the test sites indicating the the proximity to surrounding
areas which may harbor the target species. Indicate the dates of treatment.
Include all raw data used for computing the population levels. Indicate the
kind and amount of all toxic bait applied in pounds per acre. Report the
spacing between treated areas. Describe the soil type and ground cover.
Indicate the percentage of animals controlled. Submit information (mortality
by species and sex) on target and non-target kills in experimental areas.
(4) Acceptable protocols. For direct censusing of rodents, consult
reference 20 in § 96-25. For indirect censusing of pocket gophers, refer
to reference 24 in § 96-25. For indirect censusing of other rodents, refer
to § 96-30.
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§ 96-13 Rodent fumigants.
(a) General. This section lists efficacy data and test procedures for
rodent fumigants. Target animals include the Norway rat, roof rat, Polynesian
rat, house mouse, pocket gopher, and ground squirrel. Submission of efficacy
data is waived for this section.
(b) Acute toxicity studies. Submit the results of an acute inhalation
LC50 toxicity test for each target animal claimed to be controlled by a
rodent fumigant. The purpose of these studies is to assess the appropriate-
ness of dosage levels to be used in subsequent testing.
(1) Test standards. Use the wild target species. Use at least 6 males
and 6 females for each dosage level for pocket gophers and ground squirrels.
Use at least 10 males and 10 .females for each rat and mouse species. Test
animals individually in a gas-tight chamber. For acceptable equipment and
procedures in determining toxicity of fumigants, consult Section 4.3 of
reference 16 of § 96-25. Observe changes in behavior that may affect effi-
cacy. Observe gross pathological changes found at post mortem (e.g., hemor-
rhaging, edema, ulcers, discoloring). Consult a standard reference on the
subject for more detailed test standards. (See reference 9 in § 96-25.)
(2) Test information. Indicate species and origin of test
animals. Describe acclimation of animals, laboratory conditions (e.g.,
temperature, humidity, light, cage size), changes in behavior during the
fumigation test that may affect efficacy, and gross pathological changes
found at post mortem. Separate the mortality data by sex for each dosage
level. Submit a dose response curve for each sex with the computation of
the slope and LC50 with 95% confidence limits. (Check reference 5 in
§ 96-25.)
(c) Laboratory efficacy studies. Submit the results of a laboratory
efficacy test for each species claimed or implied to be controlled with a
rodent fumigant. The purpose of these studies is to assess the laboratory
efficacy of formulations.
(1) Test standards. Use the wild target species. Use at least 6 adult
males and 6 adult females for each dosage level for pocket gophers and ground
squirrels. Use at least 10 adult males and*10 adult females for each rat and
mouse species. Test the product for one hour. Test animals individually in
an artificial burrow. (See reference 18 in § 96-25.) Do not fast animals
prior to efficacy 'tests. • •
(2) Suggested performance standard. Under laboratory conditions, a
mortality of 100% is desirable; 90% is suggested.
(3) Test information. Indicate species and origin of test
animals. Describe acclimation of animals and tunnel conditions (e.g., temper-
ature, humidity). Provide a sketch with dimensions of the tunnel and exhaust
systems. Describe the type of soil surrounding tunnel systems. Record the
amount of time required to kill each individual animal. Submit all raw and
summary data including mortality according to sex of test animals. Submit
a chemical analysis of the fumigant-formulation for the percentage -of active
ingredients. Consult references 13 and. 14 in § 96-2-5.- ...
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(4) Acceptable protocols. For conducting a laboratory efficacy study
in an artificial burrow, see reference 18 in § 96-25. Others cited, in
§ 96-30 may be helpful in developing an acceptable protocol incorporating
the test standards described in paragraph (c)(2) of this section. For
methods of chemical analysis, consult references 13 and 14 in § 96-25.
(d) Field efficacy studies. Submit field efficacy tests for each
species claimed to be controlled with a rodent fumigant. The purpose
of these studies is to assess the field efficacy of the product under
actual use situations.
(1) Test standards. (i) Number of regional studies. Submit 5 field
efficacy studies for each formulation, site, method of application, major
region, and species claimed or implied to be controlled on the label.
(ii) Pre-treataent and post-treatment population censusing. Indices
of animal density can be made by live trapping, food consumption, non-toxic
tracking dust, opening or closing burrows, and smoked milk cartons. If live .
trapping is used, animals should be marked individually. If live trapping is
not one of census methods, some live trapping prior to pre-treatment census-
ing may be necessary to confirm the presence of the pest species. If food
consumption is used, there must be at least one placement inaccessible to
animals, but open to the air, to determine moisture uptake. Otherwise, all
baits should be dried to a constant weight prior to placement and after removal.
If nontoxic tracking dust or smoked milk cartons are used, some preliminary
work in the laboratory will be needed to determine how long the dust or
cartons have to be in place before the rodents begin leaving tracks. [Sug-
gested rating scale: 0 = no activity; 1 = little activity (1-3 tracks); 2 =
moderate activity (4-6 tracks); and 3 = heavy activity (7 or more tracks).]
For each study, pretreatment censusing should indicate the presence of at
least 20 animals of the target species (for treatment of buildings) of at
least 20 active burrows (for treatment of burrows) in both the test and
control areas.
(iii) Site selection. Select sites that are isolated as much as
possible from areas that may serve as reservoirs of reinfestation. Treat
all buildings and burrows at test sites.
(iv) Rate, frequency/ and method of application. All fumigants should
be tested at the rate, frequency, and method of application anticipated in
the use directions.
(v) Effect of environmental factors. Environmental factors such as
temperature and humidity may have an impact on product effectiveness.
These variables should be taken into account in the experimental test design.
(2) Suggested, performance standard. Acceptable studies are those
which show 70% or greater reduction in the target population based on the
censusing techniques outlined in this section. • . -
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(3) Test information. Report the results of all .studies.
Submit a sketch of the test and control sites, indicating the location and''
approximate dimensions of all buildings and burrows. Also, indicate where
the rodent fumigants were applied. Include all raw data used for computing
population levels. Report all data on amount of fumigant applied. Record
the weight, species, and sex of all target and nontarget animals found
dead. Perform a post mortem on these animals to determine cause of death.
Indicate the method used for marking animals.
(4) Acceptable protocols. No specific complete test protocols exist
for field evaluation of rodent fumigants. However, protocols cited in
§ 96-30 may be helpful in developing an acceptable protocol incorporating
the test standards described in paragraph (d)((2) of this section.
§ 96-14 Rodent repellents on tree seeds.
(a) General. This section indicates directions for obtaining efficacy
data and test procedures for rodent repellents applied to tree seeds.
Target species include deer mice. Submission of efficacy data is waived for
this section.
(b) Acute toxicity studies. Submit the results of toxicity studies
for each species claimed to be controlled with a repellent applied to tree
seeds. These tests shall include an acute oral LD50 test. The purpose of
these studies is to assess the appropriateness of dosage levels used in
subsequent testing.
(1) Test standards. Use the wild target species. Use at least 10
adult males and 10 adult females for each dosage level. Cage test animals
individually. Observe any changes in behavior that may affect efficacy.
Observe gross pathological changes found post mortem (e.g., hemorrhaging,
edema, ulcers, discoloring). For detailed information on test standards,
consult a standard reference on the subject. (See reference 9 in § 96-25.)
(2) Test information. Indicate the species and origin of
test animals, laboratory conditions (e.g., temperature, humidity, light,
cage size), and gross pathological changes found post mortem. Separate the
mortality data by sex for each dosage level. Submit a dose response curve
for each sex with computation of the slope and LD50 with 95% confidence
limits.
(c) Laboratory efficacy studies. Submit the results of a laboratory
efficacy test for each species claimed to be controlled with a repellent
applied to tree seeds. The purpose of these studies is to assess the
laboratory efficacy of the formulation.
(1) Test standards, (i) Use the wild target species. Use at least
10 adult males and 10 adult females for controls. Greater than 10% mortality
in the controls negates the test. Provide a free choice between the mainte-
nance ration or the standard challenge diet and the repellent-treated seeds.
Use a standard challenge diet when one has been established. For example,
see Exhibits 2 through 2'0 in § 96-30-. -' :
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(ii) The treated seeds should be of the species for which protection is
claimed or implied on the label. Fifty treated seeds should be offered daily
to the mice for 6 days. A seed is considered damaged if the seed coat has
been cracked whether or not the endosperm has been eaten. The controls re-
ceive 50 untreated seeds daily.
(iii) Repellency is calculated according to the following formula:
No. seeds damaged by test animals
% Repellency = [ 1 - ( ) ] x 100
No. seeds damaged by control animals
(iv) The consumption of the maintenance or standard diet should be record-
ed daily. The amount of maintenance or standard diet must exceed the daily -
requirement of the species tested. Do not fast animals prior to efficacy
tests. ,.- •. ...
(2) Suggested performance standards. Under laboratory conditions, a
50% or greater repellency is suggested.
(3) Test information. Indicate the species and origin of test
animals. Describe acclimation of animals and laboratory conditions. Report
daily seed damage by each animal. Provide daily maintenance or standard
challenge diet consumption data for each animal. Indicate the day of death
of each animal. Provide summary data by sex and species. Submit a chemical
analysis of the repellent-treated seed indicating the percentage of active
ingredient. (See references 13 and 14 in § 96-25).
(4) Acceptable protocols. For conducting a laboratory efficacy study
of tree seed repellents, see reference 25 in § 96-25). Other references
cited in § 96-30 may be helpful in developing acceptable protocols incorpora-
ting the test standards described in paragraph (d)(2) of this section.
(e) Field efficacy studies. Submit the results of field efficacy tests
for each species of rodents claimed to be repelled by repellent-treated tree
seed. The purpose of these studies is to assess the field efficacy of the
product under actual use situations.
(1) Test standards. (i) Number of regional studies. Submit 5 efficacy
studies for each formulation, site, method of application, major region, and
species claimed or implied to be controlled on the label.
(ii) Pre-treatment and post-treatment censusing in study areas. Use
live traps at selected points of heavy deer mouse activity in study plots for
3 to 6 nights. All deer mice should be marked and released at site of capture.
At least 12 different deer mice per 300 trap nights should be captured for ac-
ceptable test and control plots. There should be a control plot for every test
plot. Test and control.plots should be.comparable in mouse, .populations, acreage,
and topography characteristics. Control plots should be stocked with untreated
seed at the same rate as the test site. Plot sizes should be a minimum of 10
.acres and a.maximum of 100 acres,. • •
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(iii) Damage evaluation. To rate repellent effectiveness, each plot
should be. sampled by at least 64 milacre subplots spaced uniformly. All
newly germinated seedlings in the milacre should be tallied and identified.
Germination assessments should be conducted in the spring and again in the
summer.
(iv) Phytotoxicity characteristics. To determine any phytotoxic char-
acteristics of the tree seed repellent, 4 replicate lots of treated and un-
treated seeds are to be tested in a seed germinator and monitored for 4 weeks.
For additional information on phytotoxicity, consult Subdivision J.
(2) Suggested performance standard. The level of efficacy that
should be attained for an acceptable test has not been established.
However, seedling counts in the treated plots should average at least
50% higher than the control plots. Seedling counts should average at
least 1.25 seedlings per milacre sample in the test plots.
(3) Test information. Indicate the species of test animals
and tree seed in treated and control areas. Give the source of tree seeds.
Report number of animals captured and total population estimate for each
plot. Indicate the approximate age of animals. Describe plot conditions
(e.g., size, spatial arrangement, directional orientation of plots, and
soil moisture). Provide a map of the test area including directional
orientation and treatment placements. Report relevant behavior of all
animals (including nontarget species) interacting with the repellent.
Describe the procedures of the test, application method, treatment levels,
frequencies, and locations. Submit weather data (e.g., temperature, humid-
ity, wind speed and direction, and precipitation). Report the percent
• repellency for each pair of test and.:control plots. Describe and report
all statistical analyses. Report the results of phytotoxicity tests.
(4) Acceptable protocols. Reference 26 in § 96-25 and others cited
in § 96-30 may be helpful in developing an acceptable protocol incorporating
the test standards described in paragraph (e)(2) of this section.
§ 96-15 Rodent repellents on cables.
(a) General. This section indicates directions for obtaining efficacy
data and test procedures for rodent repellents applied to underground
cables. Target species include the plains pocket gopher, northern pocket
gopher, and valley pocket gopher. Submission of efficacy'data is waived for
this section.
(b) Preliminary acute and subacute toxicity studies. Submit the
results of toxicity studies for each species claimed to be controlled with
a rodent repellent applied to underground cables. These studies should
include an acute oral LD50 test and a subacute (5-day) oral LD50 test
for the Norway rat. The purpose of these studies is to determine the mode
of action of the repellent and the possibility of hazard to the target
species.
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(1) Test standards. Use the Norway rat. Laboratory strains of
this species are acceptable. Use at least 10 adult males and 10 adult
females for each dosage level. Cage test animals individually. Observe
any test changes in behavior that may affect efficacy. Observe gross
pathological changes found at post mortem (e.g., hemorrgaging, edema,
ulcers, discoloring). For detailed information on test standards, refer
to §§ 81-1 and 82-1 of Subdivision F of these document.
(2) Test information. Indicate the species, strain, and
origin of test animals. Describe acclimation of animals, laboratory
conditions (e.g.,'temperature, humidity, light, cage, size), and gross
pathological changes found at post mortem. Separate the mortality data
by sex for each dosage level, with 95% confidence limits. (See reference
5 of § 96-25.) For detailed information, refer to §§ 81-1 and 82-1 of
Subdivision F of this document.
(3) Acceptable protocols. For conducting toxicity tests, refer to
§§ 81-1 and 82-1 of Subdivision F. For presenting a dose-response curve,
consult reference 5 of § 96-25.
(c) Advanced acute and subacute toxicity studies. If either test
listed in paragraph (b) of this section yields an LD50 of 1000 mg/kg or
less, conduct tests using the target genus. The purpose of these studies
is to assess the product toxicity and mode of repellency to the target
species.
(1) Test standards. Use the wild target species. Use at least 10
adult males and 10 -adult females for each dosage level. Cage test animals
individually. Observe any changes in behavior that may affect efficacy.
Observe gross pathological changes found at post mortem (e.g., hemorrhaging,
edema, ulcers, discoloring). For detailed information on test standards,
consult a standard reference on the subject. (See reference 9 in § 96-25.)
(2) Test information. Indicate the species and origin of
test animals. Describe acclimation of animals, laboratory conditions
(e.g., temperatures, humidity, light, cage size), and gross pathological
changes found at post mortem. Separate the mortality data by sex for each
dosage level. Submit a dose response curve for each sex with computation
of the slope and LD50 with 95% confidence limits. (Consult reference 5
in § 96-25.)
(d) Small plot efficacy studies. Submit the results of small plot
efficacy studies for each species claimed to be controlled. Ihe purpose
of these studies is to assess the efficacy of formulations under small
plot conditions.
(1) Test standards. Use live traps in each study area to determine
the target species. Only use sites adjacent to acctive burrows. ("Active"
burrows.are ones that are plugged by gophers after once .being opened by the
investigator). Adjavent to an active burrow, install two 5-ft sections of
underground cable of the type to be protected. Place cable sections in
separate trenches a minimum of 3 ft apart so that treatment of one cable
does not .a'ffect an untreated cable. Use 10 .replications .per treatment,. ....
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Keep records on all methods, of application and the dosage-rates. Do ..not..
reuse trenches. Test cable-sections' under both cool (less than 5°C) and
warm (more than 20°C) weather. Test cable sections where soil is saturated
with water and where soil is well drained, because efficacy of certain
products is susceptible to leaching. After treatment, continue to monitor
cables at least twice a week for 12 weeks. Check both treated and untreat-
ed cables. Also, according to a predetermined method acceptable to the
Agency, rate the damage to the cables using teeth marks as an index.
(2) Suggested performance standard. Although no level of efficacy
for an acceptable test has been established, 70% fewer teeth marks and
opened circuits on treated cables, compared to untreated cables, is
suggested.
(3) Test information. Indicate the target species for each
study area. Include a map of all study areas, denoting location of active
burrows and treated and untreated cables. Indicate the distance between
all cables. Report the method and frequency of application, formulation,
dosage rates, type of cable (number of circuits, type of coating, and
diameter), the temperature, precipitation, and soil moisture. For each
check of cables, include the raw and summary data with respect to cable
damage. Describe all statistical methods used to evaluate efficacy.
(4) Acceptable protocols. References cited in § 96-30 may be helpful
in developing an acceptable protocol incorporating the test standards de-
scribed in paragraph (d)(2) of this section.
(e) Field efficacy studies. Submit the results of field efficacy
studies for each species claimed to be controlled. The purpose of these
studies is to assess the efficacy of the product under actual use conditions.
(1) Test standards. Use live traps at selected points of heavy gopher
activity along the length of cable to determine the target species.
(i) Number of regional studies. For each formulation, dosage rate,
method of application, and species claimed or implied to be controlled on
the label, submit data from at least 50 miles of treated and 50 miles of
untreated cables that are in actual use.
(ii) Pre-treatment and post-treatment population censusing. At least
6 study areas along both treated and untreated cable should be established.
These areas should include 3 areas of moderate gopher pressure and 3 areas
of heavy gopher pressure. Two of the areas of moderate pressure and 2 of
the areas of heavy pressure should be in well-drained soil. One of the areas
of moderate pressure and one of the areas of heavy pressure should be in
poorly drained soil. A quantitative definition of moderate and heavy pres-
sure must be established, based on the number of active burrows per unit
area. (An "active" burrow is one that is plugged by gophers after being
opened by the investigator.) At periodic intervals (e.g., every 2 months
for 12 months), evaluate the gopher pressure at these sites.
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(iii) Damage assessments. Damage assessments will be made at the same
time that census assessments are made. Indicate the number of circuits open-
ed using a volt-ohm meter. Also, according to a predetermined method, rate
the damage to the cables using the teeth marks as an index. Finally, maintain
data on the number of "blowouts" that occur along treated and untreated cable.
(iv) Records required. Keep records on the method and frequency of
application, dosage rates, temperature, soil moisture, and cable type.
(2) Suggested performance standard. Though no level of efficacy for
an acceptable test has been established, 70% fewer "blowouts" or number of
teethmarks and opened circuits for treated cable, compared to untreated
cable, is suggested.
(3) Test information. Indicate the target species at selected
points along the cable. Include a map with dimensions of all study areas
along treated and untreated cable. Submit a detailed map of the study areas.
Indicate the method and frequency of application, formulation, dosage rates,
temperature, precipitation, and soil moisture. Provide all raw and summary
data upon which the censusing and damage assessments were made. Describe all
statistical methods used to evaluate efficacy.
(4) Acceptable protocols. References cited in § 96-30 may be helpful
in developing an acceptable protocol incorporating the test • standards listed
above.
§ 96-16 Rodent reproductive inhibitors.
(a) General. This section provides efficacy data and test procedures
for rodent reproductive inhibitors applied as dry baits. Target animals
include the Norway rat, roof rat, Polynesian rat, house mouse, and certain
species of Microtus and Peromyscus. Submission of efficacy data is waived
for this section.
(b) Acute and subacute toxicity studies. Submit type results of an
acute oral LD50 test and subacute (5-day) oral LD50 test for each target
animal claimed to be controlled with a rodent dry bait reproductive inhibitor.
The purpose of these studies is to assess the appropriateness of dosage levels
used in subsequent testing.
(1) Test standards. Use the target animals. Laboratory strains of
target species are acceptable. Cage the test animals individually. Test
both juvenile animals and adult animals (sexually mature). For each sex and
age group, use at least 10 animals for each dosage level. Observe changes
in behavior that may affect efficacy and gross pathological changes found at
post mor.tem (e.g., testicular or ovarian atrophy, hemorrhaging., edema, ulcers-) ••
For detailed test standards, consult a standard reference on the subject.
(See reference 9 in § 96-25.) ,/
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(2) Test information. Indicate the species, strain, and origin
of test animals. Describe acclimation of animals, laboratory con-
ditions (e.g., temperature, humidity, light, and cage size), changes in
behavior during the test that may affect efficacy, and gross pathological
changes found at post mortem, including information on reproductive in-
hibition. Separate the mortality data by age and sex for each dosage
level. For each sex, indicate the number dying at each dosage level.
Submit a dose response curve with the computation of the slope and 1050
with 95% confidence limits.
(c) Laboratory acceptance studies. Submit the results of laboratory
acceptance studies for each formulation and species claimed to be controlled
with a dry bait rodent reproductive inhibitor. The purpose of these studies
is to assess the laboratory acceptance of the reproductive inhibitor in the
presence of a standard challenge diet and to provide subjects for subsequent
breeding studies.
(1) Test standards. Use the wild target animal. Use at least 20 males
and 20 females per age group (juvenile and adult) for each test. Use at
least 10 males and 10 females per age group as controls. If the reproduc-
tive inhibitor is sex specific, use only the appropriate sex. A standard
challenge diet is provided whenever the animals are exposed to dry bait
reproductive inhibitor. (See Exhibits 2-20 in § 96-30.) Cage animals
individually. Test both juvenile animals and adult animals (sexually
mature). Observe changes in behavior that may affect efficacy. Observe
gross pathological changes found at post mortem, including information on
reproductive inhibition. Ihe exposure period should not exceed 15 days.
(2) Test information. Indicate the species and origin of
test animals. Describe laboratory conditions. Submit the raw data on
daily consumption of reproductive inhibitor and challenge diets. Indicate
the weights of the animals at the beginning and end of the test, and the
day of death for each animal. Provide summmary data showing the mortality
(if any) and the total amount and percentage of reproductive inhibitors
and challenge bait consumed. Submit a chemical analysis of the test ration
for the percentage of active ingredient. (See references 13 and 14 in
§ 96-25.)
(3) Acceptable protocols. For acceptable methods of chemically
analyzing products, consult references 13 and 14 in § 96-25. References
cited in § 96-30 may be helpful in developing an acceptable protocol
incorporating the test standards described in paragraph (c)(2) of this
section.
(d) Laboratory breeding studies. Submit the results of laboratory
breeding studies for each formulation and species claimed to be controlled
with a dry bait rodent reproductive inhibitor, the purpose of these studies
is to assess the reproductive success of test and control rodents.
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(1) Test standards. Use the rodents tested in the laboratory feeding
tests above. Use wild target animals for controls. Use at least 10 males
and 10 females for each breeding test. Place each breeding pair in a sepa-
rate cage with a nest box. If the reproductive inhibitor is sex-specific,
treat only the appropriate sex. Breed at least 10 males and 10 females
that had been treated with a reproductive inhibitor as juveniles. Breed at
least 10 males and 10 females that had been treated with a reproductive
inhibitor as adults. Breed at least 10 males that had been treated with a
reproductive inhibitor as juveniles and 10 females that were untreated.
Breed at least 10 males that had been treated with a reproductive inhibitor
as adults and 10 females that were untreated. Breed at least 10 females that
had been treated with a reproductive, inhibitor as adults and 10 males..that
were untreated. Breed at least 10 untreated males and 10 untreated females
to serve as controls. Observe changes in behavior that may affect efficacy.
Observe gross pathological changes found at post mortem (e.g., testicular
or ovarian atrophy, hemorrhaging, edema, ulcers). Continue breeding test
for 90 days or until 3 separate litters have been born per breeding pair.
To insure that reproductive inhibition is due to the treatment and not to
sexual immaturity, juvenile subjects must be raised to sexual maturity be-
fore use in breeding tests. Sacrifice all progeny 7 days after parturition.
(2) Test information. Report the number of litters and progeny (if
and when produced) from all the mating combinations specified above.
Record individual weight, number, and sex of all progeny per litter at
sacrifice date. Report morphological abnormalities of progeny at sacri-
fice date. Describe breeding cages, nest boxes, and laboratory conditions.
Report the elapsed time in days from establishment of breeding pairs to
the birth of the first, second, and third litters. Report weekly weights
of all test and control animals from establishment of breeding pairs to
end of test. Summarize the amount of reproductive inhibitor consumed ac-
cording to: number of litters and progeny produced by age group; mortality
(if any); and the number of litters and progeny produced, treated males,
treated females, and both males and females treated. Also summarize the
number of litters and progeny produced by controls. Submit a copy of all
raw data.
(3) Suggested performance standards. Although an acceptable level
of efficacy has not been established, a 90% reduction in reproductive
success (total progeny produced), when compared to controls, is suggested.
(e) Field efficacy studies. Submit the results of field efficacy
tests for each species claimed to be controlled with a reproductive inhib-
itor, the purpose of these studies is to assess the field efficacy of the
product under actual use situations.
(1) Test standards. (i) Indoor and outdoor studies. If an indoor
use is claimed, only the indoor studies are necessary. For outdoor studies,
the following types of sites are acceptable: sheds (covered., non-enclosed
structures), small.'dumps .that are not being continually reinfested, and
small isolated stockyards (all placements in tamper-proof bait boxes or
in rodent burrows.).
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(ii) Number of regional studies. Norway rat: five indoor studies
(one in each of the five major regions of the country) and three outdoor
studies (each in a different region). Roof rat: two indoor studies (in
different regions) and two outdoor studies (in different regions). House
mouse: five indoor studies (one in each region) and three outdoor studies
(in different regions). Microtus sp.: five outdoor studies (one in each
region). Peromyscus sp.: five outdoor studies (one in each region).
(iii) Pre-treatment and post-treatment population censusing. For
reproductive inhibitors, at least two methods of pre-treatment and post-
treatment censusing are needed, one of which must be the mark and release
method. (See § 96-30, Field Census Techniques). Some methods that will
be acceptable for the second census method are food consumption, non-toxic
tracking dust, smoked milk cartons, mapping of rodent signs, tracking, and
electronic remote censusing. All live-trapped animals should be marked indi-
vidually. If food consumption is used, there should be at least one placement
inaccessible to animals, but open to the air, to determine moisture intake.
Otherwise, all baits should be dried to a constant weight prior to placement
and after removal. If non-toxic tracking dust or smoked milk cartons are
used, some preliminary work in the laboratory will be needed to determine
how long the dust or cartons have to be in place before the rodents begin
leaving tracks. A suggested rating system is: 0 = no activity; 1 = little
activity (1-3 tracks); 2 = moderate activity (4-6 tracks); and 3 = heavy
activity (7 or more tracks). The test and control sites should be censused
immediately before treatment and on a monthly basis thereafter for a year.
(iv) Site selection. Select sites that are isolated as much as
possible from areas that may serve as reservoirs of reinfestation. Treat ,
all buildings at the test sites.
(v) Treated and untreated areas. At least one untreated control
site should be censused for each study. The control sites should be treated
with a placebo form of the bait used at the treated sites at the same
application rates. All toxic rodenticide baits should be permanently
removed from the test and control sites 30 days before pre-treatment
censusing.
(2) Suggested performance standards. Field studies should meet the
following efficacy standards. Population levels are to be determined one
year after application of the reproductive inhibitor to the test sites.
Reproductive inhibitors that claim to both kill commensal rodents and
reduce reproduction in the survivors should meet this
parameter of efficacy.
(i) For test and control sites treated with a toxic rodenticide
3 months or less prior to application of a reproductive inhibitor in
the test site, a maximum of 30% population increase of target species
at the treated site and a minimum of 80% increase at the control site
is acceptable.
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(ii) For test and control sites treated with a toxic rodenticide more
than 3 months prior to application of a reproductive inhibitor, a reduction
of 70% or greater of the target species at the treated site (due to use of
the product) and not more than 20% reduction of the target species at the
control site is acceptable.
(3) Test information. Report the results of all studies.
Submit a sketch of the test and control sites, indicating the location and
approximate dimensions of all buildings. Also indicate where the baits and
traps were placed. Induce all raw data used for computing population levels.
Include all data on amount of reproductive inhibitor applied and consumed.
Report weight, sex, identification marks, and species of all animals live
trapped. For male rodents, record if the testicles are present in the scro-
tum or in the body cavity, and, for females, record if they are lactating
and/or are obviously pregnant. Record weight, sex, identification marks,
and species of all target and non-target animals found dead. Perform post
morterns to determine cause of death.
(4) Acceptable protocols. No specific complete test protocol exists
for field evaluation of reproductive inhibitors on rodents. References
cited in § 96-30 may be helplful in developing an acceptable protocol incor-
porating the test standards described in paragraph (e)(2) of this section.
§ 96-17 Mammalian predacides.
(a) General. This section deals with efficacy data procedures for
mamalian predacides applied as baits and fumigants. Target species include
the coyote, red fox, gray fox, feral dog, and skunk. Submission of efficacy
data is waived for this section.
(b) Acute and subacute toxicity studies. Submit the results of toxic-
ity studies of each species claimed to be controlled with a mammalian pred-
acide. For a single dose predacide, submit an acute oral LD50 test and a
subacute (5-day) oral LD50 test. For a fumigant, submit an acute inhalation
LC50 test. Ihe purpose of these studies is to assess the appropriateness
of dosage levels used in subsequent testing.
(1) Test standards. Use the wild target species. Use at least 12
sexually mature animals (6 males and 6 females). Cage test animals individ-
ually. Test animals must be in large enough cages to allow freedom of move-
ment. This requirement may be computed by using the following equation:
[length of target animal (in inches) + 6 inches]: 144 inches2/ft2 = required
floor space (ft2). (See reference 27 in § 96-25.) Use only animals that
are acclimated to test condition. Stabilize, diet .and general condition of
animals for at least 28 days prior to testing. Observe changes .in behavior
(e.g., vocal,, chemicals, or postural communication) that may affect efficacy.
Examine all affected animals for gross pathological and histological changes.
For detailed test standards., consult reference 9 in § 96-25.
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('2') Test information. Indicate the'species and origin of-tes-t•--"'•••-..,
animals. Describe acclimation of animals, 'laboratory conditions (e.g., tem-
perature, humidity, light, cage size), changes in behavior that may affect
efficacy, and gross pathological and histological changes of those animals
killed by the toxicant. Separate mortality data by sex for each dosage level.
Submit a dose response curve for each sex with the computation of the slope
and LD50 with 95% confidence limits. (See reference 5 in § 96-25.)
(c) Laboratory efficacy studies. Submit the results of laboratory
efficacy tests for each species claimed to be controlled with the predacide.
The purpose of these studies is to assess the laboratory efficacy of the
toxic formulation.
(1) Test standards. Use the wild target species. Use a minimum of
12 adult target animals (6 males and 6 females) and, if possible, 6 juveniles.
Provide the animals with a free choice between the habituated ration and the
toxic bait. References cited in § 96-30 may be helpful in developing an ac-
ceptable protocol incorporating the test standards described in this paragraph.
(2) Suggested performance standard. To date, the levels of mortality
required of each predacide test have not been established. Under laboratory
conditions, a mortality of 100% is preferred and a mortality of 90% is
suggested.
(3) Test information. Indicate the species and origin of
test animals. Describe acclimation of animals and the laboratory conditions
(e.g., temperature, humidity, light, cage size). Submit raw data for con-
sumption of toxicant bait and habituated.ration. Report the following data
for each affected animal: the latency period, poisoning symptoms, cause of
death, and gross pathology. Submit a chemical analysis of the test ration
for the percentage of active ingredient. (Consult references 13 and 14 of
§ 96-25 for existing methods of chemical analysis.)
(4) Acceptable protocols. References cited in § 96-30 may be
helpful in developing an acceptable protocol incorporating the test
standards described in paragraph (c)(2) of this section. For existing
methods of chemical analysis, see references 13 and 14 of § 96-25.
(d) Field efficacy study. Submit field efficacy tests for each
species claimed to be controlled with the predacide. The purpose of these
studies is to assess the field efficacy of the product under actual use
situations.
(1) Test standards. Precise field test standards are not well defined
because of the inherent variables (climate, animal movement, behavior modifi-
cation, availability of food, habitat types, and reproductive rates). Small
plot testing should precede general field testing.
(i) Number or regional studies. For each region where control of the
animals is anticipated, submit five field efficacy studies for each pattern
of use.
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(ii) Pre-treatment and post-treatment population censusing. Indices
of animal density in the area can be determined by the amount of food
consumed or by the animal tracks occurring at daily monitored sites.
Additional information should include data concerning livestock or wildlife
depredation or disease incidence such as rabies and plague.
(iii) Site selection. Study sites should be representative of those
areas viiere eventual use of the predacide is intended and should be rela-
tively free from animal immigration and emigration. At least one untreated
control site should be censused for each study.
(2) Suggested performance standard. Acceptable efficacy studies
are those which show sufficient predator reduction (due to use of the
product) as to bring about a cessation or satisfactory reduction in
livestock or wildlife depredation or an interruption of disease incidence
such as rabies and plague.
(3) Test information. Submit results of all studies.
