a EPA
United States
Environmental Protection
Agency
Office of Chemical Safety
and Pollution Prevention
(7101)
EPA712-C-021
January 2012
Ecological Effects
Test Guidelines
OCSPP 850.2500:
Field Testing for
Terrestrial Wildlife
-------
NOTICE
This guideline is one of a series of test guidelines established by the United States
Environmental Protection Agency's Office of Chemical Safety and Pollution Prevention
(OCSPP) for use in testing pesticides and chemical substances to develop data for
submission to the Agency under the Toxic Substances Control Act (TSCA) (15 U.S.C. 2601,
et seq.), the Federal Insecticide, Fungicide and Rodenticide Act (FIFRA) (7 U.S.C. 136, et
seq.), and section 408 of the Federal Food, Drug and Cosmetic (FFDCA) (21 U.S.C. 346a).
Prior to April 22, 2010, OCSPP was known as the Office of Prevention, Pesticides and Toxic
Substances (OPPTS). To distinguish these guidelines from guidelines issued by other
organizations, the numbering convention adopted in 1994 specifically included OPPTS as
part of the guideline's number. Any test guidelines developed after April 22, 2010 will use
the new acronym (OCSPP) in their title.
The OCSPP harmonized test guidelines serve as a compendium of accepted scientific
methodologies and protocols that are intended to provide data to inform regulatory decisions
under TSCA, FIFRA, and/or FFDCA. This document provides guidance for conducting the
test, and is also used by EPA, the public, and the companies that are subject to data
submission requirements under TSCA, FIFRA, and/or the FFDCA. As a guidance
document, these guidelines are not binding on either EPA or any outside parties, and the
EPA may depart from the guidelines where circumstances warrant and without prior notice.
At places in this guidance, the Agency uses the word "should." In this guidance, the use of
"should" with regard to an action means that the action is recommended rather than
mandatory. The procedures contained in this guideline are strongly recommended for
generating the data that are the subject of the guideline, but EPA recognizes that departures
may be appropriate in specific situations. You may propose alternatives to the
recommendations described in these guidelines, and the Agency will assess them for
appropriateness on a case-by-case basis.
For additional information about these test guidelines and to access these guidelines
electronically, please go to http://www.epa.gov/ocspp and select "Test Methods &
Guidelines" on the left side navigation menu. You may also access the guidelines in
http://www.requlations.qov grouped by Series under Docket ID #s: EPA-HQ-OPPT-2009-
0150 through EPA-HQ-OPPT-2009-0159, and EPA-HQ-OPPT-2009-0576.
-------
OCSPP 850.2500: Field testing for terrestrial wildlife.
(a) Scope—
(1) Applicability. This guideline is intended to be used to help develop data to submit to
EPA under the Toxic Substances Control Act (TSCA) (15 U.S.C. 2601, et seq.), the
Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) (7 U.S.C. 136, et seq.), and
the Federal Food, Drug, and Cosmetic Act (FFDCA) (21 U.S.C. 346a).
(2) Background. The source materials used in developing this harmonized OCSPP test
guideline include OPP 71-5 Simulated and Actual Field Testing for Mammals and Birds
(Pesticide Assessment Guidelines Subdivision E—Hazard Evaluation: Wildlife and
Aquatic Organisms), and the Guidance Document for Conducting Terrestrial Field
Studies.
(b) Purpose. This guideline describes factors to be considered in the design and conduct of field
studies to develop data on the effects to terrestrial wildlife species from chemical substances and
mixtures ("test chemicals" or "test substances") subject to environmental effects test regulations.
The Agency will use these and other data to assess the risk to terrestrial wildlife that these
chemicals may present through environmental exposure. The purpose of the field study is either
to provide quantification of the effects that would occur to individuals, populations, or
communities of terrestrial wildlife or refute the assumption that risks will occur under conditions
of actual use of the test substance (primary consideration for pesticides) or occur under the
pattern of production, use, disposal, or accidental release of industrial chemicals in the terrestrial
environment.
(c) Definitions. The definitions in OCSPP 850.2000 apply to this guideline.
(d) General considerations—
(1) Summary of the test. The Agency uses terrestrial field studies to evaluate only those
test substances in which significant questions on risks to nontarget wildlife exist. The
test substance may be applied in a variety of ways; the selected method should support
the specific study objective. The study is performed under natural conditions and in the
environment in which the test substance would be either applied and/or disperses to under
normal use practices for pesticides or would occur under the pattern of production, use,
disposal, or accidental release for industrial chemicals. Specific objectives and
associated qualitative and quantitative decision statements establishing measurement
endpoints and their accuracy and precision should be provided as part of the study plan.
Specific protocols should be developed as needed and submitted to the Agency for
review prior to conduct of the study.
(2) General test guidance. In contrast to laboratory tests which are generally amenable
to a high degree of standardization, field study protocols are more flexible reflecting the
case-by-case nature of issues and decisions a given field study is designed to address.
Additionally standardization of field studies is made difficult by variability in such
factors as chemical mode-of-action, species density and diversity, and for pesticides
differences in use pattern, crop type, and method of application. This guideline provides
a general outline of factors to consider in the conduct of field studies; specific protocols
should be developed and submitted to the Agency for review. Despite the variability
Page 1 of 46
-------
among field studies, several key elements common to most field studies can be identified.
This guideline was prepared to identify and discuss these elements as they pertain to
terrestrial vertebrates, and to provide a better understanding of the purpose of such field
studies. There are two types of field studies, screening and definitive. The type of field
study conducted (screening or definitive) depends on the available data on the test
chemical or substance in question and the terrestrial wildlife population and community
dynamics. Environmental and exposure conditions under which a field study is
conducted should resemble the conditions likely to be encountered under actual use,
production, disposal, or fate of the test substance or chemical. Pesticides should be
applied to the site at the rate, frequency, and by the method specified on the label. The
general guidance in the OCSPP 850.2000 guideline applies to this guideline, except as
specifically noted herein.
