iPA-540/9-82-024
October 1962
Pesticide Assessment Guidelines
Subdivision, E
Hazard Evaluation:
Wildlife and Aquatic Organisms
Prepared by
Ecological Effects Branch
Hazard Evaluation Division
Office of Pesticides Programs
Guidelines Coordinator
Robert K. Hitch
Hazard Evaluation Division
Off tea of Pesticide Programs
U.S. Environmental Protection Afenev
Offica of Pesticides and Toxic Sybstaneas
Washington, D.C, 20460
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EPA
October, 1982
PESTICIDE ASSESSMENT GUIDELINES
SUBDIVISION E
HAZARD EVALUATION:
WILDLIFE AND AQUATIC ORGANISMS
by
Ecological Effects Branch
Hazard Evaluation Division
Office of Pesticide Programs
Guidelines Coordinator:
Robert K. Hitch
Hazard Evaluation Division
Office of Pesticide Programs
U. S. Environmental Protection Agency
Office of Pesticide and Toxic Substances
Washington, D. C. 20460
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Foreword
Subdivision E describes protocols which may be used to perform
fish and wildlife effects testing to support the registration of
pesticides under the Federal Insecticide, Fungicide and Rodenticide
Act (FIFRA). It is a nonregulatory companion to 40 CFR Part 158,
Data Requirements for Registration. Subdivision E has been the
subject of comment at a series of public meetings, the last of which
occurred in July, 1982. Data requirements established by 40 CFR
Part 158 are discussed in Subdivision E so that it can be read as a
complete package and so that fish and wildlife testing procedures
can be explained in their proper context.
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Subdivision E Hazard Evaluation: Wildlife and Aquatic Organisms
TABLE OF CONTENTS
DISCUSSION
Series 70: GENERAL INFORMATION AND REQUIREMENTS
§
§
§
§
70-1
70-2
70-3
70-4
General information
Definitions
General test standards
Reporting and evaluation of data
Series 71 : AVIAN AND MAMMALIAN TESTING
§
§
§
§
§
71-1
71-2
71-3
71-4
71-5
Avian single-dose oral LD50 test
Avian dietary LC50 test
wild mammal toxicity test
Avian reproduction test
Simulated and actual field testing
and birds
for mammals
Series 72: AQUATIC ORGANISM TESTING
§
§
§
§
§
§
§
72-1
72-2
72-3
72-4
72-5
72-6
72-7
Acute toxicity test for freshwater
Acute toxicity test for freshwater
invertebrates
Acute toxicity test for estuarine
organisms
Fish early life-stage and aquatic
life-cycle studies
Life-cycle tests of fish
fish
aquatic
and marine
invertebrate
Aquatic organism accumulation tests
Simulated or actual field testing for aquatic
organisms
Page
I. ORGANIZATION and PHILOSOPHY OF SUBDIVISION E 1
II. MAJOR ISSUES 4
GUIDELINES
21
25
26
29
33
37
43
43
57
66
69
72
76
79
81
83
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1
I. ORGANIZATION AND PHILOSOPHY OF SUBDIVISION E
A- Purpose.
Subdivision E provides guidelines for testing and information
on data submission concerning the effects of pesticides on wildlife
and aquatic organisms. Data developed according to the guidelines
in this Subdivision and submitted to support the registration of a
pesticide product/ will be used by the Agency for determining
potential hazards to nontarget birds, wild mammals, fish and aquatic
invertebrates.
The tests in §§ 71-1, 71-2, 72-1, and 72-2 provide basic toxicity
information which serves as a starting point for pesticide hazard
assessments. These data are used principally for the following evalua-
tions:
- To establish acute toxicity levels of the active ingredient to the
test organisms;
- TO compare toxicity information with measured or estimated pesticide
residues in the environment to assess potential impacts to fish
and wildlife;
- To provide data which determine the need for (and support the
wording for) precautionary label statements to minimize the
potential adverse effects to wildlife and aquatic organisms; and
- To indicate the need for further laboratory and/or field studies.
Additional tests (i.e., avian, fish, and invertebrate repro-
duction and life-cycle studies) are provided when basic data and
environmental conditions suggest possible problems. Data from
these tests are used principally:
— To estimate the potential for chronic impacts, taking into account
the measured or estimated residues in the environment; and
- To determine if additional field or laboratory data are necessary
to further evaluate hazards.
Simulated field and/or actual field tests are provided so
that data can be developed to examine acute and chronic adverse
effects on captive or monitored fish and wildlife populations in
natural or near-natural environments. These data are needed
only when predictions as to possible adverse effects in less exten-
sive studies cannot be made, or when the potential for adverse
effects from the use of a pesticide is likely to be high.
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2
B. Approach.
1. Organization. The toxicity tests in Subdivision E have been
grouped into two broad areas-: tests for pesticidal effects on birds
and mammals (§§ 71-1, -2, -3, -4, -5); and tests for pesticidal
effects on fish and aquatic invertebrates (§§ 72-1, 2, -3, -4,
-5, -6, and -7). Each of these groups has been further subdivided
into short-term acute and subacute, reproduction, simulated field,
and full field studies.
The tests are arranged in a hierarchical or tier system which
progresses from the basic laboratory tests to the applied field
studies. The results of each tier of tests should be evaluated to
determine the potential of the pesticide to cause adverse effects and
to determine whether further testing should be considered. It is
expected that each sequential test will be performed far less often
than the preceding test. Figures 1, 2 and 3 of § 70-1(b) illustrate
the tier testing system for avian wildlife, wild mammals, and aquatic
organisms, respectively.
2* Content of paragraphs within sections. Within Subdivision E,
each section provides guidance for a particular test or group of
related tests, and contains the following provisions:
- A provision refering to 40 CFR § 158.145 to determine when the
data are required, and containing additional guidance on when
the data are required to support the registration of the manu-
facturing-use product or formulated product or both;
- Provisions outlining test standards that should be complied with
in conducting the studies;
- Provisions outlining standards for reporting and evaluating
test data; and
- Examples of acceptable protocols, references to published
documents and technical journals, and other aids that will
help in planning and conducting toxicity tests.
3' ^Porting standards. Section 70-4 "Reporting and Evaluation
of Data" provides guidance on the general reporting and evaluation
standards for this subdivision. In addition, each section contains
a paragraph entitled "Reporting and evaluation of data" which sets
forth the Agency's particular standards that apply to each test.
4* Acceptable protocols and references. Each section includes
paragraphs which generally provide the following kinds of information:
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3 • . . •
- Examples of acceptable test protocols;
- References that may provide useful background information in
developing acceptable protocols; and
- References that outline useful statistical procedures for handling
data.
The examples of acceptable protocols are either those which are not
available in any other published form or which are of widespread
interest because they provide examples of tests which would be
routinely performed for a significant number of products. The Agency
believes that publishing the acceptable protocols and references with
each section is the most efficient way of providing this useful
information to all potential users.
References to standard :practices established by the American
Society for Testing and Materials for conducting freshwater fish
acute toxicity studies, freshwater aquatic invertebrate acute toxicity
studies, and marine mollusc shell deposition and embryolarval toxicity
studies, appear in the appropriate aquatic test sections as examples
of acceptable protocols. Various subcommittees within ASTM are
currently preparing standard practices for other studies required by
this subdivision, such as the avian single-dose oral LD50, avian
reproduction, and aquatic accumulation studies. When these standard
practices are completed, the Agency will review them, and consider
including them in appropriate sections of this subdivision as accept-
able test protocols.
5. Relation of Subdivision E to other subdivisions. Subdivision E
provides standards for testing and data submission concerning the
effects of pesticides on nontarget birds, mammals, fish, and aquatic
invertebrates. The data developed according to Subdivision E and
required by 40 CFR § 158.145 represent only a portion of the data
considered in evaluating hazards to nontarget organisms. The
Agency also routinely examines other information submitted in
response to other sections of 40 CFR Part 158 requirements. In
evaluating the potential hazards, the Agency also considers data
developed according to Subdivision D, entitled "Chemistry Product
Chemistry," Subdivision F, entitled "Hazard Evaluation: Humans and
Domestic Animals," and Subdivision N entitled "Chemistry Environmental
Fate."
Data required in 40 CFR § 158.135 and developed according to
Subdivision F are normally adequate to indicate hazard to wild
mammals. Under certain conditions, these data are not sufficient
to assess the potential hazards to wild mammals and further tests
should be conducted. Section 71-3 provides examples of circum-
stances when additional tests and data on wild mammal toxicity may
be needed.
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Together, the data required by 40 CFR Part 158 are used by
the Administrator to make the determination, required by FIFRA
sec. 3(c)(5), whether a pesticide "...will perform its intended
function without unreasonable adverse effects on the environment"
and thus whether the pesticide may be registered. The data are
also used by the Administrator to make the "incremental risk"
finding required by FIFRA sec. 3(c)(7) (conditional registration).
Subdivision E currently does not provide testing guidelines to
address the effects of pesticides on nontarget amphibians and
reptiles. At the'present time, the Agency assesses hazards to
these nontarget organisms from the use of pesticides on a case-by-
case 'basis; using all available and appropriate data; The Agency
is currently gathering literature on the effects of pesticides on
amphibians and reptiles, and on the appropriate test methods needed
to measure these effects. -After a review of the available litera-
ture, the Agency may determine: ' ! :
- That the data required by 40 CFR Part 158.145 and developed ac-
cording to Subdivision E of the guidelines are sufficient to
determine hazards to nontarget amphibians and reptiles; or
- That additional data are needed in order to determine hazards to
these nontarget organisms. .'•"•• ;—-'-
The latter would necessitate the establishment of data requirements
in 40 CFR § 158.145 and the development of guidelines:for testing
and data submission concerning the effects of pesticides on nontarget
amphibians and reptiles.
II. MAJOR ISSUES
Public comments and Agency review of the 1978 proposed Subpart
E guidelines identified a number of major issues which are presented
in this discussion. Many comments covering both major and minor
points were adopted by the Agency, and corresponding changes in the
guidelines were made. Discussions of the major issues brought up by
public comments are presented in the following paragraphs.
A. Data Requirements for Manufacturinq^Use Products.
- In the Preamble to the 1978 proposed Guidelines, EPA asked for
public comment on the question as to whether or not the data require-
ments of this subdivision should be extended to manufacturing-use
products. After serious consideration of this issue, the Agency
has concluded that extending the data requirements to such products
is appropriate. The Agency was influenced by the views of commenters
on this issue who generally favored a data submission requirement
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which makes the basic manufacturer of an active ingredient responsible
for providing most of the effects data.
Therefore, 40 CFR § 158.50, entitled "Formulaters' Exemption"
requires a registrant of a manufacturing-use prpductto submit (or
cite) any data pertaining to the safety of an active ingredient in
its product if the same data are required to support the registration
of an end-use product that could legally be produced from the regis-
trant 's manufacturing—use products. (An end-use product is a
pesticide product bearing label directions for immediate end-use as
a pesticide.) Section 158.50 also provides that such data must be
submitted by an applicant for registration of the end-use product,
except that the producerof the end-use product will generally not
have to submit or cite data pertaining to registered products which
the end-use producer purchases and uses to formulate the end-use
product. This decision reflects the Agency's expectation that
manufacturing-use product registrants will be the major source of
registration data, and that end-use product formula tors will, in
most cases, need to suply much less data. This decision is consis-
tent with the provisions of, and Congressional intent behind, sec.
3(c)(2)(D) of FIFRA, which provides that:
No applicant for registration of a pesticide who
proposes to purchase a registered pesticide from
another producer in order to formulate such purchased
pesticide into an end-use 'product shall be required to —
(i) submit or cite data pertaining to the safety
of such purchased product; or
(ii) offer to pay reasonable compensation
otherwise required [§ 3(c)(1)(D) of FIFRA] for
use of any such data.
Implicit in sec. 3(c)(2)(D) is Congress' expectation that it
would be the registrant of the manufacturing-use product who would
provide significant amounts of data pertaining to the safety of its
product. (See, e.g., Sen. Rep. No. 334, 95th Cong., 1st Sess., pp.
8-9.)
Moreover, if data requirements were imposed solely on registrants
of end-use products, sec. 3(c)(2)(D) might be read to prevent the
Agency from obtaining data on the grounds that the data pertain to
the safety of a purchased product.
B. Degree of Specificity in the Guidelines.
In responding to the proposed version of the guidelines, ccmmen-
ters argued both that the guidelines were too specific and that they
were too general. The Agency has sought to strike a balance between
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these concerns. The Agency has rejected a step-by-step or "cookbook"
description of test procedures because it recognized that any specific
detailed method may not be suitable for the evaluation of all pesti-
cides, and that new test procedures are constantly being developed.
For example, the Agency believes that testing guidelines should.
have the flexibility to take into account differences in test
equipment, personnel, animals at various laboratories, and charac-
teristics of the pesticide actually being tested. Consequently, .
the Agency has sought to establish test standards which possess
the specificity necessary for data comparison, but .retain the
flexibility to, accommodate the other considerations'noted above.
C. Reporting of Data.
Several commenters suggested that not all the information listed
under proposed § 163.70-l(c) Reporting of data needs to be submitted
with each report. They recommended that information such as the
physical and chemical properties of the test substance, assay
methods, and identity of the test substance should be submitted to
the Agency only once with each application. The Agency agrees,
and does not request duplicative submission of data. Therefore,
references to data furnished in response to the requirements of
40 CFR Part 158 will be accepted, provided that the complete text
of the data is actually supplied elsewhere in a sutmittal and is
properly marked or headlined for reasonable accessibility.
EPA has also revised the general reporting standards in Sub-
division E in response to suggestions that the standards which do
not apply to all or most of the tests be deleted from this
section. Accordingly, the Agency has revised and relocated these
reporting standards. They now appear in the sections prescribing
reporting requirements for specific tests. A few reporting standards
that apply to a majority, but perhaps not all, of the tests in Sub-
division E have been retained in § 70-4 with appropriate qualifying
statements. For example, the standard proposed in § 163.70-l(c)(7)
(iii) now reads [at § 70-4(c)(7)(ii)] "Calculation of the LD50,
LCSO, or ECSO, and the 95 percent confidence interval, when suffi-
cient doses and test organisms are used to establish a dose-response
line." Proposed § 163.70-1(c)(2) Environmental conditions has also
been revised [at § 70-4(c)(6)] so that the data reporting standards
for tests conducted with mammals or birds and those conducted with
fish or aquatic invertebrates now appear in separate paragraphs.
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7 >'; '•.,
D. Test Substance.
There was considerable discussion of the Agency's choice of the
test substance that should be used in the proposed Subpart E tests.
Some commenters argued that only the formulated product should be
tested because isolation of the active ingredient is not always
feasible. They also argued that neither the manufacturing-use pro-
duct nor the active ingredient in technical form poses an environmental
hazard to wildlife or aquatic organisms when it is used or distributed
in commerce. Other commenters, however, felt that testing of the
technical grade of each active ingredient would be sufficient be-
cause the toxicity of a pesticide product depends almost exclusively
on the concentration of the active ingredient in the product. They
felt that the "inert" ingredients in a pesticide (those which do
not cause the intended pesticidal effect) usually have no .independent
toxic effect on wildlife or aquatic organisms.
The Agency has Considered both sides of the issue, and has
determined that short-term acute toxicity studies should be performed
on the active ingredient of the pesticide. Therefore, the fish and
wildlife guidelines now provide that all tests except simulated
and actual field studies (§§ 71-5, 72-6 and -7) should be conducted
with the technical grade of active ingredient. (The simulated and
actual field studies should be conducted with the end-use for-
mulation.) This position is based on the Agency's conclusion that,
in most cases,, the toxicity of an end-use formulation is directly
proportional to the toxicity of the active ingredients in the product
and their 'amounts. The Agency is aware that the acute toxicity of
the technical grade of an active ingredient can differ significantly
from that of the end-use formulation containing that active ingredient.
The Agency also recognizes that the various inerts (e.g., emulsifiers,
surfactants, binders, etc.) in an end-use formulated product may
affect the toxicity of the active ingredient. Nevertheless, the
Agency believes that short-term acute toxicity tests conducted with
the active ingredient will provide adequate information for identify-
ing end-use products which may potentially pose risks to wildlife
or aquatic organisms. These risks can be further evaluated in
additional studies, including pen or field tests, if necessary.
In addition, short-term acute toxicity tests conducted with the
technical grade. Of the active ingredient yill have less of an
economic impact; than short-term acute toxicity tests conducted
with each endwise formulated product.
There will be circumstances where an exception to the above
rule is appropriate. Testing end-use formulations, certain inert
ingredients, or other product components of concern, in short-term
tests, will be required according to 40 CFR § 158.75(b) on a non-
routine as-needed basis. Past experience shows that data from
these tests conducted with end-use products are only required in
unusual cases. Testing with end-use formulations in short-term
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8
aquatic tests is required by 40 CFR § 158.145 only under the fol-
lowing s ituations: ,.
- When the end-use product is expected to be introduced directly
into the aquatic environment when used as directed;
- When an ingredient in the end-use product other than the active
ingredient is expected to enhance the toxicity of the active
ingredient; or ;
- When a specified EC50 of the technical grade of the active
ingredient approximates the expected residue level in the aquatic
environment.
,'.' :i • ' •..•Yi. . ,•'.'. •.- . ',,-''•
The Agency is aware that a single registration of pesticide
products sometimes encompasses multiple formulations (i.e., same
active ingredient at same concentration but varied inert ingredient
components and concentrations). Agency concern about multiple
formulations ;is confined principally to formulation testing in §§71-
5 and 72-7. Under these two studies, the Agency permits test results
to be obtained from testing a representative formulation for the
registration under consideration. This is because, in most instances,
the overriding adverse effect would be due to the- active ingredient,
which is the ingredient of principal concern. The Agency according
to 40 CFR § 158.75(b) may, on a case-by-case basis, require testing
of more than" one of'the multiple formulations under a single regis-
tration; but this will not Occur often. In these instances, the
concern would be with evaluation of possible adverse effects that
may be attributed to the various inert ingredients that would
differ from formulation to formulation under the same registration.
E. Endangered Species.
Several commenters argued that the guideline standards which
recommend against the conduct of pen and field studies in areas
containing, or suspected to contain, threatened or endangered
plants or animals [current §§ 71-5(b)(4) and 72-7(b)(4)J, are
excessively restrictive and could place unnecessary and unrealis-
tic limitations on field testing. One conmenter stated that large
areas of the U.S. could not be used if this policy were strictly
enforced, especially for endangered animals that are mobile, far-
ranging, or migratory. Another ccinmenter would like to see this
guideline standard deleted because it pertains to the preservation
of wildlife rather than the evaluation of chemicals.
With regard to endangered species that are mobile, far-rang-
ing, or migratory, the Agency recommends that field tests not be
conducted in areas determined to be "critical habitat" by the
Office of Endangered Species of the U.S. Fish and Wildlife Service
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or in areas known, or suspected; 1s6; contain individuals of these
endangered species.