Indicate the size of area involved and test site legal locations using
U.S. Geological Survey map coordinates. Identify target species and
their relative population densities and locations. Report the number
and amount of toxic baits and/or devices placed per acre, square mile,
or township. Indicate the number and distribution of target and non-
target species found dead or seen affected. Perform post morterns to
determine cause of death. Submit all raw data used for computing
population levels, livestock depredation, and disease incidence.
(4) Acceptable protocols. No specific complete test protocol
exists for field evaluation of toxic chemicals for predator management.
References cited in § 96-30 may be helpful in developing an acceptable
protocol incorporating the test standards described in paragraph (d)(2)
of this section.
§ 96-18 Domestic dog and cat repellents.
(a) General. This section deals with efficacy data and test procedures
for domestic dog and cat repellents. Target species are the domestic
dog and cat. Submission of efficacy data is waived for this section.
(b) Preliminary acute and subacute toxicity studies. Submit the
results of toxicity studies for each species claimed to be controlled
with a repellent. Ihese studies should include an acute oral LD50 test
and a subacute (5-day) oral LD50 test for the Norway rat. The purpose
of these studies is to assess the possibility of hazard to the target
species. ' ' .
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(1) Test standards. Use the Norway rat. Laboratory strains of this
species are acceptable. Use at least 10 adult males and 10 adult females
for each dosage level. Cage test andmals individually. Observe any changes
in behavior that may affect efficacy and note any gross pathological changes
found at post mortem (e.g., hemorrhaging, edema, ulcers, discoloring). For
detailed information on test standards, refer to §§ 81-1 and 82-1 in Subdivi-
sion F of this document.
(2) Test information. Indicate the strain and origin of test
animals. Describe acclimation of animals, laboratory conditions (temperature,
humidity, light, cage size), and gross pathological changes found at post
mortem. Separate the mortality data by sex for each dosage level. Submit
a dose response curve for each sex with computation of the slope and LD50
with 95% confidence limits. For detailed information on test information,
refer to §§ 81-1 and 82-1 in Subdivision F of this document.
(3) Acceptable protocols. For conducting toxicity tests, refer to
§§ 81-1 and 82-1 in Subdivision F of this document. For presenting a dose-
response curve, consult reference 5 in § 96-25.
(c) Advanced acute and subacute toxicity studies. If either test con-
ducted as required in paragraph (b) of this section yields and LD50 of 1000
mg/kg or less, conduct the tests using the target species. The purpose of
these studies is to assess the hazard to the target species.
(1) Test standards. Use the target species. For each sex, use at
least six animals for each dosage level. Cage test animals individually.
Place animals in large enough cages to provide freedom of movement.
[See § 96-17 (b)(2).] Stabilize diet, and general condition of animals for
at least 28 days prior to testing. Observe any gross pathological changes
found at post mortem (e.g., hemorrhaging, edema, ulcers, discoloring). For
detailed test standards, consult a standard reference on the subject. (See
reference 9 in § 96-25.)
(2) Test information. Indicate the species, breed, and origin
of test animals. Describe acclimation of animals, laboratory conditions,
(e.g., temperature, humidity, light, cage size), and gross pathological
changes found at post mortem. Separate mortality data for each sex at each
dosage level. Submit a dose response curve with computation of the slope
and LD50 with 95% confidence limits. (See reference 5 in § 96-25).
(d) Pen efficacy studies. Submit the results of a laboratory efficacy
test for each formulation and target species claimed to be controlled. The
purpose of these studies is to assess the pen efficacy of the formulation.
(1) Test standards. (i) A separate test protocol should be used for
each type of behavior claimed to be reduced (e.g., chewing and tearing
garbage bags; feeding from garbage cans; and urinating, defecating,
walking, and lying in specified areas).
(ii) Use the target species. Use at least 8 test animals. An equal
ratio of males and females is recommended except when testing the claim
to reduce urination on established scen,t posts by dogs, in which case
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only males should be used. Test animals that have had previous repellent
experience may be used.
(iii) The tester must define repellency in quantitative terms. An
example of an acceptable definition is: the apparent percent reduction of
a specific behavior that is the result of the treatment but not as the
ratio between observations on the untreated object versus treated objects.
(iv) Animals should be aclimated prior to testing. Extensive pre-
treatment data collection should be made to establish the baseline behavior
against which to evaluate the treatment.
(v) The following factors should be taken into consideration in designing
an experimental protocol: pens, test animals, test days, and treatments.
(vi) Only one formulation and treatment level can be evaluated during
each test. Each test should be replicated.
(vii) Two breeds of the target species should be tested. When dogs are
the target species, a complete test should be conducted with at least 2 breeds.
At least one breed should be from one of the following categories of breeds
designated by the American Kennel Club: hound, sporting dog, toy, terrier,
working dog, or non-sporting dog. Mongrels may be considered a separate
"breed".
(viii) Females in estrous or proestrous may be housed a minimum of
400 m (437 yd) from the test pens, or be housed indoors.
(ix) When cats are the target species, a complete test should be conducted
on at least 2 breeds. The alley cat (mixed breed) can be considered one breed.
(x) The protocol should be designed so that no significant repellent con-
tamination of the test pen remains from previous tests.
(xi) The number of days a test should be conducted will vary according
to the number of animals used. However, it probably should not exceed 7 days
per test.
(2) Suggested performance standard. Using the definition of repellency
stated in paragraph d(2)(iii) of this section, a minimum repellency of 50%
due to use of the product would be acceptable, although no mandatory standards
have been set. The statistical analysis should consist of a one-tailed
t-test to determine if the formulation's performance if significantly greater
than a set percentage (minimum of 50%).
(3) Test information. Indicate the species, breed (if known),
and source of .the test animals. Indicate the number, sex, and approximate
age of each animal. Describe pen conditions (size, spatial arrangement, and
directional or.ientation of pens). Describe the protocol ,in detail, including
but not limited to the following: procedures of test, treatment level, pen
layout with dimensions, treatment locations, and statistical design. Report
weather data (temperature, humidity, wind, speed and direc'fcion., and precipi.ta-
..tion). ..Define the nature of'the product's repellent action.; Report treat-
ment 1'evel and formulation, repellency for each tesb and breed, and complete
statistical analysis.-. ... - • :" . . • '•-.••"'
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(4) - Acceptable protocols. Refer to Exhibit 21 in § -96-3.0 for .a..pr.p-.
'tocol to"evaluate the efficacy of aerosol dog repellents that claim to
reduce damage to garbage bags. With minor modification, this method could
be used to evaluate granular products to be used around garbage bags and for
products claiming to reduce feeding in garbage cans. Also, see reference 28
in § 96-25. »
(e) Field efficacy studies. Submit the results of field efficacy tests
for each formulation, behavior listed in paragraph (d)(2)(ii) of this section,
and for each target species claimed to be controlled (repelled). In addition,
for products to be applied on or near plants, consult Subdivision J for
further data guidance on phytotoxicity. Ihe purpose of these studies is to
assess the field efficacy of the product under actual use conditions.
(1) Test standards. Use the target species. Repellency should be quan-
titatively defined. Repellency can be defined as: the apparent percent reduc-
tion of a specific behavior that is the result of treatment but not as the
ratio between observations on the untreated versus treated objects. Extensive
pre-treatment data collection should be made to establish baseline behavior
against which to evaluate post-treatment data. Ihe test should be designed
for effective statistical analysis. Tests should be conducted in 2 different
areas (e.g., rural, urban, and suburban). Separate tests should be performed
during cool (mean tempature less than 5°C) and warm weather (mean temperature
more than 20°C. Census the population level of the target species before
and after treatment. See reference 29 in § 96-25 for dog population census
methods. Studies should be conducted under actual use conditions.
(2) Suggested performance standard. While no repellency standard has
been established for field studies, at least 50% repellency due to use of
the product would be needed.
(3) Test information. For each study, indicate the formulation,
specific location, kind of site (e.g., garbage bags, garbage cans, lawns),
behavior discouraged, experimental design, test procedures, weather conditions
during study, areas (rural, urban, suburban), and other relevant information.
Report relevant behavior observed of all animals seen interacting with the
repellent. Provide a map of the test area, including directional orientation,
treatment placements, and dimensions. Provide all raw data and summary data
used to evaluate effectiveness. Describe method of calculating repellency.
Report percent repellency for each observation period and each test. Provide
complete statistical analysis.
(4) Acceptable protocols. No protocols for field evaluation of dog
and cat repellents are presently available. Prior consultation with the
Agency on proposed protocols is strongly recommended. For censusing dog
populations, consult reference 29 in § 96-25.
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§ 96-19 Browsing animal repellents.
(a) General. This section gives information on efficacy data and test
procedures for browsing animal repellents used on conifers, nursery trees,
and orchard trees. Target animals include deer, elk, and lagomorphs.
Submission of efficacy data is waived for this section.
(b) Preliminary acute and subacute toxicity studies. Submit the results
of toxicity studies for each species claimed to be controlled with a browsing
repellent. These studies include an acute oral LD50 test and a subacute
(5-day) oral test for the Norway rat. The purpose of these studies is to
assess the possibility of hazard to the target species.
(1) Test standards. Use the Norway rat. Laboratory strains of this
species are acceptable. For each sex, use at least 10 adult males and 10
females for each dosage level. Cage test animals individually. Observe any
changes in behavior that may affect efficacy, and any gross pathological
changes found at post mortem (e.g., hemorrhaging, edema, ulcers, discoloring).
For detailed information on test standards, refer to §§ 81-1 and 82-1 in
Subdivision F.
(2) Test information. Indicate the species, strain, and origin of
test animals. Describe acclimation of animals, laboratory conditions
(e.g., temperature, humidity, light, cage size), and any gross pathological
changes found at post mortem. Separate the mortality data by sex for each
dose level. Submit a dose response curve for each sex with computation
of the slope and LD50 with 95% confidence limits. (See reference 5 of
§ 96-25.) For details on test information, refer to §§ 81-1 and 82-1
in Subdivision F of this document.
(3) Acceptable protocols. For conducting the toxicity tests, refer
to §§ 81-1 and 82-1 in Subdivision F. For presenting a dose response
curve, check reference 5 in § 96-25.
(c) Advanced acute and subacute toxicity studies. If either test
conducted under paragraph (b) of this section yields an LD50 of 1000 mg/kg
or less, conduct the following tests using the target species. The purpose
of these studies is to assess the hazard to the target species.
(1) Test standards. Use the target species. Use at least 6 mature
animals of each sex per dose level. Cage or pen test animals individually.
Place animals in large enough cages or pens to provide freedom of movement.
Stablize diet and general condition of animals for at least 2 weeks prior to
testing. Observe any changes in behavior that may affect efficacy. Observe
any gross pathological changes found at post mortem (e.g., hemmorrhaging,
edema, ulcers, discoloring.). For detailed test standards, consult a standard
reference such as reference 9 in § 96-25.
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(2) Test information. Indicate the species and origin of test "•'•'
animals. Describe acclimation of animals, cage or pen conditions (e.g., tem-
perature, humidity, light, cage size), and note changes in behavior during
the test that may affect efficacy. Separate mortality data by each dosage
level. Submit a dose response curve for each sex with the computation of
the slope and LD50 with 95% confidence limits as described in reference 5 in
§ 96-25.
(d) Cage or pen efficacy tests. Submit the results of a cage or pen
efficacy test for each species claimed to be controlled. The purpose of
these tests is to assess the cage or pen efficacy of the product.
(1) Test standards. (i) Use the target species. Use at least 15
test animals, as suggested in reference 30 of § 96-25. Test animals that
have had previous repellent experience may be used.
(ii) Repellency should be quantitatively defined. Repellency can be
defined as the apparent percent reduction of a specific behavior that is
the result of treatment.
(iii) Test animals should be acclimated to test conditions for at least
2 weeks prior to testing. Pre-treatment data collection (browse damage)
should be made to establish baseline behavior against which to evaluate the
treatment.
(iv) The recommended pen size is 1 acre for lagomorphs, 2 1/2 acres
for deer, and 5 acres for elk.• The area should be enclosed with poultry
wire to reduce predation on lagomorphs. All vegetation that would ordin-
arily supply cover should be removed. Artificial cover for individual
lagomorphs should be provided throughout the enclosure.
(v) For testing deer and elk, the enclosures should contain suitable
cover for loafing, but should have little natural food.
(vi) Animals are to be fed a formulated diet as described in reference
31 of § 96-35. For testing lagomorphs, a balanced ration should be supplied.
(vii) For conifers, randomize block within animal enclosures (ten 30-
by 48-foot rectangles contaning 16 rows of 10 trees planted at 3-foot inter-
vals). Select trees in each block to reduce the row-feeding effect of penned
animals. Use an equal number of treated and untreated trees.
(viii) To test any phytotoxic characteristic of the repellent, 25
trees should be simultaneously treated and evaluated throughout a growing
season for foliar effects. For additional information of phytotoxicity
data, consult Subdivision J of this document.
(ix) Conduct 2 repellent tests, each containing 8 treatments of 100,
conifers or 30 orchard trees. Examine plots periodically until the untreated
trees show 60 to 80% animal damage. Then terminate the test. Assess damage
by noting whether or not test trees have been clipped, browsed, or pulled
out of the ground. The percentage damage for each treatment is based on
the ratio of damaged to undamaged trees for each 10-tree group per block.
Conduct an analysis of 'variance as described in reference 30 of § -96—25•
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(2) Suggested performance standard. The level of efficacy that should
be attained for an acceptable test has not been established. However, a 50%
reduction in damage due to use of the product is suggested.
(3) Test information. Indicate the species of test animals
and trees in treated and control areas. Give the source of test animals
and trees. Report the number of test animals of each sex. Indicate the
approximate age of test animals and trees. Describe pen conditions (e.g.,
size, spatial arrangement, and directional orientation of pens). Describe
the procedure of the test, treatment level, and locations. Submit weather
data (e.g., temperature, humidity, wind speed and direction, and precip-
itation) . Report the percent damage for each treatment group of 10 trees
per block, and the percent repellency for all tests. Indicate the treatment
level, formulation, and application method. Report the complete statistical
analysis (analysis of variance). Provide the results of phytotoxicity tests.
(4) Acceptable protocols. References 30 and 31 in § 96-25 and others
cited in § 96-30 may be helpful in developing an acceptable protocol incorpor-
ating the test standards described in paragraph (d)(2) of this section.
(e) Field efficacy studies. Submit the results of a field efficacy
test for each species claimed. The purpose of these studies is to assess
the field efficacy of the product under actual use conditions.
(1) Test standards. (i) Number of regional studies. For each formu-
lation, method of application, major region, type of, tree, and species claimed
or implied to be controlled on the label, submit the results of at least 5
acceptable field effiacy studies. Two of these studies should be conducted
during winter, and at least 2 should be conducted during early spring.
(ii) Repellency. Repellency should be quantitatively defined. Repellency
can be defined as: the apparent percent reduction of a specific behavior
that is the result of the treatment.
(iii) Site selection. For conifers, conduct the test on a reforested
site where seedlings are under 2 years of age. For orchard and nursery
trees, conduct the test on sites that are experiencing animal damage.
(iv) Pre-treatment and post-treatment population censusing. Extensive
pre-treatment data collection should be made to establish baseline damage *•'•
calculations against which to evaluate post-treatment data (i.e., the amount
of tree damage before and after treatment). Lay out a series of line tran-
sects at least 1 mile (1.6 km) in length, and establish plots in treated and
untreated areas as described in reference 32 in § 96-25. Also, refer to
Field census techniques in § 96-30.
(v) Damage evaluation. Assess damage by noting whether or not test
trees have been clipped, browsed, or pulled out of the ground. The percent-
age damage for each treatment is based on the ratio of damaged to undamaged
trees for each transect. Use appropriate statistical analysis. Report
the percent repellency for all tests. Report the results of phytotoxicity
tes ts.
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(2) Suggested performance standard.' The level of efficacy .that-should
be attained for.an acceptable test has not been-established. However, a 50%
reduction in damage due to the use of the product is suggested.
(3) Test information. Indicate the target species and its relative
abundance in the treated and untreated areas. Report the percentage of
repellency attained for each formulation. Describe weather conditions during
the study. Report the method of calculating repellency. Compare seedling
browsing damage in treated and untreated areas. Provide a map of the test
area, including directional orientation and teatment placements. Report
relevant behavior of all animals seen interacting with the repellent. Provide
all raw and summary data used to evaluate effectiveness, and a complete stat-
istical analysis.
(4) Acceptable protocols. Reference 32 in § 96-25 and others cited
in § 96-30 may be helpful in developing an acceptable protocol incorporat-
ing the test standards described in paragraph (e)(2) of this section.
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§ 96-25 References for §§ 96-2 through -19.
1. Stephan, C.E., 1975. Chairman, Committee on Methods for Toxicity
Tests with Aquatic Organisms. Methods for Acute Toxicity with Fish, Macro-
invertebrates, and Amphibians. U.S. Environ. Prot. Agency, Ecol. Res. Sec.
EPA 660/-3-75-009. 61 pp.
2. Marking, L.L. 1975. lexicological Protocol for the Development of
Pesticides. Pp. 26-31 in Rehabilitation of Fish Populations with Toxicants:
A Symposium. P.H. Eschmeyer, ed. North Central Div., Amer. Fish Soc. Spec.
Pub. No. 4.
3. Marking, L.L., and C.R. Walker. 1973. The use of fish bi.oassays
to determine the rate of deactivation of pesticides. Pp. 357-366 in
Bioassay Techniques and Enviornmental Chemistry. Ann Arbor Science Pub.,
Inc.: Ann Arbor, Mich.
4. Marking, L.L. and V.K. Dawson. 1972. The half life of biological
activity of antimycin determined by fish bioassay. Trans. Amer. Fish Soc.
101(1):100-105.
5. Litchfield, J.T., Jr., and F.A. Wilcoxon. 1949. A simplified
method of evaluating dose-effects experiments. J. Pharmacol. Exp. Therap.
96:99-113.
6. Cherry, D.S., K.L. Dickson, and H. Cairns, Jr. 1975. Temperature
selected and avoided by fish at various acclimation temperatures. J. Fish
Res. Bd. Can. 32:485-491.
7. Hansen, D.J., S. Matthews, S.L. Nail, and D.P. Dumas. 1972.
Avoidance of pesticides by untrained mosquitofish, Gambusia affinis. Bull.
Environ. Contarn. Toxicol. 8(1):46-51.
8. University Federation for Animal Welfare, The. 1967. The UFAW
Handbook on the Care and Management of Laboratory Animals. Chapters 50 -
Reptiles, 51 - Frogs and Toads, 52 - Newts and Salamanders. 3rd Ed.
1015 pp. E. & S. Livingston: Edinburgh.
9. Anon. 1975. Standard Recommended Practice for Determining Acute
Oral LD50 for Testing Vertebrate Control Agents. Annual Book of ASTM
Standards, Part 46. Designation: E555-75. Amer. Soc. for Testing and
Materials: Phila., Pa.
10. Draize, J.H. 1965. Appraisal of the safety of chemicals in food,
drugs, and cosmetics - Dermal Toxicity. Assoc. of Food and Drug Officials
of the U.S., Topeka, Kansas. Pp. 46-59.
11. Heath, R..G., and L.F. Stickel. 1965. Protocol for Testing the
Acute and Relative Toxicity of Pesticides .on Fish and Wildlife. U.S. Dept.
Int., Fish and Wildl. Serv. Circ. 226.
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338
12. Anon. 1975. Standard Method of Test for Efficacy of Acute"
Avicides. Annual Book of ASTM Standards, Part 46. Designation: E551-75.
Araer. Soc. Testing and Materials: Phila, Pa.
13. Bontoyan, W.R., ed. 1975. Manual of Chemical Methods for
Pesticides and Devices. U.S. Environ. Prot. Agency: 401 M Street, S.W.,
Washington, D.C.
14. Anon. 1975. Official Methods of Analysis of the Association of
Official Analytical Chemists. 12th Ed. Assoc. Offic. Anal. Chenu Washington,
D.C.
15. Starr, R.I., J.F. Besser, and R.B. Brunton. 1964. A laboratory
method for evaluating chemicals as bird repellents. J. Agr. Food Chem.
12:342-344.
16. Anon. 1975. Standard Method of. Test for Efficacy of Acute
Mammalian Predacides. Annual Book of ASTM Standards, Part 46. Designation:
E552-75. Amer. Soc. for Testing and Materials: Phila., Pa.
17. Leftwich, B.H. 1972. Population dynamics and behavior of the
eastern mole, Scalopus aquaticus machrinoides. Ph.D. Thesis, Univ. Mo.
(unpubl.) 103 pp. (Dissertation Abstr. Order No. 73-21, 435).
18. Fuhr, i. and S.D. Silver. 1947. Simulated burrow systems for
studies with rodent pests. J. Wildl. Manage. 11(2):150-153.
19. University Federation of Animal Welfare, The. .1967. The UFAW
Handbook on the Care and Management of Laboratory Animals. Chapter 31 -
Bats. 3rd ed. 1015 pp. E. & S. Livingston: Edinburgh.
20. Overton, W.S., and D.E. Davis. 1969. Estimating numbers of
animals in wildlife populations. Pp. 403-455 in Wildlife Management
Techniques by R.H. Giles. Wildlife Society, Wash., D.C.
21. Horsfall, F., Jr. 1956. Pine mouse control with ground-sprayed
endrin. Proc. Amer. Soc. Hort. Sci. 67:67-74.
22. Byers, R.E., and R.S. Young. 1975. Pine vole control with
anticoagulant baits. J. Amer. Soc. Hort. Sci. 100(6):691-694.
23. Byers, R.E. 1976. New compound (RH 787) for use in control of
orchard voles. J. Wildl. Manage. 40(1):169-171.
24. Sargeant, A.B., and B.R. Peterson. 1964. Pocket gopher control
in Minnesota with the mechanical burrow builder. U.S. Bur. Sport. Fish
& Wildl., Branch of Predator & Rodent Control in coop, with Minn. Dept.
Agr., St. Paul, Minn. 19 pp.
25. Passof, P.C., R.E. Marsh, and W.E. Howard. 1964. Alpha-
naphylthiourea as a conditioning repellent for protecting conifer seed.
Proc. Sixth Vert. PestConf., Anaheim, Calif. 6:280-292.
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26. Dimock, E.J., II. 1957. A Comparison of Two Rodent Repellents
in Broadcast Seeding Douglas-fir. Pac. NW For. & Range Exp. Sta. Res.
Paper No. 20. 17 pp.
27. Anon. 1976. 9CFR Chapter 1. Subchapter A - Animal Welfare,
Part 3, Subpart A, Sect. 3.4. (Feb.)
28. Heubner, R.A., and J.D. Morton. 1964. An evaluation of the
efficacy of commercial canine repellents. Vet. Med. Small Animal din.
59(10): 1016-1020.
29. Beck, A.M. 1973. The Ecology of Stray Dogs —A study of. Free-
Ranging Urban Animals. York Press: Baltimore.
30. Dodge, W.E., C.M. Loveless, and N.B. Kverno. 1967. Design and
analysis of forest-mammal repellent test. For. Sci 13(3):333-336.
31. Wood, A.J., H.C. Nordan, and I. McT. Cowan. 1961. The care
and management of wild ungulates for experimental purposes. J. Wildl.
Manage. 25:295-305.
32. Besser, J.F., and F.F. Welch. 1959. Control of mammal damage to
plants by chemical repellents. Trans. N. Amer. Wildl. Conf. 24:166-173.
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§ 96-30 Methods and protocols.
(a) Scope. This section represents an appraisal of current methodology
for evaluating the efficacy of vertebrate control agents. The classes of
agents scrutinized are toxicants, repellents (including aversive conditioners),
reproductive inhibitors, anesthetizing chemicals, and devices. The mode of
action of the control agents could not be examined on an agent-by-agent basis,
especially with respect to the chemical senses. The vertebrate species in-
clude fish, amphibians, reptiles, birds and mammals. The text is arranged
in phylogenetic order, with any necessary introductory explanation about
the methods preceeding each target species.
The appraisal primarily evaluates, or references, methods that have been
tested and published in journals and technical bulletins; some information
was derived from unpublished reports and manuscripts. The fact that only
limited research has been conducted to test many vertebrate control agents
accounts for the paucity of citations in some areas. Also, control agents
for some species, such as bears, have not been extensively studied because
the damage caused by the animal to human property is limited.
Most target groups lack well documented, sound experimental protocols
at this time. A great deal more research is needed in some areas before
complete acceptable testing protocols can be defined.
I. Fish toxicants, repellents, and reproductive inhibitors.
A review of the needs to control fish and possible physical, biolog-
ical, and chemical controls has been published by Lennon (1970). More
specific reviews on fish toxicants and the information necessary for
registration of a product have also been published (Lennon et al., 1970;
Schnick, 1972, Marking, 1975; and Lennon and Walker, 1964).
References.
Lennon, R.E. 1970. Fishes in pest situations. Pp. 6-41 in Vertebrate
Pests: Problems and Control, Vol. 5. Univ. Calif.: Davis.
Lennon, R.E., and C.R. Walker. 1964. Laboratories and Methods for
Screening Fish Control Chemicals. U.S. Dept. Int., Bur. Sport Fish.
and Wildl. Invest, in Fish Control. Report No. 1.
Lennon, R.E., J.B. Hunn, R.A. Schnick, and R.M. Burress. 1970. Recla-
mation of Ponds, Lakes and Streams with-Fish Toxicants: A Review.
FAO Fish. Tech. Paper No. 100.
Marking, L. 1975. Toxicological Protocol for the Development of Pesticides.
Pp. 26-31 in Rehabilitation of Fish Population with Toxicants: a Symposium.
P.H. Eschmeyer, ed. North Central Div., Amer. Fish Soc. Pub. No. 4.
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Schnick, R.A. 1972. A Review of Literature on TFM (3-trifluoromethyl-4-
nitrophenol) as a Lamprey Larvicide. U.S. Dept. Int., Bur. Sport Fish.
and Wildl. Invest, in Fish Control No. 44, 31 pp.
Stephen, C.E., Chairman, Committee on Methods for Toxicity Tests with
Aquatic Organisms, 1975. Methods for Acute Toxicity Tests with Fish,
Macroinvertebrates and Amphibians. U.S. EPA Ecol. Res. Sec. EPA
660/3-75-009. 61 pp.
A. Fish toxicants.
. - Laboratory studies permit the.establishment of baseline data on a
product under controlled conditions. Possible synergistic and/or antag-
onistic action between the ingredients in the product can be evaluated
without the variables expected in field conditions.
Guidelines for aquatic toxicity testing are published jointly by the
American Public Health Association, the American Water Works Associations,
and the Water Pollution Control Federation (1976). These guidelines have
been carefully evaluated and revised frequently over the past 50 years.
Several detailed test methods have been published in Stephan et al. (1975).
Deviations from these methods should not be made unless there is considerable
reason to do so. Until fairly recently, the static toxicity test was the
only technique considered, but now flow-through toxicity tests are the
preferred method. The latter method affords less difficulty in maintaining
an adequate supply of dissolved oxygen, permitting only low metabolite
accumulation for the test animals, and greater ability to maintain constant
toxicant concentrations and constant water temperatures (Sprague, 1969;
Sinley, 1973a). Continuous-flow bioassays require more equipment, but
trouble-free equipment has been designed (Mount and Brungs, 1967; Sprague,
1969; Sinley, 1973b). A labor-saving jar rinser, a jar emptier, and an
automatic liquid measuring vessel have been described for static bioassay
(Hesselberg and Burress, 1967).
The referenced methods suggest -using .the receiving water as the diluent,
but in preliminary testing of a piscicide this requirement would not be
necessary. However, the researcher should consider the effect of water
quality on fish health and on the toxicity of the piscicide being tested
so that misleading results will not be obtained (Doudoroff, 1957; Doudoroff
and Katz, 1950 and 1953; Hoar and Randall, 1971; Applegate and King, 1962;
Slonim and Slonim, 1973; Murphy, 1970). Factors that should be considered
in laboratory testing are listed below.
1. Because temperature will have an important effect, bioassays
should be conducted at several different temperatures (Macek et al., 1969).
2. All fish for a given trial should be. obtained from the same source.
Fish from .the same source should have been exposed to the same environmental.
conditions (.pesticide levels, water quality, temperature, etc.) and similar
handling procedures. Species or s.trains of'fish known to be pesticide-
resistant should not be used (Dziuk and Plapp, 197.3; Ferguson and Bingham,
1966). , . . . .
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3. Although all life stages of the fish should'be tested eventually
(Olson and Marking, 1973; Garrison, 1968), only fish of nearly equal size
should be tested in a given trial (Murphy, 1971).
4. Because mud will alter the toxicity of the compound in field trials
(Ferguson et al., 1965), it may be desirable to conduct some tests in con-
tainers with varying amounts of mud (Loeb and Stackey, 1966).
5. In addition to testing the toxicity to the target fish, the effect
on other vertebrates (Brooks and Price, 1961; MacPhee and Ruelle, 1969b),
on representative invertebrates (Schoettger and Olive, 1961) and plants
should also be determined.
6. The rate of deactivation of the piscicide should be determined
(Marking and Walker, 1973; Marking and Dawson, 1972; Lee et al., 1971;
and Loeb and Engstrom-Heg, 1971).
Suggested reviews of toxicity tests on specific piscicides include
Berger et al., 1969; Herr et al., 1967; Haag, 1931; and MacPhee and
Ruelle, 1969b.
In acute toxicity test research, results require special statistical
treatment. Probit analysis is commonly used (Finney, 1947; and Litchfield
and Wilcoxon, 1949), but a discussion of other statistical analyses has
been written by Finney (1964). Rachlin and Perlmutter (1968) present an
approach for establishing maximum permissible concentrations.
Some of the testing being required in this portion of the Product
Performance Subdivision is required in Subdivisions D, E, and F, which
should be reviewed for additional information. For instance, tests
designed to evaluate the basic efficacy of the active ingredients in the
laboratory can be used to determine the toxicity classification of the
pesticide. The "References" paragraph in this section lists several
excellent publications that provide information as to test methods and
data requirements (Stephen et al., 1975; Standard Methods, 1976; Lennon
and Walker, 1964).
Field studies.
Protocols for field evaluations have been developed by the U.S. Fish
and Wildlife Service Fish Control Laboratories (Gumming, 1972). In addi-
tion, Lennon and Berger (1970) and Gilderhaus et al., (1969) have summar-
ized field testing conducted to gain registration of a particular piscicide.
Exhibit I of this section is a "Proposed Recommended Practice for
Determining Efficacy of Fish Toxicants in Static Freshwater Field Trials".
It was prepared by the ASTM E-35.18 task group.dealing with aquatic verte-
brate pest control products. This group, composed of U.S. Dept. of Int.
Fish and Wildlife Service and EPA personnel, and other interested indivi-
duals, is working through the ASTM E-35 Committee on Pesticides to develop
a consensus method for evaluating aquatic vertebrate pest control agents.
This draft will undergo some further revision before becoming a standard.
However, it is being included in this section as a guide to registrants
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343
and Agency registration personnel because it is the only known practice
that discusses the subject in detail. Equipment for adding chemicals
and monitoring the effect has been described by several researchers
(Anderson, 1962; Price and Haas, 1963; Ziebell, 1966; Richard, 1962).
A major problem in field applications is the effect that different
water qualities have on the toxicity. Howell and Marquette (1962) sug-
gest that a test should first be conducted on a sample of each body of
water before adding the toxicant to the treatment area; other researchers
have added the toxicant on the basis of laboratory studies and obtained
erratic results (Huish, 1961; Mayhew, 1960; Callahan and Huish,.1968).
Other problems in field applications are discussed in. publications by•
Gushing and Olive (1956), Torblaa (1968), Kiser et al., (1963), Hughes and
Lee (1973), Koeppe (1961), Bonn and Holbert (1961), and Cohen et al. (1960).'
Because such a variety of organisms are involved in field applications,
samples may have to be preserved for later analysis at perhaps several lab-
oratories. Cope (1960) suggests methods for collection. Problems of homoge-
nization of samples have been discussed by Benville (1970) and Daniels et al.
(1965). Problems with extraction of pesticides are indicated by Hesselberg
and Johnson (1972), Hughes et al., (1970), and Benevue and Beckman (1966).
References.
American Public Health Association, American Water Works Association, and
Water Pollution Control Federation. 1976. Standard Methods for the
Examination of Water and Wastewater. 14th Ed. American Public Health
Assoc., Washington, D.C. 1193 pp.
Anderson, G.A. 1962. Three portable feeders for metering chemical into
streams for control of sea lamprey. Prog. Fish-Cult. 24(4):190-192.
Applegate, V.C., and E.L. King. 1962. Comparative toxicity of 3-tri-
fluoromethyl-4-nitrophenol (TFM) to larval lampreys and eleven species
of fishes. Trans. Am. Fish. Soc. 91 (4):342-345.