(3) Environmental chemistry methods. Procedures and validity elements for
independent laboratory validation of environmental chemistry methods used to generate
data associated with this study can be found in OCSPP 850.6100. Elements of the
original addendum as referenced in 40 CFR 158.630 for this purpose are now contained
in OCSPP 850.6100. These procedures, if followed, would result in data that would
generally be of scientific merit for the purposes described in 40 CFR 158.630.
(4) Screening field study. If the available effects data is limited to laboratory toxicity
data on a limited number of species, a screening field study may be appropriate to
determine if hazards or risks extrapolated to individual animals, populations and
communities from the laboratory data are occurring in the field and, if so, to what species
before conducting a definitive field study. "Pass-fail" methods are used to determine
whether impacts occur. These methods may include carcass searching, residue analysis
of species collected on study plots, residue analyses of wildlife food sources found in and
adjacent to the area of application, behavioral observations, and enzyme analysis.
(5) Definitive field study. If a screening study indicates impacts are occurring, or if
other available data suggest that deleterious effects have occurred or are extremely likely,
the study design should be quantitative, evaluating the magnitude of the impacts in a
definitive study. A quantitative field study focuses on the species affected in the
screening phase. For some test substances or chemicals it may be appropriate to proceed
directly to a definitive study without the screening phase. Careful consideration needs to
be given to the likelihood of impacts occurring in order to determine which approach to
use. At the quantitative level (definitive study), the objectives should include estimating
the magnitude of acute or secondary mortality caused by the application, the existence
and extent of reproductive effects, and the influence of chemical use on the survival of
species of concern. Methods that can be used to address these objectives include mark-
recapture, radio telemetry, line transect sampling, nest monitoring, territory mapping, and
measuring young to adult ratios.
(6) Endangered species. Studies should not be conducted in critical habitats or areas
where endangered or threatened species could be exposed.
Page 2 of 46
-------
(e) Test standards—
(1) Test substance. For industrial chemicals the substance to be tested should be
technical grade unless the test is designed to test a specific formulation, mixture, or end-
use product. For pesticides unless specified otherwise, data is derived from testing
conducted with an end-use product. If the pesticide product is applied in a tank mixture,
dosages of each active ingredient (a.i.) should be reported with identification and
formulation for each product in the tank mix. The OCSPP 850.2000 guideline lists the
type of information that should be known about the test substance before testing, and
discusses methods for preparation for use in testing.
(2) Residue levels. When the test substance is applied under field condition testing,
residues should be determined in selected tissues of organisms collected in and around
the study area and in vegetation, soil, water, sediments, and other appropriate
environmental components. If methods to analyze for the test substance are not
available, the submitter should consult with the Agency before beginning the test.
(3) Sampling and experimental design. While examples of acceptable experimental
designs are given, it is beyond the purpose of this guideline to cover the fundamentals of
this topic. Paragraph (f) of this guideline provides a general outline for a field study
protocol to be submitted to the Agency for review. Paragraphs (e)(4) and (e)(5) of this
guideline discuss points to be considered in designing screening and definitive field
studies, respectively.
(i) A study designed to refute hazard is unusual in biological research. Typically,
an investigator is more concerned about concluding with a high degree of
confidence that an effect occurred, not that it failed to occur. FIFRA specifies
that a pesticide is to be registered only if EPA determines it will not cause
unreasonable adverse effects. The difference between an objective of "will
cause" and "will not cause" substantially influences study design and the
evaluation of data.
(ii) The adverse effects to wildlife that can result from the use of pesticides or
under the pattern of production, use, disposal, or accidental release of industrial
chemicals can be classified as those that affect populations of wildlife and those
that affect individuals but not the entire population. Either of these effects may
warrant regulatory action, including mitigation, cancellation or suspension of a
pesticide use, or controls on production/use/disposal of an industrial chemical.
An adverse effect that results in a reduction in local, regional, or national
populations of wildlife species is clearly of great concern. A chemical that can
repeatedly or frequently kill wildlife is also of concern even if these repeated kills
may or may not affect long-term populations. The terrestrial field study,
accordingly, is designed to assess both of these types of effects.
(iii) For pesticides, the field study should be designed to provide data that show
whether wildlife species will not be affected significantly by a pesticide under
normal use practices. For industrial chemicals produced, used, or disposed of in
the terrestrial environment, these data are also sought. To achieve such objectives
fully at the population level, it is necessary that detailed knowledge of the
Page 3 of 46
-------
population dynamics and varying environmental conditions for each species
potentially at risk be available. The theoretical aspects of population dynamics
are well documented in the literature. However, empirical data are available for
only a few species (see paragraph (h)(8) of this guideline). A study designed to
provide the needed data would include information on age structure, age-specific
survival and reproductive rates, and the nature and form of intrinsic and extrinsic
regulatory mechanisms. Such a study, when coupled with the influence of the
chemical use (or production/use/disposal pattern) on these parameters, could
require several years in order to give meaningful results.
(iv) The essential question is: How can these studies be performed in a practical,
economical manner and still provide data that can show that the chemical under
study will not reduce or limit wildlife populations or repeatedly kill wildlife?
(v) The question can be answered by examining the potential influence test
substances can have on wildlife. These effects include:
(A) Direct poisoning and death by ingestion, dermal exposure, and/or
inhalation.
(B) Sublethal toxic effects indirectly causing death by reducing resistance
to other environmental stresses such as diseases, weather, or predators.
(C) Altered behavior such as abandonment of nests or young, change in
parental care, or reduction in food consumption.
(D) Reduced food resources or alteration of habitat.
(E) Lowered productivity through fewer eggs laid, reduced litter size, or
reduced fertility.