F. Selection of Avian Species.
Several conmenters suggested using avian species other than mallard,
pheasant, and bobwhite quail for various toxicity tests. The test species
that were suggested included domestic chickens and ducks, Japanese
quail, and songbirds. Some ccmmenters felt that there were no practical
differences between Japanese quail and bobwhite quail for pesticide
hazard evaluation purposes. They believed, moreover, that Japanese
quail is as sensitive to toxic effects as the bobwhite, and, like
the bobwhite, there is an existing data base on the effects of
pesticides on this species. Other ccmmenters felt that songbirds
were more sensitive and more representative of the North American
avifauna than the recommended species and, therefore, should be
used. Finally, according to some ccmmenters, domestic ducks and
chickens have not been demonstrated in the published literature
to be less sensitive than the preferred species, and therefore
ducks and chickens should be equally acceptable as test species.
In response to the very emphatic and numerous comments on this
topic, the Agency's determinations and rationales pertaining to
preferred species for use in avian tests follow in the next several
paragraphs. Generally, the species named in the 1978 proposal for
the avian test were retained for the current guidelines.
Ideally, the evaluation of the toxicity and hazards of a pesti-
cide to wildlife would be made under all field conditions in which
the pesticide may be used, and would be made using as many species
as would likely be exposed. However, the number of species to be
evaluated, the number of random, uncontrollable variables, and the
size and complexity of some application sites, combine to make such
testing completely impractical on a routine basis. Thus, for purposes
of hazard evaluation, it is necessary to limit investigations to
those species which will yield the most useful information at a cost,
in time and money, that is not prohibitive.
Recognizing, therefore, that it would be impossible to test
every potentially exposed species, EPA has recommended one or more
species which would provide data most useful for hazard evaluation
and consistent comparison among chemicals. The following criteria,
which are not listed in order of importance, have been considered in
choosing species to be tested:
- (A) The test species should be one which has demonstrated
sensitivity to the effects produced by known toxic chemicals;
that is, it should be a species about which the Agency possesses
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10
substantial historical information on the dose/response of the
test organism to a variety of pesticides. With such information,
EPA can be relatively confident that the species will also be
sensitive to toxic effects caused by a previously unevaluated
chemical. In addition, such baseline data allow EPA to compare
the toxicity of different pesticides, the results obtained in
different test facilities, and the results obtained in the same
test facility over a period of time. The latter two comparisons
are one way'in which EPA can check the quality of the data which
it receives. The first comparison is useful in hazard evaluation.
(B) The test species should be ecologically significant, i.e.,
one that occurs naturally in large numbers and in widespread
habitats. Since hazard evaluation is made according to the use
pattern, the most appropriate test animals will be those that
occur naturally in the ecological communities associated with
the use pattern. If only a limited number of test species are
to be recommended, then a substantial benefit is gained by
testing a species that is abundant and widely distributed,
because birds from that species are likely to be exposed to
pesticides from a variety of use patterns. A number of species
might be suitable simply on the basis of sensitivity, but with
widely distributed species there is an additional advantage in
that a more direct extrapolation from'laboratory tests to field
situations' can be made. ' :
(C)" For the purposes of this Subdivision, it is desirable to use
a test species which is aesthetically or economically valuable
to man. This is particularly appropriate, considering that many
Agency decisions are based on risk-benefit analyses.
(D) Test organisms should be readily available for test purposes.
Moreover, they should not be endangered or threatened species,
they should be amenable to captivity, and they should be econo-
mical to maintain in a laboratory environment. For a test
species to meet these criteria, relatively extensive information
about the nutritional, habitat, and behavioral characteristics
of the natural population should be known. The source and
history, including origin of breeding stock, genetic composition,
and history of disease should be .accessible for documentation.
(E) The characteristics of test organisms should be appropriate
to the types of tests being conducted. They should demonstrate
observable effects within a reasonable period of time. For
example, they should have a life cycle short enough to accommodate
reasonably short (1-2 years) chronic and reproductive tests.
They should also be appropriate for acute, dietary, reproductive,
and simulated field tests so that the comparisons of the various
tests can be made within the species. They should be amenable
to an experimental design that can be statistically analyzed.
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11
For example, birds that lay only two eggs per year are generally
not appropriate for reproduction tests. Finally, it is desirable
that the test organisms not be overly susceptible to disease,
parasites, and handling.
Not surprisingly, no single species completely satisfies all of
the above criteria for every kind of test. The available literature
dealing with the toxicity of pesticides to avian species indicates
that, for the majority of pesticides tested, mallard and bobwhite are
as sensitive or more sensitive than Japanese quail and other domestic
birds. If Sensitivity were the only criterion for the selection of
test species, the choice between the species would be difficult
to make. However, many other factors (as mentioned above) influence
the selection of acceptable test species.
After much debate and after considering the criteria presented
above, the Agency believes that, at least for the present, allowing
the use of Japanese quail for toxicity tests is not appropriate. The
primary advantage to using Japanese quail is that they are easily
and economically maintained and bred in the laboratory. However,
the Japanese quail is generally no more sensitive than bobwhite
quail and has a smaller toxicity data base than bobwhite quail.
Furthermore, the Japanese quail has not become a part of any North
American ecological community, and consequently we know little '
about how it would behave if used in field studies. Therefore,
some species other than Japanese quail would probably be required
for higher tier studies, and EPA would lose the advantages of
comparing data from different kinds of studies on the same species.
The same observations apply similarly to the domestic duck and
chicken. Accordingly/ the Agency believes that these three Species
are not as suitable as the ones selected for these guidelines.
However, it will review the question over the next several years
as new data become available.
The Agency agrees that songbirds may often be more sensitive to
pesticides than the species required by the guidelines. The Agency
also recognizes that many of the nontarget species exposed to a pesti-
cide may be songbirds and, therefore, they may be more representative
of the exposed avian species than the species required by these
guidelines. However, there are no data which show that results from
testing with a species of songbird are more useful than bobwhite or
mallard for predicting the effects of a pesticide on a large number
of bird species. In the future, the Agency may include songbirds in
its test scheme. However, the Agency recognizes that the feasibility
of using songbirds for testing on a regular basis has not been
demonstrated except possibly for the acute oral toxicity test.
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G. Age of Birds.
Several ccmmenters recommended changes in the age range proposed
in the avian testing standards of §§ 163.71-Kb) (3), 163.71-2(b)(4),
and 163.71-4(b)(4). Commenters suggested that younger birds, 10 to
17 days old, should be used in the avian single dose oral test because
they are more sensitive, more uniform in response, more available.,
less expensive, and easier to house and handle. They felt that use
of the same age birds for both acute and dietary tests would provide
greater consistency and comparability. Concerning the avian dietary
test, coamenters felt that there was too much flexibility allowed in
the current age requirement of 10 to 17 days. Some recommended ages
were: at the time of yolk: sac absorption, 10-16 days for quail, and
5-11 days for mallards. The latter was
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13
- Because the Agency can be reasonably assured that the birds have
not been previously exposed to pesticides as adults;
- Because variability is markedly reduced when all test birds are
of the same age;
- Because the age of immature birds approaching their first breeding
season can be determined more accurately than the age of adult
birds; and
- Because first-year birds would be of lower cost than second or
third-year birds.
H« Avian Test Doses and Concentrations - Criteria for LD50 and
LC50 Determinations.
Two sections of the proposed guidelines, § 163.71-1 "Avian
single-dose oral LD50 test" and § 163.71-2 "Avian dietary LC50 test,"
provided that substances not particularly toxic to birds need not be
tested as thoroughly as more toxic chemicals. Specifically, these
sections stated that the applicant may submit data showing that the
avian LD50 is greater than 2,000 mg/kg, or that the avian.LC50 is
greater than 5,000 ppm, instead of determining precise LD50 or LC50
values. Several conmenters wanted to know the Agency's rationale
for selection- of these values.
The Agency provided these "cut off" levels as an option to
the determination of -precise LD50 and LC50 values for relatively
innocuous compounds. These values (2,000 mg/kg and 5,000 ppm) are
^arbitrary. However, the Agency believes that few (if any) pesticides
will be applied at a label rate that will result in residues equal
to or greater than these values. In addition, these "cut off" levels
have a history of use in the literature on avian toxicology, as
evidenced by:
- (A) Tucker, R.K., and D.G. Crabtree. 1970. Handbook of Toxicity
of Pesticides to Wildlife. Fish and Wildlife Service Resource
Publication 84. U.S. Dept. Interior, Washington, D.C., 131 pp.;
and
- (B) Hill, E.F., R.G. Heath, J.W. Spann, and J.D. Williams.
1975. Lethal dietary toxicities of environmental pollutants to
birds, u.s. Fish and Wildlife Service Special Scientific Report
- Wildlife No. 191. U.S. Dept. Interior, Washington, p.C. 6t pp.
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14
I. Avian Single-Dose Oral LD50 (§ 71-1).
A number of commenters questioned the need for the avian
single-dose oral LD50 test (proposed § 163.71-1). Various reasons
were given by the different commenters:
- The acute oral test is artificial and not representative of field
situations;
- The acute oral test often provides a misleading picture of hazards
through underestimation of the toxicity of accumulative chemicals
or overestimation of the toxicity of easily degraded chemicals;
- Data from the dietary LC50 tests provide a reliable basis for
assessing potential hazards, even for granular formulations;
and .
- There are too many bird tests in the guidelines.
Finally, another commenter suggested that performing this test is
only appropriate if the dietary LC50 is less than 500 ppm or if
other data indicate an unusual hazard.
The Agency recognizes that the avian acute oral test does not
represent actual exposure in field situations, except in the case
of granular formulations, and baits. The consumption of pesticide
granules and baits by birds is an exposure that is very similar to
the exposure presented by the laboratoryLD50 test. The Agency'
contends that either the acute oral test or the dietary test, used
alone, may be misleading in evaluating a pesticide that has cumulative
effects, or one that is easily degraded. One or the other, however,
is likely to represent acute toxicity, and the two in concert should
adequately define the potential hazard to birds.
The Agency rejects the contention that dietary tests are reliable
indicators for granular formulations, particularly when one or a few
granules exceed the avian LD50 level. Many N-methyl carbamates, for
example, are formulated into granules and are much more toxic to
birds in acute oral doses than in dietary doses. Published data and
data in Agency files indicate substantial disparity between acute and
dietary toxicities of carbamate pesticides. This disparity, which
also exists for a few other pesticides, is great enough that reliance
on the dietary toxicity alone could result in unsound hazard
assessments.
The Agency also rejects the contention that the guidelines con-
tain an excessive number of avian tests. The two avian tests that
are routinely required by 40 CFR § 158.145 are relatively inexpen-
sive and easy to perform, and they are essential to assess hazards
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15
to various avian species. The costs of these tests in 1982 are
estimated to be $1800 for the avian single-dose oral LD50 test,
and $3600 for both avian dietary LC50 tests.
The Agency uses data from the avian acute oral LD50 test to
establish the acute toxicity level of a pesticide to avian species.
The test also provides additional information that is useful for
determining-hazards of a pesticide to birds. For example, symptomo-
logy is used to establish the toxic action of the pesticide to
avian species. The slope of the avian LD50 dose-response line
provides information on safety factors for acute effects; a gradual
slope requires a higher safety factor for acute effects than does
a steep slope.
J. Mammalian Acute Toxicity (.$ 71-3).
Based on the number and nature of comments received in regard
to proposed § 163.71-3 (formerly "Mammalian Acute Toxicity,- now
Wild Mammal Toxicity"), the Agency has concluded that this section
needed revision to eliminate misunderstandings regarding the guidance
when such data would be required and what test procedures would be
acceptable. ,
The guidelines now expressly state that data from this test are
not routinely required by 40 CFR § 158.145, since mammalian data
required by 40 CFR § 158.135 and developed according to Subdivision
P XHazard Evaluation: Humans and Domestic Animals) are normally
S "Sh!:1* f°r.aS*:SSing thS ha2ardS t0 wild *ammals. m addition,
? % ? re<^ed" paragraph has been revised to reduce the unin-
tended emphasis on ruminants. The testing standards and protocols
have also been modified and expanded in order to address the diver-
sity of situations in which additional testing might be necessary
to *«* ^ m°re guidance for Applicants in developing methods
to address specific situations. Additional minor changes have
been made in the standards for reporting and evaluation of data.
*" well-d<^eloped and tested methods for deter-
the toxicity to laboratory animals, toxicity testing has
not been extensively performed with wild mammals. The Agency wel-
"'
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16
K. Avian Reproduction Test (§ 71-4)
Several years ago the Agency recognized that pesticides were
causing reproductive impairment in some bird populations. Con-
sequently, in § 162.82 (cM2)(ii)(A) of the 1975 proposed guide-
lines and in § 163.71-4 of the 1978. guidelines, the Agency decided
to provide avian reproduction tests for chemicals which had the
potential to affect reproduction in birds. The design of these
tests, as with most test protocols, is dynamic. The Agency is
constantly striving to update and improve these protocols.
In the past, these guidelines have recommended a testing design
which placed groups of birds in each pen (i.e., mallards: 2 males,
5 females; and bobwhite: 1 male with 2 females). With this arrange-
ment the number of replicates (pens) needed for each treatment was.
5 and 12 for mallard and bobwhite, respectively. At the 1980
FIFRA Scientific Advisory Panel meeting, the Agency concurred with
the option of pairs testing to accomodate various segments of the
scientific ccmmunity who support such a design. Now that data are
available, the Agency has analyzed the efficiencies of both testing
designs. The group design provided sufficient sensitivity to detect
a statistically significant reproductive impairment of 20% or more
using the recommended number of replicates. For the pairs testing
to achieve this sensitivity, more than 12 replicates are necessary;
calculations have indicated that"as many as 25 replicates may be
necessary.
Based on our analysis, the Agency continues to recommend the
group-pen design. This is consistant with the previous proposed
guidelines. A registrant, in consultation with a testing facililty,
may wish to use the pair-test design. In this case an attempt should
be made to design a test which provides sufficient sensitivity to
detect statistically significant reproductive impairment of 20% or
more. A reference for calculating the number of replicates needed
for a particular level of sensitivity is now included in the guide-
lines .
This change will result in a more consistent determination of
reproductive impairment, regardless of test design. There will be
no increase in cost if the group-pen design is selected; however, a
moderate increase in cost for the pair-pen test design may be in-
curred due to the increased number of replicates.
L. Small Pen Field Test (§ 71-5) - Protocol.
Commenters of proposed § 163.71-5 recommended deleting the small
pen field test, citing numerous deficiencies in the protocol for such
tests. Among the problems noted were:
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17
- That such tests do not accurately predict the hazard to wild
birds whose behavior differs from that of domestic birds;
- That the pen must be moved frequently so that the birds can be
exposed to the pesticide and adequate food sources; and
- That the birds are vulnerable to attack from predators.
The Agency recognizes the limitations in the design of small pen
tests referred to above, some of which were mentioned in the preamble
to the 1978 proposed guidelines. In that preamble, the Agency also
requested suggestions on how to improve the test design to avoid
these problems, but received no useful suggestions from the public.
The Agency again welcomes suggestions that can be supported by data,
literature, or specific rationales.
Until a better small pen test is developed and tested for
validity, feasibility, and comparability, the Agency has deleted the
existing small pen protocol from the guidelines. The existing large
pen protocol, with some modifications, will be used in its place.
Data produced by using the large pen test have proven to be more
useful to the Agency than those from small pen tests.
M. Actual Field Test (§ 71-5).
Commenters have stated that the actual field test for mammals
and birds is too comprehensive, complex, expensive, and time-consum-
ing, and therefore such tests should be eliminated. They have
also pointed out problems with specific aspects of the protocol in
proposed § 163.71-5. The Agency fully recognizes the complexity
and cost of a full-scale field test, and consequently, the Agency
provides this test only to evaluate those products which pose
significant risks to nontarget birds or mammals. The studies
conducted in previous tiers are designed to yield specific infor-
mation which forms the basic foundation of a hazard assessment.
Because of their design, however, the lower tier studies cannot
always be used to adequately predict the effects of the pesticide
in real use situations. Therefore, it may become necessary to
perform the actual field test when the previous laboratory or cage
studies demonstrate toxicity at normal use rates or indicate poten-
tial long-term adverse effects. The actual field study results
would then either support earlier indications and alter plans for
certain uses or for prospects of obtaining a registration, or they
would disprove the potential hazards evinced in the earlier tests
and thus maintain proposed uses and improve prospects of obtaining
product registration.
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18
EPA notes that each actual field test should be specifically
tailored to address the concerns that necessitate it. In developing
a test methodology, applicants are advised to consult with appropriate
EPA scientists to assure that the information obtained is pertinent
and sufficient to satisfy the Agency's hazard evaluation requirements.
In view of the diversity of potential study areas, (e.g., forests,
rangelands, rights-of-way, wetlands; and major croplands such as
cotton, wheat, corn, etc.) field studies should be designed on a case-
by-case basis. Consequently, the previously published protocol has
been deleted. In its place are a number of selected references that
can be used as guidance for developing actual field studies.
N. Aquatic Test Concentrations - Criteria for LC50 Determination.
Several sections of the guidelines for acute toxicity testing
with aquatic organisms provide that substances which are not parti-
cularly toxic need not be tested as thoroughly as more toxic chemi-
cals. Specifically, these sections state that the applicant may
conduct a range-finding study, and if the LC50 is greater than 300
mg/1 or is greater than 300,000 times the expected environmental
concentration, the determination of a precise LC50 value is not
necessary. [See §§ 72-Kb), -2(b), -3(b).] Several conmenters
suggested that these "cut-off" levels are too high.
In light of public response and the Agency's experience in
working with the previously established levels, the Agency has de-
cided to reduce the levels. The intent of the "cut-off" levels is
to preclude the need for definitive acute tests when the range-
finding tests indicate the chemical/pesticide to be relatively
non-toxic. After using the previous "cut-off" levels for several
years, the Agency has not had to request additional acute studies.
Accordingly, the Agency has decided to reduce the levels to a
concentration of 100,000 times the EEC or 100 mg/1. If, after
using the new levels, the Agency finds they do not provide adequate
data for hazard assessment, additional changes will be made. Note
that the standard for testing 30 organisms at either of these
criteria with less than 50 percent mortality remains unchanged.
0. Selection of species for freshwater fish acute toxicity studies.
Considerable public comment focused on the number and choice of
test species for freshwater fish acute toxicity studies. Basically,
these comments favored use of only one species, rather than the two
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19 •••"• >••>-• • .,.,.
fish species required by the proposed guidelines, § 163.72-1. In
addition, the conunenters ' suggested a number of factors which should
be considered in selecting the species to be tested, such as the
most sensitive species, the species most likely to be exposed
under normal use conditions, or the species which would be used if
higher tier tests are required. Also, one ccmmenter recommended
the fathead minnow as a test species in the fish acute toxicity
study because the fathead is also used in the fish life-cycle
study. In the past, industry representatives and other concerned
individuals have asked for a more precise definition of the terms
"coldwater fish" and "warmwater fish." The guidelines provided for
the testing of fish from both categories, and since these categories
were loosely defined by examples, a more precise definition was
needed. The characteristics of the fish families Centrarchidae
and Salmonidae have been used to exemplify a warmwater and coldwater
fish, respectively. ..".-' : •
The Agency has defined coldwater fish as those fish which normally
spawn in the fall and winter months in Water colder than 60°F (15°C).