Bennvile, P.E., Jr. 1970. Dry ice homogenization procedure for fish
samples in pesticide residue analysis. J. Agric. Food Chem. 18(5):
948-949.
Benvenue, A., and H. Beckman. 1966. The examination of toxaphene by
gas chromatography. Bull. Environ. Contam. Toxicol. 9(1):1-5.
Berger, B.L., R.E. Lennon, and J.W. Hogan. 1969. Laboratory Studies on
Antimycin A as a Fish Toxicant. Bur. Sport Fish and Wildl. Invest, in
Fish Control 26. 21 pp.
Bonn, E.W., and L.R. Holbert. 1961. Some effects of rotenone products
on municipal water supplies. Trans. Am. Fish. Soc. .90'(3): 287-297.
Brooks, I.e..and R.W. price:. .1961.. Studies on the chronic toxicity of
Pro-Noxf.ish, -a.' propr.ia.tory synergized .rotenone fish toxicant,. Toxicol..
-Appl.. Pharmacol. 3-U ):.49-.56.
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344
Callahan, M.A. and M.T. Huish. 1968. An evaluation of'Antimyc.in as-a---', .:
selective bluegi11 toxicant under varying conditions of pH. Proc.
Southeast. Assoc. Game and Fish Comm.: 476-481.
Cohen, J.M., L.J. Kamphade, A.E. Lemke, C. Henderson, and R.L. Woodward.
1960. Effects of fish poisons on water supplies. Part 1. Removal
of toxic materials. J. Am. Water Works Assoc. 52:1551-1566.
Cope, O.B. 1960. Collection and preservation of fish and other materials
exposed to pesticides. Prog. Fish-Cult. 22(3):103-108.
Gumming, K.B. 1972. Protocol for a Field Trial of Antimycin and Thanite
to Control Carp and to Detect Long-term Effects on Biota in Max McGraw
Wildlife Foundation Ponds on October 16-17, 1972. U.S. Dept. Int. Fish
Control Laboratory. 20 pp. (Mimeo).
Gushing, C.E., and J.R. Olive. 1956. Effects of toxaphene and rotenone
upon macroscopic bottom fauna of two northern Colorado reservoirs.
Trans. Am. Fish. Soc. 86:294-301.
Daniels, S.L., L.L. Kempe, T.J. Billy, and A.M. Beeton. 1965. Detection
and Measurement of Organic Lampricide Residues. Great Lakes Fishery
Comm. Tech. Rep. No. 9. 18 pp.
Doudoroff, P. 1957. Water quality requirements of fishes and effects of
toxic substances, pp. 404-430 in The Physiology of Fishes. W. E. Brown,
ed. Academic Press. Inc.: New York.
Doudoroff, P., and M. Katz. 1950. Critical review of literature on the
toxicity of industrial wastes and their components to fish. I. Alka-
lies, acids, and inorganic gases. Sewage Ind. Wastes 22(11):1432-1458.
. 1953. Critical review of literature on the toxic-
ity of industrial wastes and their components to fish. II. The metals,
as salts. Sewage Ind. Wastes 25(7);802-839.
Doudoroff, P., B.C. Anderson, G.E. Burdick, P.S. Galtsoff, W.B. Hart,
R. Patrick, E.R. Strong, E.W. Surber, and W.M. Van Horn. 1951.
Bioassay methods for the evaluation of acute toxicity of industrial
wastes to fish. Sewage Ind. Wastes 23:1380-1397.
Dziuk, L.J., and F.W. Plapp. 1973. Insecticide resistance in mosquito-
fish from Texas. Bull Environ. Contarn. Toxicol. 9(1):15-19.
Ferguson, D.E., and C.R. Bingham. 1966. The effects of combinations of
insecticides on susceptible and resistant mosquitofish. Bull. Environ.
Contain. Toxicol. 1 (3 ): 97-1 03.
Ferguson, D.E., J.L. Ludke, J.P. Wood, and J.W. Prather/. 1965. The
effects of mud on the bioactivity of pesticides on fishes. Miss. Acad.
Sci. 11:219-228.
Finney, D.J. 1964. Statistical Methods in Biological-Assay. 2nd: Ed.
• Hafner Publ. Co.: New York. 668 pp,.
image:
345
Garrison, R.L. 1968. The toxicity of Pro-Noxfish to salmonid eggs and
fry. Prog. Fish-Cult. 30(1):35-38.
Gilderhus, P.A., B.L. Berger, and R.E. Lennon. 1969. Field Trials of
Antimycin A as a Fish Toxicant. Bur. Sport Fish, and Wildl. Invest.
in Fish Control 27. 21 pp.
Green, R.H. 1965. Estimation of tolerance over an indefinite time
period. Ecology 46(6):882.
Haag, H.B. 1931. Toxicological studies of Derris elliptica and its
constituents. J. Pharmacol. Exp. Therap. 43(1):193-208.
Herr, F., E. Greselin, and C. Chappel. 1967. Toxicology studies of
Antimycin, a fish eradicant. Trans. Am. Fish. Soc. 96(3);320-326.
Hesselberg, R.J., and J.L. Johnson. 1972. Column extraction of pesti-
cides from fish, fish food, and mud. Bull. Environ. Contam. Toxicol.
7(2/3):115-120.
Hesselberg, R.J., and R.M. Burress. 1967. Investigations in Fish Con-
trol 21. Labor Saving Devices for Bioassay Laboratories. Bur. Sport
Fish, and Wildl. Resource Publ. 38. 8 pp.
Hoar, W.S., and D.J. Randall, eds. 1971. Fish Physiology, Volume VI,
Environmental Relations and Behavior. Academic Press, Inc.; New York
559 pp.
Howell, H., and W.M. Marquette. 1962. Use of Mobile Bioassay Equipment
in the Chemical Control of Sea Lamprey. U.S. Fish and Wildl. Serv.,
Spec. Sci. Rep.-Fish. No. 418. 9 pp.
Hughes, R.A., G.D. Veith, and G.F. Lee. 1970. Gas chromatographic analysis
of toxaphene in natural waters, fish and lake sediments. Water Res. 4(8):
547-558.
Hughes, R.A., and G.F. Lee. 1973. Toxaphene accumulation in fish in lakes
treated for rough fish control. Environ. Sci. Teehnol. 7:934-939.
Huish, M.T. 1961. Toxaphene as a fish eradicant in Florida. Proc. South-
east. Assoc. Game and Fish Comm. Pp. 200-205.
Riser, R.W., J.R. Donaldson, and P.R. Olson. 1963. The effect of
rotenone on zooplankton populations in freshwater lakes. Trans. Am.
Fish. Soc. 92(1):17-24.
Koeppe, R. 1961. The toxicology and toxicity of toxaphene with respect _.
to fish and aquatic food animals.. Z. Fisch. Hilfswiss. 9:771-794.
Lee, T.H., P.H. Derse, and S.D. Morton. 1971. Effects of. physical and
chemical conditions on the detoxification of Antimycin. Trans. Am.
- Fish. Soc. 100(1):13-17. . ~"
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346
Lennon, R.E., and B.L. Berger. 1970. A Resume on Field Applications of
Antimycin A to Control Fish. Bur. Sport Fish, and Wildl. Invest, in
Fish Control No. 40. 19 pp.
Lennon, R.E., and C.R. Walker. 1964. Laboratories and Methods for
Screening Fish Control Chemicals. U.S. Dept. Int., Bur. Sport Fish.
and Wildl. Invest, in Fish Control. Report No. 1.
Litchfield, J.T., Jr., and T. Wilcoxon. 1949. A simplified method of
evaluating dose-effect experiments. J. Pharmacol. Exper. Therap.
96(2):99-113.
Loeb, H.A., and R. Engstrom-Heg. 1971. Estimation of rotenone concen-
tration by bioassay. N.Y. Fish and Game J. 18(2):129-134.
Loeb, H.A., and R.J. Stackey. 1966. Survival of buried bullheads sub-
jected to 4'-iodo-3-nitrosalicylanilide. N.Y. Fish and Game J. 13(2):
196-205.
Macek, K.J., C. Hutchinson, and O.B. Cope. 1969. The effects of tem-
perature on the susceptibility of bluegills and rainbow trout to
selected pesticides. Bull. Environ. Contarn. Toxicol. 4(3):174-183.
MacPhee, C., and R. Ruelle. 1969a. A chemical selectively lethal to
squawf ish (Ptychocheilus oregonensis and _P_. umpquae). Trans. Am.
Fish. Soc. 98(40):676-684.
• 1969b. Lethal Effects of 1888 Chemicals
upon Four Species of Fish from Western North America. Univ. of Idaho
Forest, Wildl. and Range Exp. Sta. Bull. No. 3. 112pp.
Marking, L.L., and C.R. Walker. 1973. The use of fish bioassays to
determine the rate of deactivation of pesticides. Pp. 357-366 in
Bioassay Techniques and Environmental Chemistry. Ann Arbor science
Publishers, Inc.: Ann Arbor, Mich.
Marking, L.L., and V.K. Dawson. 1972. The half life of biological
activity of antimycin determined by fish bioassay. Trans. Am. Fish.
Soc. 101(1 ):100-105.
Mayhew, J. 1960. The use of toxaphene as a fish poison in strip mine
ponds with varying physical and chemical characteristics. Proc. Iowa
Acad. Sci. 66:513-517.
Mount, D.I., and W.A. Brungs. 1967. A simplified dosing apparatus for
fish toxicology studies. Water Res. 1:21-27.
Murphy, P.G. 1970. Effects of salinity on uptake of DDT, DDE, and ODD
by fish. Bull. Environ. Contain. Toxicol. 5(5):404-407.
. 1971. The effect of size on the uptake of DDT
from water by fish. Bull. Environ. Contain. Toxicol. 6.(1):20-23.
image:
347
Olson, L.E., and L.L. Marking. 1973. Toxicity of TFM (lampricide) to
six early life stages of rainbow trout (Salmo gairdneri). J. Fish.
Res. Board Can. 30(8):1047-1052.
Price, R.W., and J.B. Haus. 1963. Aids for stream reclamation. Prog.
Fish-Cult. 25(1):37-39.
Proceedings of Seminar on Methodology for Monitoring the Marine Environ-
ment. Environmental Monitoring Series, EPA, R & D Office 600/4-74-004,
Washington, D.C., NTIS # PB-239-052.
Rachlin, J.W., and A. Perlmutter. 1968. Fish cells.in culture for study"
of aquatic toxicants. Water Res. 2(6):409-414.
Richard, J.B. 1962. Tests on thermocline penetration by several rotenone
products. Prog. Fish-Cult. 24(4):177-181.
Schoettger, R.A., and J.R. Olive. 1961. Accumulation of toxaphene by
fish-food organisms. Limnol. Oceangr. 6(2):216-218.
Sinley, J.R. 1973a. Determining a TL50 for a Pollutant. Colo. Div.
Wild. Fish information Leaflet No. 24. 3 pp.
. 1973b. Constructing a Two-liter Proportional
Diluter. Colo. Div. Wildl. Fish Information Leaflet No. 25. 4 pp.
Slonim, C.B., and A.R. Slonim. 1973. Effect of water hardness on the
tolerance of the guppy to beryllium sulfate. Bull. Environ. Contain.
Toxicol. 10(5):295-301.
Sprague, J.B. 1969. Measurement of pollutant toxicity to fish - 1.
Bioassay methods for acute toxicity. Water Res. 3:793-821.
Stephan, C.E., Chairman, Committee on Methods for Toxicity Tests with
Aquatic Organisms. 1975. Methods for Acute Toxicity Tests with Fish,
Macroinvertebrates, and Amphibians. U.S. E.P.A., Ecol. Res. Ser.
under EPA. 660/3-75-009, 61 pp.
Torblaa, R.L. 1968. Effects of Lamprey Larvicides on Invertebrates in
Streams. U.S. Fish and Wildl. Spec. Sci. Rept-Fish. No. 572. 13 pp.
Ziebell, C.D. 1966. A floating field laboratory for estuarine fish
toxicity studies. . Prog. Fish-Cult. 28(3):180-182.
B. Fish repellents.
Basic toxicity of the repellent to target and nontarget species can
be determined using methods described above for piscicides such as static
jar and flow-through techniques (Stephan et al. 1975). There is little
information 'published on fish repellents concerning individual and group
behavior, and rate, duration, and significance of .response. There is
even less information available on. standa-ras-. and. protocols .for
'fish.repellents:* •-..... .-.-..'•.. -••.--.-. . l^-..-•.-••"•-.".• ... . '= '
image:
348
Summerfelt and Lewis (1967) published a summary .on fish repellents.
Their paper, along with one by Hansen et al. (1972), described^apparatus
for laboratory testing of repellents. Four other papers describe a mix-
ture of laboratory and field testing of repellents to migrating salmon
(Brett and MacKinnon, 1954; Alderdice et al., 1954; Idler et al., 1956;
Idler et al., 1961), but none of these constitute a protocol for testing
repellents. The-alternative is to adapt the protocols developed for
piscicides (Cherry et 'al..,- 1975). ......
References. . •. " - - •
Alderdice, D.F., J.R. Brett, D.R. Idler, and U. Fagerlund. 1954. Further
•. observations on olfactory perception in migrating adult echo and spring
• salmon. -Properties of the repellent in mammalian skin. Fish.. Res.
Board Can. Prog. Rept. Pacific Coast.Sta. No. 98. pp. '10-12.'
Brett, J.R., and.b. MacKinnon.- 1954. Some aspects of olfactory per-
ception in migrating adult coho and spring salmon. J. Fish. Res.
Board Can. 11; 310-318. ' " . ; ' • "-'';~
. Cherry,'- D.S./' K'.L. bickson, and J. Cairns. 1975. Temperature selected
and avoided by fish at various acclimation:temperatures. J. Fish Res.
Board Can. 32:485-491. •" ; . ' ...
Hansen, D.J., E. Mathews, S'.L.: Nail,. arid P.P..-.Dumas. 1972. Avoidance
— of pesticides, by untrained mbsquitofish, Gambusia affinis. Bull.
Environ. Con tarn. Toxicol. 8(1 ): 46-51;,' '. -
Idler,—D.R., H.M. Fagerlund,- arid'H. Mayoh. 1956. Olfactory perception
'• in migrating'salmon. I. L-Serine, .a salmon repellent in mammalian
skin. J. Gen. Physiol. 39:889-892. "... .:• •.:".••"'•'"•'
,Idler/-D.R;', J.R. McBride, R.E.E. Jonas, and V. Tomlinson. 1961. Olfactory
perception in migrating salmon. II. Studies on a laboratory bioassay for
homestream water, and mammalian repellent. . Ca'n. J.' Biochem. Physiol.
39:1575-1584; '•- *'''•' ' ... ." '".".-.•'•• ", ..-:—':•":"" '-"'
Summerfelt, R.C., and W.M. Lewis. 1967'. '.Repulsion of gree'n sunfish by
certain chemicals. J. Water Pollut. Control Fed. 39(12):2030-2038.
- C. Fish reproductive inhibitors.
No publications pertaining directly to fish Reproductive inhibitors
have been identified. Basic information on the. reproductive physiology
of fishes is available in 2 publications (Hoar and Randall, 1969; and
Schreck, 1974). Although the research to control sex in fish discussed by
Schreck was not very successful, such, control would inhibit reproduction.
References. ..'.-. ' .
Hoar, W.S., and D.J. Randall, eds. 1969. Fish Physiology. Vol. Ill,
Reproduction and Growth, 3i.oluminescence, Pigments,, and Poisons.
Academic Press, Inc..-..New York. 485/pp.
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349
Schreck, C.B., ed. 1974. Control of Sex in Fishes. Ext. Div., Va.
Poly. Inst. and State Univ. 106 pp.
EXHIBIT 1 - Proposed recommended practice for determining efficacy
of fish toxicants in static freshwater field trials
1. Scope.
1.1 This recommended practice is to be used in testing the efficacy
of fish toxicants in actual use conditions according to the intended use
pattern. Field trials should be conducted only after laboratory tests
for efficiacy and safety have been performed. Local authorities must be
contacted and proper permission obtained before a field trial should be
conducted. s
2. Choice of trial site.
2.1 The trial site should be chosen to expedite the objectives of
the test. Testing of a particular agent must be done in at least three
widely separated geographical areas. Sites should be chosen to permit
evaluation of biological, chemical, and physical variables on the efficacy
of the fish toxicant.
2.2 Trial sites should be restricted to those in which public access
can be completely controlled and in which results can be easily evaluated.
Ponds which can be completely drained are preferred. All trial sites
should have physical characteristics which will permit effective means of
fish population sampling with electrofishing gear and nets, if the site
cannot be drained.
2.3 Precautions should be taken to insure the isolation of the trial
site from immigration by outside species of fish. Natural or other existing
barriers should be used in preference to block nets or similar temporary
barriers.
3. Site characteristics.
3.1 Field trials of this kind should not be conducted where popula-
tions of endangered species exist. Consult State Fish and Game agencies
for specific information.
3.2 An accurate map of the surface outline of the lakes or ponds
used should be obtained from existing sources or constructed by appropriate
methods (1).
3.3 The volume of lakes shall be computed by the contour method (1).
The volume of ponds may be computed by the contour method or.by multiplying
the surface area by the mean depth. The flow rate, if any,, through the
impoundment must be determined. The frequency of seasonal turnover should
be documented. .
3.4 The percent of bottom composed .of. different .soil,types should ., •
be estimated (2). - -
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350
3.5 The immediate watershed and tributaries affecting the trial site
should be identified.
4. Water characteristics.
4.1 Chemical characteristics. General chemical analyses of water
should be conducted within 14 days of treatments. These analyses should
include those for pH, dissolved oxygen, carbon dioxide, total alkalinity,
total hardness, and any other ion or molecule suspected of influencing the
activity of the toxicant under study. A sample of water taken at the time
of treatment should be saved for more detailed analysis to help define the
cause of any highly aberrant results in the trial. The pH should be measured
near the surface, at mid-depth, and near the bottom. Dissolved oxygen should
be measured at appropriate depth intervals to define the zones which have
adequate oxygen for fish (5 mg/1 or more), marginal oxygen for fish (less
than 5 mg/1), and anaerobic zones (concentration of oxygen lower than the
accuracy of the method of analysis). Oxygen concentration and pH should be
monitored until the desired activity of the chemical is complete. Monitoring
of pH should be continued during any longer studies on the-degradation of
the toxicant. Chemical analyses are to be done according to Standard methods
(3) with the exceptions that pH and dissolved oxygen may be measured with
electronic meters. The methods should be accurate to within 0.2 units for
pH and 1.0 mg/1 for oxygen.
4.2 Physical characteristics. Pretreatment measurements should include
temperature, transparency (as indicated by Secchi disk), color (visual), and
conductivity. Temperature should be recorded to nearest 1°C at appropriate
intervals of depth to document the gradient from surface to bottom and define
the bounds of a thermocline if present. Conductivity should be measured at
the surface, corrected to 25°C. Water temperatures should be monitored
throughout the period of the trial.
4.3 Pollution. Sources of pollution in the trial site waters must be
documented and must be avoided if possible, unless they are included in the
practice objectives.
5. Target fish species.
5.1 Field trials of this kind must not*be conducted where populations
of endangered species exist. Consult local game and fish authorities for
specific information and permission. .
5.2 The existing fish population must be assessed prior to treatment
by appropriate methods (4) and (5) in terms of species, relative numbers,
and sizes. Sampling methods generally include electrofishing, gill netting,
fyke netting, and seining. The sampling before any trial should be adequate
to define the most resistant target fish present. The most resistant target
fish is usually one criterion by which the concentration of chemical is
chosen. For purposes of assessing results of a trial, one or more of the
following methods may be used:
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351
(1) Comparison of catch per unit of effort before and after treatment
by one or more types of gear in selected locations.
(2) Marking and restocking of fish captured from the existing popula-
tion and comparing number recovered after treatment with total number marked.
(3) Stocking known numbers of new fish which are either marked or of a
species not existing in the body of water, and comparing numbers recovered
after treatment with numbers stocked. Use of some caged fish can be a val-
uable addition to any of these methods.
5.3 General toxicants. The fish sampling for a trial of a general
toxicant should be much more intensive after treatment than before because
it is difficult to capture fish if only a few remain alive.
5.4 Selective toxicants. The sampling effort will generally be more
intensive in a trial where a selective kill by species or size is desired
than in one where a total kill is desired. The trial of a selective toxicant
should include adequate sampling to determine the percentage of the population
consisting of the target fish before and after treatment.
5.5. Safe water check. Generally it is desirable to determine when
the chemical has been degraded, inactivated, or diluted to nontoxic levels
so the water may be restocked with fish or declared safe for other uses.
Fish are caged at selected depths to determine when the entire water column
is nontoxic. Small bluegills, rainbow trout, fathead minnows, or the most
sensitive native fish in the area are used in these tests.
Ten or more specimens are caged at each depth. Care must be taken to
assure gradual acclimation of the fish to the test conditions. Survival
of 70-80% of the test specimens in each cage for 24 to 96 hours (depending
on water temperature and the activity of the chemical) generally is suffi-
cient evidence that the water is safe for fish.
6. Nontarget organisms.
6.1 Gross visual observations of acute effects on nontarget organisms
should be made during all periods of observation. Generally, to satisfy
requirements for•registration, at least one field trial of a toxicant must
include a detailed study of the effects on aquatic invertebrates.
6.2 Trials to determine effects on nontarget organisms must be conduct-
ed in the same manner as those used to determine efficacy to target organisms.
In most cases, these two trials can be conducted simultaneously in similar
areas on both target and nontarget animals.
.6.3 The evaluation of effects of a toxicant on aquatic invertebrates
will vary according to the nature of the toxicant, the anticipated effects,
and the types of organisms present. Evaluation studies should be designed
to define:
image:
352
(1) the significant acute effect occurring within two weeks after
treatment,
(2) the rate of recovery of the population in the event that there
are signicant effects, and
(3) long-term effects such as disappearance of major species or group.
The samples to define (1) (above) should be made within 14 days of treatment
and at about 1, 7 and 14 days after treatment. Sampling to define (2) and
(3) (above) should be started during the earliest time of normal population
peaks after treatment.
The peaks occur in the spring and in the fall. Sampling in mid-April,
mid-May, mid-June, and again in mid-September and mid-October should give
valid samples of most major organisms included in northern climes. The
schedule should be modified to accomodate the population cycles in southern
areas. As an example, a trial conducted in late October would include pre-
treatment and post-treatment sampling of 1, 7, and 14 days from the treatment
date. If significant changes in number or diversity occur, the next sampling
would be done in mid-April, mid-May, and mid-June of the following year. If
the latter samples show sustained or recovered populations on the basis of
numbers of individuals, numbers of taxa, and species diversity, further
sampling would not be necessary. If necessary, sampling should be continued
until 1 year after treatment, in this case to include mid-September and mid-
October of that year.
6.3.1 Plankton should be sampled and analyzed using (1) and (7) as
guidelines. Any modifications.of these methods must be documented. The
same methods must be used for all samples taken in a single study. Samples
should be taken in representative areas of the body of water. In shallow
or unstratified impoundments, samples should be taken subsurface and near
the bottom. In stratified lakes, additional samples should be taken just
above and below the thermocline. A minimum of 20 liters of water should be
filtered for each sample. A diversity index (7), based on lowest possible
taxon, shall be used as one of the parameters by which to compare samples
from different time intervals.
These peaks occur in the spring and in the fall. Sampling in mid-April,
mid-June, and again in mid-September and mid-October should give valid
samples of most major organisms included in northern climes. The schedule
should be modified to accommodate the populations cycles in southern areas.
As an example, a trial conducted in late October would include pre-treatment
and post-treatment sampling of 1, 7 and 14 days from the treatment date. If
significant changes in number or diversity occur, the next sampling would
be done in mid-April, mid-May, and mid-June of the following year. If the
latter samples showed sustained or recovered populations on the basis of
numbers of individuals, numbers of taxa, and species diversity, further
sampling would not be necessary. If necessary, sampling should be continued
until 1 year after treatment, in this case to include mid-September or mid-
October of that year. Primary productivity and periphyton techniques should
be used in a field test involving a.toxicant that may be persistent, hazard-
ous, or both, to nontarget organisms.
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353
6.3.2 Benthic invertebrates should be sampled on the same schedule as
plankton. The preferred samplers are Petersen or Ponar dredges. Sampling
and analysis of samples should be done using (1 ) and (7) as guidelines, and
the methodology should remain constant during any one study. Enough samples
should be taken from different substrates and depths to permit statistical
comparisons between sampling days. Pooled samples used for computation of
species diversity should contain a minimum of 100 organisms. Diversity index
(7), based on the lowest possible taxon, shall be used as one comparison
between sampling periods.
6.4 Visual observations should be made for any effect of the toxicant
on amphibians, reptiles, birds, and mammals inhabiting the immediate area of
the trial site.
7. Application of toxicant.
7.1 Approximate range of concentration of the toxicant for a particular
field trial will be determined from previously obtained laboratory data on
the activity of the chemical in relation to water quality and species of fish
present. Ideally, a series of two or three similar ponds should be treated
at different concentrations to closely bracket the correct concentrations
for that particular set of environmental variables and fish present, if
there are unusual water qualities or species of fish present, or if there
is considerable doubt as to effective concentrations, the proper concentra-
tion should be determined in field bioasssays by the methods of Burress (6)
or a comparable method. Field bioassays are usually needed to determine
the lowest effective and the highest safe concentration for a selective
treatment. All field testing should be preceded by laboratory testing.
7.2 Compute the amount of chemical needed based on the concentration
indicated by the field bioassay and the volume of water in the target area.
The concentration of toxicant in the water at the test site should be report-
ed based on active ingredient and also on total formulation.
7.3 The formulation of the toxicant used in field trials should be the
identical formulation intended for registration. Data on one formulation is
not usually applicable to registration of another similar formulation.
7.4 Special care should be taken to get the best possible horizontal
and vertical distribution of the toxicant in the water column.
8. Equipment.
8.1 The equipment used to apply the toxicant should be well suited to
the particular formulation. A complete list of equipment needed for the
various phases of the trial shpuld be prepared in advance. Include all items
of personal, sampling, application, safety, and bioassay gear.
8.2 A complete documentation of equipment used in each phase of the
field trial should be reported. This report should include, if. possible,
evidence of calibration and standardization consistent with ASTM Committee
E-35.22.
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354
9. Evaluation of results.
9.1 The remaining fish populati6n may be evaluated in several ways.
The most reliable method for small ponds is drainage of the entire basin,
which virtually assures the capture of all remaining fish. Another reliable
method is to treat the water again using a toxicant whose efficacy is well
established. Enough time should be allowed between the treatments so the
fish killed by the second cannot be confused with those killed by the orig-
inal treatment. Methods for partial sampling of the remaining fish popula-
tion are outlined in section 5.2.
9.2 Criteria of efficacy. Failure to capture live fish with extensive
sampling effort is reasonable evidence (but never absolute assurance) of a
complete kill. The post-treatment sampling should include two types of
sampling gear. Refer to sections 5.2f 5.3 and 5.4 for sampling gear, methods
of assessment, and sampling effort. A trial site which has not been drained
or retreated with a known toxicant should be periodically observed for a year
for evidence of remaining target fish.
10. Reporting. ,
10.1 Reports of field trials should include all pertinent data and
detailed descriptions of methods in a format which permits ease of review.
References. ' . •
1. Welch, P.S. 1948. Limnological Methods. McGraw-Hill Book Company:
New York, N.Y. 381 pp. .
2. . 1952. Limnology. McGraw-Hill Book Company,
New York, N. Y. 538 pp.
3. Standard Methods for the Examination of Water and Wastewater. 1971.
American Public Health Association, New York, N. Y.
4. Lagler, K.F. 1956. Freshwater Fishery Biology. Win. C. Brown Company:
Dubuqu e, Iowa.
5. Rounsefell, G.A., and Everhart, W.H. 1953. Fishery Science. John
Wiley and Sons, Inc.: New York, N. Y. 444 pp.
6. Burress, R.M. 1975. Resume of On-Site Bioassay Experiments. U.S.
Dept. Int., Bur. Sport Fish, and Wildl. Invest, in Fish Control
No. 68. 8 pp.
7. Weber, C.I. 1973. Biological Field and Laboratory Methods for
Measuring the Quality of Surface Waters and Effluents. National
Environmental Research Center; Office of Research and Development,
U.S. Environmental Protection Agency, Cincinnati, Ohio.
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355
II. Amphibian toxicants and repellents.
Many repellents and toxicants for amphibians could be tested following
the methods used for fish, since in most cases problems caused by them are
associated with fish production. Tadpoles, for example, are no problem
where there are bass present in ponds, but in hatchery situations they may
multiply rapidly and interfere with the growth of desirable fish (Helms,
1967). Terrestrial amphibians have been documented as creating problems
for bee keepers (Eckert, 1934).
Laboratory studies^
It is necessary to know the complete life history of the particular
amphibian which one wishes to control, since the laboratory studies.would
vary according to the phase of the life cycle the toxicant or repellent is
to control. Testing to determine LC50 value at 24, 48, and 72 hr. (Helms,
1967) was done in 15-gal aquaria. Three to 5 test animals were placed in
each aquarium and then the test chemical was added. A sixth aquarium was
maintained as a control. Bullfrog and leopard frog tadpoles and a few
species of toad tadpoles were tested. Seven species of fish were also
tested under identical conditions.
Field studies.
After testing in aquaria, Helms conducted field studies. The method-
ology varied depending on the life history of the organism. In the case
of tadpoles, 9 treatments were carried out in 6 different ponds. Following
treatment, the tadpole numbers were checked by seining to determine the
completeness of kill and whether nontarget species were affected.
References.
Anonymous. 1974. The Bufo plague. Time 104(6):42.
Cherry, D.S., K.L. Dickson, and J. Cairns, Jr. 1975. Temperatures
selected and avoided by fish at various acclimation temperatures.
J. Fish Res. Board Can. 32:485-491.
Eckert, J.E. 1934. The California toad in relation to the hive bee.
Copeia 2:92-93
Pitzwater, W. D. 1974. Reptiles and amphibians—a management dilemma.
Proc. Sixth Vert. Pest. Conf., Anaheim, Calif. :178-183.
Hansen, D.J., E. Mathews, S.L. Nail, and D.P. Dumas. 1972. Avoidance
of pesticides by untrained mosquitofish, Gambusia affinis. Bull.
Environ. Contam. Toxicol. 8(1):46-51.
Helms, D.R. 1967. Use of formalin for selective control :of tadpoles
in the presence of fishes. Prog. Fish-Cult. 29(1):43-47.
image:
356
Hutchens, L.H., and R.C. Nord. 1955. Fish Culture Manual. U.S. Fish
and Wildlife Service. 220 pp. (processed).
Kaplan, H. M., and J. G. Overpeck. 1964. Toxicity of halogenated
hydrocarbon insecticides for the frog, Rana pipiens. Herpetologica
20(3):163-169.
Klussmann, W.G., M.A. Champ, and J.T. Lock. 1969. Utilization of an-
hydrous ammonia in fisheries management. Proc. S.E. Assoc. Game and
Fish Conun. 23:512-519.
Laycock, G. 1966. The Alien Animals. The Natural History Press:
Garden City, N.Y. 240 pp.
Lennon, R.E. 1970. Control of freshwater fish with chemicals. Proc.
Fourth Vert. Pest Conf., West Sacramento, Calif. 4:129-137.
Mulla, M.S. 1962. Frog and toad control with insecticides. Pest
Control 30(10):20-64.
Noble, G.K. 1931. The Biology of the Amphibia. McGraw-Hill Book Co.:
New York, N.Y. 577 pp.
Sanders, H.O. 1970. Pesticide toxicities to-tadpoles of the western
chorus frog (Pseudacris triseriata) and Fowler's toad (Bufo fowleri).
Copeia 2:246-251.
Stephan, C.E. Chairman, Committee on Methods for Toxicity Tests with
Aquatic Organisms. 1975. Methods for Acute Toxicity Tests with Fish,
Macroinvertebrates and Amphibians. U.S. E.P.A. Ecol. Res. Ser. EPA
660/3-75-009. 61 pp.
A. Terrestrial amphibian and reptile toxicants and repellents.
Lizards seldom need control. Turtles can be controlled by trapping,
fishing, or shooting (DilJLar^t, ^1973). The hunting or taking of alligators
is regulated by Federal andj^tate law. Poisonous snakes in various geogra-
phic areas may cause human "death or injury aria therefore may be identified
as pests. Many snakes are beneficial or neutral because they consume rodents
and insects... However, some "reptiles may require control.
Toxicants. No test'methods for reptile toxicants were identified for review.