(vi) These effects can manifest themselves in a population through reduced
survival and/or lower reproductive success. However, if a field study shows that
actual use of a chemical does not affect survival and/or reproductive success or
that only minor changes occur, it would seem reasonable to conclude that the use
of the chemical will not significantly impact wildlife. Further, if a field study
provides estimates on the magnitude of survival and reproductive effects, it is
possible to make reasonable projections on the meaning of the effects to nontarget
populations by using available information on the species of concern and basic
theories of population dynamics. While less than ideal, field studies that collect
information on survival and reproductive effects and use these data to address
population parameters should provide a reasonable basis for evaluating potential
impacts. This is not to imply that effects on populations are the only concern,
however, a study adequate to assess these effects will also assess the degree of
risk to individual wildlife.
(vii) This guideline emphasizes avian and mammalian wildlife. The Agency is
also concerned about other terrestrial organisms such as nontarget plants,
invertebrates, amphibians, and reptiles. Plants and invertebrates are excluded
Page 4 of 46
-------
here from direct study, except as sources of food or pesticides to wildlife. Testing
guidelines for nontarget insects and plants are in Groups C and D, respectively, of
the OCSPP 850 guideline series. Established protocols, especially for acute and
chronic toxicity testing, are available for birds and mammals, but not for reptiles
and are limited for amphibians. Occasionally, however, it may be necessary to
adapt these field techniques to apply specifically to reptiles and/or amphibians.
(4) Screening study—
(i) Objective and scope.
(A) The screening study is designed primarily to demonstrate that the
hazard suggested by lower tier laboratory or pen studies does not exist
under actual use conditions (primary consideration for pesticides) or occur
under the pattern of production, use, disposal, or accidental release of
industrial chemicals. The interpretations of screening study results, in
most cases, are limited to "effect" versus "no-effect" determinations. If
the study indicates that the chemical has caused little or no detectable
adverse effect, it may be reasonable to conclude that potential adverse
effects are minor. When effects are demonstrated, determining the
magnitude of the effects is done with additional testing (see paragraph
(e)(5) of this guideline). Therefore, when information already available
shows that a chemical has caused adverse effects under normal use
conditions (primary consideration for pesticides) or under the pattern of
production, use, disposal, or accidental release of industrial chemicals, the
screening study may be of limited value. It may be appropriate to proceed
directly to a definitive field study in cases where analysis of laboratory or
other data strongly suggest that adverse effects are likely to occur, and are
unlikely to be attenuated by field conditions.
(B) Screening studies are limited to addressing the potential for acute toxic
effects, such as direct poisoning and death, and sublethal toxic effects
potentially affecting behavior and/or survival. In most instances, a
screening study would not address reproductive effects or effects such as
changes in density or diversity of populations.
(C) Further laboratory and/or pen studies may be useful prior to
proceeding to the field, or may be necessary to interpret results of the field
study. For example, additional toxicity data on species that are expected
to be exposed from the normal use pattern (primary consideration for
pesticides) or under the pattern of production, use, disposal, or accidental
release of industrial chemicals may indicate which species are more
susceptible to the chemical, allowing the study to be designed to monitor
those species in greater depth as well as to provide insight into field results
that show that some species were affected more than others, and additional
laboratory studies may be unavoidable. If residue concentrations in
resident species are being used to indicate potential problems, the
relationship between tissue levels and the doses that cause adverse effects
Page 5 of 46
-------
is estimated. If secondary poisoning is of concern, feeding secondary
consumers (held in captivity) prey items collected in the field following
application of the test substance can be useful to evaluate this potential
exposure route. Laboratory toxicity tests for secondary consumers
coupled with residue analysis of prey items can indicate the potential for
secondary poisoning of nontarget species. In designing field studies, the
utility of laboratory and/or pen tests should not be neglected, and where
appropriate their use is encouraged.
(ii) Geographic area selection.
(A) Studies should be performed in representative biogeographic areas
where the chemical could be used (primary consideration for pesticides) or
occur under the pattern of production, use, disposal, or accidental release
of industrial chemicals taking into account the diversity and variability in
wildlife species and habitats involved. To keep the number of geographic
areas at a manageable level while still accomplishing the purpose of the
field study, geographical area selection should emphasize situations likely
to present the greatest risk.
(B) A careful review of the species and habitats in the various
geographical areas where the pesticide product could be used or the
industrial chemical occur under the pattern of production, use, disposal, or
accidental release is necessary to identify the areas of highest concern. A
sound understanding of the biology of the species that are found in
association with the potential pesticide or industrial chemical exposure
sites is essential. Identifying these areas may require an extensive
literature review and consultation with experts familiar with the areas and
species of concern. The study area selected should be frequented by those
species that would have high exposure, based on their feeding or other
behavioral aspects. If exposure and fate (e.g., degradation) parameters
vary geographically, study area selection also should be biased towards
maximizing residues available to wildlife. In some circumstances
preliminary monitoring of candidate areas may be appropriate to
determine which ones should be selected for detailed study.
(iii) Study site selection.
(A) In general, study sites should be selected from what is considered to
be a typical or representative environment in which the chemical would be
either applied and/or disperses to under normal production/use/disposal
practices, but at the same time, study sites should contain the widest
possible diversity and density of wildlife species. Identifying potential
study sites may require consultation with experts familiar with the areas
where studies are proposed, and preliminary sampling.
(B) To maximize the hazard, the sites selected should have associated
species that would be at highest risk from exposure, as well as a good
diversity of species to serve as indicators for other species not present at
Page 6 of 46
-------
that specific location. The choice of study sites that are as similar as
possible in terms of abundance, diversity, and associated habitat will
facilitate an analysis of the results.
(C) Field surveys of a number of sites are used to identify which sites
should be selected for detailed study. Even when high risk species can be
identified, preliminary surveys may be needed to determine which sites
have adequate numbers of the high risk species as well as a good diversity
of other species.
(D) In the initial evaluation of potential study sites, edge effect may
indicate which sites support the larger and more varied wildlife
populations. As stated by Aldo Leopold (see paragraph (h)(23) of this
guideline), "The potential density of game of low radius requiring two or
more types is, within ordinary limits, proportional to the sum of the type
peripheries." (Type is defined as the various segments of an animal's
environment used for food, cover, or other requirements.) If study sites
are selected to maximize edge effect the potential for high density and
diversity should be increased. One quantitative measure of edge and edge
effect (see paragraph (h)(15) of this guideline) is the distances around
individual plant communities in relation to the unit area of the community.