Warmwater fish are defined as those fish which normally spawn in the
summer months in temperatures greater than 60°F. Examples, as
previously stated, are the Centrarchids and Salmonids for warmwater
and coldwater fish. The preferred warmwater species is the bluegill
sunfish (Lepomis macrochirus) and the preferred coldwater fish is the"
rainbow trout (Salmo gairdneri). Data from two species^ are used to
indicate the extent of variation in sensitivity between;the different
species. In addition, by testing two species, the Agency will have
data available on fish representative of a much larger geographical
range than if only One species were tested. These considerations
outweigh the small additional costs associated with testing a second
species. Finally, while the Agency would not reject data from tests
using the fathead minnow, it is not as sensitive to most insecticides
as the bluegill or rainbow trout, and thus is not generally preferred
for this studv.
P. Marine Toxicity Tests. . :
Commenters objected to the testing provided in proposed § 163.72-3
for developing data on the acute toxicity of a pesticide to marine and
estuarine organisms. They stated that this testing is not needed every
time a pesticide is applied directly to marine or estuarine environments.
They argued that EPA should consider the concentration of a pesticide
in these environments resulting from actual use before determining
the need for this testing. For example, they suggested that if
the estimated environmental concentration of a pesticide is less
than the LCSO or EC50 of that pesticide for freshwater organisms,
the data would not be needed.
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20
Conmenters also questioned the Agency's need for data on the
toxicity of a pesticide-to marine species in light of newly-published
literature showing a correlation between toxicity data from tests
with freshwater fish (rainbow trout) and tests with saltwater fish.
Based on this relationship, the cemmenters suggested that the data
from marine studies is not needed if a high concentration of a
pesticide, produced no effect in a test using freshwater fish.
The Agency is firmly convinced that it needs data on the acute
toxicity of a pesticide to estuarine and marine organisms. With it,
we evaluate the hazard of that pesticide when it is applied to a
marine or estuarine site. Direct applications to these environments
are made for the'i purpose of reducing the population of one or more
pest organisms existing there. Thus, there is every reason to
expect that the pesticide will be present in quantities which may
be toxic to npritarget ,organisms as well.
The Agency rejects the suggestion to use data from freshwater
fish toxicity studies to predict the hazards to saltwater fish. The
predictive equation that has been developed based on the correlation
between known rainbow trout LC50 values and known saltwater fish LC50
values for a number of chemicals cannot be used by the Agency. The
95 percent confidence limits of the predicted saltwater fish LC50
values are equal to +_ T-3, or 2.6 orders of magnitude. Saltwater
fish LC50 values with confidence limits of this magnitude do not
permit EPA to judge whether a particular pesticide will pose
unacceptable risks when it enters the marine environment.
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21 • •-,
SUBDIVISION E - HAZARD EVALUATION: WILDLIFE AND
AQUATIC ORGANISMS
Series 70: GENERAL INFORMATION AND REQUIREMENTS
§ 70-1 General information.
*a* Scope and purpose. This subdivision provides guidelines
for testing and data submission concerning the effects of pesti-
cides on nontarget birds, wild mammals, fish, and aquatic inverte-
brates. Each section contains standards for acceptable testing,
guidance on evaluation and reporting of data, examples of acceptable
testing protacols, and further guidance on when data are required.
The data produced by following these guidelines will be used by
the Agency for determining potential hazards to nontarget organisms
resulting both from direct and indirect exposure to pesticides.
(b) Organization. (1) Categories of tests by organism
This subdivision contains two broad categories of tests:
(i) Tests for pesticidal effects on birds and mammals
(§§ 71-1, -2, -3, -4, and -5); and
(ii) ' Tests for pesticidal effects on fish and aquatic in-
vertebrates (§§72-1, -2, -3, -4, -5, -6, and -7).
(2) Tier tests. (i) The Tier 1 tests within each of the
categories named above, document the acute and subacute pesticidal
effects on birds, mammals, fish and aquatic invertebrates. These
tests are required by 40 CFR § 158.145 to support the registration
of most pesticide products. Tier 1 tests on birds and mammals
appear in §§ 71-1 and 71-2. Tier 1 tests on fish and aquatic
invertebrates appear in §§ 72-1 and 72-2.
(ii) The "When required" paragraphs in §§ 71-3, -4, and -5
and 72-3, -4, -5, -6, and -7 refer to 40 CFR § 158.145 and provide
further guidance as to when data, in addition to Tier 1 test results,
will be required to support the registration of a pesticide product.
(iii) Figures 1, 2 and 3 (below) illustrate the tier testing
system for avian wildlife, wild mammals, and aquatic organisms,
respectively. These figures, however, do not constitute requirements.
The applicant should refer 40 CFR Part 158 for the actual requirements,
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22
Figure 1. Avian Testing Flow Chart.
Tier 1
SINSLE-DOSE ORAL
LD50 - § 71-1
RELATED INFORMATION
such as use pattern,
chemistry, and human
toxicology
AVIAN DIETARY
LC50 - § 71-2
Tier 2
SPECIAL TESTS
§ 70-1
! I
V
REPRODUCTION TEST
§ 71-4
Tier 3
SIMULATED (PEN) FIELD TEST
§ 71-5
Tier 4
I
I
V
ACTUAL FIELD TESTING
§ 71-5
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23
Figure 2. Wild Mammal Testing Fl&w Snir't.
Tier 1
Tier 2
RELATED INFORMATION
such as use pattern,
chemistry, and human
toxicology
WILD MAMMAL
TOXICITY TESTS
§ 71-3
Tier 3
REPRODUCTION TESTS
see Subdivis ion F
§ 83-4
SPECIAL TESTS
§ 70-1
SIMULATED (PEN) FIELD TEST
§ 71-5
Tier 4
V V
ACTUAL FIELD TESTING
§ 71-5
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Tier 1
24
Figure 3. Aquatic Organism Testing Flow Chart.
ACUTE TOXICITY FOR
FOR FRESHWATER FISH
. § 72-1
RELATED INFORMATION
such as use pattern,
~> chemistry, and human *•"
toxicology
ACUTE TOXICITY
TEST FOR FRESHWATER
AQUATIC INVERTEBRATES
§ 72-2
Tier 2
I
V
FISH EARLY LIFE-
STAGE AND AQUATIC
INVERTEBRATE LIFE-
CYCLE TESTS
SPECIAL
TESTS
§ 70-1
/
Tier 3
FISH LIFE-
CYCLE TESTS
§ 72-5
I
I
Tier 4
ACUTE TOXICITY
TEST FOR ESTUARINE
AND MARINE ORGANISMS
§ 72-3
AQUATIC ORGANISM
ACCUMULATION TEST
§ 72-6
V
SIMULATED* OR ACTUAL FIELD
TESTS OF AQUATIC ORGANISMS
§ 72-7
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25
(c) Range-finding tests. Unless the approximate toxicity of
a test substance is already known, it is usually desirable to
conduct a range-finding test to determine the concentrations of test
substances that should be used in the definitive tests in this
subdivision. -
(d) Special test requirements. In addition to the testing
guidance provided in all succeeding sections of this subdivision,
data derived from additional tests may, under unusual circumstances,
be required by 40 CFR § 158.75(a) the Agency for maki-ng judgments
regarding safety to wildlife and aquatic organisms. Methods may
be derived from tests already described or cited in this or other
subdivisions of these guidelines. Such data requests may relate
to a proposed pattern of use, a toxicological mode of action, or a
unique chemical property. The data requested will be specific to
the problem. Examples of tests for these unusual circumstances
include but are not limited to:
(1) Avian acute dermal LD50;
(2) Certain toxicity data from Subdivision F, such as rabbit
dermal LD50, eye irritation, and acute inhalation LC50;
(3) Fish or bird cholinesterase tests;
(4) Metabolism studies;
(5) Secondary toxicity studies on birds or mammals;
(6) Domestic animal safety studies (see Subdivision F, § 85-2);
and
(7) Pesticide efficacy testing, as outlined in Subdivision
G, regarding pest control that may have major impact on the food
supply or food chain of a rare or endangered species.
§ 70-2 Definitions.
(a) Terms used in this subdivision have the meanings
set forth in 40 CFR § 162.3 and at § 60-2 of Subdivision D.
(b) In addition, for the purposes of this subdivision:
(1) The term "coldwater fish" means those fish which normally
spawn in the .fall and winter months in water colder than 60°F
(15°C), e.g., fish from the family Salmonidae.
(2) The term "continuous exposure" means an extending or
prolonged exposure usually resulting from a single application of
a persistent pesticide.
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26
(3) The term "ECSO" (median effective concentration) is
the concentration of a substance producing a specific effect or
response in SO percent of the test organisms. This determination
is usually made when some effect other than lethality is being
studied. The effect should be one which is easily observed and
which is usually associated with lethality, e.g., failure of
daphnids to demonstrate swimming response to light stimulus.
(4) The term "estimated environment concentration" (EEC)
means an estimate of the concentration of a pesticide occurring
in or on various media (i.e.* soil, water, vegetation) after
pesticide application, as determined from the results of environ-
mental fate testing described in Subdivision N.
(S) The term "maximum expected environmental concentration"
(MEEC) means the highest concentration of a pesticide that would
occur [usually immediately after application(s)] at a site or in a
medium (e.g., water, vegetation, or soil) contaminated with a
pesticide, as determined from the pesticide application rate.
(6) The term "-typical end-use product" means an end-use
product representative of a major formulation category (e.g.,
emulsifiable concentrate, granular product, wettable powder)
containing the active ingredient of the applicant's product.
(7) The term "warmwater fish" means those fish which
normally spawn in the summer months in temperatures greater than
60°F (15°C), e.g., fish from the family Centrarchidae.
§ 70-3 General test standards.
(a) General policy. This section provides general test
standards which apply to all tests in this subdivision. If, for
a particular chemical, there should arise a conflict between the
general standards and the standards for a particular test, the
more specific standards should apply.
(b) Test methods. (1) Toxicity tests should be conducted
according to uniform methods, whenever possible, to maximize the
number of reliable comparisons that can be made concerning relative
toxicity and relative sensitivity.
(2) Tests should include a concurrent control group. If
a vehicle (carrier, solvent, or diluent) is used, the concurrent
control1group should be treated with that vehicle. Vehicles known
to be toxic, synergistic, or antagonistic should not be used.
(3) All control groups should be maintained in the same
manner as the test groups. However, control groups should be
protected from airborne or other contamination by the test
substance(s).
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27
(4) In no case should the results of range-finding test(s)
and definitive test(s) be combined in calculating the LD50, LC50/
or EC50. Results of two or more definitive tests may be combined
to calculate the LD50, LC50, or EC50 only if. the tests are run
concurrently using test animals which are from the same source
and which were raised under the same conditions.
(<=) Test substance. (1) Each section of Subdivision E
provides guidance concerning the substance to be tested, i.e.,
whether the data submitted in support of an application for regis-
tration should be derived from tests conducted with the technical
grade of the active ingredient, the end-use product, or a typical
end-use product.
(2) The technical grade of the active ingredient is
commonly the same substance as the manufacturing-use product
for which registration is sought or which is used to produce the
formulated pesticide product for which registration is sought.
In this case, a sample of the manufacturing-use product may be
tested in lieu of testing the technical grade of the active
ingredient.
(3) In addition to or in lieu of testing otherwise provided
by this subdivision, the Administrator may, in accordance with 40
CFR § 158.75(b), require testing to be conducted with:
(i) An analytically pure grade of an active ingredient;
(ii) The technical grade of an active ingredient;
(iii) The inert ingredient of a pesticide end-use formulation;
(iv) A contaminant or impurity of an active or inert
ingredient;
(v) A plant or animal metabolite or degradation product
of an active or inert ingredient;
(vi) The pesticide end-use formulation;
(vii) The pesticide end-use formulation plus any recommended
vehicles and adjuvants;
(viii) Any additional substance which could act as a
synergist to the product for which registration is sought; or
(ix) Any combination of the above substances.
(4) The test substance should be within the limits, if any,
certified in accordance with the requirements of 40 CFR § 158.110.
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28
(5) The test substance should be stored under conditions
that maintain its stability.
(6) If a vehicle is used to dissolve or dilute the test
substance, it should be chosen to possess as many of the fol-
lowing characteristics as possible: ;
(i) It should not interfere with absorption, distribution,
metabolism, or retention of the test substance;
(ii) It should not materially alter the chemical properties
of the test substance and not materially enhance, reduce, or
alter the toxic characteristics of the test substance;
(iii) It should not materially affect the food and water
consumption of the test organisms; and
(iv) At the levels used in the study, it should not produce
physiological effects or have local or systemic toxicity. In
addition, such a vehicle should, if possible, have a solvent
polarity similar to the vehicle to be used in the end-use
formulation.
(d) Test organisms. (1) All data submitted in support
of an application for registration should be derived from tests
conducted in accordance with good laboratory or field practices
for handling and caring of test organisms. Only healthy
organisms may be used, and they should be kept in conditions
conforming to good husbandry or cultural practices. (See Animal
Welfare Act, Pub. L. 94-279.)
(2) The organisms in each test should, as nearly as
practicable, be of uniform weight, size, and age.
(3) Organisms should be randomly assigned to test groups
to minimize bias and assure comparability of pertinent variables.
(4) The number of organisms tested per concentration,
and the number of concentrations or dosage levels evaluated should
be sufficient to yield statistically sound data.
(5) Organisms selected for testing should be species of
wildlife and aquatic organisms currently established in the United
States and its coastal waters, and should be phenotypically
indistinguishable from wild species.
•(6) In no circumstances shall threatened or endangered
species be used as test organisms.
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29
(7) Observations. Observations should be made as frequently
as necessary to record all signs of intoxication and abnormal
behavior.
§ 70-4 Reporting and evaluation of data.
(a) General policy. This section establishes general
guidance for reporting and evaluation of data which apply to all
tests in this subdivision. In the case of conflict between general
and specific guidance for reporting and evaluation of data, the
latter will govern.
(b) Basic standards for reporting and evaluation. (1)
Each test report should include all information necessary to
provide a complete and accurate description of test procedures
and evaluation of the test results.
(2) Each test report should include a summary of the
data, an analysis of the data, enough data for the Agency to
verify calculated statistical values, and a statement of con-
clusions drawn from the analysis. The summary should be of
sufficient detail to permit the reader to independently under-
stand the conclusions.
(3) The metric system should be used in test reports;
the English system may also be used.
(c) Standards for specific asaects of reporting and evalua-
tion. In addition to the guidance provided by other sections of
this subdivision, each test report should include or reference the
following information!
(1) Test method. (i) Statement of test method used
and a full description of the experimental design and procedures
[see also paragragh (c)(8) of this section];
(ii) The length of the study and the dates on which the
study began and ended;
(iii) The name and address of the laboratory performing
the test;
(iv) The location where the test was performed; and
(v) Name(s) of the principal investigator(s).
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30
(2) Test substance, (i) Identification of the test
substance/ including composition, chemical name and percentage of
active ingredient, molecular structure of the active ingredient,
and qualitative and quantitative description of the chemical
composition including the results of the analysis referenced
in § 70-3(c)(4).
(ii) Manufacturer, lot and sample numbers of the test
substance.
(3) Test organisms, (i) Identification of test organisms
(scientific names);
(ii) Rationale for selection of species, if the species
used is different than that preferred under the different sections
of this subdivision.
(iii) Age, sex, size, life stage, and/or weights, as provided
by the different sections of this subdivision;
(iv) Source of supply of the organisms;
(v) Method used in assigning test organisms to test and
control groups;
(vi) Number of organisms per dose or concentration level
and in control group;
(vii) Description of any pretest conditioning, including
diet; and
(viii) History of colony, including record of observed
diseases and disease treatments.
(4) Dosing or treatment. (i) Identification of method,
route, and frequency of administration of test material, and
randomization plan for treatments (when appropriate) .
(ii) Length of treatment period;
(iii) Rationale for selection of method, route, or frequency,
if different from that•reconmended in this subdivision;
(iv) Total volume of material administered (test substance
plus carrier) to each test organism or in feed or water at each
time administration is made;
(v) Any vehicle used to dissolve or dilute the test
substance, or used to mix the test substance with the feed;
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31
s
(vi) Doses or concentrations of test substances
administered (i.e., milligrams of test substance per kilogram of
body weight of the organisms or parts per million of the test
substance in the feed or water) and method of determination of
dose or concentration levels;
(vii) Dosing or treatment of control organisms; and
(viii) A description of the steps taken to maintain the
concentration s) , if the test substance is administered in the
feed or water and data from other subdiyision(s) suggest that
the actual concentrations over the length of the test may
decrease by 30 percent or more.
(5) Observations . (i) Frequency, duration, and method
of observation, including: - '•
(ii) Detailed description of the nature, incidence, time
of occurrence, severity, and duration of all observed toxic
effects, including death and any other abnormal or unusual signs
and symptoms; and
(iii) If gross pathological examinations are performed,
a description of all abnormalties observed.
(6) Environmental conditions . (i) Wildlife species
(mammals and birds). A description of the housing conditions
during and, if known, prior to the test, including:
(A) Number of animals per cage (see Animal Welfare Act,
Pub. L. 94-279);
(B) Ambient temperature and humidity;
(C) Photoperiod and lighting;
(D) A description of the diet, including identification
and composition;
(E) Source of animal feed and water; and
•i
(F) Dimensions of test pen(s).
Aquatic species (fish and invertebrates). A description
of the test chamber conditions , including:.
(A) Number of organisms per test chamber;
(B) The highest, lowest, and average water temperature;
(C) Photoperiod and lighting;
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32
(D) A description of the diet, including identification
and composition, and.''sources of feed;
(E) The source of dilution water, its chemical characteris-
tics, and description of any pretreatment; •
(F) Methods used for, and results of, all chemical analyses
of water and all toxicant concentration determinations at
beginning, during, and at end of tests, including validation studies
and reagent blanks, if there is reason to suspect that the con-
centrations administered to the test water do not approximate the
actual concentrations;
(G) The depth and volume of solution and dimensions of
aquatic test chambers; and
(H) A description of the toxicant delivery system and flow
rate, expressed as the average number of water volumes of test
solution passing through each test chamber in 24 hours (recommended
for flow-through aquatic tests).
<7) Data analysis. (i) Tabulation of the response data
at.each treatment level, including:
(ii) Calculation of the LD50, LC50, EC50, and the 95 per-
cent confidence intervals when sufficient doses and test organisms
are used to establish a dose-response line;
(iii) No-observed-effect level; and
(iv) Statistical method(s) used for analyzing data and
a reference to it (them) in published literature. If there is a
deviation from the published method, an explanation of the change
and a rationale for its use should be included.