Repellents. Some of the literature points out that repellents may be accep-
table control measures. Cowles and Phelan (1958) have shown that snakes are
very sensitive to chemical stimuli. Crawford and Gross (1969) have shown
that snakes show heart and respiration rate responses to chemical stimuli.
Porter and Czaplicki (1974) have shown that both water snakes and garter
snakes are sensitive to chemical cues produced by themselves or other species.
The authors conducted a series of experiments to test the attractiveness or
the repellency of snakes or others of the same species or other species. A
method for screening potential snake repellents has been developed by Jenkins
(1962).
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357
References.
Brock, E.M., and W.E. Howard. 1962. Control methods for snakes. Proc.
First Vert. Pest. Conf., Sacramento, Calif. 1:18-31.
Conant, R. 1958. A Field Guide to Reptiles and Amphibians. Houghton
Mifflin Co.: Boston. 366 pp.
Cowles, R.B., and R.L. Phelan. 1958. Olfaction in rattlesnakes. Copeia
2:77-83.
Crawford, T., and G.W. Gross. 1969. Conditioning of heart and respira-
tion rate in snakes to a chemical stimulus. Proc. 12th Ann. Mtg. of
the Soc. for the Study of Amphibians and Reptiles.
Dillard, J.G. 1973. Turtle control. Missouri Cons. 34{6):18-19.
Fitzwater, W.D. 1974. Reptiles and amphibians—a management dilemma.
Proc. Sixth vert. Pest Conf., Anaheim, Calif. 6:178-183.
Jenkins, J.H. 1962. Snake repellent. Patent No. 3,069,314. U.S.
Patent Office, U.S. Dept. Commerce, Washington, D.C.
Porter, R.H., and J.A. Czaplicki. 1974. Responses of water snakes
(Matrix £. rhombifera) and garter snakes (Thamnophis sirtalis), to
chemical cues. Animal Learning and Behavior 2(2):129-132.
Rudd, R.L., and R.E. Genelly. 1956. Pesticides: Their Use and Toxicity
in Relation to Wildlife. Game Bull. No. 7. Calif. Dept. of Fish
and Game. 209 pp.
Stickel, W.H. 1953. Control of Snakes. U.S. Bur. Sport Fish and wildl.
Leafl. No. 345.
Swaroop, S., and B. Grab. 1954. Snakebite mortality in the world. Bull.
W.H.O. 10(D:35-76.
Uhler, F.M. 1949. Facts About Snakes. U.S. Bur. Sport Fish and Wildl.
Leafl. No. 257.
Watkins, J.F., F.R. Gehlbach, and J.C. Kroll. 1969. Attractant-repellent
secretions of blind snakes (Leptotyphlops dulcis) and their army ant prey
(Neivamyrmex nigrescens). Ecology 50:1098-1102.
B. (Reserved).
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358
.III. Avian toxicants, repellents and reproductive inhibitors and
anesthetizincr chemicals.
No specific laboratory or field test protocols exist for the efficacy
evaluation of avian toxicants, repellents, reproductive inhibitors and
anesthetizing chemicals. Publications by Lee (1970), Ochs (1972), and
Schafer and Guarino (1970) provide brief descriptions of, and the rationale
behind, some tests. Since many of the laboratory tests necessary to estab-
lish efficacy are described in detail in Subdivisions E and F of these
guidelines, this discussion will elaborate on laboratory tests only when
it is necessary to provide additional information relative to avian control.
The primary emphasis will be directed toward efficacy determinations in
the field.
Discussions relative to laboratory studies for toxicants, repellents,
reproductive inhibitors and anesthetizing chemicals have been combined
under one heading ("laboratory methods"), since a majority of the labora-
tory tests must be conducted on all chemicals irrespective of the proposed
use. References to special studies or special emphasis relating to individ-
ual proposed uses are made in the text.
Discussions relative to field studies are divided into four major
groups, since evaluation procedures in the field vary considerably. Field
test protocols for bird control agents are still in the developmental stages.
Since many of the referenced tests were conducted before the most recent
legislative action on FIFRA, as amended, most of them are lacking in one or
more of the studies; now required. A number of areas can and should be
strengthened with regard to' all tests:
1. Better knowledge of cultural practices and bird populations in
test areas,-
2. Use of larger experimental units;
3. Strict adherence to treatment schedules;
4. Use of controls during the same test period;
5. Use of more replications per test;
6. Proper application of statistical techniques; and
7. More adequate appraisal of hazards to nontarget species.
It must also be recognized that the field evaluation of efficacy of any
bird control agent depends on conducting the test in the proper area at the
proper time. Birds and biologists are not always able to mesh their activi-
ties in well controlled or ideal test situations; therefore, a proposed
protocol must be adaptable to existing test situations.
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359
References.
Lee, J.O. 1970. Outlook for rodenticides and avicides registration.
Proc. Fourth Vert. Pest Conf., West Sacramento, Calif. 4:5-8.
Ochs, P. 1972. Efficacy Testing of vertebrate pest control agents.
Proc. Fifth Vert. Pest Conf., Fresno, Calif. 5:138-141.
Schafer, E.W., Jr., and J.L. Guarino. 1970. Problems in developing
new chemicals for bird control. Proc. Bird Control Seminar, Bowling
Green, Ohio 5:7-10.
A. Avian toxicants, repellents, reproductive inhibitors and anesthe-
tizing chemicals; Laboratory methods.
Acute toxi ci ty.
The various methods for determining acute oral and dermal toxicity in
birds are described in this Appendix; however, since control agents are
seldom developed for use on the test species recommended in Part VII (i.e.,
ducks and quail), it will be necessary to conduct LDgQ evaluations on the
target species and on selected nontarget species which may be affected by
the proposed control agent. The moving point interpolation method described
by Thompson (1947), Thompson and Weil (1952), and Weil (1952) is particularly
appropriate for this purpose. Descriptions of the use of this method for
wild birds are provided by DeCino et al. (1966), Schafer (1972), Schafer
et al. (1973), and Tucker and Crabtree (1970).
Subacute toxicity. : '
The exact technique used to determine subacute toxicity will depend on
the proposed application of the control agent. Tests should be conducted
on target and selected nontarget species. For proposed applications result-
ing in short-term exposure, the 5-day dietary UCe,0 test protocol developed
by Heath and Stickel (1965) .as modified by Hill (1972) may suffice. Since
many applications of control agents (i.e., repellents) may result in longer
exposure, longer term tests should be conducted to approximate the maximum
period of exposure. The 7- to 40- day test procedures described by Schafer
and Marking (1974), Schafer et al. (1975), and Tucker and Crabtree (1970)
may suffice for most applications. It may, however, be necessary to conduct
studies for as long as 90 days; in this case the methodology described by
Hayes (1967) may be acceptable. Again, the size of test populations will
necessarily vary with the availability of the test species.
Inhalation toxicity.
Data on inhalation toxicity will be.necessary on the target .species
and on representative nontarget species if the proposed use of the control.
agent results in respirable gas, fumes, aerosols, or dusts. Because there
are no published.records of laboratory tests using wild .aviah species, modi-
fied mammalian, tests, should be used.. -... ••• .
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360
Secondary hazards. ...... -•
Since the use of avian control agents in urban and suburban areas
presents a potential hazard to carnivorous avian and mammalian predators,
indicative data related to the proposed use should be gathered to define the
degree of hazard to canines, felines, or mustelids, at least one species of
hawk, and one species of scavenger bird. Schafer et al. (1974a) describes
some test methods. Because of the nature of the tests and availability of
suitable test animals, small test populations will be acceptable unless
hazards are encountered. If hazards are found, more extensive testing may
be required.
Repellents.
The repellency (or acceptance) of a compound can be determined by a
number of different techniques. The initial evaluation may utilize the
individual response-no choice method described by Star et al. (1964) and
modified by Schafer and Brunton (1971). Although this method usually over-
estimates the amount of chemical needed to produce a repellent response under
field conditions, it does provide data on the maximum treatment levels need-
ed to produce protection. To determine comparative repellency (or accept-
ance) of a chemical in individual birds and groups the methodology described
by Hill (1972), Luckwill and Weaver (1965), McLean (1972), or Ridsdale and
Granett (1969) may be acceptable. Although indications of the repellency
(or acceptance) of chemicals can also be obtained when the chemical is in-
corporated into drinking water (Duncan 1963), tests on foods are preferred
unless water is the item to be protected.
Reproductive inhibitors. • > '•• . ."..''• . . " , .-•'.'
Few laboratory methods have been published on wild avian reproduction.
Since most wild birds are difficult to breed in captivity, domesticated
species have generally been used. The application of a potential reproduc-
tive inhibitor to fpod is the principal treatment method, assuming the pro-
posed application uses oral ingestion. Methododology for determining efficacy
in domesticated species is described by Elder (1964), Powell (1966), and
Wentworth (1970) for chronic feeding; Jones et al. (1972) for acute treatment
of males; and Wentworth et al. (1968) for the treatment of eggs.
Anesthetizing Chemicals
Laboratory methods for testing a chemical's effectiveness as an anesthe-
tizing chemical are described by Cline and Greenwood (1972), Peek (1966),
Schafer et al. (1967), and Schafer and Cunningham (1972).
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361
References.
Anon. 1975a. Standard Guidelines for Use and Development of Strychnine
as an Avicide. Annual Book of ASTM Standards, Part 46. Designation:
E554-75. Amer. Soc. for Testing and Materials, Phila., Pa.
. I975b. Standard Method of Test for Efficacy of Acute
Avicides. Annual Book of ASTM Standards, Part 46. Designation:
E551-75. Amer. Soc. for Testing and Material. Phila. Pa.
. 1975c. Standard Recommended Practice for Determining
Acute Oral LDgQ for Testing Vertebrate Control Agents. -Annual Book of
ASTM Standards, Part 46. Designation: E555-75. Amer. Soc. for Testing
and Materials, Phila., Pa.
Cline, D.R., and R.J. Greenwood. 1972. Effect of certain anesthetic
agents on mallard ducks. J. Am. Vet. Med. Assoc. 161:624-633.
Crase, F.T., and R.W. Dehaven. 1977. A cage test for the evaluation of
house finch repellents for grapes. Pp. 173-176 in Test Methods for
Vertebrate Pest Control Management Materials, Vol. 1, STP 625. W. B.
Jackson and R.E. Marsh, eds., Amer. Soc. for Testing and Materials.,
Phila., Pa.
DeCino, T.J., D.J. Cunningham, and E.W. Schafer. 1966. Toxicity of
DRC-1339 to starlings. J. Wildl. Manage. 30(2):249-253.
Duncan, D.J. 1963. The response of the feral .pigeon when offered the
active ingredients of commercial repellents in solution. Ann. App.
Biol. 51:127-134.
Elder, W.H. 1964. Chemical inhibitors of ovulation in pigeons. J. Wild.
Manage. 28:556-575.
Hayes, W.J. 1967. The 90-dose LD50 and a chronic factor as measures of
toxicity. Toxicol. App. Pharmacol. 11:327-335.
Heath, R.G., and L.F. Stickel. 1965. Protocol for testing the acute and
relative toxicity of pesticide to penned birds. Pp. 18-24 in The Effects
of Pesticides on Fish and Wildlife. U.S. Dept. Int., Fish and Wildl.
Serv., Circ. 226.
Hill, E.F. 1972. Avoidance of lethal dietary concentrations of insect-
icide by house sparrows. J. Wildl. Manage. 36(2):635-639.
Ingram, C.R. 1977. Efficacy Experiments Involving Avian Pest Control
Materials: Design Considerations Imposed by the Scientific Method.
Pp. 215-224 in Test Methods for Vertebrate Pest Control and Management
Materials., Vol. 1, STP 625. W.B. Jackson and R.E. Marsh, eds. Amer.
Soc. for Testing and Materials, Phila., Pa.
Jones, P.., E».-Kominkova, and H. Jackson. .1972..- .Effects of an.tif.er/tili-ty.- - •
•-substances on male Japanese quail. • J. .Reprod.-. FertlLt .29;71-78. . .....
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362
Lee, J.O. 1970. Outlook for rodenticides and avicides registration.••••
Proc. Fourth Vert. Pest Conf., West Sacramento, Calif. 4:5-8.
Luckwill, L.C., and F.S. Weaver. 1965. Techniques for the study of bird
repellents. Ann. Rpt. Long Ashton Res. Sta., Great Britain. (pp. 64-72).
McLean, R.G. 1972. Acceptance of apholate-treated bait by pigeons. Amer.
Mid. Nat. 87:527-530.
Ochs, P. 1972. Efficacy testing of vertebrate pest control agents. Proc.
Fifth vert. Pest Conf., Fresno, Calif. 5:138-141.
Peek, J.M. 1966. Chlordiazepoxide and Pentobarbital as tranquilizers
for cowbirds and coturnix quail. J. Am. Vet. Med. Assoc. 149:950-952.
Powell, J.E. 1966. The effects of 20,25-diazacholestanol diHCl (SC-12937)
on fecundity of Japanese quail and parakeets. MS Thesis. Univ. of Mass.,
Amherst. 181 pp.
Ridsdale, R., and P. Granett. 1969. Responses of caged grackles to chemi-
cally treated and untreated foods. J. Wildl. Manage. 33:678-681.
Schafer, E.W. 1972. The acute oral toxicity of 369 pharmaceutical, pesti-
cidal, and other compounds to wild birds. Toxicol. Appl. Pharmacol.
22:315-330.
Schafer, E.W., Jr., and R.B. Brunton. 1971. Chemicals as bird repellents:
Two promising agents. J. Wildl. Manage., 35:569-572.
Schafer, E.W., Jr., R.B. Brunton, and N.F. Lockyer. 1974a. Secondary
hazards to animals feeding on red-winged blackbirds killed with
4-aminopyridine baits. J. Wildl. Manage. 38(3):424-426.
Schafer, E.W., Jr., R.B. Brunton, N.F. Lockyer, and D.J. Cunningham.
1974b. The chronic toxicity of methiocarb to grackles, doves, and
quail, and reproductive effects on quail. Bull. Environ. Contain.
Toxicol. 14:541-647.
Schafer, E.W., Jr., R.B. Brunton, N.F. Lockyer, and JoW. DeGrazio.
1973. Comparative toxicity of seventeen pesticides to the quelea,
house sparrow, and red-winged blackbird. Toxicol. Appl. Pharmacol.
26:154-157.
Schafer, E.W., Jr., and D.J. Cunningham. 1972. An Evaluation of 148
Compounds as Avian Immobilizing Agents. U.S. Fish and Wildl. Serv.,
U.S. Dept. Int., Spec. Sci. Rep.-Wildlife No. 150. 30 pp.
Schafer, E.W., Jr., and J.L. Guarino. 1970. Problems in developing
new chemicals for bird control. Proc. Bird Control Seminar, Bowl-
ing Green, Ohio 5:7-10.
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363
Schafer, E.W.., Jr., and L.L. Marking. 1974. The susceptibility of birds
and fresh water fish to repeated or continual exposure to 4-aminopyridine.
U.S. Dept. Int., Fish and Wildl. Serv., Wildl. Res. Center, Denver, Col-
orado. 9 pp. i
Schafer, E.W., Jr., R.I. Starr, D.J. Cunningham, and T.J. DeCino. 1967.
Substituted phenyl N-methylcarbamates as temporary immobilizing agents for
birds. J. Agr. Food Chem. 15:287-289.
Starr, R.I., J.F. Besser, and R.B. Brunton. 1964. A laboratory method for
evaluating chemicals as bird repellents. J. Agr. Food Chem. 12:342-344.
Thompson, W.R. 1947. Use of moving averages and interpolation to estimate
median effective dose. I. Fundamental formulas, estimation of error, and
relation to other methods. Bacteriol. Rev. 11:115-145.
Thompson, W.R., and C.S. Weil. 1952. On the construction of tables for
moving average interpolation. Biometrics 8:51-54.
Tucker, R.K., and D.G. Crabtree. 1970. Handbook of Toxicity of Pesticides
to Wildlife. U.S. Dept. Int., Fish s Wildl. Serv. Res. Pub. No. 84.
Weil, C.S. 1952. Tables for convenient calculation of median effective dose
(LD50 or ED50) and instructions in their use. Biometrics 8:249-263.
Wentworth, B.C. 1970. Sterility and reproductive inhibition of Japanese
quail.induced by Mestranol ingestion. J. Wildl. Manage. 34:1477-1484.
Wentworth, B.C., B.C. Hendricks, and T.J. Sturdivant. 1968. Sterility
induced in Japanese quail by spray treatment of eggs with Mestranol.
J. Wildl. Manage. 32:879-887.
B. Avian toxicants - Field methods.
There are numerous published methods that can be used to appraise the
efficacy of oral bird toxicants under different application situations.
Although the methodology described covers basic needs to determine efficacy,
more effort needs to be expended with regard to nontarget species.
Acceptable test methodology for determining the efficacy of dermal tox-
icants applied as sprays is readily available: Jackson and Park (1973);
Lefebvre and Seubert (1970); Marsh (1964); and U.S. Fish and Wildlife Service
(1973). Although these methods may be used for spray applications, they are
deficient in information regarding hazards to non-target species. A single
test for contact toxicants is available (Schafer et al., 1969); again, addi-
tional data on hazard to non-target species may need to be provided.
A single published test is available for determining the efficacy of
inhalation toxicants (Devore et al., 1966).
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364
References.
Anon. 1975a. Standard Guidelines for Use and Development of Strychnine as
an Avicide. Annual Book of ASTM Standards, Part 46. Designation: E554-75.
Amer. Soc. for Testing and Materials, Phila., Pa.
. 1975b. Standard Method of Test for Efficacy of
Acute Avicides. Annual Book of ASTM Standards, Part 46. Designation:
E551-75. Amer. Soc. for Testing and Materials, Phila., Pa.
. 1975c. Standard Recommended Practice for Determining
Acute Oral LDgg for Testing Vertebrate Control Agents. Annual Book of
ASTM Standards, Part 46. Designation: E555-75. Amer. Soc. for Testing
and Materials, Phila., Pa.
Besser, J.F., W.C. Royall, Jr., and J.W. De Grazio. 1967. Baiting starlings
with DRC-1339 at a cattle feedlot. J. Wildl. Manage. 31(1):48-51.
Crunden, C.W., A. Zajanc, J. Keck, D. Graves, and J. DeHoop. 1965. Test at
McDougal's Livestock Company Feedlot, Solano County. Pp. 39-47 in Progress
Report on Starling Control. C.C. Seibe, ed. Univ. of Calif.: Davis.
Devore, A.L., D.W. Maxson, J.L. Albright, and R.W. Taylor. 1966. The Use
of Anhydrous Ammonia for the Control of Birds in Free Stall Housing.
Purdue Univ. Res. Prog. Rept. 260. 2 pp.
Graham, F. 1957. Control of native hens in Tasmania. Tasm. J. Agric.
25:368. ' - , . .
Guarino, J.L., and E.W. Schafer, Jr. 1967. Magpie Reduction in an Urban
Roost. U.S. Dept. Int. Fish and Wildl. Serv. Spec. Sci. Rept. - Wildl.
No. 104. 5 pp.
Jackson, J.J., and P.O. Park. 1973. The effects of fenthion on a nesting
population of quelea during experimental control by aerial spraying.
Proc. Bird Control Seminar, Bowling Green, Ohio 6:53-73.
Larsen, K.H., and J.H. Dietrich. 1970. Reduction of a raven population of
lambing grounds with DRC-1339. J. Wildl. Manage. 34(1):200-204.
Lefebvre, P.W., and J.L. Seubert. 1970. Surfactants as blackbird stressing
agents. Proc. Fourth Vert. Pest Conf., West Sacramento, Calif. 4:156-161.
Marsh, R.E. 1964. Blackbird control trials. Pp. 51-58 in Progress Report
on Starling Control. C.F. Kelly, ed. Univ. of Calif.: Davis.
Royall, W.C., Jr., T.J. DeCino, and J.F. Besser. 1967. Reduction of a
starling population at a turkey farm. Poult. Sci. 46(6):1494-1495.
Savidge, I.R. 1974. A model for management predictions of territorial
bird populations. Ohio J. Sci. 74(5):301-312.
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365
Schafer, E.W., Jr., R.R. West, and D.J. Cunningham. 1969. New starling
toxicant: DRC-1347. Pest Control 37(9):22,24,30.
Snyder, D.B. 1961. Strychnine as a potential control for red-winged
blackbirds. J. Wildl. Manage. 25(1):96-99.
U.S. Fish and Wildlife Service. 1973. Instructions for Use of PA-14 Avian
Stressing Agent (effective 1 Jan. 1974 to 31 Dec. 1974). U.S. Dept. Int.,
Fish & Wildl. Serv., Patuxent Research Center, Laurel, Maryland. 6 pp.
(Mimeo).
C. Avian repellents; Field methods.
The determination of the field efficacy of avian repellents is based
on 3 main factors—changes in behavior, reduction to bird numbers, and a
reduction in damage. There are 2 major types of chemical repellents-gusta-
tory and tactile; each requires somewhat different evaluation procedures.
The gustatory repellents (those acting through some physiological
mechanism during or after oral ingestion) have been the most thoroughly
investigated, and appraisals exist for sprouting agricultural seeds
(Besser, 1973; Frank et al., 1970; Guarino and Forbes, 1970; Herman and
Kolbe, 1971; Ingram et al., 1973; Stickley, and Guarino, 1972; West and
Dunks, 1969; and West et al., 1969); sprouting horticultural seeds (Abbot,
1958; Royall and Ferguson, 1962); and ripening grain (DeHaven et al.,
1973; Dolbeer et al., 1973; Guarino et al., 1973, 1974; and Stickley and
Ingram, 1973). It is particularly important that minimum plot sizes be
established according to the recommendations of Guarino (1972) and Herman
and Kolbe (1971.), and that replications be used.
Other gustatory repellents are those that induce a behavioral response
in a very small proportion of birds causing damage. Four published apprais-
als contain information: Besser et al. (1973); DeGrazio et al. (1971,
1972); and Stickley et al. (1972). Again, test plot size and replications
are of prime importance.
There are no published acceptable procedures for evaluating tactile
avian repellents.
References.
Anon. 1975a. Standard Guideline for Use and Development of Strychnine
as an' Avicide.- Annual Book of ASTM Standards, Part 46. Designation:
E554-75. Amer. Soc. for Testing and Materials, Phila., Pa.
. 1975b. Standard Method of Test for Efficacy of
Acute Avicides. Annual Book of ASTM Standards, Part 46. Designation:
E551-75. Amer. Soc. for Testing and Materials, Phila., pa.
• ., 1975c. Standard Recommended Practice for Deter-
mining Acute Oral LD'gQ .for Testing Vertebrate Control .Agents.. -Annual
Book of ASTM Standards., Part 46-. Designation: E555-75... Amer. Soc.
for Testing .and Materials, Phila., pa. . ' . .•-„
image:
366
Abbott, H.G,. 1958. Application of avian repellents to eastern 'white: pine '
seed. J. Wildl. Manage. 22(3):304-306
Besser, J.P. 1973. Protecting seeded rice from blackbirds with methiocarb.
Int. Rice Comm. Newsl. 22(3):9-14.
Besser, J.F., J.W. DeGrazio, and J.L. Guarino. 1973. Decline of a blackbird
population during seven years of baiting with a chemical frightening agent.
Proc. Bird Control Semin., Bowling Green, Ohio 6:12-14.
Bollengier, R.M., J.L. Guarino, and C.P. Stone. 1973. Aerially applied
methiocarb spray for protecting wild lowbush blueberries from birds.
Proc. Bird Control Semin., Bowling Green, Ohio 6:216-220.
DeGrazio, J.W., J.F. Besser, T.J. DeCino, J.L. Guarino, and E.W. Schafer,
Jr. 1972. Protecting ripening corn from blackbirds by broadcasting
4-aminopyridine baits. J. Wildl. Manage. 36(4):1316-1320.
DeGrazio, J.W., J.F. Besser, T.J. DeCino, J.L. Guarino, and R.I Starr.
1971. Use of 4-aminopyridine to protect ripening corn from blackbirds.
J. Wildl. Manage. 35(3) :565-569.
DeHaven, R.W.., J.L. Guarino, F.T. Crase, and E.W. Schafer, Jr. 1971.
Methiocarb for repelling blackbirds from ripening rice. Int. Rice Comm.
Newsl. 20(4)-.25-30.
Dolbeer, R.A., C.R. Ingram, and'A.R. Stickley, Jr. 1973. Bird damage to
Michigan blueberries. Proc. Bird Control Semin., Bowling Green, Ohio
6:28-40.
Duncan, D.J., E.N. Wright, and M.G. Ridpath. 1960. A review of search for
bird repellent substances in Great Britain. Ann. Epiphyties 11:206-212.
English, W.S. 1953. Preventing bird damage to fruit trees. Agriculture
60:426-429.
Frank, F.R., E.W. Schafer, Jr., and J.L. Guarino. 1970. Laboratory and
field studies with an avian repellent for sprouting seeds. Proc. Bird
Control Semin., Bowling Green, Ohio 5:86-89.
Goodhue, L.D., and F.M. Baumgartner. 1965a<. Applications of new bird
control chemicals. J. Wildl. Manage. 29(4):830-837.
Goodhue, L.D., and F.M. Baumgartner. 1965b. The Avitrol method of bird
control. Pest Control 33(7):16-17,46,48.
Griffin, D.N., and F.M. Baumgartner. 1959. Evaluation of certain chem-
icals as bird repellents. Proc. Oklahoma Acad. Sci. 39:78-82.
Guarino, J.L. 1972. Methiocarb, a chemical bird repellent: A review
of its effectiveness on crops. Proc. .Fifth vert. Pest Conf., Fresno,
Calif. 5:108-111.
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367
Guarino, J.L., and J.E. Forbes. 1970. Preventing blackbird damage to
sprouting corn with a carbamate repellent. New York Fish and Game J.
17(2):117-120.
Guarino, J.L., W.F. Shake, and E.W. Schafer, Jr. 1974. Reducing bird
damage to ripening cherries with methiocarb. J. Wildl. Manage.
38(2):338-342.
Guarino, J.L., C.P. Stone, and W.F. Shake. 1973. A low-level treatment
of the avian repellent methiocarb on ripening sweet cherries. Proc. Bird
Control Semin., Bowling Green, Ohio 6:24-27.
Herman, G., and W. Kolbe. 1971. Effect of seed coating with Mesurol for
protection of seed and sprouting maize against bird damage, with consid-
eration to varietal tolerance and side effects. Pflanzenschutz-Nachrichten
Bayer 24(2):279-329. (English Edition)
Ingram, C.R. , R.I. Mitchell, and A.R. Strickley, Jr. 1973. Hopper box
treatment of corn seed with methiocarb for protecting sprouts from
birds. Proc. Bird Control Seminar, Bowling Green, Ohio 6:206-215.
Ingram, C.R. 1977. Efficacy Experiments Involving Avian Pest Control
Materials: Design Consideration Imposed by the Scientific Method.
Pp. 215-224 in Test Methods for Vertebrate Pest Control and Management
Materials, Vol. 1, STP 625. W.B. Jackson and R.E. Marsh, eds. Amer.
Soc. for Testing and Materials, Phila., Pa.
Laflin, T., and W.S. English. 1960. Bird damage to buds of fruit trees.
Exp. Hort. 3:13-19.
Metzer, R., and W.C. Royall, Jr. 1961. Field Tests of Three Chemicals
as Bird Repellents on Mature Grain Sorghum. Texas Agr. Exp. Sta.
MP524, College Station, Texas. 6 pp.
Neff, J.A., and B. Meanley. 1956. A review of studies on bird repellents.
Prog. Rept. No. 1—Research in Bird Repellents. U.S. Dept. Int., Fish and
Wildl. Res. Center, Denver. 13 pp. (Mimeo).
Przygodda, W. 1955. Experiments on house sparrows with two repellents:
Morkit and Morkit Cone. Hofchen-Briefe 8:251.
Royall, W.C., Jr., and E.R. Ferguson. 1962. Controlling bird and mammal
damage in direct seeding loblolly pine in east Texas. J. Forestry
60(1):37-39. .
Savidge, I.R. 1974. A model for management predictions of territorial
bird populations. Ohio J. Sci. 74(5): 301-312.
Southon, T.F. 1959. Trials with thiram as seed and ripening grain.
protectant. H.Z. J. .Agric. 99:1-03.
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368
Stickley, A.R., Jr., and J.L. Guarino. 1972. A repellent corn .seed from . .
blackbirds and crows. J. Wildl. Manage. 36(1):150-152.
Stickley, A.R., and C.R. Ingram. 1973. Two tests of the avian repellent,
methiocarb, in Michigan sweet cherry orchards. Proc. Bird Control Seminar,
Bowling Green, Ohio 6:41-46.
Stickley, A.R., Jr., R.T. Mitchell, R.G. Heath, C.R. Ingram, and E.L.
Bradley, Jr. 1972. A method for appraising the bird repellency of
4-aminopyridine. J. Wildl. Manage. 36(4):1313-1316.
West, R.R. 1968. Protecting sprouting corn from pheasants with DRC-736.
P. 12 in Trans. 13th Ann. Conf. Central Mountain and Plains Section
Wildl. Soc.
West, R.R., R.B. Brunton, and D.J. Cunningham. 1969. Repelling pheasants
from sprouting corn with a carbamate insecticide. J. Wildl. Manage.
33(1):216-219.
West, R.R., and J.M. Dunks. 1969. Repelling boat-tailed grackles from
sprouting corn with a carbamate compound. Texas J. Sci. 21(2):231-233.
Wright, E.N. 1962. Experiments with anthraquinone and thiram to protect
germinating maize against damage by birds. Ann. Epiphyties 13:27-31.
Young, W.R., and D.C. Zevallos. 1960. Studies with chemical seed treat-
ments as bird repellents for the protection of germinating maize in
the Mexican tropics. FAQ Plant Prot. Bull. 8(4):38-42.
D. Avian reproductive inhibitors; Field methods.
Only a few tests on efficacy of reproductive inhibitors have been conduct-
ed to date. The variety of species evaluated is limited and the adequacy of
test methodology in individual studies is not established. Efficacy deter-
minations require measuring fertility rates of treated and untreated birds
and effects on breeding behavior, production, and population levels.
Test procedures have been developed for evaluating the effects of repro-
ductive inhibitors in various blackbird species (Bray et al., 1974; Fringer
and Granett, 1970; Messersmith, 1971; Peek, 1972; and Vandenbergh and Davis,
1962). In addition to the production studies common to each paper, histo-
logical evaluations of treated birds (male and/or female)'are outlined by
Vandenbergh and Davis (1962), and behavioral aspects are described by Bray
et al. (1974) and Peek (1972). Effects on nontarget species should also be
included in the test design.
Test procedures are less well developed for field evaluations in pigeons
(Shortemeyer and Beckwith, 1970; Wofford and Elder, 1967; and Woulfe, 1968)
and should be augmented with data on productivity, histology, breeding
behavior, and effects on nontarget species.-
Procedures are also available for gulls (Wetherbee, 1967).
image:
369
References.
Anon. 1975a. Standard Method of Test for Efficacy of Acute Avicides.
Annual Book of ASTM Standards, Part 46. Designation: E551-75.
Amer. Soc. for Testing and Materials, Phila., Pa.
1975b. Standard Recommended Practice of Determining Acute Oral
LD5Q for Testing Vertebrate Control Agents. Annual Book of ASTM Standards,
Part 46. Designation: E555-75. Amer. Soc. for Testing and Materials,
Phila., Pa.
Bray, O.E., J.J. Kennelly, and J.L. Guarino. 1974. Fertility of eggs
produced on territories of vasectomized red-winged blackbirds. Wilson
Bull. 87(2):187-195.
Davis, D.E. 1959. Effect of triethylenemelamine on testes of starlings.
Anat. Rec. 134(3):549. (Abstr.)
. 1961. Principles for population control by gametocides.
Trans. N. Am. Wildl. and Nat. Resources Conf. 26:160-167.
Fringer, R.C., and P. Granett. 1970. The effects of Ornitrol on wild
populations of red-winged blackbirds and grackles. Proc. Bird Control
Semin., Bowling Green, Ohio 5:163-176.
Messersmith, D.H. 1971. TFM, potential control for red-winged blackbirds.
Pest Control 39(2);35-41.
Peek, F.W. 1972. The effect of tranquilization upon territory maintenance
in the male red-winged blackbird. Anim. Behav. 20:119-122.
Shortemeyer, J.L., and S.C. Beckwith. 1970. Chemical control of pigeon
reproduction. Trans. N. Am. Wild, and Nat. Resources Conf. 35:47-55.
Vandenbergh, J.C., and D.E. Davis. 1962. Gametocidal effects of triethyl-
enemelamine on a breeding population of red-winged blackbirds. J. Wildl.