Population densities, in general, are positively related to the number of
feet of edge per unit area of community. Study sites chosen to maximize
the ratio of edge to core may serve to indicate sites with higher densities
and diversities of wildlife species.
(E) While this ratio can be helpful in selecting study sites, the other
characteristics of edge should not be neglected in screening potential study
sites. Density and diversity of wildlife species are also influenced by the
variety in the composition and arrangement of the edge component cover
types and its width. Also, interspersion (the plant types and their
association with one another) influences densities of wildlife species. The
edge effect is the sum of all the characteristics of edge and hence each
component needs to be considered. An agricultural field with a relative
high edge to core ratio may not have as high a density and diversity as one
with a lower ratio but greater variety, width, and interspersion. In general,
edge characteristics can be used to screen potential study sites; however,
preliminary sampling of prospective study sites will be needed to identify
study sites with adequate density and diversity of wildlife species.
(iv) Number of sites.
(A) The number of sites needed can be estimated using the binomial
theorem. Briefly, the rationale is that for each study site there are two
possible outcomes, either effect or no-effect. Trials of this type are known
as binomial trials and, when repeated, the results will approximate a
binomial distribution. In this case, to use the binomial theorem, it is
necessary first to define the expected probabilities that birds or mammals
Page 7 of 46
-------
on a site are affected or not affected, after which the probability of the
discrete binomial random variable x for n replications can be used to
determine the minimum number of sites at a certain level of significance.
(B) For purposes of illustration, a problem exists if some specific mortality
rate or level of some other variable occurs on more than 20 percent (20%)
of the potential application sites. Translated into binomial probabilities,
there is a 0.2 probability of a site showing an effect and a 0.8 probability
of a site not showing an effect. Therefore, if the results from the field trial
show that the number of sites affected is significantly lower than 0.2, it
can be concluded that potential impacts will be below the stated level of
concern.
(C) To calculate the minimum number of sites necessary to show a
significant difference between the observed and expected, Equation 1 for
the probability of the binomial random variable x can be used (see
paragraph (h)(37) of this guideline) with rearrangement.
Equation 1
where:
x = number of sites showing effects,
n = number of sites,
p = probability of a site showing an effect, and
q = probability of a site not showing an effect.
(D) For the illustration in paragraph (e)(4)(iv)(B) with the probability of x
= 0, no sites showing effects, set at the desired alpha level (i.e.., P(x = 0) =
a), Equations 2 through 5 show the sequential steps in rearranging and
solving Equation 1 for n.
Equation 2
"
a = q Equation 3
Equation 4
n = Equation 5
Page 8 of 46
-------
(E) The minimum number of sites can be determined using Equation 5.
Continuing with the discussion example of 20% occurrence of an effect
(i.e.., a 0.2 probability of an affected site, a 0.8 probability of a nonaffected
site) at an alpha (a) level of concern of 0.05 sample size would be
calculated using Equation 5 as demonstrated in Equations 6 with a
resulting n = 13.4.
flogO.05^
n = — - =13.4 Equation 6
(F) Therefore in the example provided, 14 is the minimum number of sites
needed such that the probability is not greater than 0.05 that all sites
surveyed would be unaffected. In other words, if 20% of the application
sites are actually affected, there is only a 5% chance of finding all 14 sites
unaffected when n = 14. Moreover, if 20% of the application sites are
actually affected, we expect to find 1, 2, 3, and 4 sites affected with
probabilities of 0.15, 0.25, 0.25, and 0.17, respectively, when n = 14.
(G) Under many circumstances, conducting this number of replications
may not be practical. However, the number of sites can be reduced if site
selection is biased toward hazard. While arguable, it seems logical that if
the worst cases are sampled, a less stringent level of significance could be
accepted. While this must be determined on a case-by-case basis, the
Agency believes a minimum acceptable level of significance under worst-
case conditions is 0.2 rather than 0.05 under average or normal use
conditions. At this level, eight sites showing no effect would be required
to conclude at the 0.2 level of significance that the effect occurred on less
than 20% of the application sites or — there is less than a 20% chance that
all eight sites will be judged unaffected when n = 8 sites. Under some
circumstances, this may not seem adequately protective. It should be
noted, however, that based on this same design, it could be concluded that,
at the 0.1 level of significance, effects occur on less than 30% of the
application sites, and at the 0.05 level of significance, effects occur on less
than 40% of the application sites. Hence, with eight sites, it could be
concluded with a relatively high degree of confidence that effects would
occur on less than 40% of the application sites. Because worst-case study
sites were used, the Agency could have additional confidence that adverse
effects would occur on less than 20% of all normal application sites.
(H) Under some circumstances, particularly if endangered species could
be exposed from the proposed use, additional replication may be desirable.
Under these conditions, a high degree of confidence that an effect was a
rare occurrence would be required. (Under no circumstances should field
studies on chemicals be conducted in areas where endangered species
could be exposed.)
(I) The calculations under paragraphs (e)(4)(iv)(C) through (e)(4)(iv)(G)
of this guideline are for when x = 0, no effects are observed on any site. A
Page 9 of 46
-------
similar approach can be used to estimate the number of sites necessary to
show a significant result for a critical value of x > 0. Again the formula
for the probability of the binomial random variable can be used summing
the probabilities of x and all outcomes of less than x. Then by using
increasing values of n, the number of replications required to show
statistical significance may be determined for a given level of significance
for individual x values.