(8) References. (i) The Agency will accept references to
the data as long as the complete text of the referenced data is
readily available for Agency review.
(ii) The applicant should submit citations of protocols
used for testing, or list references to any published literature
and copies of unpublished literature used in developing the test
protocol, performing the testing/ making and interpreting obser-
vations, and compiling and evaluating the results.
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Series 71: AVIAN AND MAMMALIAN TESTING
§ 71-1 Avian single-dose oral LD50 test.
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34
(2) Mean body weights for each test and control group
at initiation and termination of test;
(3) Diet used;
(4) Description of any antibiotics, vitamins, or food
additives added to the feed preceding or during testing;
(5) Pen dimensions;
(6) Whether the pen is indoors or outdoors;
(7) Weather conditions, if pen is outdoors;
(8) Total feed consumption for each test and control
group;
(9) Preparation of test material;
(10) Amount'of test material dosed per bird;
(11) Amount of diluent dosed per bird, if used;
(12) Number of birds per treatment level;
(13) Number of controls used;
(14) LD50 in mg/kg, with 95 percent confidence limits;
(15) Method used for calculation of LD50;
(16) Slope of the dose response line;
(17) Time and date of mortalities;
(18) All signs of intoxication and regurgitation if any
occurs; and
(19) Any gross pathological changes as noted by gross
necropsies, if conducted.
(d) Acceptable protocol. The following is an example
of an acceptable protocol for conducting an avian single-dose
oral LD50 study. This study is a modification of a study that
appears in an unpublished draft report to EPA from the American
Institute of Biological Sciences (AIBS), titled Analysis of
Specialized Pesticide Problems, Volume VI, Wildlife Toxicology
Study, Pages 10 to 16. This report is dated October, 1974 and
was funded under EPA Contract No. 68-01-2457.
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35
Species and history. The test species of choice
are pen-reared bobwhite and mallards not less than
16 weeks of age at initiation of test. Birds
should not have been mated before being placed on
test. A history of rearing practices such as
photoperiod, medication, and type of food should be
included in the report. All lots of birds should
be healthy and of uniform size, weight, and parity.
All lots should be weighed, then maintained for a
minimum of 15 days prior to the test. If tested
indoors, temperature and humidity should be con-
trolled, with freedom from drafts and sudden noises
which could disturb the test birds. Any lots of
birds that suffer an abnormal .weight loss or suffer
more than 10 percent mortality during the holding
period should not be used.
Cages. All birds should be caged by treatment
level groups or subgroups under acceptable animal
husbandry practices.
Weight. All birds should be weighed at the
beginning of the test, and on day 14 after
administration of the test substance. Also, birds
should be weighed at the termination of the test
if it extends beyond 14 days.
Diet and fasting. Feed should be withheld
from the control "and test birds (bobwhite and mal-
lard) for 15 hours prior to oral administration of
the chemical. At all other times, birds should
have free access to feed. The diet should be
standard feed ration known to be adequate for game
birds. Feed consumption should be determined for
treated and control birds. The feed consumed should
be calculated as average daily food consumption for
each dose level.
Preparation of test material. 1. The test
material should be administered without the use of
a vehicle, if possible. The test material should
be accurately weighed and put directly in gelatin
capsules. If the chemical is first dissolved in
an evaporative vehicle (e.g., acetone, methylene
chloride) in preparation for use with capsules, the
evaporative vehicle should be completely evaporated
at room temperature before the capsules are used.
2. If a vehicle is required, the preferred
vehicle is distilled water. Other acceptable
vehicles include table grade corn oil, propylene
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36
glycol, 1 percent carboxymethylcellulose, and gum
arable (acacia). The reccmmended maximum amount
of vehicle per dosage is 0.1 to 1 percent of body
weight. The total volume for all dosage levels
should approximate a constant volume-to-body weight
factor. .',, - • , , ..v. .,,.-. .. , ,,•
3. A concurrent vehicle control is required.
Vehicles known to be toxic, synergistic, or antagonistic
should not be used. The vehicle should be administered
to the control birds at the same volume as the admini-
stered to the test animals receiving the vehicle and
the .pesticide. . . . ; ,
Methods of administration. The test chemical should
be placed by oral intubation into the crop or proventri-
culus.
Number of birds per dose level. The number of
test birds per treatment level and the number of controls
should be no less than ten.
Dosage-mortality data. The factor between dosage
levels used to calculate the acute LD50 should be based
on a geometric or logarithmic scale. After preliminary
screening, there should be a minimum of 5 dosage
levels used in calculating the acute oral LD50. The
calculation of an acute oral LD50 should follow any
generally acceptable method. Two examples of
acceptable methods are Thompson and Weil (1952) and
Litchfield and Wilcoxon (1949).
In cases where the LD50 is found to be in excess
of 2000 mg/kg, based upon the 10 or more birds per
dosage level, data should show that less than half
of the birds died at the 200Q mg/kg dosage level. If
any birds die at this level, sequentially lower levels
should be tested to provide a dose-response series
which includes at least one level at which no
mortality occurs.
Period of observation. The test animals should
be observed for a.minimum of 14 days. The observation
period should be longer if signs of toxicity are
still evident. The time of all deaths should be
recorded by day. If more than 1 control bird (i.e.,
10 percent) dies during the test period, the test may
be considered invalid, depending on the circumstances
and apparent cause of mortality.
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37
Gross necropsy. Gross necropsies can give valuable
• additional data and they are encouraged, but are not
required. When gross necropsies are conducted, they
should be conducted on all dead birds and sufficient
numbers of surviving birds to characterize any gross
lesions in dead birds. Gross necropsies include general
inspection of digestive tract, liver, kidneys, heart,
and spleen. Any gross pathological changes shall be
reported.
Recording of signs of intoxication. All signs
of intoxication should be recorded as they occur.
(e) References. The following references can provide useful
background information in developing acceptable protocols; some
outline useful statistical procedures for handling data.
(1) Litchfield, L.J., and F. Wilcoxon. 1949. A simplified
method of evaluating dose-effect experiments. J. Phannacol. Exp.
Therap. 96(2) :99-133.
(2) Schafer, E.W., R.B. Bruntox, N.F. Lockyer, and J.W.
Degrazio. 1973. Comparative toxicity of seventeen pesticides to
the quelea, house sparrow and red-winged blackbird. Toxicol. Appl.
Pharmacol. 26:154-157 (See also page 279.)
(3) Thompson, W.R. , and C.S. Weil. 1952. On the
construction of tables for moving average interpolation. Biometrics
8:51-54. ' ~~ ~~
(4) Tucker, R.K. , and D.G. Crabtree. 1970. Handbook on
Taxicity of Pesticides to Wildlife . Fish and Wildlife Service
Resource Publication 84. U.S. Dept. Interior, Washington, D.C.,
(See esp . pp 6 & 7.)
(5) Tucker, R.K., and M.A. Haegele. 1971. Comparative
acute oral toxicity of pesticides to six species of birds. Toxicol.
Appl. Pharmacol. 20:57-65. (See esp. pp. 57-59.)
§ 71-2 Avian dietary LC50 test.
(a) When required. (1) Data on the avian dietary toxicity
of a pesticide are required by 40 CFR § 158.145 to support the
registration of an end-use product intended for outdoor application,
and to support each application for registration of a manufacturing-
use product which may be used to make such an end-use product.
(2) Data on the avian dietary toxicity of a pesticide are
required by 40 CFR § 158.145 on a case-by-case basis, to support the
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38
registration of an end-use product intended solely for indoor
application, and to support each application for registration
of a manufacturing-use product which may be used to make such an
end-use product.
(3) See 40 CFR § -158.50, " Formula to rs' exemption," to de-
termine whether these data must be submitted. Section II-A of
this Subdivision provides an additional discussion on this subject.
(b) Test standards. :Data sufficient to satisfy the
requirements 40 CFR § 158.145 should be derived from tests which
comply with the general test standards in § 70-3 and the following
test standards: . .-!....-.-
(1) Test substance. Data shall be derived from testing
conducted with the technical grade of each active ingredient
in the product. ,
(2) Species. Testing should be performed on two avian
species: one species of wild waterfowl (preferably the mallard)
and one species of upland game bird (preferably the bobwhite).
One of the two species selected for these studies should be the
same species selected for the avian acute oral LD50 study, § 71-1.
(3) Age. Bobwhite quail used in this study should be
from 10 to 14 days old and mallards should be 5 to 10 days old
at the beginning of the testing period. Within a given test,
all birds should be from the same hatch.
(4) Determination of LC50. Satisfactory data should
establish that tne 8-day dietary (5 days treated diet and 3
days clean diet) LC50 is greater than 5000 ppm or establish
an LC50 value and corresponding 95 percent confidence intervals.
(c) Reporting and evalution of data. In addition to
the information provided in § 70-4, the test report should contain
the following information:
(1) Age of birds (days);
(2) Breeding history of test birds;
(3) Source and strain of test birds;
(4) Mean body weights for each test and control group at
initiation and termination of test;
(5) Test diets; .
(6) Description of any antibiotics, vitamins, or food
additives added to the feed preceding or during testing;
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39
(7) Number of birds per concentration;
(8) Signs of intoxication (including unusual responses
to chemical) by test birds;
(9) Total food consumption for each test and control
group;
(10) LGSO determinations (ppm) with 95 percent confidence
limits;
(11) Specific method used to calculate LCSO's; and
(12) Slope of concentration response line.
(d) Acceptable protocols. The following are examples of
acceptable protocols for conducting an avian dietary LCSO study.
(1) This protocol is a modification of a protocol that
appears in an unpublished draft report to EPA from the American
Institute of Biological Sciences (AIB:S) , titled Analysis of Spe-
cialized Pesticide Problems, Volume YI, Wildlife Toxicology Study,
pages 17 to 22. This report is dated! October, 1974 and was funded
under EPA Contract No. 68-01-2457. '
Test. This method determines a chemical's
toxicity to birds expressed as the median lethal
concentration (LCSO) of the chemical (technical
material) in dry diet [the LCSO being the parts
per million (ppm) toxicant in ad libitum diet
expected to produce 50 percent mortality among
birds in 8 days (5 days oh treated diet followed
by at least 3 days on untreated diet)].
Species. This protocol is appropriate, with
modification (e.g., acclimation to test diet), for
most bird species, but it addresses primarily the
mallard and bobwhite. ' • *
Source of strain of birds. Test birds should
be pen-reared and phenotypically indistinguishable
from wild birds. It is recommended that only
birds be used from colonies that have had breeding
histories maintained for them, it is also preferred
that test birds be from the same hatch.
Age and sex. Bobwhite quail should be from TO
to 14 days old and mallard should be 5 to 10 days old
at the initiation of the test, it should be noted
that LCSO determinations may be affected by the age of
the birds at the time of testing. Therefore, within a
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40
given test all birds should be the same age. A random
selection of birds without regard to sex may be used.
Health of birds. Only birds in apparent good
health should be used. Birds with obvious abnor-
malities, injuries, or sickly appearance shall be
excluded. Test birds should be preconditioned to
the test facilities and untreated test diet for as
long as possible prior to the beginning to the test.
Pen facilities. Pen conditions should conform
to good husbandry practices. The facilities as
described in the following paragraphs are con-
sidered to be satisfactory.
(1) Size. Bobwhite (about 10 birds per pen)
may be tested in commercial brooder units with
individual pens measuring approximately 35 x 100 x
24 cm high. Waterfowl (mallards) may be tested in
pens 70 x 100 x 24 cm high. Floors and external
walls can be of wire mesh, while ceilings and common
walls may be of galvanized sheeting.
••• C2T ~T-emperature and relative humidity. For
bobwhites and mallards, brooder temperature should
be about 35°C, It can be maintained with thermo-
statically controlled central heating. Temperature
outside the brooder may range from 22-27°C. The
relative humidity should generally be not less than
30 nor more than 80 percent. Adequate ventilation
should be maintained.
(3) Lighting. Fluorescent or incandescent
lighting may be supplied with varying light
regimes. A diurnal photoperiod is recommended;
however, lighting maintained continuously, i.e.
24 hours per day, is also acceptable.
(4) Pen locations. Generally, studies
conducted indoors provide better control of
environmental conditions, and, if predators are
a problem, better protection from predation.
However, studies can be conducted outdoors and
still provide suitable data.
(5) Test diets. A standard commercial game
bird diet (mash form) or its equivalent should be
used. Water should be continuously available. The
test material should be added to the diet without the
use of a vehicle, if possible. If a vehicle is used,
the preferred vehicle is distilled water. However,
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41
when a test chemical is not water-soluble, the
test material may be dissolved in a reagent grade
evaporative vehicle (e.g., acetone, methylene
chloride) and mixed with the test diet. The vehicle
must be completely evaporated at room temperature
prior to feeding. Other acceptable vehicles include
table grade corn oil, propylene glycol, 1 percent
carboxymethy1cellulose, and gum arabic (acacia).
The vehicle and the dissolved test material should
be mixed thoroughly with dry commerical mash
in a ratio of 2 parts of solution to 98 parts of
feed by weight. An equivalent amount of vehicle
should be added to untreated! diets. .Diets can be
mixed by commercial mechanical food mixers.
Mashes and; toxicant should be freshly mixed in
small quantities. Then mixtures should :be ccm-
bined with larger quantities of mash to provide
uniformity in..the final dietary concentration
of pesticide in, the mash. : ,
Concentrations and dosage.mortality data.
Generally, each chemical should be administered in
at leas.t 4 dietary concentrations (but 5 :or 6 are
preferred) spaced geometrically over a stian
intended to produce mortality ranging frcin 10 to
90 percent. It is advisable to perform a range-
finding study prior to selecting concentrations
for testing.
Ten birds per concentration and 10 birds
for the control group are recommended. Experimental
variation may require more than 10 birds, and in
some cases fewer than 10 birds may be used.
In cases where the LC50 is found to be in excess
of 5000 ppm by dietary exposure, based upon the 10
or more birds per dosage level, data should show that
less than half the birds died at the 5000 ppm dosage
level. If any birds die at this level, sequentially
lower levels should be tested to provide a dose-
response series which includes at least one level
in which no mortality occurs.
Observations on signs of intoxication. Through-
out the test, all signs of intoxication and abnormal
behavior should be noted. Any unusual manifestation
of the chemical to the test birds should be recorded.
The period after the 5-day exposure of birds to
treated diets should be extended for at least 3 days
or for as long as test birds exhibit toxic signs
and continue to die.
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42
Food consumption. Estimates of average food
consumption should be made for each pen of birds
in each test. Care should be taken to see that
feed spillage or air contamination by volatile
chemicals from pen to pen does not take place.
Statistical procedure for handling data.
The LC50 values and associated statistics can be
derived by methods of probit analysis as described
by Finney (1971) and programmed for computer by
Daum and Killcreas (1966). The application of
the basic proJait method by Litchfield and
Wilcoxon (1949), Miller and Tainter (1944), or
other appropriate sources may be utilized*
(2) ASTM standard E-857-81, Standard practice for
conducting subacute dietary toxicity tests with avian species.
American Society for Testing and Materials, 1916 Race Street,
Philadelphia, PA 19103.; .••;-'
(e) References. The following references can provide
useful background information in developing acceptable protocols;
some outline useful statistical procedures for handling data.
(1) Daum, R.J. 1970. Revision of two computer programs
for probit analysis. Bull. Entomol. Soc. Am. 16(1):10-15.
(2) Daum, R.J. , and W. Killcreas. 1966. Two ccmputer
programs for probit analysis. Bull. Entomol. Soc. Am.
12(4):365-369.
(3) Finney, D.J. 1971. Probit Analysis. 3rd Ed.
Cambridge University Press: London and New York.
(4) Heath, R.G., J.W. Spann, J.R. Kreitzer, and C. Vance.
1970. Effects of polychlorinated biphenyls on birds. Presented
at XV International Ornithological Congress, The Hague, 30 Aug. -
5 Sept., 1970. Pp. 475-485 in Proceedings of the XV Int. Ornith.
Congress. K.-H. Voous, ed. Published by E.J. Rill, Leiden.
(5) Heath, R.G., and J.W. Spann. 1973. Reproduction and
related residues in birds fed mirex. Pp. 421-435 in Pesticides
and the Environment: A Continuing Controversy. Win. B. Deichman,
ed. Intercontinental Medical Book Corp. N.Y.,-N.Y. (Selected
Papers, 8th Inter-Amer. Cong, on Toxicol. & Occupat. Med., Univ.
of Miami, School of Med., Miami, Florida.)
(6) Hill, E.F. , R.G. Heath, J.W. Span, and J.D. Williams.
1975. Lethal dietary toxicities of environmental pollutants to
birds. U.S. Fish and Wildlife Service Special Scientific Report -
Wildlife No. 191. U.S. Dept. Interior, Wash., D.C. 61 pp.
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43
(7) Litchfield, J.T., Jr., and F. Wilcoxin. 1949. A
simplified method of evaluating dose-effect experiments. J.
Pharmacol. Exp. Therap. 96(2):99-133. ~
(8) Miller, L.C., and M.L. Tainter. 1944. Estimation of
ED50 and its error by means of logarithmic-probit graph paper.
Proc. Soc. Exp. Biol. 57:261-264.
(9) Weil, C.S. 1952. Tables for convenient calculation of
median-effective dose (LD50 or ED50) and instructions in their
use. Biometrics 8:249-262.
§ 71-3 Wild mammal toxicity test.
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(1) Test substance, (i) Generally, data shall be derived
from testing conducted with the technical grade of each active
ingredient in the product.
(ii) In special circumstances, data from testing conducted
-with the applicant's end-use formulation or a typical end-use pro-
duct may be required by 40 CFR § 158.75(b) to support the registra-
tion of an end-use product. Examples of such circumstances include,
but are not limited to:
(A) When effects of the product on the rumen fermentation
process need to be determined, or
(B) When secondary toxicity studies need to be performed
(e.g., product is introduced into prey species which is fed to
predator species).
(2) Species. Testing should be performed on a mammalian
species representative of those found in the area(s) likely to
be affected by the proposed use pattern(s). Test animals may
be pen-reared or wild captured, but should be phenotypically
indistinguishable from wild mammals. In no case should endangered
or threatened animals to be used for testing.
(3) Toxicity determination. When the animals are large
or the species is relatively scarce, a study which determines
only the approximate maximum tolerated dosage for the test species
may be acceptable. In all other cases, the acute oral LD50, dietary
LC50, or dietary no-effect level should be determined, following
consultation between the Agency and the registrant.
(c) Reporting and evaluation of data. In addition to
the information provided in § 70-4, test reports should contain
the following information:
(1) Age of each animal and how determined;
(2) Mean body weight for each test and control group at
initiation and termination of test;
(3) Number of animals of each sex of animal tested;
(4) Total food consumption for each test and control group;
(5) Test diet;
(6) Dose schedule;
(7) Mortality;
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.-• 45 •'••.-•
(8) Number and circumstanced o£ accidental deaths or
injuries;
(9) LD50 (in mg/kg) or LC50 (in ppm) with 95 percent
confidence limits, or estimated maximum tolerated dose;
(10) Specified methods used to calculate LD50 or LC50; and
(11) Slope of the dose-response line.