Manage. 26(4):366-371.
Wetherbee, O.K. 1967. Population control of herring gulls by the embryocide,
sudan black. M. Cummings, ed. Proc. Third Vertebrate Pest. Conf., San
Francisco, Calif. 3:61-67.
Wofford, J.E., and W.H. Elder. 1967. Field trials of the chemosterilant
SC-12937 in feral pigeon control. J. Wildl. Manage. 31(3):507-515.
Woulfe, M.R. 1970. Reproduction inhibitors for bird control. Proc
Fourth Vert. Pest Conf., Fresno, Calif. 5:168-170.
E. Avian anesthetizing chemicals; Field methods.
The most important, measures of;the.field efficacy evaluation are
percent.capture, mortality, the time until anesthesia., .and the duration - ^ ,
of anesthesia. For control programs, population reduction by capture
and removal is also important. .• "''•-'•'•.'
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370
Methods for evaluating the effects of anesthetizing chemicals on upland
game birds have been described by Mosby and Cantner (1956), Williams (1966,
1967), Williams and Phillips (1972), and Williams et al. (1966). However,
additional information, such as hazards to nontarget species and body residues
in treated birds, may also be required. Accurate scientific description of
measurement parameters must also be used (i.e., g/kg bait, not g/cup).
Methods for using anesthetizing chemicals to capture waterfowl are also
available (Crider and McDaniel, 1966, 1967, 1968, and Crider et al., 1968);
however, these methods are not complete, and additional information may be
necessary. Additional methodology is described for other species of birds
commonly found near water (Smith, 1967 and Williams and Phillips, 1973).
Methodology for urban pigeon and sparrow control with anesthetics is
well detailed by Murton et al. (1963), Ridpath et al. (1961) and Thearle et
al. (1971), although certain aspects (e.g., hazards to domestic animals)
may need more documentation. Methodology for the use of anesthetizing
chemicals in rural situations is best described by Martin (1967), Murton et
al. (1963), Ridpath et al. (1961), and Williams and Phillips (1972); however,
hazards to predatory animals may need more documentation.
References.
Anon. 1975a. Standard Method of Test for.Efficacy of Acute Avicides.
Annual Book of ASTM Standards, Part 46. Designation: E551-75. '
Amer. Soc. for Testing and Materials, Phila., Pa.
. 1975b. Standard Recommended Practice for Determining
Acute Oral LDgQ for Testing Vertebrate Control Agents. Annual
Book of ASTM Standards, Part 46. Designation: E555-75. Amer. Soc.
for Testing and Materials, Phila., Pa.
Borg, K. 1956. Use of chloralose for destruction of injurious birds.
2. Jagdwissensch 2(3):180-182.
Condy, J.P. 1965. A technique for capturing Abdim's storks. Ostrich
36(3):121-122.
Crider, E.D., and J.C. McDaniel. 1966. Technique for capturing Canada
geese with alpha-chloralose. Proc. S.E. Assoc. Game and Fish Comm.
20:226-233.
1967. Alpha-chloralose used to capture Canada geese.
J. Wildl. Manage. 31 (2) -.258-264.
1968. Oral drugs used to capture waterfowl. Proc. S.E.
Assoc. Game and Fish Comm. 22:156-161.
Crider, E.D., V.D. Stotts, and J.C. McDaniel. 1968. Diazepam and alpha-
chloralose mixtures to capture waterf.owl. Proc. S.E. Assoc. Game and
Fish Comm. 22:133-141.. •• "~~r"
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371
Lindan, L. 1962. Experiments with tranquilizers in birds. Nord. Vet.
Med. 14(Suppl. 1):112-117.
Martin, L.L. 1967. Comparison of methoxymol, alpha-<-:hloralose and two
barbiturates for capturing doves. Proc. S.E. Assoc. Game and Fish Comm.
21:193-200.
Mosby, H.L., and D.E. Cantner. 1956. The use of avertin in capturing wild
turkeys and as an oral-basal anesthestic for other wild animals. Southwest.
Vet. 9(2):132-136.
Moser, C.M. 1965. Anesthetize pest birds with tribromoethanol for humane
removal. Amer. City 80:30.
Murray, R.E., and D. Dennet. 1963. A preliminary report on the use of
tranquilizing compounds in handling wildlife. Proc. S.E. Assoc. Game
and Fish Comm. 17:134-139.
Murton, R.K. 1962. Narcotics v. wood pigeons. Agriculture 69(7);336-339.
1963. Stupefying wood pigeons. Agriculture 70(1 0):500-501 .
Murton, R.K., A.J. Isaacson, and N.J. Westwood. 1963. The use of baits
treated with alpha-chloralose to catch wood pigeons. Ann. Appl. Biol.
52:271-293.
1965. Capturing columbids at the nest with stupefying baits.
J. WiId1. Manage. 29(3):647-649.
Potts, R.M., and D.J. Womeldorf. 1960. Pigeon control in Fresno, Calif.
Vector Views 7(10).-59-62.
Ridpath, M.G., R.J.P. Thearle, D. McCowan, and P.J.S. Jones. 1961.
Experiments on the value of stupefying and lethal substances in the
control of harmful birds. Ann. Appl. Biol. 49:77-101.
Smith, N.G. 1967. Capturing seabirds with avertin. J. Wildl. Manage.
31(3):479-483.
Thearle, R.J.P. 1960. The use of narcotics in catching harmful birds.
Ann. Appl. Biol. 48:414-415.
1968. Urban bird problems. Pp. 181-197 in Symposium of the Institute
of Biology No. 17: The Problems of Birds as Pests. R.K. Murton and E.N.
Wright, eds. Academic Press: N.Y.
1969a. The use of stupefying baits to control birds. Pp. 10-16
in The Humane Control of Animals Living in the Wild. UFAW Symposium,
23 January 1969. UFAW, Potters Bar, Great Britain.
1969b. Some problems involved in the use of stupefying baits
to control birds. Pr.oc. Br.. Insect. Fungi.- Conf. 5:458-464.
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372
Thearle, R.J.P., R.K. Murton, M.M. Senior, and D.S^.Malam.'• 1971. Improved
stupefying baits for the control of town pigeons. Internat. Pest. Cont.
13(2):11-14, 16,19.
Tomlinson, J.T. 1967. Sedatives interfere with walking more than flying.
Wilson Bull. 79(2):242-243.
Weigand, G. 1965. Orienting experiments in the control of crows with alpha-
chloralose. Nach richtenbl. Dtsch. Pfanzenschutzdienstes (Braunschw.)
17(7):108-110.
Williams, L.E., Jr. 1966. Capturing wild turkeys with alpha-chloralose.
J. Wildl. Manage. 30(1):50-56.
^ . 1967. Preliminary report on methoxymol to capture
turkeys. Proc. S.E. Assoc. Game and Fish Comm. 21:189-193.
Williams, L.E., Jr., D.H. Austin, and J. Peoples. 1966. Progress in
capturing turkeys with drugs applied to baits. Proc. S.E. Assoc.
Game and Fish Comm. 20:219-226.
Williams, L.E., Jr., and R.W. Phillips. 1972. Test of oral anesthetics
to capture mourning doves and bobwhites. J. Wildl. Manage. 36(3):
968-971.
. 1973. Capturing sandhill
cranes with alpha-chloralose. J. Wildl. Manage. 37(1):94-97.
Wright, H.M. 1953. A suggested method of capturing birds with a
narcosis producing drug. Proc. Midwest Wildl. Conf. December, 1953.
Woronecki, P.P., J.L. Guarino, and J.W. DeGrazio. 1967. Blackbird
damage control with chemical frightening agents. Proc. Third Vert.
Pest. Conf., San Francisco, Calif. 3:54-56.
IV. Bat toxicants and repellents.
Bats are beneficial in the United States in that they consume great
quantities of insects. Bats can present local nuisances, however, when
they inhabit the attics of private homes or the rafters of public buildings.
Because bats can be vectors of rabies, chemical bat control may become
necessary in circumstances where the likelihood of bat-human contact is high.
The goal of bat control, whether by toxicant or by repellent, is the elimin-
ation of the problem. Bat control with pesticides in buildings should reduce
the target populations to near complete eradication. Products with marginal
efficacy are essentially useless in solving bat problems. Therefore, products
should produce 90% control or better in efficacy tests.
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373
To demonstrate efficacy, it is necessary to develop a reliable means
of estimating the number of bats in the target area or, at the very least,
of assessing the level of bat activity. Direct counts of bats leaving
premises have proven to be useful (Greenhall and Stell, 1960). When using
this method, it is necessary that all openings used by bats be visible at
all times. A team of observers may be required. If the bats use few points
of entry, it may be possible to monitor activity by use of photoelectric
cells which activate electical counting devices. Such devices would not
differentiate between bats entering and bats leaving, and could, therefore,
provide only relative activity indices.
Through thorough exploration of infested areas by day, it may'be possible
to make absolute counts of roosting bats. The size of the infestation could
also be assessed through droppings counts. However, these activities may
disturb the bats, causing them to return in fewer numbers even if no bat
control agent is applied. Physical disturbances due to product application
may also exert repellent effects. Check studies (.with no chemical applied.) •"
are therefore necessary to establish the efficacy of the chemical agent it-
self. Care must be taken that physical disturbances in check and treatment
studies be equivalent.
The list of references provided below includes material on the biology
of bats, the trapping of bats, the maintenance of bats in captivity, bat
control, bats as rabies vectors, and other information which may be useful
in developing products and control strategies.
References.
Baer, G.M., and D.B. Adams. 1970. Rabies in insectivorous bats in the
United States, 1953-1965. Publ. Health Rep. 85:637-645.
Bigler, W.J., G.L. Hoff, and E.E. Buff. 1975. Chiropteran rabies in
Florida: A twenty year analysis, 1954 to 1973. Am. J. Trop. Med.
Hyg. 24:347-352.
Bonaccorso, F.J., N. Smythe, and S.R. Humphrey. 1975. Improved
techniques for marking bats. J. Mammal. 57:181-182.
Buchler, E.R. 1976. Chemiluminescent tag for tracking bats and other
small nocturnal animals. J. Mammal. 57:173-176.
Constantine, D.G. 1958. An automatic bat-collecting device. J. Wildl.
Manage. 22:17-22.
1962. Rabies transmission by non-bite route. Publ. Health
Rep. 77:287-289.
Coutts, R.A., M.B. Fenton, and E. Glen. 1973. Food intake by captive
Myotis lueifugus and Eptesicus fuscus (Cairoptera; Vesper.ti.lionidae).
J. Mammal. 54:985-990.
.Gates, W."H', ' 1936.'" -Keeping bats'-in capta.-v.ity.' . J.. MamriiaJ.V.i.7:;. 26B-2T3'., ..•• ',-'
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374
Greenhall, A.M. 1963. Use of mist nets-.and strychnine for vampire control ••
in'Trinidad. J. Mammal. 44:396-399. ';
Greenhall, A.M., and G. Stell. 1960. Bionomics and chemical control of
free-tailed house bats (Molossus) in Trinidad. U.S. Dept. Int. Fish &
Wildl. Serv. Spec. Sci. Rep. - Wildlife No. 53. 19 pp.
Humphrey, S.R., and J.B. Cope. 1977. Survival rates of the endangered
Indiana bat, Myotis sodalis. J. Mammal. 58:32-36.
Laidlaw, G.W.J., and M.B. Fenton. 1971. Control of nursery colony
populations of bats by artificial light. J. Wildl. Manage. 35:843-846..
Luckens, M.M. 1973. Seasonal changes in the sensitivity of bats to DDT.
Pp. 63-75 in Pesticides in the Environment: A Continuing Controversy.
W.B. Deichman, ed. Intercont. Med. Book Corp.: New York.
Lukens, M.M., and W.H. Davis. 1964. Bats: sensitivity to DDT. Science
146:948.
Mitchell, G.C., and R.J. Burns. 1973. Chemical control of vampire bats.
U.S. Dept. Int. Denver Wildl. Res. Center Monogr. 37.
Slaughter, B.H., and D.W. Walton. 1970. About Bats. So. Meth. Univ.
Press: Dallas.
Tuttle, M.D. 1974a. Bat trapping: results and suggestions. Bat Res.
• News 15:4-7. \ -.'.,.
Tuttle, M.D. 1974b. An improved trap for bats. J. Mammal. 55:475-477.
Wilson, D.E., and J.S. Findley. 1972. Randomness in bat homing.
Am. Nat. 106:418-424.
Wimsatt, W.A. 1970. The Biology of Bats. Vol. 1 & 2. Academic Press:
N.Y.
Wimsatt, W.A., and A. Guerriere. 1961. Care and maintenance of the
common vampire bat in captivity. J. Mammal. 42:449-455.
1962. Observations on the feeding capacities and excretory
functions of captive vampire bats. J. Mammal. 43:17-27.
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375
V. Rodenticides; commensal and non-commensal.
One of the earliest—and relatively comprehensive—publications avail-
able on rodenticides and their efficacy is "Biological Methods for the
Evaluation of Rodenticides" (Bentley, 1958). In 1963, Kverno and Hood
published a volume on the evaluation procedures and standards for screening
and developing chemicals for forest rodent control.
Tracking powders came.into use almost by accident when deaths among
house mice were observed in places where insecticides such as DDT and BHC
were used. Until its ban in 1972, DDT was used quite extensively for house
mouse control. Anticoagulant rodentcides were found effective as tracking
powders for both rats and mice and, until recently, were used more in Europe
than in the United States.
Little is available on methods for evaluating tracking powders. Bentley
(1958) devotes a section of his bulletin to tracking powders, with reference
to their evaluation. Szuber and Brodniewicz (1964) provide a drawing of
their test cage and the results achieved with some some anticoagulant track-
ing powders. More recently, Tanaka (1973) published evaluations of rodenti-
cides as tracking powders, and described the methods utilized in his studies.
Laboratory methods. The Environmental Protection Agency has developed methods
that have been used for establishing laboratory efficacy of rodenticides for
commensal rodents (See Exhibits 2-20).
Articles concerning laboratory test methodology have been published by
various researchers: World Health Organization, 1967; Bentley and Larthe,
1959; Dicke and Richter, 1946; Durbin and Robens, 1964; Emlen and Strecker,
1951; Hayes and Gaines, 1959; Hankins et al., 1973; Howard et al., 1968;
Loosjes, 1959, Palmateer, 1974, 1977; Saunders, 1955; Savarie et al., 1973;
and Ward et al., 1949.
Field methods; Emlen and Crow (1951), Miller (1953), Richens (1967), Schein
(1950), and Wood (1965) have reported on field studies which utilized tech-
niques or methodology of value to those unfamiliar with setting up field
evaluations.
The food preferences of the target species, as well as the acceptability
or palatability of the prepared bait will have a direct bearing on efficiency
in both the laboratory and field. Palmateer and McCann (1976) found a very
strong relationship between acceptance of anticoagulant rodenticides and
mortality. Thus, techniques or methods for measuring food preferences or
acceptance should be recognized as an integral part of evaluating a rodenti-
cide for any species. Bentley (1958) discussed palatability of baits in
considerable detail. His evaluations included measuring the number of visits
to the bait, duration of feeding, amount consumed, and rate of intake. He-.
also included comments on the time of onset of poison symptoms and bait
shyness.
Shumake et al. (1971) made comparisons of taste preference studies between
.laboratory and wild.Norway rats-.. , Barne'tt--an-d.- Spencer ...(1953^'Vct3ff<jQcted".sfr •' ~'"
extensive study of food preferences.of wild Norway rats, and Khan (1974)
image:
376
conducted laboratory experiments on food preferences of the black rat. Other
researchers, such as Gregson (1966), Cornwell and Bull (1967T, Beer C1964),
Patric (1970), Fitch (1954), Joy et al. (1967), and Mass and Hood (1969)
have published data which may be valuable to those trying to formulate better
rodent baits.
References.
Abbott, W.S. 1925. A method of computing the effectiveness of an insecticide.
J. Econ. Ent. 18:265-267.
Anon. 1970. Provisional instructions for determining the susceptibility of
rodents to anticoagulant rodenticides. WHO Tech. Kept. Ser. 443:140-147.
Barnett, S.A., A.H. Bathard, and M.M. Spencer. 1951. Rat populations and
control in two English villages. Ann. Appl. Biol. 38(2):444-463.
Barnett, S.A. 1958. Laboratory methods for behavior studies of wild rats.
J. Anim. Tech. Assn. 9(1):6-14.
Barnett, S.A., and M.M. Spencer. 1953. Experiments on the food preferences
of wild rats (Rattus norvegicus). J. Hyg. 51(1):16-34.
Beer, J.R. 1964. Bait preferences of some small mammals. J. Mammal.
45:632-634.
Bentley, E.W. 1958. Biological Methods for the Evaluation of Rodenticides.
Ministry of Agric. Fisheries and Food. Tech. Bull. No. 8, 35 pp.
Bentley, E.W., and Y. Larthe. 1959. The comparative rodenticidal efficacy
of five anticoagulants. J. Hyg. 57(2):135-149.
Brooks, J.E. 1964. Population responses of sewer rats following poisoning.
California Vector Views 11(7):41-46.
Byers, R.E. 1975. New compound (RH-787) for use in control of orchard
voles. J. Wildl. Manage. 40(1):169-171.
Cornwell, P.B., and J.O. Bull. 1967, Taste preference in rodenticide
development. Internal. Pest Cont. 8(35);15.
Corr, P.V. 1971. The acceptability and effectiveness of pellet baits
for poisoning rabbits. Austral. J. Exp. S Anim. Husb. (11):407-414.
DeCaire, E. 1961. A method of recording the feeding behavior of a rat
which has the choice of several feeding troughs. S. African J. Med.
Sci. 26(4):105-108.
Dieke, S.H., and C.P. Richter. 1946. Comparative assays of rodenti-
cides on wild Norway rats. I. Toxicity. Public Health Rep. 61:672-679.
Doty, R.E. 1944. Rat-trapping records show effectiveness of control
methods. Hawaiian Plant Rec. 48:73-82.
image:
377
Drununond, D.C. 1960. Partial avoidance of a. rodenticidal dust by Rattus
rattus L. Parasitica 16;1-6.
Drummond, D.C., and E.J. Wilson. 1968. Laboratory investigations of
resistance to warfarin of Rattus norvegicus Berk, in Montgomeryshire
and Shropshire. Ann. Appl. Biol. 61:303-349.
Durbin, C.G., and J.P. Robens. 1964. The Use of Laboratory Animals for
Drug Testing. U.S. Dept. Hlth. Ed. and Welfare, Food and Drug Admin.
(pp. 696-711 ).
Emlen, J.T., and J.F. Crow. 1951. A test for increased resistance in a
chronically poisoned mouse population. Am. J. Hyg. 54:71-75.
Emlen, J.T., and R.L. Strecker. 1951. The effect of laboratory confine-
ment on survival of poisoned house mice. Ecology 32(2):331-332.
Fitch, H.S. 1954. Seasonal acceptance of bait by small mammals. J_.
Mammal. 35(1}:39-47.
Gregson, R.A.M. 1966. Cross-modal matching of histograms and four
component taste mixtures with one component fixed under two pacing
conditions. Percept. Mot. Skills 23:183-190.
Hankins, W.G., J. Garcia, and K.W. Rusinak. 1973. Dissociation of odor
and taste in bait shyness. Behav. Biol. 8(3):407-419.
Hayes, W.J., Jr., and T.B. Gaines. 1959. Laboratory studies of five
anticoagulant rodenticides. Public Health Rep. 74:105-113.
Hermann, G., and W.M. Zeck. 1964. The role of industry in developing
new materials for vertebrate pest control. W. Koehler, ed. Proc.
Second Vert. Pest Conf., Anaheim, Calif. 2:9-15.
Hoffer, M.C., P.C. Passof, and R. Krohn. 1969. Field evaluation of
DRC714 for deermouse control in a redwood habitat. J. Forestry
67(3):158-159.
Howard, W.E., S.D. Palmateer, and M. Nachman. 1968. Aversion to
strychnine sulfate by Norway rats, roof rats, and pocket gophers.
Toxicol. Appl. Pharmacol. 12:229-241.
Joy, R.J.T., C.P. Emma, and J. Mayer. 1967. New rat feeding jar; use
in study of relationship of food intake and body weight. J. Appl.
Physio1. 23(4): 589-590.
Keith, J.O. 1961. An efficient and economical pocket gopher exclosure.
J. Range: Manage. 14:332-334.
Khan, J.A.' 1974. Laboratory experiments, on. the food preferences of the
black rat. Zool. J. 54.: (2V: 167.
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378
Koerker, R.M. 1974. A feeder for controlling food consumption in treat-
ment control pairs of mice. Lab, Anim. Sci. 24(2): 535-5.36.
Kverno, N.B. 1970. Standardization procedures for developing vertebrate
control agents. R.H. Dana, ed. proc. Fourth vert. Pest Conf., West
Sacramento, Calif. 4:138-139.
Kverno, N.B., and G.A. Hood. 1963. Evaluation Procedures and Standards:
Chemical Screening and Development for Forest Wildlife Damage. U.S.
Dept. int., Fish and Wildl. Serv. Bur., Sport Fish and Wildl., Branch
of Wildl. Res. GPO 836-707. 59 pp.
Laycock, W.A., and B.Z. Richardson. 1975. Long-term effects of pocket
gopher control on vegetation and soils of a subalpine grassland. J_.
Range Manage. 28(6):458-462.
Lee, J.O., Jr. 1970. Outlook for rodenticides and avicides registration.
R.H. Dana, ed. Proc. Fourth Vert. Pest Conf., West Sacramento, Calif.
4:5-8.
Lindsey, G.D., R.D. Mass, and G.L. Hood. 1971. An evaluation of bait
stations for controlling rats in sugarcane. J. Wildl. Manage.
35(3):440-444.
Loosjes, F.E. 1959. The evaluation of anticoagulants as rodenticides in
the laboratory. Tijd. Plant 65(3);116-118.
Marsh, R.E. 1968. An aerial method of dispensing ground squirrel bait.
J. Range Manage. 21(6):380-384.
Mathys, G. 1975. Guidelines for the development and biological evaluat-
ion of rodenticides. EPPO Bull. 5(1): special issue. 49 pp.
Miller, J.G. 1974. The significance of preference in laboratory bait
acceptance studies. W.V. Johnson, ed. Proc. Sixth Vert. Pest Conf.,
Anaheim, Calif. 6:78-81.
Miller, M.A. 1953. Experimental studies on poisoning pocket gophers.
Hilgardia 22(4):131-166.
Nass, R.D., and G.A. Hood. 1969. Time-specific tracer to indicate bait
acceptance by small mammals. J. Wildl. Manage. 33(3):584-588.
Ochs, p. 1972. Efficacy testing of vertebrate pest control agents. R.E.
Marsh, ed. Proc. Fifth Vert. Pest Conf., Fresno, Calif. 5:138-141.
Palmateer, S.D. 1974. Laboratory testing of albino rats with anticoag-
ulant rodenticides. W.v. Johnson, ed. Proc. Sixth Vert. Pest Conf.,
Anaheim, Calif. 6:63-72. 605
Palmateer, S.D. 1977. Rat and Mouse Acute Liquid Bait Test Method.
Pp. 61-66 in Test Methods for Vertebrate Pest Control and Management
Materials-, Vol. 1, STP 625. W.B. Jackson and R..E. Marsh, eds.
Amer. -Soc. for Testing & Materials, Phila., Pa. . ... '.
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379
Palsiateer, S.D., and J.A. McCann. 1976. Relationship of acceptance and
mortality of anticoagulant baits to rats. Bull. Environ. Contain Toxicol.
15(6):750-755.
patric, E.F. 1970. Bait preferences of small mammals. J. Mammal.
51:179-182.
Peardon, D.L. 1977. Field Testing Method Used in Evaluating Acute
Commensal Rodenticides. Pp. 67-76 in Test Methods for Vertebrate
Pest Control and Management Materials Vol. 1, STP 625. W.B. Jackson
and R.E. Marsh, eds. American Society for Testing and Materials.
Phila. Pa.
Peardon, D.L., E.E. Kilbourn, and J.E. Ware, Jr. 1972. New selective
rodenticides. Soap/Cosmetics/Chemical Specialties 12:70,74,80,82,84.
Richens, V.B. 1967. The status and use of gophacide. M.W. Cummings,
ed. Proc. Third Vert. Pest Conf., San Francisco, Calif. 3:118-125.
Rowe, F.P. 1957. The control of house mice (Mus musculus L.) with a
warfarin dust surrounding water baits. Sanitarian 66(3):183-188.
Rowe, F.P., and A.H.J. Chudley. 1963. Combined use of rodenticidal
dust and poison solution against house mice (Mus musculus L.)
infesting a food store. J. Hyg. Camb. 61:169-174.
Rowe, F.P., J.H. Greaves, R. Redfern, and A.D. Martin. 1970. Rodenti-
cides—problems and current research. R.H. Dana, ed. Proc. Fourth
Vert. Pest Conf., West Sacramento, Calif. 4:126-128.
Rowley, I. 1963. Bait materials for poisoning rabbits. II. A field
study on the acceptance of carrots and oats by wild populations of
rabbits. CSIRO Wildl. Res. (8):62-77.
Saunders, J.P., S.R. Helsey, A.D. Goldstone, and E.G. Bay. 1955.
Comparative toxicities of warfarin and some 2-acetyl-1,3-indandiones
in rats. J. Agric. Food Chem. 3:762-765.
Savarie, P.J., G.H. Matschke, E.W. Schafer, and G.J. Dasch. 1973.
UK 786 - possible species-specific toxicant for Norway rats.
Nature 241(5391):55-552.
Schein, M.W. 1950. Field test of the efficiency of the rodenticide
compound W.A.R.F. 42. Public Hlth. Rep. 65(11):368-373.
Shumake, S.A., R.D. Thompson, and C.J. Caudill. 1971. Taste preference
behavior of laboratory versus wild Norway rats.. J. Comp. Physiol.
Psycho1. 77(3):489-494.
Spencer., D.A. 1964.. 'Testing and registration of new control materials.
J.W. Koehler,, ed. Proc. Second Vert. Pest Conf., Anaheim, Calif
•-.-2:3-8. . ......
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380
Szuber, T, and A. Brodniewicz. 1964. The effectiveness of' some- anti--
coagulant rodenticide dusting powders. J. Hyg. Epidemic!. Microbiol.
Immunol. (Prague) 8:332-337.
Tanaka, I. 1973. Evaluation of rodenticides as a tracking powder. Jap.
J. Sanit. Zool. 23(3):219-223.
Ward, A.L., P.L. Hegdal, V.B. Richens, and H.P. Tietjen. 1967. Gophacide,
a new pocket gopher control agent. J. Wildl. Manage. 31 (2):332-338.
Ward, J.C., D.G. Crabtree, and F. E. Garlough. 1940. Red Squill VIII.
Further notes on bioassay methods. Amer. Pharm. ftssoc. J. 29:354-357.
Wood, J.E. 1965. Response of rodent populations to controls. J. Wildl.
Manage. 29:425-438.
World Health Organization. 1967. Toxicity Test for Rodenticides.
Specifications for Pesticides. Geneva WHO/M/14. 300 pp.
A. EXHIBITS 2-20
(I.) The methods listed below [see (III.)] are published by the Agency
in its "Manual of Biological Testing Methods for Pesticides and Devices,
Volume 1." Copies of individual test methods may be obtained by writing
to the Environmental Protection Agency, EPA Chemical and Biological
Investigations Branch, Agric. Res. Center, Bldg. 402, Beltsville, Md.
20705, £r_ Product Manager (16), Insecticide-Rodenticide Branch, (TS-767),
Environmental Protection Agency, 401 M St.,' S.W., Washington, D.C. 20460.
Users are advised that some BSFD (Benefit and Field Studies Division)
methods listed below are undergoing review and possible revision. Excerpts
from Exhibits 2 to 20 are provided in (II.) below. These excerpts call
attention to important specific testing conditions and requirements, such as
caging, challenge diets, selection of test animals, environmental conditions,
and specific requirements for particular uses.
(II.) Test standards.
(A.) Type, weight, and number of animals.
1. For a new claim use the wild target species. These may be wild-
caught or from a wild colony. For an existing claim use either the wild
target species or laboratory strains of the target species. The Wistar is
the preferred strain of the laboratory rat, and the Swiss-Webster is the
preferred strain of the laboratory mouse. Roof rat claims may be supported
by Norway rat data. Baits presented to a species in an unopened place pack
must have a test on the formulation itself and on the place pack.
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2. All animals shall be outwardly healthy, active, sexually mature,
and fall within the following weight classes (in grams):
Max. Acceptable Differences
Minimum Maximum In Mean Wts. between Sexes
Lab rats 150 300 50
Norway rats 150 400 65
Lab mice 15 35 5
House mice 10 25 3
3. The test should be replicated at least once. For each test
expose at least 10 males and 10 females to the toxicant. Use another
10 males and 10 females as controls. If a series of tests is conducted
concurrently, use at least one control group.
(B.) General environmental conditioning.
1. All animals used in these tests must be held for observation
in the laboratory for one to four weeks prior to testing. Ectoparasite
control with appropriate concentrations of carbaryl, malathion, or
pyrethrum dusts is permissible if applied externally to both test and
control animals not less than seven days prior to testing. Acclimate
animals to test conditions three days prior to testing. Animals should
not be'subjected to undue or.unnecessary stress, from noise or human
activities (e.g., movement). Human activity within the animal test room
should be minimal. Water and commercial rat or mouse diet must be avail-
able to them at all times. Do not use the standard rodent challenge diet
described below for pre-test feeding.
2. During holding and test periods the animals should be held in
areas with the following environmental conditions.
Temperature 20 to 25°C. Strong air currents
from heaters or air conditioners
shall not blow directly onto the
animals.
Relative humidity ' 50 to 55%
Light 12 hrs artificial light per day,
not to exceed 2153 Ix (200 ft
candles) at cage location.
:.•;.'..' Total reversing of natural.
. . . . photoperiods is not recommended.
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(C.) Standard rodent challenge diet.
1. A standard rodent challenge- diet must be provided whenever the
animals are exposed to a toxic bait. This challenge diet shall be composed
(by weight) of 65% cornmeal (whole yellow ground corn), 25% rolled oat groats
(ground), 5% sugar (10X powdered or confectioners, 95+% purity), and 5% corn
oil (95+% purity). Combine dry ingredients together, add oil, and thoroughly
mix. Be certain that the mixing utensils are free of contamination before
preparing the diet.
2. The whole (not degerminated) yellow ground corn shall be from the
most recently available crop and be fresh ground. Seventy-five percent
(+_ 5%) shall pass through a #10 screen (10 meshes to the inch or 2.54 cm)
and 50% (+_ 10%) be retained by a 120 screen (20 meshes to the inch). The
oats shall be steam rolled oat groats (oat seeds with hulls removed) and
coarsely ground after the rolling process. Seventy-five percent (+_ 5%) of
the ground oats shall pass through a 15 screen (5 meshes to the inch) and
50% (+_ 10%) be retained by a #20 screen. The corn oil shall be of the type
available as cooking oil, undiluted with other oils, and shall not be rancid.
3. The challenge diet shall be stored under refrigeration if it is to
be used within three days of preparation. If it is to be held for longer
periods, the diet shall be packaged in plastic containers [2.2 to 4.5 kg (5
to 10 Ib) per container], tightly closed or sealed, and maintained at -18°C
or below until used. It shall be at room temperature when offered to test
or control animals. Mixed challenge diet shall not be stored longer than
six months. .
(D.) Specific conditions for dry baits, wax blocks, and wax pellets.
1. For rats, place animals in individual cages with a minimum
bottom surface area of 0.538-2.15 sq ft (500-2000 sq cm). For mice,
place animals in a group cage with a minimum bottom surface area of
1.61-2.15 sq ft (1500-2000 sq cm). For mouse shelters, use empty cans
with one end removed and slightly flattened to prevent rolling. Use
one can per five mice. Provide shelters in both the test and control
cages.
2. The rodenticide-treated food and the standard rodent challenge
diet are each offered to the test group in separate cups in excess of the
daily food requirements. The control group is offered only the rodent
challenge diet. The gross weight of each cup and its contents is determined
daily and returned to the starting weight by addition of food. If food be-
comes fouled by urine or feces, replace food in each cup. Each day record
the quantity of food consumed during the preceding 24 hours. Spilled food
should be recovered for weighing to establish exact food consumption. Where
food spillage is damp, it shall be dried to its original moisture content
before weighing. The positions of the bait and challenge diet cups are
reversed every 24 hours to counter any feeding position preference of the
test animals.
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(E.) Specific conditions for liquid baits.