Equation 7
(J) The minimum value of n in Equation 7 occurs when P(X< r) is set to
the desired alpha level (i.e. P(X < r) = a level). Continuing the previous
example, Table 1 gives the results for x < 1 and x U2 for the previously
defined acceptable occurrence level of effect (i.e., a 0.2 probability of an
affected site, a 0.8 probability of nonaffected site). From the table, the
minimum number of sites needed when the critical value for x is set at 1,
to conclude that (at a 0.2 level of significance) effects are occurring below
levels of concern is 14. If x < 2, 21 sites are needed in order to have an
equivalent criterion. As can be seen, as x (the number of sites with
effects) increases, the number of sites required to show a statistical
significance becomes inordinately large.
Table 1.—Probabilities for Binomial Random Variable with p = 0.2 for x < 1 and x <
2 as a Function of the Number of Sites (n).
n
8
9
10
11
12
13
14
15
16
17
18
19
20
21
P(x<1)
0.5033
0.4362
0.3758
0.3321
0.2749
0.2336
0.1979
0.1671
0.1407
0.1182
0.0991
0.0827
0.0692
0.0576
P(x<2)
0.7969
0.7382
0.6778
0.6174
0.5583
0.5017
0.4481
0.3980
0.3518
0.3096
0.2713
0.2369
0.2061
0.1787
(K) When the probability of an affected site is 0.2, application of the rule
of zero-observed-affected-sites results in a declaration of "no-effect" 16.8
and 13.4% of the time for samples of size 8 and 9, respectively. It also
results in a declaration of "no-effect" 43.1 and 38.7% of the time for
samples of size 8 and 9, respectively, when the probability of an affected
site is 0.1, a value less than the criterion probability. Application of the
Page 10 of 46
-------
rule of zero-observed-affected-sites for a declaration of no-effect means
that a study is considered to be negative (shows no effect) if, and only if,
none of the sites show effects.
(L) Under any condition, it is extremely important with the binomial
approach to define the critical or threshold level for an effect, and to be
sure that the methods used are sensitive enough to detect an effect should
one occur. These assessments depend upon the species potentially at risk
as well as the parameter being sampled. It should be noted that the
measure of effect is not limited to mortality. Other parameters, such as
residue or enzyme levels, can be used. Whatever parameters are used,
defining the criteria level for an effect is extremely important, and when
designing studies this issue should be considered carefully.
(M) Using this approach, control (reference) sites are not an absolute
necessity. While the Agency encourages their use, in some cases the
additional information gained from the control sites for a screening study
may not justify the additional effort required. In most instances, control
sites would serve to protect from erroneously attributing effects due to
other causes to the chemical. However, for most chemicals, this can be
avoided by employing methods, such as residue analysis and/or
cholinesterase (ChE) inhibition tests, that can be used to indicate if the
chemical contributed to the observed effect. Further, because studies have
shown that it is a relatively rare event to locate dead or sick animals in the
wild except under unusual conditions (see paragraph (h)(18) of this
guideline), it is unlikely to find dead animals that were killed by
something other than the chemical being tested.
(N) Nevertheless, controls may be necessary when reliable methods to
confirm the cause of effect are not available. In these cases the binomial
design can be modified to a paired-plot binomial design, with a treatment
plot and a comparable control plot for each study site within an area.
When critical levels of effect and occurrence are defined, the binomial
theorem can be used for sample size determination, which gives 8 site
pairs (16 paired plots) showing less than a defined difference between
plots to conclude at the 0.2 level of significance that the effect occurred on
less than 20% of the application sites. Alternatively, a quantitative
difference or, preferably, ratio of treated to control responses could be
used to test for a treatment effect on each of the measured response
variables. (This is discussed further under paragraph (e)(5)(ii) of this
guideline.)
(v) Size of study sites. For a satisfactory field study, study sites should be large
enough to provide adequate samples. The size is dependent on the methods used,
the sensitivity required, and the density and diversity of species and their ranges.
In some cases, particularly with slow-acting poisons or where species at high risk
have relatively large home ranges, areas several times larger than the treatment
area may need to be examined. In some circumstances, several fields in an area
Page 11 of 46
-------
may be included in a single study site to account for wide-ranging species of
lower densities. Except in the unusual circumstance where fields are extremely
large (e.g., forested and range areas), the study site should never be less than an
individual field and the surrounding area. The nature of the surrounding area is
discussed further under individual methods. Another consideration is the distance
between study sites—in general, sites should be separated adequately to ensure
independence, which is dependent mainly on the range of the species that could
be exposed.
(vi) Chemical application.
(A) In general, the study conditions should resemble the conditions likely
to be encountered under actual use of the product (primary consideration
for pesticides) or occur under the pattern of production, use, disposal, or
accidental release of industrial chemicals. For pesticides, consideration
should be given to application rates and methods and in most instances the
pesticide should be applied at maximum use rates and frequencies
specified on the label. If more than one application method is specified on
the label, the method that maximizes exposure of nontarget species should
be used.
(B) This evaluation should relate wildlife utilization of the area to
exposure. For example, for pesticides if the crop is one that is used by
avian species as preferred nesting areas, feeding areas, or cover, ground
application may be the method that maximizes exposure. However, if it is
a crop with low utilization by wildlife species, but with high utilization of
its edges, aerial application where drift could increase exposure may be
more appropriate. In any case, the method of application used should be
consistent with the label.
(C) Equipment used may influence potential exposure of nontarget
species. In some instances, preliminary tests may be required to estimate
which method and equipment poses the highest exposure. For pesticides
there is a diversity of types of farming equipment that, depending on the
particular use pattern involved, could influence exposure. For example,
for pesticides applied in-furrow at planting there are several types of
covering devices employed on seeders, such as drag chains, drag bars,
scraper blades, steel presswheels, etc., in which the efficiency may vary
for covering the pesticide. In general, the various equipment normally
used for the particular pesticide application has to be evaluated to estimate
the potential influence of equipment choice on exposure.