(d) Acceptable protocols. Because the Agency intends
that toxicity tests on mammals, other than those required by
40 CFR § 158.135, be conducted only to access specific situations,
no single test methodology can provide adequate guidance for
all cases. In addition, there are no widely accepted protocols
that include husbandry practices appropriate to a diversity of
mammals. Therefore, the test methodologies should be determined
on a case-by-case basis. Appropriate tests methods should be
developed by the registrant in consultation with the Agency. The
following are offered for guidance.
(1) Dietary LG50 and no-effect level tests. Methods for
dietary tests may be adapted from the subchronic oral dosing studies
for mammals in Subdivision F, § 82-1, and/or from the avian dietary
LC50 study in this Subdivision at § 71-2. See § 71-3(e) for
references.
(2) Toxicity studies for large and relatively scarce
mammals. An example of an acceptable protocol for toxicity studies
with mammals that are large, relatively scarce, or otherwise difficult
to obtain is provided below. This protocol is a modification of
a protocol that appears in an unpublished draft report to EPA
from the American Institute of Biological Sciences (AIBS), titled
Analysis of Specialized Pesticide Problems, Volume VT, Wildlife
Toxicology Study, pages 4 to 9. This report is dated October,
1974 and was funded under EPA Contract No. 68-01-2457.
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Introduction
The purpose of this test is to compare the toxic effect(s)
of a pesticide on different species of wild mammals. As such,
precise LC50 values are not always essential; rather, the
approximate maximum tolerated dose can be estimated and the
character of the toxic syndrome determined. Careful observation
and thorough necropsy examination are required. A preliminary
range-finding test should be first:conducted to establish the
approximate lethal dose, and subsequent tests with dosing of
additional animals (in amounts determined according to the
geometric progression, indicated by the results:-of standard
mammalian acute toxicity) should be conducted to establish the
approximate maximum 'tolerated dosev A single and/or multiple
dosing schedule may be used for the test. The "-latter schedule,
if extended for 10 days or more, can be used to-indicate the
relative chronicity of the pesticide in the wild species in
place of an extended subacute to xi city: schedule;. After dos-
ing, animals should be observed for1 several days for delayed
toxic effects. Beyond simple mortality, signs:of intoxica-
tion such as loss of weight, anorexia, arid diarrhea are im-
portant. The general procedures followed in testing the
toxicity of herbicides in cattle and sheep by the U.S. Depart-
ment of Agriculture ' s Veterinary Toxicology and'Entomology
Research Laboratory (College Station, Texas), Science and
Education Administration (formerly Toxicological Investigation
laboratory, Animal Disease and Parasite Research Division,
Agricultural Research Service) are'-typical and representative
of common practice. :
Methods and Materials
The best policy to follow, within the constraints dictated by
the particular species selected, is to exercise the following sound
principles of animal selection and management. Animals should be
in healthy conditions, as free of social and environmental stress
as possible, acclimated to diet, and uninfluenced by drugs.
Normally, adults of similar age, judged at least by body weight
(but preferably by additional methods as well), should be used, and
data from both sexes should be obtained if possible. Use of reared
stock is generally preferable to live-trapped animals.
Animal conditioning. Animals selected from appropriate sources
should be allowed a prolonged period of acclimatization to the test
facilities and feed. Use of replicate controls is desirable in
tests with wild mammals.
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47
Dose selection. Dosage should be calculated on a rag/kg body
weight basis for the active ccmponent(s) of each chemical tested.
Selection of the dosage rates for each type of test animal involves
several variables. The initial dosage level should be estimated
from existing acute toxicity data, and the toxic range found by
trial and error. When a toxic dosage is found, additional dosages
above and below this rate should then be applied to the other
animals. Where a step-by-step increase of dosage indicates
increased toxicity, repetition of individual dosage is not
necessary. ';'<•.
Chemical administration. Oral dosing should be accom-
plished by placement in the stomach of gelatin capsules contain-
ing the test substance. A balling gun, or "drenching" (in
which a solution or suspension of the chemical ,is introduced
into the stomach by syringe and stomach tube), may be used.
Occasionally it is necessary to conceal the dosing in an
attractive food item. . Drenching usually involves water-
diluted preparations as compared to undiluted material placed
in capsules. Data from the U.S. Department of Agriculture's
Veterinary Toxicology and Entomology Research Laboratory
indicate general inconsistency between these forms of dosing,
but no generalization is possible favoring one over the other.
Use of a vehicle such as corn oil and its volume may also
affect the acute toxicity evaluation. For the immediate
purpose of interspecies comparisons, the solvent or dispersant
should match that used in the acute oral toxicity testing
(i.e., § 81-1 of Subdivision F guidelines). The volume admi-
nistered orally should never exceed 3 percent of the body
weight and should be constant at all dosage levels.
Absorption or effects related to the amound of feed in
the digestive tract are minimized by overnight starvation prior
to administration of the chemical dose. Feed should be
provided following dosing, and water ad libitum prior to and
following dosing. Withholding feed from ruminants serves little
purpose; it is preferable to maintain such animals on restricted
feed allotments calculated to maintain body weight.
Dosing schedule and test design. Testing may use a
single and/or a multiple dose schedule. If the single dose
schedule is used, the test animals should be observed for
approximately ten days following the dose for signs of toxic
effect, such as loss of weight, anorexia, and abnormal function
or behavior. If a multiple dose schedule is used, the test
animal should be observed during a series of 10 or more daily
administrations and a minimum of 10 days following the last
administered dose'.
(e) References. The following references can provide useful
background information in developing acceptable protocols:
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48
(1) Large and relatively scarce mammals.
Agr. Res. Service, U.S.D.A. Animal Disease and Parasite Research
Division. 1969. The toxicity of some organic herbicides to cattle,
sheep, and chickens. A.R.S. Production Research Report No. 106. U.S.
Dept. Agriculture, Wash., D.G.
(2) Small mammals LC5CJ. The following reference contains an
acceptable protocol for determining the dietary toxicity in small animals.
McCann, J.A., Teeters, W., Urban, D.J., and Cook, N. 1981. "A
Short Term Dietary Toxicity Test on Small Mammals,11 Avian and
Mammalian Wildlife Toxicology; Second Conference, ASTM STP 757,
D.W. Lamb and E.E. Kenaga, Eds., American Society for Testing and
Materials, Pp. 132-142.
§ 71-4 Avian reproduction test.
(a) When required. (1) Data on avian reproductive effects
are required by 40 CFR § 158.145 to support the registration of an ,
end-use product which meets one or more of the following criteria:
(i) Its labeling contains directions for using the product
under conditions where birds may be subject to repeated or continuous
exposure to the pesticide or any of its major metabolites or
degradation products, especially preceding or during the breeding
season. •
(ii) The pesticide or any of its major metabolites or
degradation products are stable in the environment to the extent
that potentially toxic amounts may persist in avian feed.
(iii) The pesticide or any of its major metabolites or
degradation products is stored or accumulated in plant or animal
tissues, as indicated by the partition coefficient of lipophilic
pesticides (§§ 165-3, -4, and -5 of Subdivision N) metabolic release
and retention studies (§ 85-1 of Subdivision F), or as indicated by
structural similarity to known bioaccumulative chemicals.
(iv) Any other information, such as that derived fron mammalian
reproduction studies (§ 83-4 of Subdivision F), that indicates the
reproduction in terrestrial vertebrates may be adversely affected
by the anticipated use of the pesticide product.
(2) Applicants for registration of avicides should consult
with the Agency prior to conducting this test.
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49
(3) See 40 CFR § 158.50, "Formulators' exemption," to determine
whether these data must be submitted. Section II-A of this Sub-
division provides an additional discussion on this subject.
(b) Test standards. Data sufficient to satisfy the require-
ments in 40 CFR § 158.145 should be derived from tests which comply
with the general test standards in § 70-3 and all of the following
test standards:
(1) Test substance. Data shall be derived from testing
conducted with the technical grade of each active ingredient in the
product.
(2) Species. Testing should be performed on the bobwhite
quail and mallard.
(3) Dose levels. At least two treatment level groups and a
vehicle control group should be used.
(4) Number of test animals. When other test data reveal
bioaccumulative potential, the number of test animals in the test
group should be increased sufficiently to partly offset animal
deaths or data-gathering problems associated with morbidity or with
tissue residue determinations.
(5) Age. Birds approaching their first breeding season should
be used.
(6) Duration of administration. Birds should be exposed to
treated diets beginning not less than 10 weeks before egg laying is
expected, and extending throughout the laying season.
(c) Reporting and evaluation of data. In addition to the
information provided in § 70-4, the test report should contain:
(1) Test results. The following information, reported for
all test groups:
( i) All observed abnormal behavior;
(ii) All observed morphological and physiological responses;
(iii) Post mortem autopsy. , ' '
(2) Test conditions. The following information, reported
for each treated and untreated test group:
(i) Species;
(ii) Strain;
(iii} Age;
Body weight;
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50
(v) Number of birds per test (include sex ratio);
(vi) Individual identification of birds;
(vii) Diet;
(viii) Storage;
(ix) Feed consumption (grams per day) ;
(x) Observation on palatability or repellency;
(xi) Housing conditions of test birds-, including:
(A) Space allocations for mating and nesting;
(B) Protection from weather and injuries; and
(C) Lighting program, including hours per day and wattage
or foot candles at bird level;
(xii) Diagram of test layout;
(xiii) Temperature;
(xiv) Water supply;
(xv) Pretest and test history or medical and chemical
administration; and
(xvi) Length of treatment period and observation period.
(3) Egg and hatching data. The following information, reported
for each treated and untreated test group:
(i) Egg shell thickness;
(ii) Number and percent of cracked eggs;
(iii) Eggs laid (number eggs per bird per day and per season);
(iv) Hatching egg storage data:
(A) Temperature;
(B) , Humidity;
(C) Incubation data;
(D) Eggs set; and
(E) Egg turning frequency;
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51
(V) Fertility (viable embryos);
(vi) Live 3-week embryos;
(vii) Embryos that mature, embryos that pip shell, and embryos
that liberate themselves, and a determination of hatchability;
(viii) Dead embryos; - .
(ix) Fourteen-day-old survivors;
(x) Crippled survivors;
(xi) Post-hatching mortability;
(xii) Weights of fourteen-day-old survivors; and
(xiii) Any signs of intoxication in post-hatching survivors.
(4) Feed analysis data. Levels of concentration of
pesticide in the feed used in each test, and the rationale for
choice of such levels.
(d) Acceptable protocol. Except where noted, the following
example of avian reproduction protocol is acceptable for the testing
of both bobwhites and mallards. This study is a modification of a
study that appears on pages 23 to 50 in an unpublished draft report
to EPA from the American Institute of Biological Sciences (AIBS),
titled analysis of Specialized Pesticide Programs, Volume VI,
Wildlife Toxicology Study. The report is dated October, 1974, and
was funded under EPA Contract No. 68-01-2457.
Test animals. Pen-reared birds, previously
untreated, approaching their first breeding season,
and phenotypically indistinguishable from wild birds,
should be used as test animals. If shipped, all birds
should be examined following shipment for possible
physical injury that may have been encountered in
transit. If deemed necessary, several birds may be
randomly selected for pretreatment necropsy at a diag-
nostic laboratory to assess the state of health upon
arrival. It is desirable to have a 2- to 6-week health
observation period prior to selection of birds for
treatment.
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52
A history of rearing practice for the birds to
be tested should be obtained if possible. This history
should include lighting practices during rearing,
disease record, drug and any other medication adminis-
tered, and exact age.
Test groups - Bobwhite. A minimum of 3 test
groups of bobwhite should be used. One group should
serve as a control and 2 groups as treated birds. By
random distribution, 1 male and 2 females per pen,
replicated by a. minimum of 12 pens, should be used per
group. If individual pairs (1 male and 1 female) are
to be used per pen, more pens (greater than 12) per
test group should be used to proide similar sensitivity
to the group testing design. To determine the number
of pens needed for a particular level of sensitivity,
see Waipole and Myers (1972). Control and treated
birds should be kept under the same experimental con-
ditions . •
Test groups - Mallards. A minimum of 3 test
groups of mallards should be used. One group should
serve as a control and 2 groups as treated birdss. Bv
random distribution, 2 males and 5 females per pen,
replicated by 5 or more pens, should be used per group.
If individual pairs ( 1 male and 1 female) are to be
used per pen, considerably more pens (greater than 12
per test group should be used to provide similar sen-
sitivity to the group testing design. To determine
the number of pens needed for a particular level of
sensitivity, see Walpole and Myers (1972). Control
and treated birds should be kept under the same experi-
mental conditions.
Diet preparation. Concentrations for the test
substance should be based on measured or calculated
residues expected in the diet from the proposed use
pattern(s). The concentrations should include an
actual or expected field residue exposure level and a
multiple level such as five. The highest nonlethal
level may be estimated from data developed from the
avian dietary LC50 (§71-2).
The test material should be added to table grade
corn oil or other appropriate >vehicle and premixed
with an aliquot of basal diet, utilizing a mortar and
pestle or mechanical blender. It is recommended that
the aliquot of basal diet used for the premix be
screened to remove large particles of diet before
blending in the corn oil and test material . The final
diet should be a uniformly mixed composition consisting
of 98 to 99 parts by weight of basal diet and 1 or 2
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53
parts by weight of corn oil. The basal diet should be
a commercial game bird breeder ration (or its equivalent)
that is treated with an equivalent amount of vehicle.
The premix should be stored under conditions which
maintain stability. Test diets should be analyzed for
pesticide concentrations at intervals during the tests.
If other long-term animal tests have demonstrated a
propensity for the test chemical to persist or bioac-
cumulate, the degree .of bioaccumulation in birds should
be determined by measurement of tissue residues in the
birds from an extra pen group put through the reproduc-
tion test. Two or three tissues should be selected
for residue analysis at the end of the exposure period,
based on tissues known from other studies to hold
highest residues.
Testing phase - test environment. The birds
should be housed in breeding pens of adequate size
conforming to good husbandry practices. The malla.rd
pens should be screen-bottomed or kept clean of
spilled food and excrement. It is desirable to offer
mallards water in which to bathe.
Since light is extremely important, both during
rearing and during the egg laying period, all birds
should be maintained for the first 8 weeks under a
regime of 7 hours of light per day for maximum egg
production.
The photoperiod should then be increased to
16-17 hours of light par day and either maintained
at this level or increased by 15 minutes per week
for the following 12 weeks. (The 12-week period
may vary depending upon the time required for the
onset of egg production.) An illumination intensity
of 6 footcandles at the bird level during the lighting
phase of the reproductive study is adequate. Avoid
the use of shorter wavelength "cool white" fluorescent
lights which do not emit the daylight spectrum.
Temperature and relative humidity control through-
out the reproductive test is desirable and should be
recorded. Recommended levels are 21 °C and 55 percent
relative humidity. Ventilation is necessary.
Feeding and husbandry. All birds should receive
the appropriate diet ad libitum for the duration of
the study. Water is to be provided ad libitum. The
test chemical should be administered for at least 10
weeks prior to the onset of egg laying.
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54
Body weights should be recorded at test initiation
prior to onset of laying, and at termination. During
egg laying,% body weight recording is discouraged because
of the adverse effects that handling may have on egg
production.
Food consumption should be recorded at least
at biweekly intervals throughout the study.
Mortality should be recorded by date and morbidity
(noted together with clinical signs) throughout the
test phase. Gross pathology data should be obtained
for birds that die during the course of the test phase
and for some survivors.
Egg collection, storage, and incubation. All eggs
should be collected daily, marked according to pen
from which collected, and stored at 16°C and 65 percent
relative humidity. Eggs should be set at weekly
intervals for incubation in a commercial incubator.
All eggs should be candled on day 0 for eggshell
cracks; on approximately day 11 for bobwhites and
day 14 for mallards to measure fertility and early
death of embryos; and on day 18 for bobwhite and
day 21 for mallards to measure embryo survival. For
hatching, transfer of the eggs to a separate
commercial incubator or hatcher should be made on
day 21 for bobwhites and Day 23 for mallards.
Recommended temperatures and relative humidity
during hatching phase are 39°C and 70 percent,
respectively.
Bobwhite chick observations. On Day 24 of
incubation, the hatched bobwhite chicks should be
removed, hatchability recorded, chicks housed according
to the appropriate parental grouping, and maintained
on control diet for 14 days. The time period should
be extended if mortality occurs appreciably late.
The diet should be a commercial bobwhite starter
diet or its equivalent.
Duckling observation. On Day 27 of incubation,
the hatched mallard ducklings should be removed,
hatchability recorded, ducklings housed according
to the appropriate parental grouping, and maintained
on control diet for 14 days. The time period should be
extened if mortality occurs appreciably late. The
diet should be commercial mallard starter diet or its
equivalent.
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Eggshell thickness. One day every two weeks newly
laid eggs should be collected and measured for eggshell
thickness. For consistency, the eggs used for thickness
determinations should be collected during weeks 1, 3, 5,
7 and 9 of the egg-laying period. An accepted
procedure is to crack open the eggs at the widest
portion (girth or waist), wash out all egg contents,
air-dry the shells for at least 48 hours, and then
measure the thickness of the dried shell plus the
membranes at 3 or 4 points around the girth using a
micrometer calibrated to 0.01 mm units.
Analysis. Reproductive data consists of continuous
variables (e.g., shell thickness, and body weight data)
and discrete variables (e.g., number of eggs laid or
14-day-old survivors). For continuous variables,
experimental groups should be compared: to controls by
analysis of variance. For most discrete variables,
survival percentages should be computed (e.g., 14-day-
old survivors of eggs laid) and arcsine transformed
prior to analysis of variance. Alternately, a chi
square analysis of survival (contingency tables) may
be used for discrete variables. Analyses should include:
body weight, food consumption, eggs laid, eggshell thick-
ness, eggs cracked, viable eggs, fertility, live 3-week
embryos, hatchability, number of normal•chicks or
ducklings, 14-day-old survivors (per number of eggs
hatched, per hen, and per number of eggs laid).' Sample
units are generally the pens within each group.
Withdrawal. If the test substance is toxic (re-
duced reproduction evident), then a withdrawal study
period should be added, to the test phase. The withdrawal
period need not exceed 3 weeks. Continued observations
should be made on egg production, fertility, hatchability,
and hatching survival.
Definitions:
1. Eggs laid. The total egg production during a
breeding season (which is approximately 10 weeks).
2. Eggs cracked. Eggs determined to have cracked
shells when inspected with a candling lamp; fine cracks
cannot be detected without utilizing a candling lamp
and if undetected will bias data by adversely affecting
embryo development.
3. Eggs set. All eggs placed under insubation, i.e.,
total eggs laid minus cracked eggs and those selected for
eggshell thickness analysis.