1. For rats and mice, group cage animals in solid-bottomed all-metal
cages, having a bottom surface area of 18.3-26.9 sq ft (17,000 to 25,000
sq cm. The bottom of each cage should be covered to a depth of approximately
1 inch (2.5 cm) with clean wood shavings. Each cage should contain two metal
nest boxes, approximately 14 by 14 by 4 inches (36 by 36 by 10 cm) without
bottoms.
2. Use metal or ceramic feeders designed to prevent spillage or
contamination of the diet. Provide each cage with two or more feeders,
filled daily with fresh challenge diet to provide at least 40 grams per
rat or 2 grams per mouse.
3. Use graduated 100 ml no-drip waterers fitted with ball-type
watering tubes. Do not use automatic or open-cup type waterers. Provide
each treated test group with a minimum of 12 waterers for rats or 4 for
mice. For a liquid bait, an alternate, non-toxic water source must be
provided. Fill one half of the waterers with tap water and the others
with test liquid diluted according to use directions. This arrangement
provides each rat with 30 ml or each mouse with 10 ml of both liquids,
which are their daily requirements. Replenish both liquids as necessary
so that each waterer never becomes less than one-third empty. Record
daily the total quantity of each liquid consumed during the preceding 24
hours for both the test and control groups.
(F.) Specific conditions for tracking powders.
1. For rats an acceptable test apparatus consists of two 150 gal
(568 liter) stock watering tanks connected by two square hardware cloth
tunnels 36 inches (914 mm) long with 4-1/2 inch (114 mm) sides, top, and
bottom. The tunnels are parallel and spaced 40 inches (1016 mm) apart.
A galvanized steel removable pan 12 inches (305 mm) long by 4 inches
(102 mm) wide with a 1/4 inch (6 mm) lip on both sides is centered in
each tunnel. The test animals must have free and equal access to both
tanks through the tunnels. In each tank place bowls of the standard
rodent challenge 613 diet and water ad libitum. Each tank contains a
screened-bottom nest box approximately 14 by 14 by 4 inches (35 by 10 cm).
2. For mice an acceptable test apparatus consists of two screened-
bottom 15 by 18 by 9 inches (381 by 457 by 229 mm) cages connected by
two square hardware cloth tunnels, 36 inches (914 mm) long with 4-1/2
inch (115 mm) sides, top, and bottom. The tunnels are parallel and
spaced 4 inches (102 mm) apart. A galvanized steel removable pan 12
inches (305 mm) long by 4-1/2 inches (114 mm) wide with 1/4 inch (6 mm)
lip on both sides is centered in each tunnel. The test animals must
have free and equal access to both cages through the tunnels. In each
cage place bowls of the standard rodent challenge diet and water ad
libitum. Provide shelters in both cages. Use empty cans with one end
removed and slightly flattened to prevent rolling. Use one can per five
mice. ' .
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384
3. Place ten male and ten female mice or rats in the appropriate
testing apparatus. Five days after the introduction, dust one of the
removable steel pans with the tracking powder according to label dose
rates (weight per surface area?. Any additional dust must be applied
only to this pan.
4. For tracking powder, the animals is always provided with a
non-toxic route to all sources of food and water. Maintain a daily
record of the degree of activity across the dust on the treated plate.
All previous signs of activity are covered after the daily reading.
(G.) Exposure periods. The exposure period for a single-dose
toxicant is 3 days. For multiple-dose toxicant, the exposure period
is 15 days.
(III.) List of Exhibits.
Exhibit 2 (TSD f 1.201) - Standard Norway rat anticoagulant liquid bait
laboratory test method.
Exhibit 3 (TSD # 1.202) - Standard house mouse anticoagulant liquid bait
laboratory test method. .
Exhibit 4 (TSD # 1.203) - Standard Norway rat anticoagulant dry bait
laboratory test method.
Exhibit 5 (TSD # 1.204) - Standard house mouse anticoagulant dry bait
laboratory test method. •
Exhibit 6 (TSD i 1.207) - Standard Norway rat acute liquid bait labora-
tory test method.
Exhibit 7 (TSD # 1.208) - Standard house mouse acute liquid bait labora-
tory test method.
Exhibit 8 (TSD # 1.209) - Standard Norway rat acute dry bait laboratory
test method.
Exhibit 9 (TSD # 1.210) - Standard house mouse acute dry bait laboratory
test method.
Exhibit 10 (TSD # 1.213) - Standard Norway rat anticoagulant wax block
and wax pellet laboratory test method.
Exhibit 11 (TSD # 1.214) - Standard house mouse anticoagulant wax block
and wax pellet laboratory test method.
Exhibit 12 (TSD # 1.215) - Standard Peromyscus species acute dry bait
laboratory test method.
Exhibit 13 (TSD #•1.216) - Standard Peromyscus species anticoagulant dry
bait laboratory test method.
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385
Exhibit 14 (TSD # 1.219) - Standard rat acute place-pack dry bait laboratory
test method.
Exhibit 15 (TSD # 1.220) - Standard mouse acute place-pack dry bait laboratory
test method.
Exhibit 16 (TSD # 1.217) - Standard rat anticoagulant place-pack dry bait
laboratory test method.
Exhibit 17 (TSD # 1.218) - Standard mouse anticoagulant place-pack dry bait
laboratory test method.
Exhibit 18 (TSD # 1.205) - Standard Norway rat anticoagulant tracking powder
efficacy laboratory test method.
Exhibit 19 (TSD # 1.212) - Standard house mouse anticoagulant tracking
powder efficacy laboratory test method.
Exhibit 20 (TSD # 1.211) - Standard Norway rat acute tracking powder
efficacy laboratory test method.
D. Rodent fumigants.
The information on the test methods or procedures for evaluating
fumigants (toxic gases) for rodent control is sparse.
Laboratory Methods.
It may be necessary to draw on some of the laboratory procedures
used by entomologists or to rely on the methods used to evaluate eutha-
nasia materials for laboratory animals and unwanted cats and dogs.
Glen and Scott (1973), for example, describe a simple chamber for the
use of carbon dioxide as a means of euthanasia for cats; Smith et al.
(1973) describe an apparatus for anesthetizing small laboratory rodents
which could be readily adapted to evaluate rodent fumigants under lab-
oratory conditions.
Field Methods.
A field test used to evaluate methyl bromide as a .fumigant for ground
squirrels (Berry, 1938) provides a basis for designing field test methods.
A rather comprehensive study on the effects of various fumigants on fleas
was conducted by Steward and Mackie (1938). A woodchuck control experiment
was carried out by de Vos and Merrill (1957) using gas cartridges. Some
early experiments with calcium cyanide on ground squirrels by Sanders
(1921) provide some useful field evaluation techniques. Miller (1954)
used the open hole test to evaluate a number of potential fumigants for
pocket gophers... In the course of research on a method of fumigation using
a rodenticide cartridge,, simulated natural burrow systems-, were constructed
and tested by Fuhr and Silver (1947).
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386
A test design is needed which can be used to evaluate fumigants in the -
laboratory and which would be a suitable technique to use for all rodent
species that may be controlled with fumigants. Field evaluations will differ
widely depending on the species, habitat and intended use practices.
References.
Anon. 1975a. Standard Recommended Practice for Determining Acute Oral LD50
for Testing Vertebrate Control Agents. Annual Book of ASTM Standards,
Part 46. Designation: E555-75. Amer. Soc. for Testing and Materials,
Phila., Pa.
. 1975b. Standard Guideline for Use and Development of
Sodium Cyanide as a Predacide. Annual Book of ASTM Standards, Part 46.
Designation: E553-75. Amer. Soc. for Testing and Materials, Phila., Pa.
Berry, C.E. 1938. Methyl bromide as a rodenticide. Bull. Dept. Agric.
State of Calif. 27(2):172-180.
de Vos, A., and H.A. Merrill. 1957. Results of a woodchuck control experi-
ment. J. Wildl. Manage. 21(4):454-456.
Fuhr, I., and S.D. Silver. 1947. Simulated burrow systems for studies
with rodent pests. J. Wildl. Manage. 7(2):150-153.
Glen, J.B., and W.N. Scott. 1973. Carbon dioxide euthanasia of cats.
British Vet. J. 129:471-479.
Mead-Briggs, A.R., and R.C. Trout. 1975. A field evaluation of the effec-
tiveness of a phosphine fumigant for mole control. AgroEcosysterns 2:1-13.
Miller, M.A. 1954. Poison gas tests on gophers. Calif. Agr. 8:7,14.
Sanders, G.E. 1921. Gopher control by means of calcium cyanide. Agr.
Gaz. Can. 8(6):628-629.
Smith, D.M., K.M. Goddard, R.B. Wilson, and P.M. Newberne. 1973. An
apparatus for anesthetizing small laboratory rodents. Lab. Animal
Sci. 23(6):869-871.
Stewart, M.A., and D.B. Mackie. 1938. The control of sylvatic plague
vectors. Am. J. Hyg. 28(3):469-480.
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387
C. Rodent repellents.
Although few repellents are presently used in rodent control, much
research was undertaken in the past. Examples of some of the test methods
used in evaluating some rodent repellents are discussed; others are cited
in the literature. Since repellents encompass such a wide range of effects,
a complete coverage is not easily attained and has not been attempted here.
Repellents are generally classed by their intended use, the most common
being to protect growing crops, forest or fruit trees, or ornamentals. Other
repellents have been developed for coating seeds to protect them from rodent
depredation. Further, a great deal of effort has.been expended on repellents
for commensal rats and mice to minimize damage to food packages, textiles,
and other materials in storage and shipment, and to building materials and
cables. There are also chemicals which have been explored as area repellents
to keep rodents from entering and/or occupying certain areas.
1. Rodent repellents for protecting living plants.
Although repellents have been explored over many years to protect
growing crops, forest or fruit trees, and ornamentals from various rodents
such as squirrels, porcupines, rabbits, and hares, few test methods have
been reported in the literature.
Cardinell and Hayne (1947) describe field tests for rabbit repellents
using treated twigs and apple trees. The experiments were planned to take
advantage of the analysis of variance methods of statistical comparison.
Later, Hayne (1949) described both pen and field tests for protecting
gardens from cottontail rabbits. Hooven (1966) provided a technique for
evaluating a repellent to protect Douglas fir trees from hares and rabbits.
Dodge et al. (1967) described an experiment for use under semi-field
conditions -to test deer and rabbit repellents on trees. They utilized a ran-
domized complete block design and analyzed the data by analysis of variance.
References.
Cardinell, H.A., and D.W. Hayne. 1947. Pen test of rabbit repellents.
Mich. Agric. Exp. Sta. Q. Bull. 29(4):303-315.
Dodge, W.E., C.M. Loveless, and N.B. Kverno. 1967. Design and analysis
of forest-mammal repellent tests. For. Sci. 13:333-336.
Hayne, D.W. 1949. Tests of repellents for protecting gardens against
cottontail rabbits. Mich. Agric. Exp. Sta. Q. Bull. 31(4):434-440.
Hooven, E.F. .1966. A test of thiram oil two rabbit-infested areas of
Oregon. Tree Planter's Notes 79.:3.
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388
2. Rodent repellents for protecting seed.
Laboratory methods.
Kverno and Hood (1963) published a method of screening seed protectants.
Their testing period, however, was only 3 days, which may have excluded the
discovery of many aversive conditioning repellents. Lindsey et al. (1974)'
conducted laboratory tests on the repellency of Douglas fir seeds coated
with mestranol. He expressed the amount of treated seeds consumed as a
percentage of the untreated seeds eaten. Passof et al. (1974) used the
percentage of treated seeds cracked open as an index of repellency for
deer mice in laboratory tests; less preferred alternate food was provided
to prevent starvation, should the mice be highly repelled by the treated
seeds. Radvanyi (1970) expressed repellency as "percent effectiveness" and
calculated his efficacy using the following formula:
% untreated seeds destroyed - % treated seeds destroyed
% untreated seeds destroyed
Field methods.
In field studies of conifer seed repellents, the repellency is often
measured indirectly by comparing the number of seedlings produced in an
area seeded with treated seeds to that of an area seeded with untreated
seeds (Passof et al., 1974; Lindsey et al., 1974).
Roy (1961) used a seed spot technique to test a,specific seed protectant
under field conditions. The difference in the percentage of treated and
untreated seed that survived was used to evaluate the results.
References.
Dick, J., and W.H. Lawrence. 1957. Protective Seeding with Tetramine-
coated Douglas-fir Seed. Weyerhauser Timber Co. For. Res. Notes. 10 pp.
Dimock, E.J. 1957. A Comparison of Two Rodent Repellents in Broadcast
Seeding Douglas-fir. U.S. For. Serv., Pac. NW For. Range Exp. Sta.
Res. Paper No. 20. 17 pp.
Kverno, N.B. 1954. Development of better seed protectants. J. For.
52:826-827.
Kverno, N.B. and G.A. Hood. 1963. Evaluation Procedures and Standards.
Chemical Screening and Development for Forest Wildlife Damage. U.S.
Dept. Int., Fish and Wildl. Serv., Bur. of Sport Fish and Wildl.,
Branch of Wildl. Res. GPO 836-707. 59 pp.
Lindsey, G.D., R.M. Anthony, and J. Evans. 1974. Mestranol as a repellent
to protect Douglas-fir seed from deer mice. W.V. Johnson, ed. Proc.
Sixth Vert. Pest Conf., Anaheim, Calif. 6:272-279.
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389
Lindsey, G.D. 1977. Evaluation of Control Agents for Conifer Seed Protect-
ion. Pp. 5-13 in Test Methods for Vertebrate Pest Control and Management
Materials, Vol. 1, STP 625. W.B. Jackson and R.E. Marsh, eds. Amer. Soc.
for Testing & Materials, Phila., Pa.
Passof, P.C., R.E. Marsh, and W.E. Howard. 1974. Alpha-naphthylthiourea
as a conditioning repellent for protecting conifer seed. W.V. Johnson,
ed. Proc. Sixth Vert. Pest Conf., Anaheim, Calif. 6:280-292
Redvanyi, A. 1970. A new coating treatment for coniferous seeds. For.
Chron. 46(5):3
Roy, D.F. 1957. Seed spot tests with tetramine-treated seed in northern
California. J. For. 55(6):442-445.
. 1961. Seed Spotting with Endrin-treated Douglas-fir
Seed in Northwestern California. U.S. Dept. Agr. Pac. SW For. Range
Exp. Sta., Berkeley, Calif. Tech. Paper No. 61. 12 pp.
3. Rodent repellents for protecting packaging materials.
just preceding World War II, considerable effort was exerted to develop
commensal rodent repellents or deterrents for the protection of paper and
cardboard packing materials. A number of laboratory test methods was developed
for screening and evaluating chemicals for repellency.
BeHack and DeWitt (1949) published one test method which was used ex-
tensively in early evaluations. This test used laboratory rats housed in
individual cages and offered 2 food cups: one with a standard laboratory chow
and the other with the laboratory chow containing the candidate repellent.
The test period was 4 days, and consumption from each cup was recorded daily.
Bellack and DeWitt (1949) developed an arithmetic expression that yielded a
numerical value for repellency of treated food items. Their test procedure
took into account physiological effects, random feeding, and variations of
animal weights. Welch et al. (1950) used the same technique and compounds.
Feeding tests became a useful procedure for rapidly screening potential
repellents. Barnett and Spencer (1953) used a slight variation of the usual
feeding studies by placing the deterrent below the food item, thereby attempt-
ing to measure the influence of odor alone as a repellent.
The exposure of chemically-treated materials (i.e., containers, etc.)
in large enclosures containing rat populations has been used by a number of
researchers (DeWitt et al., 1950; Welch, 1951; Welch and Duggan, 1952; Welch,
1954).
Stolurow (1948a) described a laboratory testing device called the Pitts-
burgh Obstruction. Unit. He used this unit to.evaluate the effectiveness of
repellents in preventing the destruction .of barriers. The basic principles
of this method have been used'in simpler-and less expensive designs, for
testing repellents on barriers (.Bellack and DeWitt, 1950J . .Stolurow (194Sb.)
prepared..a. .laboratory .method for. the evaluation of roden-t ;dfrbe~r.r.enits». . ..•>.••
image:
390
Analysis of the potential repellency of -various packaging and structural'
materials is discussed by Weeks (1959). Rats are offered food on the opposite"
side of a barrier and are timed to determine how long it takes them to gnaw
through. The time required for barriers treated with the test repellent is
compared to that required for untreated control barriers. Weeks (1959)
developed another repellent test called the graded strip test. A strip of
corrugated paperboard is placed over a row of peanuts so that the rat must
gnaw through the board to get to the peanuts. The further up the strip the
rat gnaws, the less the effectiveness of the repellent. Statistical evalu-
ation was based on the method of biological assay for graded responses used
by Burn et al. (1950).
Tigner and Besser (1962) devised a new quantitative method of evaluating
chemicals as rodent repellents on packing materials. Tigner (1966) later
reported on a series of compounds used to treat multiwall tarps and bags.
He devised a qualitative method of evaluating the chemicals as potential
rodent repellents.
Bull (1972) describes an approach to evaluating attractants and the
influence different odors have in inducing Norway rats to feed at given
locations. In this study the effect of certain repellents was also
examined.
References.
Barnett, S.A., and M.M. Spencer. 1953. Responses of wild rats to offensive
smells and tastes. Br. J. Anim. Behav. 1:32-37.
Bellack, S., and J.B. DeWitt. 1949. Rodent repellent studies. I. Method
for the evaluation of chemical repellents. J. Am. Pharm. Assn. 38:109-112.
. 1950. Rodent repellent studies. III. Ad-
vanced studies in the evaluation of chemical repellents. J. Am. Pharm.
Assn. 39:197-202.
Bull, J.O. 1972. The influence of attractants and repellents on the
feeding behavior of Rattus norvegicus. R.E. Marsh, ed. Proc. Fifth
Vert. Pest Conf./ Fresno, Calif. 5:154-160.
Burn, J.H., O.J. Finney, and L.D. Goodwin. 1950. Biological Standard-
ization. 2nd Ed. Oxford Univ. Press: London. (pp. 32-48).
DeWitt, J.B. 1958. Rodent control. Soap 33(6) .-87,89,92.
DeWitt, J.B., E. Bellack, and J.F. Welch. 1953. Rodent repellent
studies. IV. Properties and preparation of trinitrobenzene-aryl
amine complexes. J. Am. Pharm. Assn. 42:695-697.
DeWitt, J.B., J.F. Welch, and E. Bellack. 1950. Rodent repellency.
Mod. Packag. 23(9):123-126.
image:
391
Fern, J.E., and J.B. DeWitt. 1965. Correlation between chemical structure
and rodent repellency of benzoic acid derivatives. J. Agric. Food Chem.
13:116-117.
Stolurow, L.M. 1948a. A Standard Laboratory Method for the Evaluation
of Proposed Rodent Deterrents. Dept. Psych., Univ. Pittsburgh, Res.
and Development Contract No. W44-109-qm-1058; O.I. 4598.
. 1948b. Rodent behavior in the presence of barriers. J. Comp.
Physiol. Psychol. 41(3):219-231.
Tigner., J.R. 1966. Chemically treated multiwall tarps and bags tested
for rat repellency. J. Wildl. Manage. 30(1):180-184.
Tigner, J.R., and J.F. Besser. 1962. A quantitative method for evalu-
ating chemicals as rodent repellents on packaging materials. J. Agric.
Food Chem. 10:484-486.
Weeks, J.R. 1957. Quantitative evaluation-of repellency of chemical ' "
coatings on paperboard. J. Pharm. Exp. Therap. 119:193.
. 1959. Quantitative evaluation of repellency of chemical
coatings on paperboard. J. Agric. Food Chem. 7(3):193-196.
Welch, J.P. 1951. Rat-repellent findings. Mod. Packag. 24(9):138-140.
. 1954. Rodent control. J. Agric. Food Chem. 2(3):142-149.
Welch, J.F., J.B. DeWitt, and E. Bellack. 1950. Rat deterrents for
paper packages. Soap and Sanitary Chemicals, April, May. 6 pp.
Welch, J.F., and E.W. Duggan. 1952. Rodent-resistant vinyl films.
Mod. Packag. 25(6):130-131.
4. Rodent repellents for protecting wire and cable coatings.
Anthony and Tigner (1967) briefly describe both laboratory and field
tests of a repellent used in various types of communication wire or cables.
Field studies involved a number of geographical areas, which was essential
because of the different rodent species involved. Also, environmental condi-
tions associated with severe cable damage vary from region to region.
Howard (1953) conducted laboratory tests of pocket gophers gnawing
electric cables. His test method confined pocket gophers to one part of
a cage; a short section of cable was positioned and fastened to block the
passageway to the remainder of the cage. By this means data could be
obtained for treated and untreated cables and for cables with different
types of covering or insulations.
image:
392
References.
Anthony, C., and J.R. Tigner. 1967. Rodent repellent cable coating
development. Wire and Wire Products. Haire Publ. Company, Inc.:
New York.
Howard, W.E. 1953. Tests of pocket gophers gnawing electric cables.
J. Wildl. Manage. 17(3):296-300.
Tigner, J.R., and R.E. Landstrom. 1968. Chemical protection methods
progress. Electronic Packaging and Production, April.
D. Rodent reproductive inhibitors.
A reproductive inhibitor can be defined as an agent that can cause
permanent or temporary sterility in either or both sexes, or, through some
other physiological mechanism, reduce the number of offspring or alter the
fecundity of the offspring produced.
The numerous compounds capable of inhibiting reproduction in various
rodent species have diverse and complex modes of action (Jackson, 1959;
Saunders, 1968). Many of these compounds do not lend themselves to standard
laboratory or field evaluation methods. Both Jackson and Saunders provide
reviews of the capabilities of various compounds.
Because of the intensive search for oral contraceptives for humans, a
large number of references are available. The literature cited illustrates
representative methods of measuring the physiological effects of compounds
on inhibiting reproduction in rodents commonly used in the laboratory.
Laboratory methods.
Histological evaluations of the effects of chemicals on cellular modi-
fications or makeup of reproductive systems of rodents is a procedure used
in some laboratories. However, histological evaluation is laborious and
expensive when conducting preliminary testing or screening new compounds
(Hershberger et al., 1969). Studies of the testes in treated rats are ex-
amples of this method in determining the effects of certain chemosterilants
(Pate and Hays, 1968; Ericsson, 1970; Ericsson and Baker, 1970; Gibson et
al., 1967).
Many researchers have used the number of pregnant females, implan-
tations and/or the number of rats that ovulated to determine the success
of several orally active estrogens and progestins (Kind and Dorfman,
1965). Banik et al. (1969) made a statistical analysis of the data from
the females with ova or implantations using the four-fold contingency test
tables of Mainland and Murray (1952). The average number of ova or im-
plantation sites in each rat in the experimental series was compared with
those of the controls.
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393
Boris et al. (1971) demonstrated a method which may have merit in
establishing more precise effects of compounds on ovulation by using the
pubertal ovulation in the rat for the study of anti-ovulatory compounds.
Uterotrophic responses as measured by the mean uterine ratio (milligrams
of uterus per gram of body weight) is a means of expressing the estrogen-
icity of certain potential chemosterilants (Rooks et al., 1968; Saunders
and Rorig, 1964).
Efficacy of potential rodent chemosterilants can be analyzed in
laboratories by using some of the following indices: average number of
days from mating to the appearance of first litters; average total off-
spring produced per female; average number of litters produced per female
(Generoso et al., 1971; Howard and Marsh, 1969). Survival of the off-
spring and their reproductive capabilities may also be used to determine
efficacy expressed in the Fj_ generation (Kinel et al., 1965; Clancy and
Edgren, 1968). These are only some of the commonly used criteria for
efficacy.
Where a compound may affect spermatogenesis, efficacy of that com-
pound may be determined by using a serial mating method. This technique
involves placing the treated male with 1 or 2 females for a prescribed
number of days and then moving him on to new females for a similar period.
Hi us, by pairing the males with a series of females, it is possible to
determine the time period in which sterility occurred and its duration
(Hershberger et al., 1969).
Field tests (simulated).
Tests of a rodent chemosterilant may be conducted in large pens where
small populations can be maintained and the tests can be replicated (Kenale
et al., 1973; Gwynn, 1972a). Studies should be replicated a number of times
under conditions where evaluations of effects can be exacting.
Field methods.
While all the methods cited have value, some may be more applicable
than others. The characteristics of the compound, the proposed site, the
intended pest, and the method of application will all influence the selection
of the proper method.
Marsh and Howard (1969) evaluated mestranol as a reproductive inhibitor
in Norway rats in garbage dumps. Two dumps were treated and a third served
as a control. The age structure of the rat population and populuation
numbers were the criteria used to determine efficacy. Bowerman and Brooks
(1971) carried out pen and field efficacy studies on Norway rats using quines-
trol. Gwynn. (1972a, 1972b). published pen and field results using still a
different rodent chemosterilant.
These represent a portion of field studies which have been -con-ducted. •
Field evaluations differ slightly from one another to. meet the existing..
situations .-and. the intended .uses.of the test compounds. .... . ...•-•-"'
image:
394
Significant reproduction inhibition assumes that comparable untreated
populations are used as conrols to permit a sound statistical analysis.
The Chi square analysis of the two-way analysis of variance might be the
appropriate measure or the significant differences between the treated
and control groups. Efficacy tests should be replicated to demonstrate
a high degree of reproducibility results under various conditions of use.
Potential chemosterilants also should be tested in widely separated
geographical areas. Because evidence exists that many potential chemo-
sterilants can be quite specific in their effects (Skinner, 1968), all
species on which the product is to be used must be tested both in the
laboratory and in the field.
References.
Asdell, S.A., H. Doornenbal, S.R. Joshi, and G.A. Sperling. 1967. The
effects of sex steroid hormones upon longevity in rats. J. Reprod.
Fert. 14:113-120.
Banik, U.K., C. Revesz, and F. Herr. 1969. Orally active estrogens and
progestins in prevention of pregnancy in rats. J. Reprod. Fert.
18:509-515.
Barnes, L.E., and R.K. Meyer. 1962. Effects of ethamoxytriphetol, MRL-37,
and clomiphene on reproduction in rats. Fertil. Steril. 13:472-480.
Boris, A., T. Trmal, and E.W. Nelson, Jr. 1971. Utility of pubertal
ovulation in the rat for the study of anti-ovulatory compounds.
Contraception 4(2}-.97-103.
Borkovec, A. 1972. Safe Handling of Insect Chemosterilants in Research
and Field Use. Agr. Res. Serv., U.S. Dept. Agr. ARS-NE-2.
Bowerman, A.M., and J.E. Brooks. 1971. Evaluation of U-5897 as a male
chemosterilant for rat control. J. Wildl. Manage. 35(4):618-624.
Brooks, J.E., and A.M. Bowerman. 1969. Chemosterilants in rodent control.
Soap Chem. Spec, pp. 58, 60, 62, 64, and 80.
1971. Estrogenic steriod used to inhibit
reproduction in wild Norway rats. J. Wildl. Manage. 35(3):444-449.
Chang, M.C., and R. Yanagimachi. 1965. Effect of estrogens and other
compounds as oral antifertility agents on the development of rabbit ova
and hamster embryos. Fertil. Steril. 16:281-291.
Clancy, D.P., and R.A. Edgren. 1968. The effects of norgestrel, ethinyl
estradiol, and their combination, ovral, on lactation and the offspring
of rats treated during lactation. Intern. J. Fert. 13(2):133-141.
Coppola, J.A., and J.L. Ball. 1967. The efficacy of two non-steriodal
antifertility agents after topical administration in rats. J. Reprod.
Fert. 13:373-374.
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395
Crabo, B., and L.E. Appelgran. 1972. Distribution of [14C]alpha-chloro-
hydrin iri mice and rats. J. Reprod. Pert. 30:161-163.
Danezis, J. 1968. Antifertility agents and mammalian gonads - a proposal
for assessing the effectiveness of antifertility compounds. Intern.
J. Pert. 13(2):95-102.
Davies, A.G. 1971. Histological changes in the seminiferous tubules of
immature mice following administration of gonadotrophins. J. Reprod.
Pert. 25:21-28.
Davis, D.E. 1961. Principles for population control by gametocides.
Pp. 160-167 in Trans. 26th North Am. Wildl. and Nat. Resources Conf.,
March 6 Tech. Sessions.
Desaulles, P.A., and C. Krahenbuhl. 1964. Comparison of the anti-fertil-
ity and sex hormonal activities of sex hormones and their derivatives.
Acta Endocr. 47:44-456.
Driesbach, R.H. 1959. The effects of steriod sex hormones on pregnant
rats. J. Endocr. 18:271-277.
Drevius, L.O. 1971. The 'sperm-rise1 test. J. Reprod. Pert. 24:427-429.
Duncan, G.W., and B.B. Pharriss. 1970. Effect of nonsteroidal compounds
on fertility. Fed. Proc. 29(3):1232-1239.
Emlen, J.T., and D.E. Davis. 1948. Determination of reproductive rates
in rat populations by examination of carcasses. Physiol. Zool. 21(1):
59-65.
Ericsson, R.J. 1970. Male antifertility compounds: U-5897 as a rat
chemosterilant. J. Reprod. Fert. 22:213-222.
Ericsson, R.J., and V.F. Baker. 1970. Male antifertility compounds:
biological properties of U-5897 and U-15,646. J. Reprod. Fert.
21:267-273.
Ericsson, R.J., and N.D. Connor. 1969. Lesions of the rat epididymis and
subsequent sterility produced by U-5897 (3-chloro-1,2-propanediol).
Page 25 In Abstracts of Papers, Soc. Study of Reprod., Second Ann.
Meeting, Davis, California.
Ericsson, R.J., and G.A. Youngdale. 1970. Male antifertility compounds:
structure and activity relationships of 0-5898, U-15,646 and related
substances.. J. Reprod. Pert. 21:263-266.
Ericsson, R.J., H.E. Downing, R.E. Marsh, and W.E. Howard. 1971. Bait
acceptance by .rats of .microencapsulated male sterilant .alpha-chlorohy-
drin. J. Wildl. Manage. 35.(3) :573-576. •.-.,.
image:
396
Generoso, W.M., S.K. Stout, and S.W. Huff. 1971. Effects of alkylating
chemicals on reproductive capacity of adult female mice. Mutat. Res.
13:171-184.
Giannina, T., B.C. Steinetz, and A. Meli. 1967a. General biology of
quinestrol. Intern. J. Pert. 12(2):142-147.
. 1967b. Pathway absorption of
orally administered ethinyl estradiol and quinestrol in the rat.
Intern. J. Fere. 12(2):155-157.
Giannina, T., and A. Meli. 1969. Prolonged estrogenic activity in rats
after single oral administration of ethinyloestradiol-3-cyclopentyl
ether. J. Pharm. Pharmacol. 21:271-272.
Gibson, J.P., J.W. Newberne, W.L. Kuhn, and J.R. Elsea. 1967. Compara-
tive chronic toxicity of three oral estrogens in rats. Toxicol. Appl.
Pharmacol. 11(3):489-510.
Gilmore, D.P., R.H. Hooker, and M.C. Chang. 1971. A technique for the
collection by fistula of epididymal spermatozoa from the rat. J. Reprod.
Fert. 25:137-140.
Goulet, L.A. and R.M.F.S. Sadleir. 1974. The effects of a chemosterilant
(mestranol) on population and behavior in the Richardson's ground squirrel
(Spermophilus richardsonii) in Alberta. W.V. Johnson, ed. Proc. Sixth
Vert. Pest Conf., Anaheim, Calif. 6:90-100. .-
Gwynn, G.W. 1972a. Effects of a chemosterilant on fecundity of wild
Norway rats. J. Wild. Manage. 36(2):550-556.
. 1972b. Field trial of a chemosterilant in wild Norway rats.
J. Wildl. Manage. 36(3):823-828.
Gwynn, G.W., and S.M. Kurtz. 1970. Acceptability and efficacy of an
antifertility agent in wild Norway rats. J. Wildl. Manage. 34(3) :
514-519.
Hershberger, L.G., D.M. Hansen, and L.M. Hansen. 1969. Effects of anti-
fertility agents on male mice as determined by serial mating method
(33948). Proc. Soc. Exp. Biol. Med. 131:667-669.
Howard, W.E. 1967a. Biological control of vertebrate pests. M.W. Cummings,
ed. Proc. Third Vert. Pest Conf., San Francisco, Calif. 3:137-157.
. 1967b. Biocontrol and chemosterilants. Pp. 343-386 in Pest
Control. Academic Press Inc.: New York.
. 1968. Controlling rodents biologically and with sound. Pp.
220-241 in Asia-Pacific Interchange Proc., Rodents as Factors in Disease
and Economic Loss. Honolulu, Hawaii.