(vii) Methods. This section provides a general outline of methods appropriate for
use in a screening field study and indicates some of their limitations. While
methods described have been found to be most useful, a screening study is not
limited to these methods. If other methods are more appropriate, their use is
encouraged. Because procedures should be adapted to specific situations, the
outlines presented should not be interpreted as strict protocols. Normally,
Page 12 of 46
-------
different methods will be combined to evaluate potential impacts. Due to the
indefinite number of variables and the unpredictability of wild animals, even
normally reliable procedures can sometimes prove inadequate. The methods used
in a screening study address exposure by monitoring overt signs of toxicity such
as mortality or behavioral modifications, or by evaluating parameters that indicate
animals are under stress, such as residue concentrations in tissues or degree of
enzyme inhibition. Measurements of density and diversity of species are needed
to aid in evaluating the results. The methods in paragraph (e)(4)(vii)(A) through
(e)(4)(vii)(F) of this guideline can be useful for screening studies.
(A) Carcass searches.
(1) Searching for dead or moribund wildlife is a basic method used
in field studies to evaluate the impact of chemicals on nontarget
species. Carcass searches can roughly indicate the magnitude of
kills when adequate areas are searched and the reliability of the
search is documented. This latter point is extremely important.
Rosene and Lay (under paragraph (h)(30) of this guideline)
indicated that finding even a few dead animals suggests that there
has been considerable mortality; failure to find carcasses is poor
evidence that no mortality has occurred. The reliability of the
search is based upon the percentage of carcasses recovered by
searchers and the rate of disappearance. By knowing the
reliability, the significance of failure to find carcasses can be
assessed and the extent of the kill estimated.
(2) Finding dead animals is seldom easy, even if every animal on a
site is killed. For example, three breeding pairs of small birds per
acre is considered a large population (see paragraph (h)(18) of this
guideline), and under average cover conditions, a small bird is
difficult to detect. Small mammals may be more abundant but, due
to their typically secretive habits, they are more likely to die under
cover and can be even more difficult to find than birds. Carcass
searching specifically for mammals should be attempted only when
cover conditions permit reasonable search efficiency. However,
any vertebrate carcasses found should be collected, even if the
search is oriented primarily to one taxon.
(3) Because results may be biased by scavenging and failure to
find carcasses, the sensitivity of this procedure should be
determined. Under conditions of heavy cover and/or high
scavenger removal, other methods may be more appropriate.
(4) There are no standard procedures for carcass searches.
Paragraph (e)(6) of this guideline outlines practices that have been
used typically and should be considered in designing searches.
Page 13 of 46
-------
(B) Radio telemetry.
(1) Radio telemetry has been found to be extremely useful for
monitoring mortality and other impacts caused by chemical
exposure of wildlife. Advances in miniaturizing electronic
equipment have made it feasible to track most vertebrate animals.
Transmitters weighing a few grams have been used to track species
as small as mice. Cochran's excellent summary of this technique
provides additional details (see paragraph (h)(5) of this guideline).
(2) Radio telemetry has the advantages of providing information
on the fate of individual animals following a chemical application
and of facilitating carcass recovery for determining the cause of
death. Although the initial cost of this technique may be more than
for other methods, the increase in information obtained under some
circumstances can more than justify the cost. The method is
particularly useful with less common or wide-ranging species.
(3) In addition to mortality, radio telemetry can be used to monitor
behavioral modification as well as physiological changes.
Automatic radio-tracking systems permit continual surveillance of
the location of animals (see paragraph (h)(5) of this guideline),
which can be used to provide insight into behavioral changes such
as nest abandonment, desertion of young, or decreases in activities
such as flying or feeding. Radio telemetry equipment is also
available for the transmission of physiological data such as heart
rates or breathing rates (see paragraph (h)(25) of this guideline).
(4) While this technique can provide very useful information on
impacts of chemicals to wildlife, other points need to be
considered in addition to cost. Capturing animals alive and
unharmed requires time, skill, and motivation. For the method to
be consistently successful, the investigator must be thoroughly
familiar with the habits of the species under study and with the
various capture methods that can be used. Even for the most
experienced investigator, adequate sample sizes can be difficult to
obtain.
(5) Adequate sample size is very important. The binomial theorem
can be used to estimate minimum sample size per site, if the
question is limited to mortality. Briefly stated, to be sure that
nontarget species are not being affected by environmental
concentrations greater than, for example, an LC20, the expected
binomial probabilities would be 0.2 for mortality and 0.8 for
nonmortality. Depending on the level of significance, 8 (a = 0.2)
to 14 (a = 0.05) individuals would need to be monitored per site
(see paragraph (e)(4)(iv) of this guideline for further details on
these calculations). However, since the LC20 may differ between
Page 14 of 46
-------
species, 8 to 14 individuals would be required for each species,
unless laboratory tests have documented relative species
sensitivity. Further complications can arise if radiotagged animals
leave the area or if the movements of individuals limit their
exposure. If these complications occur at relatively low rates, a
few additional radio-tagged animals may be sufficient to overcome
these problems.
(C) Tests of Cholinesterase inhibition.
(1) Measuring cholinesterase (ChE) concentrations in animal
tissues has been found to be a very useful field technique for
evaluating exposure of nontarget animals to ChE-inhibiting-
chemicals (see paragraphs (h)(18) and (h)(19) of this guideline).
These chemicals, including organophosphates and carbamates,
affect the synaptic transmission in the cholinergic parts of the
nervous system by binding to the active site of acetylcholinesterase
(AChE), which normally hydrolyzes the neuro-transmitter
acetylcholine. Thus, ChE inhibitors permit excessive acetylcholine
accumulation at synapses, thereby inhibiting the normal cessation
of nerve impulses (see paragraphs (h)(6) and (h)(27) of this
guideline).