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56
4. Viable embryos (fertility). Eggs in which
fertilization has occurred and embryonic development has
begun. This is determined by candling the eggs 6 to
14 days after incubation has begun. It is difficult
to distinguish between the "absence of fertilization
;!and early embryonic death i! This distinction can be
made by breaking out eggs that appear infertile and
examining further. This " is';especially important when
a test compound induces early embryo mortality-
5. Live 3-week embryo. Embryo that is developing
normally after 3 weeks of incubation. This is determined
by candling the egg. '"••"
6. Hatchability. The percentage of embryos that
mature, pip the shell, and liberate themselves from their
eggs as computed from the number of fertile eggs. For
quail this generally occurs on day 23 or '"24 of incubation,
and for mallard on day 25, 26, or 27.
7. 14-day-old-survivors« Birds that survive for
2 weeks following hatch.
8. Eggshell thickness. The thickness of the
shell and the membrane of the egg at the girth after the
egg has been opened and washed out, then the shell with
membrane dried for at least 48 hours at room temperature.
(e) References. The following references can provide useful
background information in developing acceptable protocols; some
outline useful statistical procedures for handling data.
(1) Cochran, W.G. 1943. Analysis of variance for percentages
based on unequal numbers. Am. Stat. Assoc. 38:287-301.
(2) Davidson, K.L. , and J.L. Sell. 1974. DDT thins shells
of eggs from mallard ducks maintained on ad libitum or controlled-
feeding regimes. Arch. Environ. Contain. Toxicol. 2(3):222-232.
(3) Duncan, D.B. 1955. Multiple range and multiple F tests.
Biometrics 11:1-42.
(4) Heath, R.G., J.W. Spann, and J.F. Kreitzer. 1969.
Marked DDE impairment of mallard reproduction in controlled studies.
Nature 224(5215):47-48.
(5) Heath, R.G., J.W. Spann, J.R. Kreitzer, and C. Vance.
1970. Effects of polychlorinated biphenyls on birds. Presented at
the XV Internat. Ornith. Congress, The Hague, 30 Aug - 5 Sept.,
1970. Pp. 475-485 in Proceedings of XV Internat. Ornith* Congress.
K.H. Voous, ed. E.J. Brill, (pub.) Leiden.
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57 > .U ....
(6) Heinz, G. 1974. Effects of dietary levels of methyl
mercury on mallard reproduction. Bull. Snv. Cont. Toxicol. 11-386-
392. ' ' : : ' —~
(7) Longcore, J.R. , F.B. Sampson, and T.W. Whittendale, Jr.
1971. DDE thins eggshells and lowers reproductive success of
captive black ducks. Bull. Env. Cont. Toxicol. 6:485-490.
(8) Prince, H.H., F.B. Seigel, and G.W. Cornwall. 1969.
Incubation environment and the development of mallard embryos.
J. Wildlife Manage. 33:589-595.
(9) Stromborg, K.L. , 1981. Reproductive test of diazinon on
bobwhite quail, Avian and Mammalian Wildlife Toxicology: Second
conference, AS1M STP 757, D.W. Lamb and E.E. Kenaga, Eds., American
Society for Testing and Materials, pp. 19-30.
(10) Walpole, R.E. and R.H. Myers. 1972. Probability and
statistics for engineers and scientists. The MacMillan Company,
New York. Pp. 387-392.
§• 71~5 Simulated and actual field testing for mammals and birds.
(a) When required. (1) Data from any of the following kinds of
tests are required by 40 CFR § 158.145, on a case-by-case basis,
to support the registration of an end-use product intended for outdoor
application. Consultation with the Agency is advised before under-
taking these tests. Whenever data are required by 40 CFR § 158.145,
the determination will be made in writing by the Agency and will
state which properties and use patterns of the product were used
in the determination. The following criteria are provided as
further guidance:
(i) Simulated (pen) field tests are required by 40 CFR
§ 158.145 to support the registration of an end-use formulated
product if use of the pesticide is likely to result in adverse
effects on wildlife organisms exposed to the pesticide, and if pen
field tests can yield data useful in assessing such risks. A
decision as to whether such a test is needed should take into
account:
A. An analysis of the pesticide properties (e.g., persistence,
conversion to toxic metabolites);
B. Retention on food, and repellency;
C. Intended use patterns (e.g., treated habitats, expected
presence of species, and treatment amounts at toxic levels after
application); and
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58
D •The results of other laboratory tests.
(ii) Actual field tests are required by 40 CFR §.158.145
to support the registration of an end-use formulated product if
use of the pesticide is likely to result in adverse effects oh
wildlife exposed to the pesticide, and if actual field tests can
yield data useful in assessing such risks. A decision as to
whether such a test is needed should take into account:
(A) An analysis of the pesticide properties (e.g.,
persistence, conversion to toxic metabolites, retention on food,
and repellency);
(B) Intended use patterns (e.g., treated habitats, expected
presence of species, and treatment amounts at toxic levels after
application); and
(C) Evidence (e.g., reported field effects, simulated pen
studies or chronic lab studies) demonstrating acute toxicity at
normal use rates or demonstrating long-term chronic or reproductive
effects.
(2) See 40 CFR § 158.50, "Formulators' exemption," to deter-
mine whether these data must be submitted. Section II-A of this
Subdivision provides an additional discussion on this subject.
(b) Test standards. Data sufficient to satisfy the require-
ments in 40 CFR § 158.145 should be derived from tests which satisfy
the purposes of the general test standards in § 70-3 and the follow-
ing test standards:
(1) Test substance. Unless specified otherwise, data shall
be derived from testing conducted with an end-use product (or with
an end-use product whose properties are like those of products to
which the determination under paragraph (a) of this section applies).
An "end-use product" may be the applicant's own product or a typical
end-use product.
(2) Test conditions. The test conditions for conducting a
simulated pen, or actual field test should resemble the conditions
likely to be encountered under actual use of the product. Specifi-
cally, the pesticide should be applied to the site at the rate,
frequency, and method specified on the label .
(3) Endangered species. Studies should not be conducted in
critical habitats or areas containing, or suspected to contain,
endangered or threatened plants or animals which may be threatened
by the tests to be conducted.
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(4) Residue levels. When the'test substance is applied unde-
simulated or actual field condition testing, and residues of the
test substance can be detected j.n warm-blooded animals as evidenced
by tests required by 40 CFR Part 158, residues should be determined
- in selected tissues of test organisms and in vegetation, soil,
water, sediments, and other appropriate environmental components.
If residues of the test substance cannot be detected in warm-blooded
animals as evidenced by tests required by 40 CFR Part 158, then the
applicant should consult with the Agency before beginning this
test. • . 33
(5) Other standards. Additional standards for conducting
actual field tests are not delineated because of the wide variety
of mechanisms by which a pesticide may enter the environment, and
because of the great variety of food sources and habitats that may
be affected. Any additional standards for conducting these tests
will be provided by the Agency in writing following consultation
between the applicant or registrant and the Agency, and will take
into account the variety of mechanisms, food sources, and habitats
mentioned above.
(c) RePortinq and evaluation of data. In addition to the
information provided in § 70-4, the test report should contain
the following information:
(1) Simulated (pen) field tests.
(i) Test methods and materials data.
(A) Test location;
(B) Dates of beginning and end of test, and any other
significant dates of events in the test;
(C) Weather data;
(D) Test species, age and history;
(E) Medical and chemical administration history (if any);
(F) Measured body weights;
(G) Individual identification;
(H) Pesticide chemical formulation, application rate and fre-
quency, and manner of application;
(I) Vegetative cover;
(J) Measured residues in selected tissues oftest oraanisms and
in vegetation, soil, water, and sediments, when required-
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60
(K) Analytical methods for residue determinations;
(L) Housing conditions, including pen description, pen place-
ment and number of animals per pen; :
(M) Diet;
(N) Food and water supply schedule;
(O) Feed consumption;
(P) Visual signs of intoxication;
(Q) Clinical measurements for intoxication;
(R) Accidental death or injuries;
(S) Replacement schedule; and
(T) Statistical methods used.
(ii) Test results.
(A) Mortality: number, dates, and other pertinent information;
(B) Toxic signs;
(C) Body weight changes;
(D) Food consumption data;
(E) Clinical observations;
(F) Results of gross necropsy or pathological examinations,
if conducted;
(G) Residue analysis results; and
(H) Any noted effects on reproduction.
(iii) Summary and conclusion. Potential hazards to wildlife
should be identified in addition to the toxicity results per se.
(2) Actual field tests.
(,i) Test methods and materials data.
(A) Description of the study area including vegetation, topo-
graphy, and all pertinent ecological information;
(B) Study plot layout - locations and replications;
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(C) Listing of resident and migrant fauna with estimates of
population densities or relative abundance;
(D) Details of application equipment, methods, and weather
conditions;
(E) Statistical design and methods of analysis;
(F) Weather data collection methods;
(G) Analytical methods for residue determination;
(H) Clinical data methods;
(I) Reproductive study methods;
(J) Carcass search methods; and
(K) Any other methods utilized.
(ii) Test results.
(A) Mortality: number, dates, and other pertinent information;
(B) Signs of intoxication;
(C) Bird and mammal census results;
(D) Arthropod numbers and biomass;
(E) Food habits data, if any;
(F) Necropsy observations;
(G) Residue analysis results;
(H) Weather data;
(I) Inclusive dates of test; and
(J) Results of nest studies and fledgling wildlife.
(iii) Summary and conclusion. All data should be integrated
in a way which reflects the full impact of the pesticide on the
ecosystem. Potential hazards to wildlife should be estimated with
full consideration of possible indirect effects due to ecological dis-
turbances .
(d) Acceptable protocols.
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(1) Simulated (pen) field tests. The following is an example
of an acceptable protocol for conducting a simulated (pen) field
study for birds. This study is a modification of a study that
appears on pages;65 to 73 in an unpublished draft report to EPA
from the American Institute of Biological Sciences (AIBS), titled
Analysis of Specialized Pesticide Problems, Volume VI, Wildlife
Toxicology Study. This report is dated October, 1974 and was funded
under EPA contract No. 68-01-2457.
Introduction
For assessing the hazards of pesticides to bobwhite
quail, large cages enclosing portions of habitat of
0.005 ha (500 sq ft) or more may be utilized. With
modifications other species can also be tested by this
method. Tests utilizing large pens may be conducted for.
pesticides to be applied on cropland, rangeland, or wild-
lands, or for other outdoor applications such as roadsides,
rights-of-way, "waste areas," or known wildlife habitats.
This type of test is not a substitute for an actual field
study. However, a carefully designed simulated (pen)
field study serves to bridge the gap between lab studies
and a comprehensive field study.
Methods and Materials
Pens. Wire-covered, pens should be constructed cover-
ing a minimum ground area of 0.005 ha (500 ft2 per pen).
Suitable minimum pen dimensions for quail are 3.1 m by
15.2 m (10 ft x 50 ft) with the top cover at a height of
about 2.0 m (6.5 ft). Other dimensions covering more than
0.005 ha are encouraged. Larger pens will be needed for
testing larger birds such as mallards or pheasants".
For uncovered pens, birds may be wing clipped or
pinioned. When cage covers are used, they should not inter-
fere with the pesticide reaching the interior of the cage.
To reduce predation, metal flashing should be placed around
all pens both above and below the ground. In addition,
the top perimeter of the fence may be electrified.
A minimum of 3 (TO1 x 50') pens should be used for
each treatment group and control. Each pen should contain
8 pair of quail. Thus a study consisting of one treatment
and one control group would have a. total of s ix cages and
96 birds (48 pair). An independent water supply and a
small shelter should be furnished in each pen.
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Before pens are planned and constructed, wildlife
agencies and successful game farms should be consulted to
consider factors such as prevention of parasites and
disease/ soil drainage requirements, support of top cover
to prevent collapse under the weight of snow, types of
watering equipment, and similar information.
Birds. Adult birds of known history, not previously
exposed to pesticides, should be used and placed in the:pens
at least 2 weeks prior to the pesticide application(s). A
supply of replacement bobwhite should be maintained in out-
door pens near the test pens.
Test conditions and procedures. Uniformity of soil
type and field conditions are important considerations in
selecting study sites. All pens should be numbered and
locations mapped or charted. Daily recoreds should be kept
of observations, toxic signs, test bird deaths and replace-
ments (if any), complete data on pesticide formulations,
application rates and methods, weather conditions, and all
other data of value in assessing the hazard to birds. It
may be desirable to use move able pens that can be set up
over the crop or vegetation on which the pesticide is in-
tended to be applied. When permanent (non-portable) pens
are used, then the soil should be suitable for growing the
pertinent crop or vegetation. The pesticide should be
applied with the same equipment, at the same rate, timing,
number of applications, and formulations as specified on the
pesticide label. Additional treatment groups can be added
to test the effects of different application techniques
(e.g., broadcast versus soil incorporation), application
timing (e.g., pre-plant versus postemergent), or irrigation
versus nonirrigation. Spraying should be done under minimum
wind conditions and with protective shielding to prevent
contamination of adjacent sprayed pens or control pens. For
statistical purposes, it is best to randomize the test pens,
but because of the drift problem, it may be best to stratify
the treatment pen locations.
If supplemental feed is needed, the treatment rates
can be based on results of residue studies when such studies
are available. Where possible, supplemental food should
be withheld 1 to 2 days after pesticide treatment. Treated
food should be prepared within 1 day of the time the pen
environments are sprayed.
The duration of the test should be a minimum of 21
days after the final application, and longer if any birds are
showing toxic signs or other effects.
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64
Pesticide residue determinations may be included in
the study. Diet and water levels of the test chemicals
should be analyzed. Vegetation, soil, and other environmen-
tal samples may be analyzed for residues to determine persis-
tence and bioaccumulation. Test birds poisoned by the pesti-
cide and a sample of surviving birds should be analyzed for
residues in selected tissues. Gross pathology may be deter-
mined at the same time.
(2) Actual field studies for hazard to wildlife. The objective
of the actual field study is to determine the impact of pesticide
applications on wildlife populations under real-use conditions.
Effects of greatest importance include:
Direct poisoning and death (by ingestion, dermal exposure,
or inhalation);
Toxic effects indirectly causing death such as increasing
susceptibility to predation and diseases;
- Harmful reproductive effects and consequent inability to
maintain populations.
Full-scale field studies are useful for determining the effects
of a pesticide from large-scale spraying of forests, rangelands,
rights-of-way, roadsides, wetlands, and major croplands such as
cotton, wheat, corn, soybeans, rice, sorghum, or alfalfa, or other
major wildlife habitats. In view of the diversity of potential
study areas, actual field studies need to be designed on a case-by-
case basis. No standard protocol would be appropriate. Therefore,
the references that appear in § 71-5(e)(2) can be used as guidance
for developing acceptable actual field studies.
(e) References. The following references can provide useful
background information for developing acceptable field tests. Some
outline useful statistical procedures for handling data.
(1) Simulated (pen) field tests.
(i) Fink, R.J. 1979. Simulated field studies - Acute hazard
assessment, Avian and Mammalian Wildlife Toxicology, ASTM STP 693,
E.E. Kenaga, Ed., American Society for Testing and Materials, pp.
45-51.
(ii) Kreitzer, J.F., and J.W. Spann. 1968. Mortality among
bobwhites confined to a heptachlor contaminated environment. J_._
Wildl. Manage. 32(4): 874-878. ,
(iii) Schemnitz, S.D., 1981. Wildlife Management Techniques.
4th Ed.: revised. The Wildlife Society, Inc., Washington, D.C. 722
pp.
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65
(2) Full-scale field tests.
(i) Bart, 1979. Effects of acephate and sevin on forest birds.
J. Wild. Manage. 43(2): 544-549.
(ii) Bunyan, P.J., M.J. Van Den Heuvel, P.I. Stanley and E.N.
Wright. 1981. An intensive field trial and a multi-site surveillance
exercise on the use of aldicarb to investigate methods for the
assessment of possible environmental hazards presented by new
pesticides. Agro Ecosystems 7:239-262.
(iii) Caughley, G. 1977. Analysis of Vertebrate Populations.
John Wiley and Sons Ltd., A Wiley-Interscience Publication pp. 234.
(iv) De Weese, L.R., C.J. Henny, R.L. Floyd, K.A. Bobal, A.W.
Schultz. 1979. Response of breeding birds to aerial sprays of
trlchlorfon (Dylox) and carbaryl (Seven-4-oil) in forests. Special
Scientific Report Wildlife No. 224, U.S. Department of the Interior,
Fish and Wildlife Service, Washington, D.C., pp. 29.
(v) Edwards, P.J., S.M. Brown, M.R. Fletcher and P.I. Stanley.
1979. The use of a bird territory mapping method for detecting
mortality following pesticide application. Agro Ecosystems 5:271-282.
(vi) Flickinger, E.L., K.A. King, W.F. Stout and M.M. Mohn.
1980. Wildlife hazards from Furadan 3G applications to rice in
Texas. J. Wild Manage. 44(15:190-197.
(vii) Hegdal, P.L. , T.A. Gatz, K.A, Fagerstone, J.F. Glahn
and G.H. Matschke. 1979. Hazards to wildlife associated with 1080
baiting for California ground squirrels. USFWS, under agency
Agreement between EPA and FWS, EPA-IAG-D7-0449 (Unpublished final
report) .
(viii) Hegdal, P.L. T.A. Gatz. 1977. Hazards to pheasants
and cottontail rabbits associated with zinc phosphide baiting for
microtine rodents in orchards. USFWS, under Interagency Agreement
between EPA and FWS, EPA-IAG-D4-0449 (Unpublished final report).
(ix) Herman, S.G., J.B. Bulger. 1979. Effects of a forest
application of DDT on nontarget. organisms. Wildlife Monographs,
No.69, pp. 62. .
(x) Johnson, E.V., G.L. Mack and D.Q. Thompson. 1976. The
effects of orchard pesticide applications on breeding robins. The
Wilson Bull. 88(1):16-35.
(xi) Ludke, J.L., E.F. Hill and M.P. Dieter. 1975. Choli-
nesterase (ChE) response and related mortality amo fed ChE inhi-
bitors. Arch. Environ. Contain. Toxicol. 3(1): 1-21.
(xii) McEwen, L.C., C.E. Knittle, and M.L. Richmond. 1972.
Wildlife effects from grasshopper insecticides sprayed on short-
grass range. J. Range Manage. 25(3):188-194.
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66
(xiii) Moulding, J.D. 1976. Effects of a low-persistence in-
secticide on forest bird populations. Auk 93 (4): 692-708.
(xiv) Pearce, P.A., D.B. Peakall and A.J. Erskine. 1976.
Impact on forest birds of the 1975 spruce budworm spray operation
in New Brunswick. Progress Notes, Canadian Wildlife Service, No.
62, pp 7. ' . ' • . •,.,., : . : , ,-,•-, , ,. . ,.
(xv) Ralph, C.J. and J.M. Scott. Eds. 1981. Estimating
numbers of terrestrial birds. Studies in Avian Biology No. 6,
Cooper Ornithological Society, Pp. 630.