Howard, W-..E-.•, and R.E. Marsh. 1969. Mestranol as a reproductive inhibitor
in rats and voles. J. Wildl. Managev- 33:403-408. •, . . '•
'
image:
397
Jackson, H. 1959. Antifertility substances. Pharmacol. Rev. 11:135-172.
. 1973. Chemical methods of male contraception. Am.
Sci. 61(2):188-193.
Jackson, H., B.W. Fox, and A.W. Craig. 1961. Antifertility substances and
their assessment in the male rodent. J. Reprod. Pert. 2:447-465.
Jackson, H., and A.W. Craig. 1969. Effects of alkylating chemicals on
reproductive cells. Ann. N.Y. Acad. Sci. 160:215-227.
Jackson, H., and H. Schnieden. 1968. Pharmacology of reproduction and
fertility. Ann. Rev. Pharmacol. 8:467-490.
Kaltreider, D.F. 1968. Clinical evaluation of ethinylestrenol and mestranol
as a contraceptive. Fertil. Steril. 19(4):589-592.
Kendle, K.E., A. Lazarus, P.P. Rowe, J.M. Telford, and O.K. Vallance. 1973.
Sterilization of rodent and other pests using a synthetic oestrogen.
Nature 244:105-108.
Kennelly, J.J., M.V. Garrison, and B.E. Johns. 1970. Laboratory studies
of the effect of U-5897 on the reproduction of wild male rats. J. WildL.
Manage. 34(3):508-513.
Kennelly, J.J., B.E. Johns, and M.V. Garrison. 1972. Influence of sterile
males on fecundity of a rat colony. J. Wildl. Manage. 36(1):161-165.
Kind, F.A., and R.I. Dorfman. 1965. Antifertility activity of various
steroids in the female rat. J. Reprod. Fert. 10:105-113.
Kincl, F.A., A.F. Pi, M. Magneo, L.H. Lasso, A. Oriol, and R.I. Dorfman.
1965. Inhibition of sexual development in male and female rats treated
with various steroids at the age of five days. Acta Endocr. 49:193-206.
Knipling, E.F. 1959. Sterile-male method of population control. Science
130:902-904.
Knipling, E.F., and J.U. McGuire. 1972. Potential Role of Sterilization
for Suppressing Rat Populations. A Theoretical Appraisal. Agr. Res.
Serv.,. U.S. Dept. Agr., Tech. Bull. 1455. U.S. Govt. Print. Off.
Marsh, R.E., and W.E. Howard. 1969. Evaluation of mestranol as a repro-
ductive inhibitor of Norway rats in garbage dumps. J. Wildl. Manage.
33:133-138.
_- ^_ . 1970. Chemoster.ilants as.an approach to
rodent control'. R. Dana, ed. Proc.. Fourth .Vert. Pest Cbnf., West
Sacramento, Calif. 4:55-63. '. .
_^_ . • ' . .'•:... ' ..'..» 1973.. .Prospects of. ch.'emos.ter.ilan,t -and
genetic control..pof rodents.. •'.Bull... W..H.O.'.:48.:;3-09—3.T6.
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Mainland, D., and I.M. Murray. 1952. Tables for use in four-fold contingency
tests. Science 116:591.
Meli, A., D.I. Cargill, T. Giannina, and E.G. Steinetz. 1968. Studies on
the transport of estrogens by the rat small intestine in vivo (33463).
proc. Soc. Exp. Biol. Med. 129:937-944.
Mischler, T.W., P. Welaj, and P. Nemith. 1971. Biological evaluation of
two estrogenic steroids as possible rodent chemosterilants. J. Wildl.
Manage. 35(35:449-454.
Monro, J. 1963. Population control in animals by overloading resources
with sterile animals. Science 140:496-497.
Pate, B.D., and R.L. Hays. 1968. Histological studies of testes in
rats treated with certain insect chemosterilants. J. Econ. Entomol.
61(1):32-34.
Peterson, D.L., R.A. Edgren, and R.C. Jones. 1964. Steroid-induced
block of ovarian compensatory hypertrophy in harnicastrated female
rats. J. Endocr. 29:255-262.
Pimlott, D.H., and H.W. Mossman. 1959. A macroscopic ovary-sectioning
method. J. Wildl. Manage. 23(2):232-233.
Pingale, S.V., K. Krishnamurthy, and T. Ramasivan. 1967. Rats. Bull.
Grain Technol. Foodgrain Tech. Res. Assoc. of India, Hapur :(U.P.).
91 pp.
Powers, J.B. 1970. Hormonal control of sexual receptivity during the
estrous cycle of the rat. Physiol. Behav. 5:831-835.
Rooks, W.H., S.L. Kugler, and R.I. Dorfman. 1968. The relative expressed
estrogenicity of oral contraceptives. Pertil. Steril. 19(3):419-423.
Rowley, I. 1968. Studies on the resurgence of rabbit populations after
poisoning. C.S.I.R.O. Wildl. Res. 13:59-69.
Saksena, S.K., and R.R. Chaudhury. 1969. Effect of orally administered
azasteroids on implantation in rats. J. Reprod. Fert. 19:177-178.
Saunders, F.J. 1968. Effects of sex steroids and related compounds on
pregnancy and on development of the young. Physiol. Rev. 48(3) :601-643.
Saunders, F.J., and R.L. Elton. 1967. Effects of ethynodiol diacetate
and mestranol in rats and rabbits, on conception, on the outcome of
pregnancy and on the offspring. Toxicol. Appl. Pharmacol. 11:229-244.
Saunders, F.J., and K. Rorig. 1964. Separation of antifertility and
classic estrogenic effects. Fertil. Steril. 15(2):202-205.
image:
399
Skinner, W.A. 1968. QiemosterHants in rodent control. Pp. 192-201 in
Asia-Pacific Interchange Proc., Rodents as Factors in Disease and Economic
Loss. Honolulu, Hawaii.
Scrivastava, A.S. 1966. Mixture of furadantin and colchicine acts as
effective chemosterilant against male or female field rat. Sci. Technol.
4(3):178-180.
Steinetz, B.G., A. Meli, T. Giannina, V.L. Beach, and J.P. Manning. 1967.
Distribution of radioactivity in body fat and organs of rats treated with
labeled quinestrol, ethynylestradiol or 17-beta-estradiol (31678). Soc.
Exp. Biol. Med. 124:111-117.
Turner, M.A. 1971. Effects of alpha-chlorhydrin upon the fertility of
spermatozoa of the cauda epididymidis of the rat. J. Reprod. Fert.
24:267-269.
Vickery, B.H., G.I. Erickson, and J.P. Bennett. 1974. Mechanism of anti-
fertility action of low doses of alpha-chlorhydrin in the male rat.
J. Reprod. Fert. 38:1-10.
VI. Mammalian predacides, repellents, reproductive inhibitors, and
devices.
Research in predator damage control has only recently attempted to pro-
vide information on the extent of predation and possible alternative solutions
to past and present control methods. Balser (1974a, 1974b) has reviewed the
status of coyote control, data on livestock losses, and research related to
control methodology. His review and others (Cummings, 1966; Howard, 1974;
Knowlton, 1972) have pointed to the paucity of data necessary for predator
management. Methods without undesirable characteristics have not yet been
developed.
Changing land-use patterns and land development have affected the habitats
and food choices available to both predator and prey animals. Consequently,
maintenance of wild animal populations above levels sustained by available
natural foods encourages predation on domestic livestock. Control of animal
damage to protect habitats, other wild species, and domestic products is
basic to sound management and includes regulation of game and non-game popu-
lations. It is an essential element in carnivore management and must be
considered in the light of current and developing land-use patterns.
Among the larger carnivores that cause damage and resulting management
problems .are the families Canidae, Felidae, Ursidae, and Mustelidae. Bobcats
(Lynx rufus) may prey extensively at times on domestic animals and lynx
(Lynx canadensis) may occasionally cause -damage, but offending animals usually
can be managed by mechanical methods. Bears -(Oraus spp.) and mountain lions
•(.Felis concolor)- may cause extensive damage a.t •• times..but ,.a-re. typically con-
sidered game animals. Again, offending animals.-are.managed- by mechanical
methods sanctioned by State wildlife agencies. ,_. . _..
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400
The carnivores of primary interest here are coyotes (Canis latrans),
gray foxes (Urocyon cinereoargenteus), red foxes (Vulpes fulva), feral dogs
(Canis familiaris and various hybrids). Ihey are particularly difficult to
manage because of their inherent caution, adaptability, and high reproductive
rates.
Evaluation of the efficacy of various control methods is extremely diffi-
cult because of the wide variation in circumstances where controls are applied.
Heavily populated areas with small farms present a markedly different environ-
ment than open range with large land holdings and few humans. Climate and
weather, habitat types, predator populations, reproductive rates, food bases,
and feeding habits are soine of the variables that often prevent replication
of experiments under similar conditions. Another major impediment is the
lack of adequate census techniques to measure populations and the changes
induced by natural and applied controls.
Development of efficacy criteria for control methods will require cogni-
zance of time and cost factors caused by the complex nature of predator-prey
ecology and the extreme difficulty in securing uniform replicate field tests.
Several years of study over extensive geographic areas under widely-varied
climatic conditions will be needed to provide even minimal knowledge of
predator-prey economics, behavior, and ecology. A review of information and
data from field studies and other sources, such as consensus standards (e.g.,
American Society for Testing and Materials) may provide adequate guidelines
for protection of nontarget species and the environment.
To summarize, effective canid control methods vdiich meet all desired
criteria do not exist. Known methods applied to minimize undesirable
effects may provide efficacy data and essential damage control. Effective
and useful methods vary with time and circumstance and may require removal
of specific offending animals or local population reduction to achieve
adequate depredation control.
References.
Arion. 1975a. Standard Guideline for Use and Development of Sodium Cyanide
as a Predacide. Annual Book of ASTM Standards, Part 46. Designation:
E553-75. Amer. Soc. for Testing and Materials, Phila., Pa.
. 1975b. Standard Method of test for Efficacy of Acute
Mammalian Predacides. Annual Book of ASTM Standards, Part 46.
Designation: E552-75. Amer. Soc. for Testing and Materials, Phila., Pa.
. 1975c. Standard Recommended Practice for Determining
Acute Oral LD50 for Testing Vertebrate Control Agents. Annual Book of
ASTM Standards, Part 46. Designation: E555-75. Amer. Soc. for Testing
and Materials, Phila., Pa.
Balser, D.S. 1974a. A review of coyote control research. W.V. Johnson,
ed. Proc. Sixth Vert. Pest Conf., Anaheim, Calif. 6:171-174.
1974b. An overview of predator-livestock problems with
emphasis on livestock losses. Trans. 39th N. Amer. Wildl. and Nat.
Res. Conf. Wildl. Mgrat. Inst., Wash., D.C.
image:
401
Berryman, J.H. 1971. Affidavits of Jack H. Berryman. Pages 217, 219,
221, 228. Predator Control Hearings. Comm. on Agr., House of Rep.,
93rd Congress. Sept. 18, 20, 21. Serial No. 93-DD. U.S. Govt. Print.
Off., Wash., D.C.
Cain, S. A. (Committee Chairman). 1972. Predator Control - 1971.
Report to the Council on Environmental Quality and the Department
of the Interior by the Advisory Committee on Predator Control,
Washington, O.C. 207 pp.
Carley, C.J. 1973. Development of coyote census techniques. Paper
presented at Wildl. Soc. and Soc. for Range Mgmt. meeting. Dec. 7,
Englewood, Colo. 17 pp. (mimeo.)
Clark, F.W. 1972. Influence of jackrabbit density on coyote population
change. J. Wildl. Manage. 36(2):343-356.
Cook, R.D., M. White, D.O. Trainer, and W.C. Glazener. 1971. Mortality
of young vftiite-tailed deer fawns in south Texas. J. Wildl. Manage.
35(1):47-55.
Cummings, M.W. 1966. Principles of animal damage economics and control.
Pp. 235-238 in Trans. 32st No Amer. Wildl. Conf. The Wildl. Mgmt.
Inst., Wash., D.C.
Dolnick, E.H., R.L. Medford, and R.J. Schied. 1976. Bibliography on
the Control and Management of the Coyote and Related Canids with
Selected References on Animal Physiology, Behavior, Control Methods and
Reproduction. Bibliography is available upon request from U.S. Dept.
Agr., Agr. Res. Serv., Protein Nutrition Lab., Nutrition Inst.,
Beltsville, Md. 20705.
Gates, N.L. 1977. Chemosterilant Evaluation in Coyotes: Parameters
for Animal Selection and Laboratory Methodology. Pp. 106-113 in
Test Methods for Vertebrate Pest Control and Management Materials.
Vol. 1, STP 625. W.B. Jackson and R.E. Marsh, eds. Amer. Soc. for
Testing and Materials, Phila., Pa.
Gurba, J.F. 1974. Rabies vector control in Alberta. W.V. Johnson, ed.
Proc. Sixth Vert. Pest Conf., Anaheim, Calif. 6:161-170.
Howard, W.E. 1974. The Biology of Predator Control. Addison-Wesley
Module in Biol. (11). Addison-Wesley Publ. Co. Inc.: Reading, Mass.
Kadlec, J.A. 1971. Effects of introducing foxes and raccoons on herring
gull colonies. J. Wildl. Manage. 35(4):625-635.
Knowlton, F.F. 1972. Preliminary interpretation of coyote population
mechanics with some management implications. J. Wildl. Manage. 36(2):
369-382.
image:
402
. 1973a. Using population mechanics in management schemes.
Pp. 11-16 in Proc. Great Plains Wildl. Damage Control Workshop. Kansas
State Univ., Manhattan. 81 pp.
. 1973b. Coyote research newsletter. Vol. 1(1). Unit of
Pred. Studies, Bur. Sport Fish, and Wildl., Dept. Wildl. Sci., Utah
State Univ.-UMC52, Logan 84322.
Leopold, A. S. 1964. Predator and Rodent Control in the United States.
N. Amer. Wildl. and Nat. Res. Conf. 29:27-49.
Linhart, S.D., and W. Robinson. 1972. Some relative carnivore densities
in areas under sustained coyote control. J. Wildl. Manage. 53(4):
880-884.
Nesse, G.E. 1973. Predation and the Sheep Industry in Glenn County,
California. M.S. Thesis. Univ. of Calif., Davis.
Robinson, W.B. 1953. Population trends of predators and fur animals in
1080 station areas. J. Wildl. Manage. 34(2):220-227.
. 1961. Population changes of carnivores in some coyote
control areas. J. Wildl. Manage. 42(4):510-515.
Robinson, W.B., and E.F. Grand. 1958. Comparative movements of bobcats
and coyotes as disclosed by tagging. J. Wildl. Manage. 22(2):117-122.
Robinson, W.B., and M.V. Cummings. 1951. Movements of Coyotes to and
from Yellowstone National Park. Spec. Sci. Rep. Wildl. No. 11. .
U.S. Dept. Int., Washington, D.C.
Seyler, K., and C. Niemeyer. 1974. Emergency rabid skunk control in
Montana. Proc. Sixth Vert. Pest Conf., Anaheim, Calif. 6:198-203.
Timm, R.M., W.E. Howard, M.W. Monroe, Roy Teranishi, and E.L. Murphy.
1977. A Method for Evaluating Coyote Scent Baits. Pp. 145-151
in Test Methods for Vertebrate Pest Control and Management Materials.
W.B. Jackson and R.E. Marsh, eds. Vol. 1, STP 625. Amer. Soc. for
Testing and Materials, Phila., Pa.
U.S. Department of the Interior. 1974. Coyote Damage Control by
Mechanical Techniques. Div. Wildl. Serv., Bur. Sport Fish, and Wildl.
Spec. Rep. January 25. 28 pp. (mimeo.)
. 1974. Emergency relief for woolgrowers authorized (News release)
May 30. 2 pp.
Wagner, F.H., and L.C. Stoddart. 1972. Influence of coyote predation
on black-tailed jackrabbit populations of Utah. J. Wildl. Manage.
36(2):329-342.
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403
. 1973. Predator Control Study, Final Report. Four Corners
Regional Commission. June 30. Contract FCRC No. 621-366-044. Utah
State Univ., Logan. 47 pp.
Wolfe, C.J. 1973. A Literature Review on Coyote Census Techniques.
Tech. paper. Dept. Fish. Wildl. Biol., Colo. State Univ., Fort Collins.
November, 24 pp. (mimeo.)
. 1974. Siren-elicited Howling Response as a Coyote Census
Technique. M.S. Thesis. Dept. Fish. Wildl. Biol., Colo. State Univ.,
Fort Collins.
Young, S.P., and H.H.T. Jackson. 1951. The Clever Coyote. Wildl. Mgmt.
Inst., Wash., D.C. 411 pp.
A. Mammalian predacides.
Laboratory methods for evaluating the toxicology and mode of action
of candidate chemicals are well established and will not be considered
here except as related to mode of application. Consensus standards devel-
oped by the American Society for Testing and Materials may provide addi-
tional guidance in evaluation of laboratory efficacy.
Atzert (1971), Crabtree (1962), Robinson (1970), Ward and Garlough
(1936), and Ward and Spencer (1947), have summarized the toxicology and
past use patterns of toxic chemicals to control canids. Field research
data are limited. Experimental efficacy studies of the M-44 (sodium
cyanide spring-loaded ejector mechanism) have been developed in several
western states to evaluate its use in management of red foxes and coyotes.
As a result of the efficacy studies, the Environmental Protection Agency
has approved registrations for use of the sodium cyanide M-44 device.
No specific complete test protocols exist for field evaluation of
toxic chemicals for predator management. Survey research data related to
domestic livestock losses and relative effects of toxic baiting on coyote
populations have been reported by Robinson (1948, 1953a, 1953b, 1961)
and by Linhart and Robinson (1972). Effects on target and nontarget
species populations are evaluated from trap-catch data and observations.
Suggested methods for baiting to reduce hazards to nontarget species are
given. The use of more than one control method in survey areas confounds
the evaluation of a single method. The scope of the reports does provide
extremely useful information on the effects of control and animal popu-
lation trends over a time span.
Arrington and Edwards (1951) employed antelope population surveys to
evaluate effectiveness of predator reduction on antelope productivity.
Toxic chemicals were the primary reduction method, but overlapping use of
more than one chemical, as well as other methods, prevent-ed isolation of
•the effects of each method. Coyote population reduction was'reported to
result, in greatly increased antelope productivity; however, coyote popula-
tions .were not indexed. . •
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404
Beasom (1973a,b) discussed relative effectiveness and .sele.ctiv.ity of ,
various methods of predator reduction to enhance wild turkey and 'deer pro-
ductivity. High selectivity by the M-44 was noted.
Balser et al. (1968) reported results of a well-designed field study of
predator reduction and waterfowl nesting success. Several reduction methods
were used. Selectivity was not thoroughly discussed, but a similar design
would be useful in evaluating specific methods if experimental areas were
adequate.
Field tests of baits must consider food preference and availability to
target species. Bait acceptance by extremely wary individuals or species
usually requires that the chemical either be masked or be relatively tasteless.
Also seasons, weather, temperature, and other factors affect bait acceptance.
References.
Anon. 1975a. Standard Guideline for Use and Development of Sodium Cyanide
as a Predacide. Annual Book of ASTM Standards, Part 46. Designation:
E553-75. Amer. Soc. for Testing and Materials, Phila., Pa.
. 1975b. Standard Method of Test for Efficacy of Acute Mammalian
Predacides. Annual Book of ASTM Standards, Part 46. Designation:
E552-75. Amer. Soc. for Testing and Materials, Phila., Pa.
. 1975c. Standard Recommended Practice for Determining Acute Oral LD50
for Testing Vertebrate Control Agents. Annual Book of ASTM Standards,
part 46. Designation: E555-75. Amer. Soc. for Testing and Materials,
Phila., Pa.
Arrington, O.N., and A.E. Edwards. 1951. Predator control as a factor in
antelope managment. Trans. N. Amer. Wildl. Nat. Resour. Conf. 16:179-193.
Atzert, S.P. 1971. A Review of Sodium Monoflouoroacetate (Compound 1080),
Its Properties, Toxicology and Use in. Predator and Rodent Control. Spec.
Sci. Rpt. Wildl. No. 146. Div. Wildl. Serv., Bur. Sport and Fish. Wildl.,
Washington, D.C. 34 pp.
Balser, D.S., H.H. Dill, and H.K. Nelson. 1968. Effects of predator re-
duction on waterfowl nesting success. J. Wildl. Manage. 32(4):669-682.
Beasom, S.L. 1973a. Selectivity of predator control techniques in South
Texas. Pp. 333-336 in Predator Control Hearings: Comm. on Agr., House of
Rep., 93rd Congress. Sept. 18, 20, 21. Serial No. 93-DD. U.S. Govt.
Print. Off., Washington, D.C.
Beasom, S.L. 1973b. The effect of predator removal on whitetailed deer
productivity. Pp. 337-344 in Predator Control Hearings: Comm. on Agr.,
House of Rep., 93rd Congress. Sept. 18, 20, 21. Serial No. 93-DD. U.S.
Govt. Print. Off., Washington, D.C.
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405
Connolly, G.E. , and W.H. Longhursto 1975. The effects of control on
coyote populations; a simulation model. Univ. Calif. Div. Agric. Sci.
Bull. 1872. 37 pp.
Crabtree, D.G. 1962. Review of current vertebrate pesticides. Proc.
First Vert. Pest Conf., Sacramento, Calif. 1:327-361.
Dolnick, E.H., R.L. Medford, and R.J. Schied. 1976. Bibliography on the
Control and Management of the Coyote and Related Canids with Selected
References on Animal Physiology, Behavior, Control Methods and Reproduction'.
Bibliography is available upon request from U.S. Dept. Agr.', Agr. Res.
Serv., Protein Nutrition Lab., Nutrition Instit., Beltsville, Md. 20705. ...
Knowlton, F.F. 1973. Coyote Research Newsletter. Vol. 1(1). Unit of
Pred. Studies, Bur. Sport Fish and Wildl., Dept. Wildl. Sci., Utah State
Univ. UMC52, Logan.
Lewis, J.C. 1968. Use of poison bait to control rabies in Tennessee
wildlife. Public Health Rept. 83(1):69-78.
Linhart, S.B., and W.B. Robinson. 1972. Some relative carnivore densities
in areas under sustained coyote control. J. Mammal. 53(2): 880-884.
Lynch, G.M. 1972. Effect of strychnine control on nest predators of
dabbling ducks. J. Wildl. Manage. 36(2):436-440.
Matheny, R.W. 1976. Review and results of sodium cyanide spring loaded
ejector mechanism (SCSLEM) experimental programs. Proc. Seventh Vert.
Pest Conf., Monterey, Calif. 7:161-177.
Nelson, R.L., and R.L. Linder. 1972. Percentage of raccoons and skunks
reached by egg baits. J. Wildl. Manage. 36(2):1327-1329.
Newsome, A.E., L.K. Corbett, and D.R. Stephens. 1972. Assessment of an
aerial baiting campaign against dingoes in Central Australia. Aust.
C.S.I.R.O. Div. Math. Stat. Tech. Pap. 24:3-11.
Robinson, W.B. 1948. Thallium and Compound 1080 impregnated stations
in coyote control. J. Wildl. Manage. 12(3):279-295.
Robinson, W.B. 1953a. Population trends of predators and fur animals
in 1080 station areas. J. Wildl. Manage. 34(2):220-227.
. 1953b. Coyote control with compound 1080 stations in
national forests. J. For. 51 (12):880-885.
• . 1961. Population changes of carnivores in-, some coyote .
control areas. J. Mammal. 42(4):510-515.
image:
406
Robison, W.H. 1970. Acute toxicity of sodium monof luoroacetate to cattle.
J. Wildl. Manage. 34(3) : 647-648.
Seyler, K. , and C. Niemeyer. 1974. Emergency rabid skunk control in
Montana. Proc. Sixth Vert. Pest Conf., Anaheim, Calif. 6:198-203.
R. M. , W.E. Howard, M.W. Monroe, Roy Teranishi, and E.L. Murphy.
1977. A Method for Evaluating Coyote Scent Baits. Pp. 145-151 in Test
Methods for Vertebrate Pest Control Management Materials. W.B. Jackson
and R.E. Marsh, eds . Vol. 1, STP 625. Amer. Soc. for Testing and
Materials, Phila., Pa.
Tomlinson, A.R. 1954. Aerial baiting against wild dogs and foxes in
Western Australia. J. Agric. West. Aust. 3(1):37-49.
Ward, J.C., and F.E. Garlough. 1936. Strychnine IV: Lethal dose studies
on cattle and sheep. J. Am. Pharm. Assoc. 25(5) :423-426.
Ward, J.C. , and D.A. Spencer. 1947. Notes on the pharmacology of sodium
monof luoroacetate - Compound 1080. J. Am. Pharm. Assoc. 36(2):59-62.
B. Mammalian predator repellents.
Few studies of potential repellents tested on wild carnivores have
been reported. Definitive results from laboratory and pen tests are
difficult to obtain and are not necessarily representative of behavior
in wild free-ranging species under field conditions. In addition, highly
variable physical and biological factors may obscure the real effects -of
potential repellents. Preliminary screening of methods with domestic
animals may be useful before evaluation with wild species and field tests,
if such limitations are recognized.
Nesse (1971), and Swanson and Scott (1973), reported results of
limited field trails of the "Gerhardi Livestock Protector" to protect
goats and sheep from coyote depredation, but replications were not ade-
quate for accurate evaluation. Pfost (1971) conducted field trials of
the Gerhardi device to protect weeder geese from dogs and coyotes. Rep-
lications were not uniform, but the study was reasonably well designed.
Gustavson et al. (1974) attempted to induce conditioned aversion in
coyotes with lithium chloride; the discussion is not adequate for thorough
evaluation. Jankovsky et al. (1974) reported briefly on the results of
coyote repellent research in Colorado. Again, discussion of methodology
and results is incomplete, and no wildlife census techniques were employed.
References .
Anon. 1975a. Standard Guideline for Use and Development of Sodium Cyanide
as a Predacide. Annual Book of ASTM Standards, Part 46. Designation:
E553-75. Amer. Soc. for Testing and Materials, Phila., Pa.
_ . 1975b. Standard Method of Test for Efficacy of Acute Mammalian
Predacides. Annual Book of ASTM Standards , Part 46;. Designation: S552-75.
Amer. Soc. for Testing and Materials, Phila., Pa.
image:
407
. 1975c. Standard Recommended Practice for Determining Acute Oral LD50
for Testing Vertebrate Control Agents. Annual Book of ASTM Standards,
Part 46. Designation: E555-75. Amer. Soc. for Testing and Materials,
Phila., Pa.
Connolly, G.E., and W.H. Longhurst. 1975. The effects of control on
coyote populations; a simulation model. Univ. Calif., Div. Agric.
Sci. Bull 1872. 37 pp.
Dolnick, E.H., R.L. Medford, and R.J. Schied. 1976. Bibliography on the
Control and Management of the Coyote and Related Canids with Selected
References on Animal Physiology, Behavior, Control Methods and Repro-
duction. Bibliography is available upon request from U.S. Dept. Agr.,
Agr. Res. Center-East, Protein Nutrition Lab., Nutrition Instit., Belts-
ville, Md. 20705.
Gustavson, C.R., J. Garcia, W.G. Hankins, and K.W. Rusiniak. 1974. Coyote
predation control by aversive conditioning. Science 184:581-583.
Huebner, R.A. 1964. An evaluation of the efficacy of commercial canine
repellents. Vet. Med. Small Anim. din. 59(10):1016-1019.
Jankovsky, M.J., V.B. Swanson, and D.A. Cramer. 1974. Field trials of
predator repellents on spring lambs. Page 95 in Proc. 87th Ann. Res.
Conf., Colo. State Univ., Fort Collins.
Matheny, R.W. 1976. Review and results of sodium cyanide spring loaded
ejector mechanism (SCSLEM) experimental programs. Proc. Seventh Vert.
Pest Conf., Monterey, Calif. 7:161-177.
Nesse, G.E. 1971. Field Testing the Gerhardi Livestock Protector on
Angora Goats in Amador County. Project Report. Dept. Anim. Physiol.,
Univ. Calif., Davis.
Pfost, R.E. 1971. Predator repellents in geese. Proj. No. CC19.
Report, Sept. 14. Coop* Ext. Ser., Univ. of Calif., Davis.
Swanson, V.B., and G.S. Scott. 1973, Livestock protectors for sheep
predator control. Proc. West. Sect. Amer. Sec. Anim. Sci. 24:34-37.
C. Mammalian predator reproductive inhibitors.
Few studies have been directed toward management of wild canids by
controlling reproduction. A wealth of information has been accumulated
for screening tests of potential human contraceptives. These studies and
others carried out with rodents, lagomorphs, dogs, and cats provide a
reference source. Preliminary testing on domestic species to clarify
physiological effects may be necessary because of difficulty in securing
normal reproductive cycles in captive wild species. ' Pen tests with target
species are essential, since separation of physiological effects from those
caused by inadequate application techniques and other factors is extremely
.:difficult.with..highly mobile wild species under, field conditions.. , •'
image:
408
Branker (1943) and Jackson (1953) have demonstrated clinical use of
diethylstilbestrol (DES) to manage canine mismating. Kennelly (1969) de-
termined abortifacient effects of mestranol in dogs during early gestation.
Modification of Kennelly1s procedure to determine effects at specific stages
in the reproductive cycle is feasible. Balser (1964b) reported abortion
in captive coyotes induced by a single oral dose of DES.
Modification of test designs employed by Linhart and Enders (1964)
and Storm and Sanderson (1969) to determine reproductive inhibition in
captive female foxes is necessary to delineate specific states of repro-
duction affected. Additional changes are needed to evaluate chemical
effects on spennatogenesis and male fox fertility.
Linhart (1964b) reported results of field tests to determine bait
acceptance by wild foxes, but the test design does not provide population
measurements. Oleyar and McGinnes (1974) conducted field trials of DES
to reduce fox reproduction.
Balser (1964b) discussed results of field trials with DES to reduce
coyote reproduction. Linhart et al. (1968) reported results of the field
trials by Balser (1964) and several others to determine effects of DES
on reproduction of wild coyote populations. Inclusion of several trials
with the variation in methodology employed prohibits a thorough evaluation.
the authors suggest modifications of techniques and application but do not
recommend specific methods.
Improved techniques for application of reproductive inhibitors and
methods to measure populations are needed. In many, wild species, relative
indices may be the only feasible method to determine population levels and
changes.
References.
Anon. 1975a. Standard Guideline for Use and Development of Sodium Cyanide
as a Predacide. Annual Book of AS1M Standards, Fart 46. Designation:
E553-75. Amer. Soc. for Testing and Materials, Phila., Pa.
. 1975b. Standard Method of Test for Efficacy of Acute Mammalian
Predacides. Annual Book of ASTM Standards, Part 46. Designation: E552-75.
Amer. Soc. for Testing and Materials, Phila., Pa.
. 1975c. Standard Recommended Practice for Determining Acute
Oral LD50 for Testing Vertebrate Control Agents. Annual Book of ASTM
Standards, Part 46. Designation: E555-75. Amer. Soc. for Testing and
Materials, Phila., Pa.
Balser, D.S. 1964a. Antifertility agents in vertebrate pest control.
Proc. Second Vert. Pest Conf., Anaheim, Calif. 2:133-137.
. 1964b. Management of predator populations with antifertility
agents. J. Wildl. Manage. 28(2):352-358.
. 19,68. A technique for .producing antifertility- tallow baits
for predatory mammals. J. Wi'l'dT. Manage. 32(1'): 183-184. ' '
image:
409
Branker, W.M. 1943. Stilbestrol in veterinary practice. Vet. Rec.
55:461-462.
Brusman, H.H., S.B. Linhart, DoS. Balser, and L.W. Sparks. 1968. A
technique for producing antifertility tallow baits for predatory
mammals. J. Wildl. Manage. 32(1);183-184.
Davis, D.E. 1961. Principles of population control by gametoeides.
Trans. N. Am. Wildl. Nat. Resour. Conf. 26:160-167.
Dolnick, E.H., R.L. Medford, and R.J.-Schied. 1976. Bibliography on
the .Control and Management of the Coyote and Related Canids with
Selected References on Animal Physiology, Behavior, Control Methods
and Reproduction. Bibliography is available upon request from U.S.
Dept. Agr., Agr. Res. Serv., Protein Nutrition Lab., Nutrition Instit.,
Beltsville, Md. 20705.
Dunbar, M.R. 1973. Seasonal changes in testis morphology and spermato-
genesis in immature and adult coyotes (Canis la trans). Coop. Wildl.
Res. Unit, Oklahoma State Univ., Stillwater. Q. Prog. Rep. 26(2):16-17.