(2) The depression of AChE activity, when measured and
compared to controls, can indicate the degree to which an animal is
affected. Brain ChE depression of >50% in birds has been found
sufficient to assume that death is pesticide-related (see paragraph
(h)(24) of this guideline). Depressions of more than 70% are often
found in dead birds poisoned by these chemicals (see paragraphs
(h)(l), (h)(2), and (h)(33) of this guideline), although some
individual birds with less than 50% inhibition may die. A 20%
depression of brain ChE has been suggested as an indication of
exposure (see paragraph (h)(24) of this guideline). ChE
concentrations in blood can also be used to indicate exposure,
avoiding the necessity of sacrificing the animal. However, blood
ChE concentrations are influenced more by environmental and
physiological factors than are brain ChE concentrations. Because
ChE activity varies among species, the degree of depression must
be based on an estimated normal value for concurrently tested
controls of the species potentially at risk. Because of this
difference between species, each case must be considered unique
(see paragraph (h)(19) of this guideline).
(3) Although there are several calorimetric methods for
determining ChE activity, the general methods are similar. Brain
tissues (or blood samples) are taken and analyzed for ChE
concentrations. Comparisons are made between pre- and post-
treatment and between treated and untreated areas. It is important
Page 15 of 46
-------
to ensure that untreated controls have not been exposed to any ChE
inhibitors. It also should be noted that absolute enzyme levels in
the literature are derived from various different, although similar,
methods and are reported in different ways. For example, Ludke et
al. (see paragraph (h)(24) of this guideline) used a modification of
the method reported in paragraph (h)(13) of this guideline and
reported results of ChE activity as nanomoles of acetylthiocholine
iodide hydrolyzed per minute per milligram of protein, whereas
Bunyan et al. (see paragraph (h)(l) of this guideline) used their
own colorimetric method (in addition to a pH change method) and
reported the results as micromoles of acetylcholine hydrolyzed per
hour per milligram of protein. Therefore, without a tightly
standardized method, it is necessary to use concurrent controls of
the same species obtained from the general vicinity (but untreated)
of the exposed birds, rather than literature values. Because of the
greater variation in plasma ChE levels than for brain, more
controls are necessary to evaluate blood samples.
(4) Tests for ChE activity can be used to help confirm cause of
death and monitor levels of exposure. In the latter case, 5 to 10
individuals of each species are collected before treatment and at
periodic intervals following treatment. Mean inhibition of 20% or
more is considered an indication of exposure to a ChE inhibitor.
Confirmation of cause of death may be determined by analyzing
brain tissue from wildlife found dead following treatment and
comparing the activity with controls. Inhibition of 50% or more is
considered strongly presumptive evidence that mortality was
caused by a ChE-inhibiting compound. The cause-effect
relationship can be further supported by chemical analysis of the
contents of the digestive tract or other tissues for the chemical in
question.
(5) For this technique to provide accurate information, prompt
collection and proper preservation of specimens are essential. ChE
concentrations in tissues are influenced by time since death,
ambient temperatures, and whether or not reversible ChE inhibitors
are being investigated. Therefore, the response of postmortem
brain ChE to ambient conditions can seriously affect diagnosis of
antiChE poisoning. Samples must be collected shortly after death
and frozen immediately to halt changes in tissue or enzyme-
inhibitor complexes. A technique for field monitoring and
diagnosis of acute poisoning of avian species has been reviewed,
discussing sample collection, sample numbers, preservation
procedures, and sources of error (see paragraph (h)(19) of this
guideline).
Page 16 of 46
-------
(D) Residue analysis.
(1) Residue analyses of wildlife food sources provide information
about the level and duration of chemical exposure. Residue
analysis of animal tissues also can indicate actual exposure levels.
If the relationship between tissue concentrations and toxic effects
is known for the species in question, residue analyses can provide a
measure of the degree to which animals are affected. For this
application of residue analyses, laboratory trials are necessary to
establish the relationship between residue levels and toxicity. In
addition to death, these laboratory trials should include such signs
as anorexia, asthenia, asynergy, or ataxia. For chemicals that are
readily metabolized by vertebrates, residue analysis may not be
appropriate for diagnostic purposes. With many chemicals, it will
be necessary to analyze for residues of active metabolites also.
(2) For determining residues on wildlife food sources, the
investigator should collect samples of insects, seeds, leafy parts of
plants, etc., immediately after chemical application and at periods
thereafter. Samples should be analyzed for the chemical to
determine potential exposure rate and duration. The application
method needs to be considered in determining where to take
samples. If drift is likely, samples should be taken from habitats
surrounding the treatment sites as well as in the treated sites.
Because analysis can be costly, the investigator should consider
carefully the number of samples necessary to provide adequate
data. Where feasible, samples from different locations within a
site should not be pooled. Separate analysis of samples can
provide data on the range and variability of exposure as well as
mean levels.
(3) When residue analysis is used to evaluate exposure in nontarget
animals, the tissues selected for analysis differ depending on the
purpose. Heinz et al (see paragraph (h)(17) of this guideline)
indicated that for many chemicals, residues in brains of birds and
mammals can be used to determine if death is chemical-related.
The authors believe that sublethal exposure is judged better from
residues in other tissues. Therefore, they proposed that analyses of
whole body homogenates should be used to quantify the body
burden of a chemical. If this is not feasible, analysis of muscle
tissue is suggested, because muscle residues reflect body burden
more nearly than those of any other tissue, and the amount of
muscle tissue is not unduly large. For persistent chemicals, it has
been suggested that residues in liver and fat tissues could be
misleading for determining acute body burdens. Liver is a
processing organ and its residue level largely represents current
availability of the chemical. Residues in fat are greatly affected by
changes in the amount of body fat, and are not dependable
Page 17 of 46
-------
indicators of body burden of the chemical; however, for some
chemicals, liver, fat, or other tissues may be good qualitative
indicators that exposure did occur. In general, laboratory trials or
data gathered in metabolism or other studies may be necessary to
determine which tissues can provide the most useful information.
Residue analysis of eggs taken from nests in treatment areas can
indicate the degree of contamination that a treatment has caused, as
well as possible reproductive effects of the treatment.
(4) Two approaches may be used to determine the number of
samples to be collected. Frequently, residue samples will be
collected to establish a mean value and confidence limits. To
determine the number of samples to collect, it is necessary to
estimate the standard deviation and to set an arbitrary limit from
the mean value that is acceptable. Although the mean value does
not need to be estimated, it is also necessary to have some idea of
the mean so that the standard deviation can be estimated and the
limit can be set. The formula for the number of samples for 95%
probability, from the reference in paragraph (h)(34) of this
guideline, is presented in Equation 8.