(xvi) Schemnitz, S.D., ed. 1971. Wildlife Managenint Techi-
ques. 3rd Ed.: revised. The Wildlife Society, Inc., Washington,
D.C. 722 pp. '
Series 72: AQUATIC ORGANISM TESTING
§ 72-1 Acute toxicity test for freshwater fish.
(a) When required. (1) Data on the acute toxicity of a pesti-
cide to freshwater fish are required by 40 CFR § 158.145 to support the
registration of an end-use product intended for outdoor application,
and to support each application for registration of a manufacturing-
use product which may be used to make such an end-use product.
(2) Data on the acute toxicity of a pesticide to freshwater
fish are required by 40 CFR § 158.145, on a case-by-case basis to
support the registration of an end-use product intended solely
for indoor application, and to support each application for
registration of a ma.nufacturing-use product which may be used to
make such an end-use product.
(3) See 40 CFR § 158.50, "Formulators' exemption," to de-
termine whether these data must be submitted. Section II-A of
this Subdivision provides an additional discussion on this subject.
r
(b) Test standards. Data sufficient to satisfy the require-
ments in 40 CFR § 158.145 should be derived fron tests which comply
with the general test standards in § 70-3 and the following test
standards:
(. 1.) Test substance. (i) Data shall be derived fron testing
conducted with the technical grade of each active ingredient in the
product.
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67
(ii) In addition, data frcm testing with the applicant's end-
use product or a typical end-use product are required by 40 CFR §
158.145 to support the registration ,of products which meet any of
the following conditions:
(ft) The end-use pesticide will be introduced directly into an
aquatic environment when used as directed;
(B) The LC50 or EC50 of the techleal grade of active ingredient
is equal to or less than the maximum expected environmental
concentration (MEEC) or the estimated environmental concentration
(EEC) in the aquatic environment when the end-use pesticide is used
as directed; or
(C) An ingredient in the end-use product other than the active
ingredient is expected to enhance the toxicity of the active ingre-
dient or to cause toxicity to aquatic organisms.
(2) Test organisms. (i) Testing should be performed on one
coldwater fish species, preferably rainbow trout, and one warmwater
species, preferably bluegill sunfish.
(ii) Very young (not yet actively feeding), spawning, or recently
spent fish should not be used.
(iii) Fish should weigh between 0.5 and 5.0 grams and be frcm
the same year class. The standard length of the largest fish should
be no more than twice that of the shortest fish.
I
(3) Determination of LC50. (i) Satisfactory data must establish
either:
(A) A 96-hour LC5.0 value with 95 percent confidence intervals; or
(B) That the 96-hour LC50 is greater than 100 mg/1 or greater
than 100,000 times the MEEC or EEC.
(ii) If data are submitted to satisfy either criterion in
paragraph (b)(3)(i)(B) of this section, at least.30 individuals should
be tested at concentrations equal to or greater than the criterion
chosen. '
(c) Reporting and evaluation of data. In addition to information
provided in § 70-4, a report of the results of a fish acute LC50
test should include the following:
(1) LC50 data. (i) (A) Data showing the 96-hour LC50, the
corresponding 95 percent confidence intervals, slopes of the concentration
response line, and, when possible, the LC50 values at 24, 48, and 72
hours; or
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68
(B) Data showing, that the 96-hour LC50 is greater than 100,000
times the MSEC or EEC or greater than 100 mg/1.
(ii) If the data submitted in- accordance with paragraph
(c)(1)(i)(B) of this section show that the LC50 is greater than
100,000 times the MEEC or EEC of the pesticide; the basis for
calculating the MEEC or EEC should be.reported.
(2) Dilution water. Detailed description of dilution water,
including source, chemical characteristics (e.g., dissolved oxygen
content, pH, dissolved salts), and pretreatment (if any).
(3) Test description. Detailed description of the test,
including:
(i) Design;
(ii) Containers;
(iii) Water depth and volume;
(iv) Treatments;
(v) Method of exposing fish to the test substance (e.g.,
placing chemical in water which already contains fish or placing
fish in water which already contains the chemical);
(vi) Number of organisms per treatment;
(vii) Loading (weight of organisms per unit volume of water);
(viii) Lighting, acclimation, and test temperatures (averages
and range); and
(ix) Any unusual feature of the test methodology.
(4) Chemical analyses. If conducted, a description of the
methods (or references to established methods) used for all the \
=analyses of water for chemical content and toxicant concentrations,
and the results of such analyses, including validation studies and
reagent blanks.
(5) Effects of exposure. Detailed description of the effects
of exposure to the test substance including:
(i) The criteria used to determine the effects;
(ii) Percentages of organisms that died or showed effects of
treatment; and
(iii) A summary of these observations.
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69 .
(6) Additional information. Any additional relevant informa-
tion about the. test or its results that would assist in the determi-
. nation of hazard potential.
(d) Acceptable protocols. Examples of acceptable protocols for
conducting a freshwater fish acute toxicity study are found in the
following references. Fish species listed in these publications are
acceptable with the exception of goldfish.
(1) ASTM Standard E 729-80, Practice for Conducting Acute
Toxicity Tests with Fishes, Macroinvertebrates , and Amphibians.
American Society for Testing and. Materials, 1916 Race "street,
Philadelphia, PA 19103.
(2) Committee on Methods for Toxicity Tests with Aquatic
Organisms. 1975. Methods for acute toxicity tests with fish,
macroinvertebrates, and amphibians. U.S. Environmental Protec-
tion Agency, Ecol. Res. Series, EPA 660/3-75-009. 61pp.
(e) References. The following references can provide useful
background information in developing acceptable protocols:
(1) Weber, C. E. (ed.) 1973. Biological field and laboratory
methods, for measuring the quality of surface waters and'effluents .
U.S. Environmental Protection Agency, Environ. Monit. Series, s?A-
670/4-73-001.
(2) Anonymous. 1981. Standard Methods for the Examination of
Water and Wastewater. 15th Ed. American Public Health Assoc.,
Washington, D.C. 1134 pp.
§ 72~2 Acute toxicity test for freshwater aquatic invertebrates.
(a) When required. (1) Data on the acute toxicity of a
pesticide to freshwater aquatic invertebrates are required by 40
CFR § 158.145 to support the registration of an end-use product
intended for outdoor application, and to support each application
for registration of a manufacturing-use product which may be used
to make such an end-use product.
(2) Data on the acute toxicity pf a pesticide to freshwater
aquatic invertebrates are required by 40 CFR § 158.145, on a case-by-
case basis to support the registration of an end-use product in-
tended solely for indoor 'application,, and to support each application
for registration of a manufacturing-use product which may be'used
to make such an end-use product.
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70
(3) See 40 CPR § 158.50, "Formulators' exemption," to deter-
mine whether these data must be submitted. Section II-A of this
Subdivision provides additional discussion on this subject.
(b) Test standards. Data sufficient to satisfy the require-
ments in 40 CFR § 158.145 should be derived from tests which comply
with the general test standards in § 70-3 and the following test
standards.
(1) Test substance. (i) Data shall be derived from testing
conducted with the technical grade of each active ingredient in the
product.
(ii) In addition, data from testing with the applicant's end-
use product or a typical end-use product are required by 40 CFR §
158.145 to support the registration of products which meet any of
the following conditions:
(A) The end-use product will be introduced directly into an
aquatic environment when used as directed; I ' '
(B) The LC50 or EC50 of the technical grade of active ingredient
is equal to or less than the maximum expected environmental concentra-
tion (MSEC) or the estimated environmental concentration (EEC) in
the aquatic environment when the end-use product is used as directed;r
or
(C) An ingredient in the end-use product other than the active
ingredient is expected to enhance the toxicity of the active ingredient
or to cause toxicity to aquatic organisms.
(2) Test organisms. Immature invertebrates should be used
whenever possible. Among fresh-water organisms, daphnids should be
in the first instar; amphipods, stoneflies, and mayflies in an early
instar; and midges in the second or third instar.
(3) Determination of EC50 or LC50. (i) Satisfactory data
should establish either:
(A) An EC50 or LC50 value with 95 percent confidence intervals;
or
(B) That the EC50 or LC50 is greater than 100 mg/1 or greater
than 100,000 times the MSEC or EEC.
(ii) If data are submitted to satisfy either criterion in para-
graph (b)(3)(i)(B) of this section, at least 30 individuals should be
tested .at concentrations equal to or greater than the criterion
chosen.
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71
(4) Duration of tests. Daphnids and midge larvae should be ex-
posed to the test substance for 48 hours in static tests. All other
organisms should be exposed for 96 hours in static tests. For flow-
through tests, all organisms tested under this section should be
exposed for at least 96 hours.
(c) Reporting and evaluation of data, m addition to informa-
tion provided in § 70-4, a report of the results of an acute toxicity
test for aquatic invertebrates should include the following:
(1) LC50 data. (i) (A) Data showing the ECS0 or LC50, the
corresponding 95 percent confidence intervals, slope of the concen-
tration reponse line, and when possible, the EC50 or LC50 values
at 24-hour intervals for the duration of the test; or
(B) Data showing that the EC50 or LC50 is greater than 100,000
tunes the MEEC or EEC or greater than 100 mg/1.
(ii) If the data submitted in accordance with paragraph (c)(1)-
(i)(B) of this section show that the LC50 or EC50 is greater than
100,000 times the MEEC or EEC of the pesticide, the basis for
calculating the MEEC or EEC should be reported.
(2) Dilution water. Detailed description of dilution water, in-
cluding source, chemical characteristics (e.g., dissolved oxygen
content, pH, dissolved salts), and pretreatment (if any).
(3) Test description. Detailed .description of the test, in-
cluding:
(i) Design;
(ii) Containers;
(iii) Water depth and volume;
(iv) Treatments;
(v) Method of exposing organisms to the test substance (e.g.,
placing chemical in water which contains organisms or placing
organisms in water which contains chemical);
(vi) Number of organisms per treatment;
(vii) Lighting, acclimation, and test temceratures (averages
and range); and
(viii) Any unusual feature of the test methodology.
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72
(4) Chemical analyses. If conducted, a description of the
methods (or references to established methods) used for the analyses
of water for chemical content and toxicant concentrations, and the
results of such analyses, including validation studies and reagent
blanks.
(5) Effects of exposure. Detailed description of the effects
of exposure to the 'test substance, including:
(i) The criteria used to determine the effects;
(ii) Statement of percentages of organisms that died or showed
effects of treatment; and
(iii) A summary of these observations.
(6) Additional information. Any additional relevant infor-
mation about the test or its results that would assist in the de-
termination of hazard potential.
(d) Acceptable protocols. Examples of acceptable protocols
for conducting a freshwater aquatic invertebrate acute toxicity
study are found in the following references:
(1) ASTM Standard E 729-80, Practice for Conducting Acute
Toxicity Tests with Fishes, Macroinvertebrates, and amphibians.
American Society for Testing and Materials, 1916 Race Street,
Philadephia, Pa. 19103.
(2) Committee on Methods for Toxicity Tests with Aquatic
Organisms. 1975. Methods for acute toxicity tests with fish,
macroinvertebrates, and amphibians. U.S. Environmental Protection
Agency, Ecol. Res. Series, EPA 660/375-009. 61 pp. (Aquatic
invertebrate test temperatures in this publication are acceptable
with the exception of 17"C for Daphnia spp. Daphnia should be
tested at 20°+ 1°C.)
(e) Reference. The following publication can provide useful
background information in developing acceptable protocols:
Anonymous. 1981 . Standard Methods for the Examination of
Water and Wastewater. 15th Ed. American Public Health Assoc.,
Washington, D.C. 1134 pp.
§ 72-3 Acute toxicity test for estuarine and marine organisms.
(a) When required. (1) Data on the acute toxicity of a pes-
ticide to estuarine and marine organisms are required by 40 CFR
§ 158.145 to support the registration of an end-use product intended
for direct application to the estuarine or marine environment or
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•73
expected to enter this environment in significant concentrations
because of its expected use or mobility pattern.
(2) See 40 CFR § 158.50, "Formulators' exemption," to deter-
mine whether these data must be submitted. Section.II-A of this
Subdivision provides an additional discussion on this subject.
(b) Test standards. Data sufficient to satisfy the require-
ments in 40 CFR § 158.145 should be derived from tests which comply
with the general test standards in § 70-3 and the following test
standards:
(1) Test substance, (i) Data shall be derived from testing
conducted with the technical grade of each active ingredient in the
pr oduct.
(ii) In addition, data from testing with the applicant's end-
use product or a typical end-use product are required by 40 CFR
§ 158.145 to support the registration of products which meet any
of the following conditions:
(A) The end-use product will be introduced directly into an
aquatic environment when used as directed;
(B) The EC50 or LC50 of the technical grade of active ingre-
dient is equal to or less than the maximum expected environmental
concentration (MEEC) or the estimated environmental concentration
(EEC) in the aquatic environment when the end-use product is used
as directed; or
(C) An ingredient of the end-use product other than the active
ingredient is expected to enhance the toxicity of the active ingre-
dient or to cause toxicity to aquatic organisms.
(2) Test organisms and test duration. The 96-hour LC50 should
be determined for shrimp and an estuarine or marine fish. Also, the
48-hour EC50 for oyster embryolarvae or 96-hour EC50 shell deposition
data should be determined on a'representative mollusc, such as the
American oyster.
(3) Determination of EC50 or LC50. (i) Satisfactory data
should establish either:
(A)
or
An SC50 or LC50 value with 95 percent confidence intervals;
(B) That the EC50 or LC50 is greater than 100 mg/1 or greater
than 100,000 times the MEEC or EEC.
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74
(ii) If data are submitted to satisfy either criterion in
paragraph b)(3)(i) of this section, at least 30 individuals should
be tested at concentrations equal to or greater than the criterion
chosen.
(c) Reporting and evaluation of data. In addition to informa-
tion provided in § 70-4, a report of the results of an acute toxicity
test for estuarine and marine organisms should include the following:
(1) LC50 data. ( i) (A) Data showing the LC50 or EC50 , the
corresponding 95 percent confidence intervals, slope of the concen-
tration-response line, and, when possible, the LC50 values at
24-hour intervals for the duration of the test; or
(B) Data showing that the LC50 or EC50 is greater than
100,000 times the MEEC or EEC or greater than 100 mg/1.
(ii) If the data submitted in accordance with paragraph (c)(1)-
(i)(B) of this section show that the LC50 or EC50 is greater than
100,000 times the MEEC or EEC of the pesticide, the basis for
calculating the MEEC or EEC should be reported.
(2) Dilution water. Detailed description of dilution water,
including source, chemical characteristics (e.g., dissolved oxygen
content, pH), and pretreatment (if any).
(3) Test description. Detailed description of the test,
including:
(i) Design; • .
(ii) Containers;
(iii) Water depth and volume;
(iv) Treatments;
(v) Method of exposing organisms to the test substance (e.g.,
placing chemical in water which contains organisms or placing
organisms in water which contains the chemical);
(vi) Number of organisms per treatment;
(vii) Loading (weight of organisms per unit volume of water);
(viii) Lighting;
(ix) Acclimation and test temperatures (average and range);
(x) Salinities; and
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75
(xi) Any unusual feature of the test.
(4) Chemical analyses. If Conducted, a description of methods
(or references to established methods) used for the analyses of water
for chemical content and toxicant concentrations, and the results of
such analyses, including validation studies and reagent blanks.
(5) Effects of exposure. Detailed description of the effects
of the exposure to the test substance, including:
(i) The criteria used to determine the effects;
(ii) Statement of percentages of organisms that died or showed
effects of treatment; and -
(ill.) A summary of these observations.
(6) Additional information. Any additional relevant information
about the test or its results that would assist in the determination
of hazard potential.
(d) Acceptable protocols.
( 1) Acute estuarine and marine fish, and shrimp toxicity '•
tests. Examples of acceptable protocols are found in the following
references:
(i) Banner, L.H., C.D. Craft, and D.R. Nimmo. 1975. A salt- -
water flow-through bioassay method with controlled temperature
and salinity. Prog. Fish-Cult. 37 ( 3 ): 126-1,29 .
(ii) Committee on Methods for Toxicity Tests with Aauatic
Organisms. 1975. Methods for acute toxicity tests with fish,
macroinvertebrates, and amphibians. U-S. Environmental Protection
Agency, Ecol. Res. Series, EPA 660/375-009. 61 pp. (Marine and
estuarine species listed in this publication are acceptable.)
(2) Marine mollusc shell deposition and embryolarvae toxicity
tests. Examples of acceptable protocols are found in the following
references: •
(i) Anonymous. 1981. Standard Methods for the Examination
of Water and Wastewater. 15th Ed. American Public Health Associ-
ation, Washington, D.C. 1134 pp.
(ii) ASTM Standard E 724-80, Practice for Conducting Static
Acute Toxicity Tests with Larvae of Four Species of Bivalve Molluscs.
American Soci.ety for Testing and Materials, 1916 Race Street,
Philadelphia, PA 19103.
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76
(iii) Woelke, C.E. 1967. Measurement of water quality with
the Pacific oyster embryo bioassay. Pp. 112-120 in Water Quality
Criteria, ASTM, STP 416. American Society for Testing and Materials,
Philadelphia, Pa. '
(e) References. The following references can provide useful
background information in developing acceptable protocols:
(1) Anonymous. 1.378. Bioassay Procedures for the Ocean Disposal
Permit Program. U.S. Environmental Protection Agency, Office of Res.
and Dev. EPA-600/9-78-010. 121 pp.
(2) Clark, J.R., and R.L. Clark, eds. 1964. Seawater systems
for experimental aquariums. U.S. Dept. Int., Fish. S Wild. Serv.
Bur. Sport. Fish. Wild. Res. Rep. #63. 192 pp.
(3) DeBen, E.A. 1970. Design and construction of saltwater
environment simulator. Fed. Water Qual. Admin., Pacific N.W. Water
Lab., Working Paper 71:1-30.
(4) Strickland, J.D.H., and T.R. Parsons. 1968. A practical
handbook of seawater analysis. Fish. Res. Board Can. Bull. No. 167.
311 pp.
(5) White, D.B., R.R. Stickney, D. Miller, andL.H. Knight.
1973. Seawater systems for aquaculture of estuarine organisms at the
Skidaway Institute of Oceanography. Ga. Mar. Sci. Center, Technical
Rep. Ser. No. 73-1. 18 pp.
(6) Wood, L. 1975. A controlled condition system (CCS) for
continuously flowing seawater. Limnol. Oceanoqr. 10:475-477.
§ 72-4 Fish early life-stage and aquatic invertebrate, life-cycle
studies.