Gates, N.L. 1977. Chemosterilant Evaluation in Coyotes: Parameters
for Animal Selection and Laboratory Methodology. Pp. 106-113 in Test
Methods for Vertebrate Pest Control and Management Materials. Vol. 1,
STP 625. W.B. Jackson and R.Eo Marsh, eds. Amer. Soc. for Testing
and Materials, Phila., Pa.
Hamlett, W.D. 1938. The Reproductive Cycle of the Coyote. Bull. No.
616. U.S. Dept. Agri., Wash., D.C.
Howard, W.E. 1967. Biological control of vertebrate pests. Proc. Third
Vert. Pest Conf., San Francisco, Calif 3:137-157.
Jackson, W.F. 1973. Chemical methods of male contraception. Am. Sci.
61(2):188-193.
Jackson, W.F. 1953. Management of canine mismating with diethylstilbes-
trol. Calif. Vet. 22:29.
Kennelly, J.J. 1969. The effects of mestranol on canine reproduction.
Biol. Reprod. 1(3):282-288.
Linhart, W.B. 1963. Control of sylvatic rabies by antifertility agents -
principles and problems. N.E. Fish and Wildl. Conf., Portland, Maine.
April 14-17. 16 pp. (Mimeo)
. .1964. Acceptance by wild foxes of certain baits for administering
antifertility agents. N.Y. Fish and Game J. 11(2):69-77.
f
Linhart, S.B.., and R.K- Enders. 1964. Some effects of .die thyls tilbes troL
on reproduction- in captive red foxes. J. Wildl. Manage. 28-(.2).:.358-3€3.
image:
410
Linhart, S.B., and J.J. Kennelly. 1967. Fluorescent bone ..labeling o.f
coyotes with dimethyl-chlortetracycline. J. Wildl. Manage. 31(2):
317-321.
Linhart, S.B., J.J. Brusman, and D.S. Balser. 1968. Field evaluation of
an antifertility agent, stilbestrol, for inhibiting coyote reproduction.
Trans.N. Am. Wildl. Nat. Resour. Conf. 33:316-327.
Linhart, S.B., G.J. Dasch, J.D. Roberts, and P.J. Savarie. 1977. Test
Methods for Determining the Efficacy of Coyote Attractants and Repell-
ents. Pp. 114-122 in Test Methods for Vertebrate Pest Control and
Management Materials. Vol. 1, STP 625. W.B. Jackson and R.D. Marsh, -
eds. Amer. Soc. for Testing and Materials, Phila., Pa.
Marsh, R.E., and W.E. Howard. 1970. Chemosterilants as an approach to
rodent control. Proc. Fourth Vert. Pest Conf., West Sacramento, Calif.
4:53-63.
Nelson, R. 1972. Development of techniques for distribution of baits
to raccoon for chemosterilant studies. M.S. Thesis. So. Dakota State
Univ., Brookings.
Nelson, R., and R.L. Linder. 1972. Percentage of raccoons and skunks
reached by egg baits. J. Wildl. Manage. 36(4):1327-1329.
Oleyar, C.M., and B.S. McGinnes. 1974. Field evaluation of diethylstil-
bestrol for suppressing reproduction in foxes. J. Wildl. Manage.
38(1):101-106.
Storm, G.L., and G.C. Sanderson. 1969. Effect of medroxyprogesterone
acetate (Provera) on productivity in captive foxes. J. Mammal.
50(1):147-149.
Timm, R.M., W.E. Howard, M.W. Monroe, Roy Teranishi, and E.L. Murphy.
1977. A Method for Evaluating Coyote Scent Baits. Pp. 145-151 in
Test Methods for Vertebrate Pest Control and Management Materials.
Vol. 1, STP 625. W.B. Jackson and R.E. Marsh, eds. Amer. Soc. for
Testing and Materials, Phila., Pa.
D. Mammalian predator control devices.
The Agency does not register devices per se. Devices are registered
when they are used to deliver a pesticide to a target animal (e.g., the
use of the M-44 to deliver sodium cyanide to coyotes). Published research
reports on control devices are few. Extensive overlap with toxic chemicals
occurs since chemicals are employed in mechanical devices. Devices are also
often used simultaneously with other methods. Separation of effects, there-
fore, is difficult.
image:
411
Beasom ( 1973a,b) presents data and discusses the use of the M-44,
shooting, steel traps, and strychnine baits to remove predators and enhance
productivity of wild turkeys and white-tailed deer in specific study areas.
Simultaneous use of several methods reduces the value of the data for de-
termining effectiveness and selectivity of the various methods. However,
high selectivity was noted for the M-44 and shooting when compared to traps
and baits. Predator track counts and rodent snap trap transects were uti-
lized to establish relative population indices, but specific census tech-
niques were not provided.
Robinson (1943) discussed the use of steel traps and the "Humane"
Coyote Getter" for predator control. Ihe report compares efficiency and
selectivity, seasonal and weather effects, age of animals removed, experi-
mental areas, and defects of the methods. Census techniques are not pro-
vided except indirectly by indices based on trap catch data. Hey (1964)
provided a limited amount of data regarding use and selectivity of the
"Humane Coyote Getter" in comparison with other control methods, and
discussed hazards to nontarget species. No census or index methods are
discussed.
Robinson (1953, 1961) and Linhart and Robinson (1972) provide data
indication selectivity of steel traps in relation to species populations,
and discuss population trends of target and nontarget species in predator
control areas. Population indices are taken from trap-catch data. These
reports do not provide the protocols, but they include highly useful in-
formation for developing test methodology.
Field methods reported by Balser et al. (1968) require adequate
experimental area and population indices.
References.
Anon. 1975a. Standard Guideline for Use and Development of Sodium Cyanide
as a Predacide. Annual Book of ASTM Standards, Part 46. Designation:
E553-75. Amer. Soc. for Testing and Materials, Phila., Pa.
. 1975b. Standard Method of Test for Efficacy of Acute Mammalian
Predacides. Annual Book of ASTM Standards, Part 46. Designation: E552-75.
Amer. Soc. for Testing and Materials, Phila., Pa.
_^ . 1975c. Standard Recommended Practice for Determining Acute
Oral LD50 for Testing Vertebrate Control Agents. Annual Book of ASTM
Standards, Part 46. Designation: E555-75. Amer. Soc. for Testing
and Materials, Phila., Pa.
Arrington, O.E., and A.E. Edwards. 1951. Predator control as a factor in
antelope management. Trans. N. Am. Wildl. Nat. Resour... Conf. 16:179-193.
Balser. D.S., H.J. Dill., and H.K. Nelson. 1968. Effect of predator re-
""'duction on waterfowl nesting success. J. Wildl. Manage. 32-(4) :669—632.
image:
412
Beasom, S.L. 1973a. Selectivity of predator control techniques in south
Texas. Pp. 333-336 in Predator Control Hearings: Comm. on Agr., House of
Rep., 93rd Congr. Sept. 18, 20, 21. Serial No. 93-DD. U.S. Govt. Print.
Off., Wash., D.C.
. 1973b. The effect of predator removal on whitetailed deer produc-
tivity. Pp. 337-344 in Predator Control Hearings: Conun. in Agr., House
of Rep., 93rd Congr. Sept. 18, 20, 21. Serial No. 93-DD. U.S. Govt.
Print. Off., Wash., D.C.
Connolly, G. 1973. Economics of Coyote Fencing. Regional Project W-123.
Evaluation Management of Predators in Relation To Domestic Animals. Sept.
5, 6. Univ. Calif., Hopland Field Station, Hopland, Calif. 3 pp. (mizneo.)
Chesness, R.A., M.M. Nelson, and W.H. Longley. 1968. The effect of predator
removal on pheasant reproductive success. J. Wildl. Manage. 32(4):683-697.
Dolnick, E.H., R.L. Medford, and R.J. Schied. 1976. Bibliography on the
Control and Management of the Coyote and Related Canids with Selected
References on Animal Physiology, Behavior, Control Methods and Reproduction.
Bibliography is available upon request from U.S. Dept. Agr., Agr. Res.
Center-East, Protein Nutrition Lab., Nutrition Instit., Beltsville, Md.
20705.
Fitzwater, W.D. 1964. Report, on Spring-Ejection Getters. U.S. Dept. Int.,
Bur. Sport Fish, and Wildl., Branch of Predator and Rodent Control,
Albuquerque, N. Mex. Spec. Rep. 17 pp.
Hey, D. 1964. The control of vertebrate problem animals in the Province of
the Cape of Good Hope, Republic of South Africa. Proc. Second Vert. Pest
Conf., Anaheim, Calif. 2:57-73.
Linhart, S.B., and W.B. Robinson. 1972. Some relative carnivore densities
in areas under sustained coyote control. J. Mammal. 53(2):880-884.
Matheny, R.W. 1976. Review and results of sodium cyanide spring-loaded
ejector mechanism (SCSLEM) experimental programs. Proc. Seventh Vert.
Pest Conf., Monterey, Calif. 7:161-177.
Robinson, W.B. 1943. The humane coyote-getter vs. the steel trap in
control of predatory animals. J. Wildl. Manage. 7(2):179-189.
. 1953. Population trends of predators and fur animals in 1080
station areas. J. Mammal. 34(2):220-227.
. 1961. Population changes of carnivores in some coyote-control
areas. J. Mammal. 42(4):510-515.
. 1962. Methods of controlling coyotes, bobcats, and foxes.
Proc.. First Vert. Pest Conf., Sacramento, Calif. 1:32-56.
U.S. Department of the Interior. 1974. Coyote Damage Control by
Mechanical Techniques. Div. Wildl. Serv., Bur. Sport Fish, and
Wildl. Spec. Rep. Jan. 25. 28 pp. (mimeo.) . '
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413
E. Domestic dog and cab repellents and reproductive inhibitors.
The number of protocols in the literature for tests of repellency with
dogs and cats is limited. A comprehensive search would require consultation
with the workers in this field.
Repellents.
Huebner and Morton (1964) published a tentative test of efficacy of 5
commercially-available dog repellents. Papendeck (1968) conducted laboratory
research on capsaicin by spraying the test dogs' muzzles and eyes. Ihe
agent produced an aversive response in dogs in the laboratory. Histological
examinations showed that the spray did not cause lasting damage to the eyes.
The Environmental Protection Agency has developed a method that has been
used as an efficacy test for aerosol dog repellents. Refer to proposed effi-
cacy test TSD #1.504 (Exhibit 21).
1. Exhibit 21.
The method listed below is published by the Agency in its "Manual of
Biological Testing Methods for Pesticides and Devices, Volume 1." A copy may
be obtained by writing to the Product Manager #16, Insecticide-Rodenticide
Branch (TS-767), Environmental Protection Agency, Washington, D.C. 20460.
Users are advised that this BFSD (Benefits and Field Studies Division) method
is subject to review and possible revision by the Agency and by the ASTM
Committee E-35 on Pesticides.
Exhibit 21 (TSD # 1.504) is a proposed efficacy test for aerosol dog
repellents that are designed to reduce damage to plastic garbage bags.
2. Reproductive inhibitors.
Balser (1964) specified the attributes of an ideal reproductive inhibitor.
It must be effective in a single oral dose, offer a wide margin of safety
between the effective dose (ED) and lethal dose (LD), remain chemically
stable at an ED of less than 500 mg, be relatively tasteless and odorless,
and have a sterility effect for one breeding season. The author's protocol
was tried with the synthetic estrogen diethylstilbestrol.
Yasmuth et al. (1970) tested ovarian suppressants for dogs. They sub-
cutaneously administered 2 ml medroxyprogesterone acetate to 60 free-roaming
females. Blood tests, counts of mortality, and frequency of litters were
taken. All females had at least 1 litter before treatment. Observation
were made for a year.
Lunnen et al. (1974) published a laboratory protocol for inducing impot-
.ency in male dogs. Male beagles were immunized with a purified bovine luten-
izing hormone. The dogs were sacrificed at the end. of .fch<e experiment. The
testes., epididymides and -prostate., adrenal., thyroid,/ and. anterior .pituitary
glands were excised and measured, for weigh.t. differences.
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414
References.
Ambrose, A.M., P.S. Larson, J.F. Borselleca, and G.R. 'Henninger, Jr. 1970.
Toxicological studies on diethy!-!-!-(2,4-dichlorophenyl)-2-chlorovinyl
phosphate. Toxieol. Appl. Pharmacol. 17(2):323-336.
Anon. 1975. Indices of Predatory Abundance in the Western United States.
U.S. Dept. Int., Fish and Wildl. Serv., Denver Wildl. Res. Center,
Denver, Colo.
Balser, D.S. 1964. Management of predator populations with antifertility
agents. J. Wildl. Manage. 28(2):352-358.
Beck, A.M. 1971. Ihe Life and Times of Shag, a Feral Dog in Baltimore.
Nat. Hist. 80(8):58-65.
Berencsi, F., T. Weger, A. Antol, and J. Borsy. 1969. The influence of
pesticides on the effect of muscle relaxants. Arzneimittel-Forsch.
19(9):598-603.
Fox, M.W., A.M. Beck, and E. Blackman. 1975. Behavior and Ecology of
a Small Group of Urban Dogs (Canis familiaris). Appl. Anim. Ethology
1:119-137.
Huebner, R.A., and J.D. Morton. 1964. An evaluation of the efficacy of
commercial canine repellents. Vet. Med. Small Anim. Clin. 59(10):
1012-1016. .
Lunnen, J.E., L.D. Faulkner, M.L. Hopwood, and B.W. Pickett. 1974.
Immunization of dogs with bovine luteinizing hormone. Biol. Reprod.
10:453-460.
Oehm. F.W. 1970. Species differences: the basis for and importance of
comparative toxicology. Clin. Toxieol. 3(1):5-10.
Papendeck, U. 1968. Chemical and histological investigation on the action
of a dog-repelling spray containing capsicum (aversive agent). Inaugural
dissertation (in German)...Der Tierarztlichen Fakultat (#79): Klinische
and histologische Utersuchungen uber die Wirkung eines Capsaicin-haltigen
Hunde-Abuchrsprays: 69pp. (Eng. sum. pp. 65-66.)
Peardon, D.L., E. Kilbourn, and J.E. Ware, Jr. 1972. New selective rodenti-
cides. Soap Cosmet. Chem. Spec. 48(12):70.
Royal, L., and D. Tainturier. 1973. Rational use of anovulatory steroids
in the canine species. Rev. Med. Vet. 124(7):909-928 (in French).
Scott, M.D. 1971. The Ecology and Ethology of Feral Dogs in East-Central
Alabama. Ph.D. Thesis. Auburn Univ., Auburn, Ala. 213 pp.
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415
Snider, C.C. , and J.A. McCann. 1977. Proposed Efficacy Test for Aerosol Dog
Repellents that are Designed to Reduce Damage to Garbage Bags. Pp 123-125
in Test Methods for Vertebrate Pest Control and Management Materials. Vol.
1, STP 625. W.B. Jackson and R. E. Marsh, eds. Amer. Soc. for Testing and
Materials, Phila., Pa.
Yasmuth, D., T.O. Rowe, T.C. Doege, and H.N. Bangxang. 1970. Ovarian
suppressants in dogs. Lancet 7660:1312-1315.
F. Browsing animal repellents.
Animal repellents are now widely used in the United States. In general,
two types of repellents have been used for deer: odor repellents and taste
repellents.
There are several methods for testing the efficacy of repellents on
vegetation in relation to deer damage. The oldest method has been to lay
out line transects and establish plots in treated and untreated areas
(Besser and Welch, 1959). The most widely used method (Duncan and Whitaker,
1959), however, is to survey transects in the field on treated and untreated
areas. These methods require a long period of time and the results are
difficult to evaluate.
Another method involves pen tests where materials are treated and then
presented to the test animals. An additional modification consists of pro-
viding pens with captive animals and planted blocks of treated and untreated
seedlings. These pen tests yield more controlled data. In these experiments,
the seedlings are planted in 2-1/2- and 1-acre (1 and 0.4 ha) enclosures
holding deer and hares. A control and a repellent planting are used to
evaluate the test chemical. Dodge, Loveless, and Kverno (1967) modified the
techniques for the planted enclosures. These tests were randomized complete
block design in which 10 blocks with 10 trees per block for each of the treat-
ments were used. The untreated control pen must be observed routinely until
60 to 80% of the reference seedlings sustain animal damage.
Campbell and Bullard (1972) devised a preference test design using an
apparatus **iich presents 2 food samples. The arrangement can be quickly
changed to offer 2 more small dishes of a test material. These presentations
are made so that at least 2 completely different sets of experiments can be
executed at one time. Four to 10 deer are routinely tested once or twice
daily for 1 or 2 weeks. The test consists of at least 10 to as high as 30
choices. Normally a deer can complete 20 choices in about 6 minutes. The
animals are tested against a repellent standard. One objective of the test
is to obtain marginal acceptance.
References.
Anon. 1973. Controlling deer. U.S. Fish and Wildlife Service. Wildlife
Leaflet No. 404, Twin Cities, Minn. 2 pp.
Besser, J. P., and. J.F. Wel.ch. 1959. Chemical, repellents .for the control
of . mammal-• damage, to plants. Trans. N. Amer.. Wildl. Na.t-. .•Res'our-. -Con-f •«•
24:.166-173, . . ... ...
image:
416
Black, H.C., E.J. Dimock II, W.E. Dodge, .and W.H. Lawrence. 1969. Survey
of animal damage on forest plantations on Oregon and Washington. Trans.
N. Amer. Wildl. Nat. Resour. Conf. 34:388-408.
Bullard, R.W., and D.L. Campbell. 1968. Evaluation of adhesives for foliar
application of chemicals. For. Sci. 14(1):39-44.
Campbell, D.L. 1969. Plastic fabric to protect seedlings from animal damage.
Pp. 87-88 in Proc. Wildl. and Reforestation in Pacific Northwest. Oregon
State Univ., Corvallis.
Campbell, D.L., and R.W. Bullard. 1972. A preference testing system for
evaluating repellents for black-tailed deer. Proc. Fifth Vert. Pest
Conf., Fresno, Calif. 5:65-63.
Campbell, D.L., and J. Evans. 1977. Guidelines for Field Evaluations of
Repellents to Control Deer Damage to Reforestation. Pp. 45-151 in Test
Methods for Vertebrate Pest control and Management Materials. Vol. 1, STP
625. W.B. Jackson and R.E. Marsh, eds. Amer. Soc. for Testing and Materi-
als, Phila., Pa.
Carpenter, M. 1967. Control of deer damage. Virginia Wildlife. May, 1967.
Reprint D-19. Richmond, Va. 2 pp.
Crawford, J.C., and D.C. Church. 1971. Response of black-tailed deer to
various taste stimuli. J. Wildl. Manage. 32(5):210-215.
Crouch, G.L. 1969. Animal damage to conifers on national forests in the
Pacific Northwest region. U.S. Forest Service, PNW For. and Range Exp.
Sta. Res. Bull. PNW-28. 18 pp.
Dodge, W.E., C.M. Loveless, and N.B. Kverno. 1967. Design and analysis of
forest mammal repellent tests. For. Sci. 13(3):333-336.
Duncan, D.A., and L.B. Whitaker. 1959. Repellents reduce cattle browsing
on pines. U.S. Forest Service, South. For. Exp. Sta., South. Forestry
Note 119.
Gauditz, Illo. 1977 Bioassay Methods Guilding the Development of a Big
Game Repellent. Pp. 34-46 in Test Methods for Vertebrate Pest Control and
Management Materials. W.B. Jackson and R.E. Marsh, eds. Vol. 1, STP 625.
Amer. Soc. for Testing and Materials, Phila., Pa.
Hall, E.R., and K.R. Kelson. 1959. Ihe Mammals of North America. Ronald
Press Company: New York. (2 vols.).
Lawrence, W.H. 1958. Wildlife-damage control problems of Pacific North-
west tree farms. Trans. N. Amer. Wildl. Nat. Resour. Conf. 23:146-151.
Radwan, M.A. 1971. Differences between Douglas-fir genotypes in relation
to browsing by black-tailed deer. Can. J. For. Res. 2:250-255.
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417
. 1972. Occurrence and genotype differences of chlorogenic acid
in Douglas-fir foliage. U.S. Forest Service, PNW For. and Range Exp. Sta.,
Res. Note 173. 6 pp.
Rochelle, J.A., and W.H. Lawrence. 1971. Control of wildlife damage with
chemicals. Pp. 79-88 in Pesticides, Ecology & Natural Resource Management.
Washington State Univ.: Pullman.
Siegel, S. 1956. Non-parametric Statistics for the Behavioral Sciences.
McGraw-Hill Co.: New York.
Welch, J.F. 1967. Review of animal repellents. Proc. Third Vert. Pest
Cpnf., San Francisco, Calif. 3:36-40
VII. Field census techniques.
Population censusing under field situations is essential to adequately
evaluate many vertebrate pest control agents. Some means must be used in
determining the number of animals before treatment and again at post-treatment
to establish efficacy. Censusing techniques are fundamental to all population
studies designed to measure the influence of chemicals on the target or non-
target populations.
Censusing techniques for the orders Rodentia and Lagomorpha are numerous
and varied. Probably no technique is perfect; each has its strengths and
weaknesses. The researcher must make every effort not only to determine
which methods of population estimation are best suited to his purposes but
also to apprise himself of all the weaknesses of his chosen methods. This
enables the best interpretation of the results within the framework of his
outlined test methods or procedures. In a general vein, Murray (1957) warns
of the danger of error in most censusing techniques due to the selective
distribution of animals according to habitat type within an area being cen-
sused. Censusing methods which in themselves affect the population (for
example, fatal trapping, shooting, etc.) should not be used unless statistical
procedures can be applied to correct for these effects. When practical, more
than one censusing procedure should be used.
Many comparisons of techniques are available. Edwards and Eberhardt
(1963) tested various methods of estimating a population of cottontail rabbits
(Sylvilagus floridanus) from live-trapping data against a known population.
They suggested, with some qualifications, that linear regression or maximum
likelihood estimation (MLE) methods were the most useful. When several pro-
cedures were tried on grey squirrels (Sciurus carolinensis) in Maryland,
Flyger (1959) found that a Schnabel (1938) population es-timate., based on what
•Flyger termed "trap-sight records," was best for. estimating tbe population.
He used visual sightings because he felt'.that it:lessened bias injected by
trap-shy or trap-prone animals. The Schnabel method is .a.variation of the
Lincoln Index that uses the average of a series of- ra-tios' rather than one,
and is considered., therefore, to be less prone to- sampling error*. Nixon ;e£r ,
a-1. (1967) also felt that population estimates.'of/squirrels (J. carolinensis -
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418
and ^. niger).from the Schnahel metho.d were low because of capture frequency
variation for different individuals. They preferred the simplified MLE method
(Edwards and Eberhardt, 1967).
Robinette et al. (1974) conducted tests of 10 different types of strip
censuses. Ihe 3 most satisfactory methods were those of Kelker (1945),
Anderson and Pospahala (1970), and King. [King's method was described by
Leopold (1933) in his book on game management.] Hayne (1949) presents a
critical evaluation of the King method.
Beaver census techniques are reviewed and evaluated in light of an
extensive census operation by Hay (1958). After checking several census
methods, he found aerial counting of food caches to be the most practical
and reliable.
Forest rodent censusing to determine conifer seed depredation potential
provides an interesting example of a situation where the techniques used are
not aimed toward absolute enumeration, but toward population indices for use
in evaluating rodent control operations. Lines of live or snap traps are
often used for these determinations. However, some researchers feel that the
"seed-spot" census method is more useful to foresters than trapping because
of its relative ease in use (Tevis, 1956; Moore, 1950). Tevis discusses the
method thoroughly. It is based on the assumption that the percentage of piles
of seeds placed in an area that are disturbed is proportional to the number
of seed eaters in that area. Moore gives a key for determining what species
disturbed each seed spot.
Emlen et al. (1949) reviewed Norway rat (Rattus'norvegicus) census
methods in some detail and discussed the shortcomings of some of the various
methods. Chitty (1942) and Bentley et al. (1957) used wheat consumption as
a population index for Norway rats in sewers. Rohe (1966) gives guidelines
for censusing of roof rats (Rattus rattus); he used snap-traps and nontoxic
paraffin-grain bait blocks to test for the presence of rats and their relative
numbers. Nelson and Clark (1973) published a method for correcting trapping
results by taking into account sprung traps in calculating catch effort.
Emlen et al. (1957) and Tester and Emlen (1960) tested dropping boards as a
method of obtaining population indices.
Census methods for pocket gophers (Geomys bursarius) and moles (Talpa
europa) are presented by Beck and Hansen (1966) and Mead-Briggs and Woods
(1973), respectively. Estimating pocket gophers by counting new signs was
also reported by Reid et al. (1966).
When faced with the task of censusing a population, 2 sources for back-
ground information are Dice (1941) and Davis (1956). Dice reviews many of
the methods for dealing with mammalian populations. Davis gives an outline
of field and laboratory methods for accumulating and analyzing data on rodent
populations. In addition to these sources, Chitty and Shorten (1946) present
a compilation on the techniques for the study of Norway rats. And, finally,
Tepper (1967) compiled a bibliography entitled, "Statistical Methods in Using
Mark-Recapture Data for Population Estimations."
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419
References.
Adams, L. 1951. Confidence limits for the Petersen or Lincoln index used
in animal population studies. J. Wildl. Manage. 15(1):13-19.
Anderson, D.R., and R.S. Pospahala. 1970. Correction of bias in belt
transect studies of immobile objects. J. Wildl. Manage. 34(1):141-146.
Arnold, F., and H.G. Reynolds. 1943. Droppings of Arizona and antelope
jack rabbits and the "pellet census." J. Wildl. Manage. 7(3):(327-332.
Bailey, N.T.J. 1952. Improvements in the interpretation of the recapture
data. J. Animal Ecol. 21:120-127.
Beck, R.F., and R.M. Hansen. 1966. Estimating plains pocket gopher abundance
on adjacent soil types by a revised technique. J. Range Manage. 19:224-225.
Bentley, E.W., A.H. Bathard, and J.D. Riley. 1957. The rates of recovery
of sewer rat populations after poisoning. J. Hyg. 57(3):291-298.
Blair, W.F. 1941. Techniques for the study of mammal populations. £.
Mammal. 22(1):148-157.
Calhoun, J.B. 1955. A technique for investigating the distance parameter
of home range. J. Mammal. 36(l):42-52.
Chitty, D. 1942. A relative census method for brown rats (Rattus norvegi-
cus). Nature 150:59-60.
Chitty, D., and M. Shorten. 1946. Techniques for the study of the Norway
rat (Rattus norvegicus). Nature 150:59-60.
Davis, D.E. 1956. Manual for Analysis of Rodent Populations. Edwards
Bros.: Ann Arbor, Mich. 82 pp.
Davis, D.E., and C. Zippin. 1954. Planning wildlife experiments involving
percentages. J. Wildlife Manage. 18(2):170-178.
Dean, F.C., and G.A. Gallaway. 1965. A fortran programme for population
study with minimal computer training. J. Wildl. Manage. 20:892-894.
Dice, L.R. 1931. Methods of indicating the abundance of mammals.
J. Mammal. 12(4):376-381.
1941. Methods for estimating populations of mammals. J.
Wildl. Manage. 5(4) .-398-407.
Edwards, WYK., arid: L. Eberliardt. 1967. .Estimating, cotfcen.tail 'abundance.
from livetrapping data. J. Wildl. Manage. 31 (.1.):8.7-96.
Emlen., J.T.,. A.W. Stokes., and D.E. Davis. 1949. Methods for estimating
populations of brown rats in urban habitats. Ecology 30'-(:4.),':430-442.
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420
Etnlen, J.T., R.L. Hine, W.A. Fuller, and P. Altenso. 1957. Dropping boards
for population studies of small mammals. J. Wildl. Manage. 21(3}:300-314.
Evans, F.C., and R. Holdenried. 1943. A population study of the Beechey
ground squirrel in central California. J. Mammal. 24(2):231-261.
Flyger, V.F. 1959. A comparison of methods for estimating squirrel popu-
lations. J. Wildl. Manage. 23(2) .-220-223.
Friley, C.E. 1965. Criteria for estimating fall fox squirrel populations.
J. Wildl. Manage. !9(1):89-93.
Graham, S.A. 1940. The intersection method of counting animals. J. Wildl.
Manage. 4:313-314.
Hay, K.G. 1958. Beaver census methods in the Rocky Mountain region. J_,_
Wildl. Manage. 22(4):395-402.
Hayne, D.W. 1949a. An examination of the strip census method for esti-
mating animal populations. J. Wildl. Manage. 13(2):145-157.
. 1949b. Two methods for estimating population from trapping
records. J. Mammal. 30(4):399-411.
Hayne, D.W., and D.Q. Thompson. 1965. Methods for estimating microtine
abundance. N. Amer. Wildl. Nat. Resourc. Conf. 30:393-400.
Holgate, P. 1966. Contributions to the mathematics of animal trapping.
Biometrics 22(4):925-936.
Keith, L.B., E.G. Meslow, and O.J. Rongstad. 1968. Techniques for snow-
shoe hare population studies. J. Wildl. Manage. 32(4):801-812.
Kelker, G.H. 1945. Measurement and interpretation of forces that determine
populations of managed deer. PhD Thesis, Univ. Mich., Ann Arbor. 422 pp.
Leopold, A. 1933. Game Management. Chas. Scribner's Sons: N.Y. 281 pp.
Leslie, P.H., D. Chitty, and H. Chitty. 1953. The estimation of population
parameters from data by means of the capture-recapture method. Biometrika
40(1,2) :137-169.
Linn, I. 1964. A theory of small mammal index trapping. Proc. Int. Conor.
Zool. 16:268.
MacLulich, D.A. 1951. A new technique of animal census with examples.
J. Mammal. 32(3):318-328.
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Marten, G.G. 1972a. Censusing mouse populations by means of tracking.
Ecology 53(5)-.959-967.
. 1972b. The remote sensing approach to censusing. Res. Popul.
Ecol. 14(l):36-57.
Mead-Briggs, A.R., and J.A. Woods. 1973. An index of activity to assess
the reduction in mole numbers caused by control measures. J. Appl. Ecol.
10:837-845.
Moore, A.W. 1950. Forest tree seed eaters and methods used to measure
their populations in Pacific Northwest Douglas-fir region. Univ. Wash.
For. Club Quart. 23(1):7-11.
Murray, K.F. 1957a. Problems of small animal sampling. Calif. Vector
Views 4(6):85,89»
1957b. Some problems of aplied small mammal sampling in
western North America. J. Mammal. 38(4):441-451.
Nelson, L., Jr., and F.C. Clark. 1973. Correction of sprung traps in
catch/effort calculations of trapping results. J. Mammal. 54(1):295-298.
Nixon, C.M., W.R. Edwards, and L. Eberhardt. 1967. Estimating squirrel
abundance from live trapping data. J. Wildl. Manage. 31(11:96-101.
Overton, W.S., and D.E. Davis. 1969. Estimating numbers of animals
in wildlife populations. Pp. 403-455 in Wildlife Management Techniques.
R.H. Giles, ed. Wildlife Society, Washington, D.C.
Reid, V.H., R.M. Hansen, and A.L. Ward. 1966. Counting mounds and earth
plugs to census mountain pocket gophers. J. Wildl. Manage. 30(2): 327-334.
Robinette, W.L., C.M. Loveless, and D.A. Jones. 1974. Field tests of strip
census methods. J. Wildl. Manage. 38(l):81-96.
Rohe, D.L. 1966. Survey methods for roof rat populations in sanitary sewers<
Calif. Vector Views 13(10).-75.
Schnabel, Z.E. 1938. The estimation of the total fish population in a lake.
Am. Math. Monthly 45(6):348-352.
Seber, G.A.F. 1962. ftie multi-sample single recapture census. Biometrika
49(3,4):339-350.
. 1965. A note on the multiple recapture census. Biometrika
52(1,2):249-259.
197-3. . lhe Estimation of -Animal. Abundance. Hafher Press:
New York. 506.pp.
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422
Shorten, M., and F.A. Courtier. 1955. A population study of the grey
squirrel (Sciurus carolinensis) in May 1954. Ann. Appl. Biol.
43(3):493-510. . .
Smith, M.H. 1968. A comparison of different methods of capturing and
estimating numbers of mice. J. Mammal. 49(3):455-462.
Southern, H.N. 1965. The trap line index to small animal populations.
J. Zool. London 147:217-221.
Stickel, L.F. 1946. Experimental analysis of methods for measuring small
mammal populations. J. Wildl. Manage. 10(2):150-159.
. 1948. The trap line as a measure Of small mammal populations.
J. Wildl. Manage. 12(2) :153-161.
Taylor, W.P. 1930. Methods of determining rodent pressure on the range.
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