(a) When required. (1) Data from fish early life-stage tests
or lifecycle tests with aquatic invertebrates (whichever species
is most sensitive to the pesticide as determined from the results
of tests performed in §§ 72-1, -2, and -3) are.required by 40 CFR
§ 158.145 to support the registration of an end-use product intended
to be applied directly to water or expected to be transported to
water from the intended use site, and when any of following condi-
tions apply:
(i) If the pesticide is intended for use such that its pre-
sence in water is likely to be continuous or recurrent regardless
of toxicity, as revealed by studies required by 40 CFR § 158.130;
or
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77
, (ii) If any LC50 or EC50 value determined in testing required
by §§ 72-1,. -2, or -3 is less than 1 mg/1; or
/ (iii) If the estimated environmental concentration in water is
equal to or greater than 0.01 of any EC50 or LC50 determined in acute
testing for aquatic organisms required by 40 CFR § 158.145; or
(iv) If the actual or estimated environmental concentration in
water resulting from use is less than 0.01 of any EC50 or LCSO de-
termined in acute testing for aquatic organisms required by 40
CFR § 158.145 and any of the following conditions exists:
' "" (A) Studies of other organisms indicate the reproductive ohy-
siology of fish and/or invertebrates may be affected; or
(B) Physiochemical properties indicate cumulative effects; or
(C) The pesticide is persistent in water (e.g., half-life
in water greater than 4 days).
(2) See 40 CFR § 158.50, "Formulators ' exemption," to
determine whether these data must be submitted. Section II-A of
this Subdivision provides an additional discussion on this subject.
(t>) Test standards. Data sufficient to satisfy the require-
ments in 40 CFR § 158.145 should be derived from tests which comply
with the general test standards in § 70-3 and the following test
standards:
(1) Test substance. Data shall be derived from testing
conducted with the technical grade of each active ingredient in the
product.
( 2} Duration of tests.
(i) Fish early life-stage test. Fish should be exposed to
the test substance through the embryolarvae phase (e.g., a fish
"egg-fry" test) but not all stages of life-cycle of one generation
of the species.
Invertebrate life-cycle test. Invertebrates should be ••
cultured in the presence of the test substance from one stage of
its life-cycle to at least the same stage of the next generation
(e.g., egg to egg).
(3) Species. ..The applicant should consult with the Agency
regarding the appropriate species and test methodologies. The choic-
of species and test methods will be tailored to the pesticide's
characteristics .
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78
(4) Concentration analysis. The concentration of the test
substance in the water should be determined at the start of the study,
and periodically throughout the study to verify concentrations.
(c) Reporting and evaluation of data. ,,In addition to the basic
information provided in § 70-4, the test report should contain the
following information (where appropriate): : ^ ,
(1) Reproductive effects;
(2) Detailed records of spawning, egg numbers, fertility, and
fecundity; •
(3) No effect level;
(4) Mortality data;
(5) Statistical evaluation of effects;
(6) Locomotion, behavioral, physiological, and pathological
effects;
(7) Definition of the criteria used to determine effects;
(8) Summary of general observation of signs of intoxication or
other effects;
(9) Stage of life cycle in which organisms were tested;
(10) Duration of the test; and
(11) Concentration analysis.
(d) Acceptable protocols.
.(1) Fish early life-stage test. Examples of acceptable
protocols are found in the following references:
(i) National Water Quality Laboratory Committee on Aquatic
Bioassays. 1971. Recommended bioassay procedure for fathead
minnow Pimephales promelas (Rafinesque) chronic tests. (Revised
January, 1972). Pp. 15-24 in Biological Field and Laboratory
Methods. U. S. Environmental Protection Agency, Office of Res.
and Dev. EPA-670/473-001.
(ii) 1971. Recommended bioassay procedure for brook
trout Salvelinus fontinalis .(Mitchell) partial .chronic tests.
(Revised January, 1972). Pp. 25-33 _in Biological Field and
Laboratory Methods. U.S. Environmental Protection Agency,
Office of Res. and Dev. EPA-6 70/4-73-0 01 .
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nrotJ2? Inv*rtebrate Hfe-cvcle test. Examples of acceptable
protocols are found in the following references:
'*' 1974(aK ^°cgdure for Daphnia maona
tanding system. U.S. Environmental .
1 L fieSin^r' .K'E- 1974(b> ' ^ocedure for Daphnia magna
!!StS ln flowin? system. U.S. Environmental Protectiof^
(iii) Nimmo, D.E., T.L. Hamaker, and C.A. Sommers. 1978.
Entire li.e-cycle toxicity test using mysids (Mysidopsis bahia) ir
S^ngrf "- **' 64~68 ^ Bi°aSSa y Procedures for^he 3c^T '
OfSr l1,P!rmt Pr09ram- U'S- Environmental Protection Agency,
Ut^ice of Res. and Dev. SPA-600/9-78-010 .
(e) Reference. Additional information mav be : found in ^he
rollowing reference: "
i^oth^'/;^ 1974(C)< ^^uring methods for Daphnia and
in otner cladocerans. U.S. Environmental Protection-Agency
72~5 Life-cycle tests of fish.
(a) When required. ( 1 ) Data obtained from a life-cvcle
test or fish are retired by 40 CFR § isa.145 to support thf
registration of an end-use product intended to be allied dL^
site ard°rHeX?eCted ^ tranS?°rt to «ter from the' intended use
Site, and when any of the following conditions apply:
SStimated environmental concentration is eaual
fish -
. e
provides an additional discussion on ^,
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(b) Test standards - Data sufficient to satisfy 'the requirements
in 40 CFR § 158.145 should be derived from tests which comply with
the general test standards in § 70-3 and the following test standards:
(1) Test substance. Data shall be derived from testing con-
ducted with with the technical grade of each active ingredient in
the product.
(2) Duration of tests. Fish should be cultured in the presence
of the test substance from one stage of the life cycle to at least ~
the same stage of the next generation (e.g., egg to egg).
(3) Species. Testing should be performed on a freshwater
fish ie.g., fathead minnow). An estuarine species (e.g., sheep-
shead minnow) may be used if the pesticide is expected to enter
the estuarine environment.
(4) Concentration analysis. The concentration of the test
substance in the water should be determined at the start of the study
and periodically throughout the study to verify concentrations.
(c) Reporting and evaluation of data. In addition to the basic
information provided in § 70-4, the test report should contain the
following information (where appropriate):
(1) Reproductive effects;
(2) Detailed records of spawning, egg numbers, fertility, and
fecundity;
(3) No-effect level, and mortality data;
(4) Statistical evaluation of effects;
(5) Locomotion, behavioral, physiological, and pathological
effects;
(6) Definition of the criteria used to determine
effects;
(7) Summary of general observation of signs of intoxication
or other effects;
(8) Stage of life cycle in which organisms were tested;
(9) Duration of the test; and
(10) Concentration analysis.
(d) Acceptable protocol.
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M> /reshwater fish life-cycle test. AH example of an acceptable
protocol is found in the following reference:
1971 NaRi™pn^^Uality Labo"tory Committee on Aquatic Bioassays.
1971. Reconmended bioassay procedure for fathead minnow Pimephales
promelas (Raf inesque ) chronic tests. (Revised January, 1972T - PpT
15-24 -in Biological Field and Laboratory Methods. U. S. Environmental
Protection Agency, office of Res. and Dev. EPA-670/4-73-001 .
(2) Sstuarine fish life-cycle test. Examples of acceptable
protocols are found in the following references:
(i) Schimmel, S.C. , and D.J. Hansen. 1974. Sheepshead min-
now Cyprinodon variegatus; an estuarine fish suitable for chronic
(entire li recycle )bioassays. Proc. 28th Ann. Cong. S.E. Assoc.
Game-Fish Cotnm. Pp. 392-398. ~ ' - -
(ii) Hansen, D.J., P.R. Parrish, S.C. Schimmel, and L.R.
Goodman. 1978. Life-cycle toxicity test using sheepshead minnows
(Cyprinodon variegatus) . Pp. 109-116 in Bioassay Procedures for
the Ocean Disposal Permit Program. U.S. Environmental Protection
Agency, Office of Research and. Development. EPA-600/9-78-0 10 .
§ 72-6 Aquatic organism accumulation tests.
(a) When required. Data from aquatic organism accumulation
testing are required by 40 CFR § 158.145 on a case-by-case basis
to support the registration of an end-use product whose use is
likely to result in residues in an aquatic environment and which
may accumulate in aquatic organisms to toxic levels. Consultation
with the Agency is advised before undertaking this test. The
determination that a product meets these conditions should be made
when any of the following conditions apply:
(1) If the active ingredient or its principal degradation
product(s):
(i) Has water solubility less than 0.5 mg/1 and an octanol/water
partition coefficient greater than 1000; and
(ii) Is 'persistent in water (e.g., a half-life greater than
four days); or
(2) If the active ingredient or its principal degradation
product(s) accumulates in the organs and tissues of mammals or avian
species.
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(b) Test standards. Data sufficient to satisfy the requirements
in 40 CFR § 158.145 should be derived from tests which comply with
the general test standards in § 70-3 and the following test standards:
(1) Test, substance. Data shall be derived from testing
conducted with the technical grade of each active ingredient in the
product (studies using radioisotopes require analytical grade) or the
purest available form pf the principal degradation products, whichever
meets the general or specific conditions set forth in paragraph (a)(i-)
and (ii). / .' : . ':•-:; ",' '•, . '•' , •" . /., -• "
(2) Test organisms. (i) Consultation with the Agency is
advised before selection of species is made. One or more of the
following species may be used in accumulation testing:
(A) A typical bottom-feeding fish (e.g., catfish or carp);
(B) A cold-water fish, a warm-water fish, or marine fish (e.g.,
brook trout, rainbow trout/ bass, bluegill, northern pike, walleye,
or sheepshead minnow);
(C) Molluscs (e.g., oyster or freshwater clams);
(D) Crustaceans (e.g., Daphnia spp., shrimp, or crayfish); or
(E) Insect nymphs (e.g., mayfly).
(ii) The following factors should be considered
in selecting species:
(A) The use pattern of the formulated product;
(B) The relative sensitivity of the different species to toxic
effects; and
(C) Data on route of exposure and method of uptake.
(c) Reporting and evaluation of data. In addition to the in-
formation provided in § 70-4, specific data reporting and evaluation
guidance should be determined by consultation with the Agency.
(d) References. The following references can provide useful
background information in developing protocols. The conditions under
which an accelerated aquatic organism test [reference (d)(4)] may be
an acceptable substitute for a full length test [references (d)(1)-
(3)] should be determined by consulting with the Agency.
(1) Johnson, B.T. , and R.A. Schoet'tger. 1975. A biological
"model for estimating the uptake, transfer, and degradation of
xenobiotics in a food chain. Fed. Regis. 40 (123): 26906-26909.
(June 25, 1975.)
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83
(2) Macek, K.J., M.E. Burrows, R.F. Frasny, and s.H. Sleight,
III. 1975. Bioconcentration of 14C pesticides by bluegill sunfish
during continuous exposure. Pp. 119-142 in Structure-activity
correlations in studies of toxicity and bioconcentration with aauatic
organisms. Proceedings of a Symposium, Burlington, Ontario, March
11-13, 1975. G.D. Veith and D.E. Konasewich, eds. Sponsored by
Standing Committee on Scientific Basis for Water Quality Criteria of
the International Joint Commission's Research Advisory Board.
(3) Schimmel, S.C., J.M. Patrick Jr., and A.J.?Wilson. 1977.
Acute toxicity to and bioconcentration of endosulfan by estuarine
animals. Pp. 241-252 in Aquatic Toxicology and Hazard Evaluation.
F.L. Mayer and J.L.. Hamelink, eds. STP #634, American Society for
Testing and Materials, Philadelphia, Pa.
(4) Branson, D.R., G.E. Blau, H.C. Alexander, and W.B. Neely.
1975. Bioconcentration of 2,2',4,4'-tetrachlorobiophenyl in-rainbow
trout as measured by an accelerated test. Trans. Am. Fish. Soc.
104(4) :785-792. ~ :
72~7 Simulated or actual field testing for aquatic organisms
(a) When required. (1) Data from any of the following kinds
of tests .are required by 40 CFR § 158.145, on a case-by-case basis,
to support the registration of an end-use product intended for- out-
door application. Consultation with the Agency is advised before
undertaking these tests. Whenever data are required by 40 CFR
§ 158.145, the determination will be made in writing by the Agency
and will state which properties and use patterns of the product
were used in the deterziination. The following criteria are provided
as further guidance; ,
(i) Data frctn a short-term simulated field test (or an actual
short-term field test) are required by 40 CFR § 158.145 to support
tne registration of an end-use product which is likely to cause
adverse short-term or acute effects on fish or aquatic invert°-
brates. The short-term simulated field test (where confined
populations are observed) should be selected if it can yield data
useful in assessing such risks. An actual short-term field test
(where natural populations are observed) may be needed if a s^-
mulated test would not suffice. The determination of test selec-
tion or whether either should be conducted should take into account
available laboratory toxicity data, use pattern information, and
exposure information.
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(ii) Data from a long-term simulated field test (or an actual
long-term field test) are required by 40 CFR § 158.145 to support
the registration of an end-use product which is likely to cause
adverse long-term, cumulative, or life-cycle effects in fish or
aquatic invertebrates. The long-term Simulated field test (where
growth and reproduction:of confined populations are observed)
should be selected if it:can yield data useful in assessing such
risks. An actual long-term field test (where growth and reproduction
of natural populations are observed).may be needed if the simulated
test would not suffice.: The determination of test selection or
whether either should be conducted should take into account available
laboratory .toxicity data, use pattern.information, and exposure
information. :. : : . : :
(2) See 40 CFR § 158.50, "Formulators' exemption," to determine
whether these data must be submitted. Section II-A of this
Subdivision provides an additional discussion on this Subject.
(b) Test standards. Data sufficient to satisfy the require-
ments in 40 CFR § 158.145 should be derived from tests which comply
with the general test standards in § 70-3 and the following test
standards:
(1) Test substance. Unless specified otherwise, data shall
be derived from testing conducted with an end-use product [or with
an end-use product whose properties are like those of products to
which the determination under paragraph (a) of this section applies].
An "end-use product" may be the applicant's own product or a typical
end-use product.
(2) Concentration analysis. The concentration of the test
substance in the water should be determined at the start of the study
and collected periodically for analysis to verify concentrations.
(3) Test conditions. The test conditions for conducting field
tests should resemble the conditions likely to be encountered under
actual use. Specifically, the pesticide should be applied according
to the rate, frequency, and method specified on the label.
(4) Endangered species. Studies should not be conducted in
critical habitats or areas containing, or suspected to contain,
endangered or threatened plants or animals which may be threatened
by the.tests to be conducted.
(5) Residue levels. When the test substance is applied under
simulated or actual field condition testing, residues should be
determined in appropriate vegetation, soil, water, sediments, and
other environmental components, and in selected tissues of test
organisms.
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85
„. (6] Other sta"dards. Any additional standards for conducting
uhese tests will be provided by the Agency in writing following
consultation between the applicant and the Agency, and will take into
account the mechanise by which a pesticide may enter the envS^n?,
and the food sources and habitats that may be affected.
(c) Reporting and evaluation of data. m addition to the
information provided in § 70-4, specific data reoorting and evalua-
tion guidance should be determined by consultation with the Agency.
(d) References . The following references can provide useful
background information for conducting a simulated or actual field
study for aquatic organisms.
(1) Coppage, D.L. 1971. Characterization of fish brain acetyl-
cholinesterase with an automated PH stat for inhibition studies.
Bull. Environ. Contam. Toxicol. 6(4 ) :304-3 10.
(2) Klngsbury, P.O. 1976. studies of the impact of ae-ial
applications of the synthetic pyrethroid NRDC-143 on aquatic
ecosystems. Chemical Control Research Institute, Department o*
the Envornment, Ottawa, Ontario. Report CC-X-127 (unpublished
^ (3) Macek, K.J., D.F. Walsh, J.W. Hogan and D.D. Holz. 1972.
Toxicity of the insecticide Dursban® to fish and aouatic inve-e-
fcrates in ponds. Trans . Am. Fish. Soc. 1 0 1 ( 3 ): 420-427 .
(4) Nicholson, H.P., H.J. Webb, G.J. Lauer, R.E.. o' Brian, A.R.
farm T $'"' Shankli- 1962« --cticide contamination In a
farm pond. Part I - Origin and Duration; Part II - Biological
Effects. Trans. Am. Fish Soc. 91 (2 ): 2 13222.
4th Ed^ SChSmftZ' S'D-' ed' 198°- Wildlife Management Techniques .
4th Ed.: revised. The Wildlife Society, Inc., Washington, D.C."
/ Z. £ P •
1974 E^rt2' M'E", PWW'1 3°rthwick' G'H- Cook and D.L. Coppaae.
1974. ^.fects or ground applications of Malathion on salt marsh'
environments in northwestern Florida. Mosauitp_News 34 (3 ) : 309^ 3 15.
^enedh °f Interi°r' 1977' National Handbook of Recom
Reston, VA "' ^ ^ AC^isition- *'** Geological Survey,
1972 Ric'<'' > Amed' J-D'.I4n» »<* K.G.'whitesell.
" mos^lto c°ntrol studies with low volume Dursban®
sor.v u
nonSr ^ ^^ ' Californi- ™ Effects upon aquatic
nontarget organisms. Mosquito News 32 (4 } -53 1-537
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86
(9) Weber, C.I., Ed. 1973. Biological field and laboratory
methods for measuring the quality of surface waters and effluents.
U.S. Environ. Protect. Agcy, ORD, Environmental Moritoring Series,
EPA-670/4-73-001 (July 1973).
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["REPORT DOCUMENTATION ; i... REPORT No.
i PAGE ]
Subdivision E - Hazard Evaluation: Wildlife and
Aquatic Organisms.
I 7. Auinordj '• . . -
I 5- Seppn One
October 1982
Ecological Effects Branch, HED, OP
9. P.rforrrun; O'giniulion film, »nd Aacr.ss
Office of Pesticide Programs
! U.S. Environmental Protection Agency
i Washington, D.C. 20460
; 10. Proj*cS/TjiX/Wofh Unit No.
i
,' 11. ConUaciiC; cc Grantt'G) No.
12. Sponsoring Orjan.zj :;an Njm. and Aaoress
Office of Pesticide Programs
j U.S. Environmental Protection Agency
Washington,.D.C. '20460
! Guideline
15. SupciemenUry Natcl
Guidelines Project Manager: Robert K. Hitch
Subdivision S is a guideline package which is intended to support
the FIFHA (Federal Insecticide Fungicide and Rodenticide Act) data'
•requirements in 40 CFR Part 158. Subdivision E provides test protocols
for identifying the effects of pesticides on nontarget fish and:wild-
life . The guidelines state when a test is required, the testing standards
that should be met, the data that should be reported, and references to
appropriate test methods.
Subdivision E is only 1 Volume of a twelve-part FIFRA guideline
series published by the National Technical Information service.
L7. Oocument An
S. icientifiers/Open-Endec! Tcrmi
=. COSA7; rTeid/G.-o
l.tjr Sijt«meru
;ThcS Rtoon)
21. No. of Peg*
ty Ciui (TMi ?age
22. Price
(S« ANSI-I39.13)
. FQflM 272 ("4-771
NT1S-35J
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