. 2 /
/? = 4cr /2 Equations
/ -/-/
where:
G = the standard deviation, and
L = the allowable limit around the mean.
(5) For example, to calculate the residue concentrations on
vegetation within ±10 ppm, with an estimate of the standard
deviation of 20 ppm, then n = 4(20)2/(10)2 means 16 samples are
required to have a 95% probability that the sample mean value will
be within ±10 ppm of the true mean.
(6) In some situations, there may be little information useful for
estimating the standard deviation, or the standard deviation may be
rather large, thus requiring a very large sample size. For some
types of samples, such as residues in nontarget wildlife carcasses,
the sample size cannot be increased to permit more precision. The
mean value of a parameter certainly has utility, but it also is very
important to establish confidence limits around the mean. In
general, the Agency will use the 95% confidence limits (usually
the upper boundary, as in the case of residues) in the assessment of
the data. This approach will substantially reduce the impact of
outliers but will still incorporate the range of reasonable values
into the assessment. In addition, the use of confidence limits
reduces the necessity for taking a large number of samples.
Page 18 of 46
-------
Because the width of the confidence intervals decreases with
increasing sample sizes, investigators should take samples that are
as large as feasible.
(7) Since the sample size will nearly always be less than 30, the
calculation of confidence limits should be based on Student's t-
distribution. The t values are derived from tables available in most
statistics books, and the 95% confidence limits are:
Equation 9
where
s = the standard deviation estimated from the sample of
size n.
(8) Alternatively, the binomial approach may be used for
determining if residues, typically in collection of live nontarget
animals, exceed a particular threshold value that indicates an
effect. The required sample size is the same as presented for the
binomial approach in determining the number of study sites.
Specifically, in the preceding example a minimum of 8 samples
with none exceeding the threshold value or 14 samples with one or
none exceeding the threshold value indicates no effect atp = 0.2 in
20% of the samples. This approach requires the establishment of
threshold values which are determined on a case-by-case basis. In
general, residues reflecting a 20% lethal concentration (LC2o) level
of exposure would seem to be a maximum acceptable effect
concentration for a screening study. A median lethal concentration
(LCso) should be determined in the laboratory for each species
analyzed for residues, after which a group of animals would be
exposed to an LC20 concentration to determine the mean threshold
concentration of residues. Since this approach is impractical for a
screening study, it is suggested that the mean residue concentration
in bobwhite and/or mallards exposed to an LC20 dietary
concentration would provide an indication of threshold levels.
(9) The number and timing of collection periods should be based
on the persistence of the specific chemical under study. Where
persistence in the field has not been adequately determined, it may
be necessary to sample at regular intervals (e.g., days 0, 1, 3, 7, 14,
28, 56) to provide data on degradation rates.
(E) Behavioral observations. Observations of behavior sometimes can
be an extremely important indicator of treatment effects. Such
observations might include characteristic signs of toxicity or behavioral
changes seen in test animals exposed to the chemical in the laboratory.
Page 19 of 46
-------
Other abnormal behavior (e.g., territorial males abruptly ceasing singing,
birds not feeding, reduced avoidance of humans) also may be important.
(F) Density and diversity estimates.
(1) It is necessary to know the number of individuals and variety of
species on and around a study site in order to indicate which
species could have been exposed and to aid in evaluating the
significance of mortalities or other findings. In addition,
preliminary information on density and diversity is necessary for
site selection and to determine the size of study sites. Under some
circumstances, comparisons of density estimates between
treatment and control sites, or between before and after treatments,
may be used to indicate chemical impacts. In general, the
usefulness of these comparisons is limited in a screening study due
to the relatively small acreage involved. If mortality occurs,
replacement from outside is likely to be so rapid that losses are
replaced before censuses are completed. In addition it is necessary
to consider seasonal changes, such as migration, molt, or
incubation, which can affect real or apparent densities.
(2) Several techniques may be used to estimate the density and
diversity of wildlife species, including counts of animal signs,
catch-per-unit effort, mark-recapture, and line-transect sampling.
Although the methods selected depend on the species of concern,
for the screening field test line transect methods are likely to be the
most useful for birds.
(3) The major advantage of line-transect sampling is that it is
relatively easy to use in the field once a proper sample of lines has
been chosen. However, line-transect-sampling is not applicable to
all species, particularly those that are not easily observed.
Individuals using line-transects must be extremely competent in
species identification.
(4) In the line transect method an observer walks a distance (L)
across an area in nonintersecting and non-overlapping lines,
counting the number of animals sighted and/or heard (N), and
recording one or more of the following statistics at the time of first
observation:
(a) Radial distance from observer to animal.
(b) Right-angle distance from the animal sighted or heard
to the path of the observer or angle of sighting from the
observer's path to the point at which the animal was first
sighted or heard.
Page 20 of 46
-------
(5) Although the field procedures are simple, they must be
understood adequately and implemented well to obtain good
estimates of density (see paragraph (h)(3) of this guideline). The
authors provide a thorough review of the theory and design of line-
transect-sampling and this monograph should be reviewed for
details.
(6) For mammals, density and diversity estimates from capture
data may be the most practical for a screening study. There are
several ways of estimating the populations from capture data, some
relatively simple, that may provide adequate information for a
screening study. Davis and Winstead (see paragraph (h)(7) of this
guideline), review the various methods available, explaining their
advantages and disadvantages.
(viii) Interpretation of results.
(A) The numerous variables involved in field studies makes a meaningful
discussion of the interpretation of results somewhat tenuous, particularly
with the almost inexhaustible array of results that could occur. Each study
must be considered unique and therefore will require a case-by-case
analysis that incorporates not only the actual study but other relevant
information that is available.
(B) In general, the results of the screening field study should provide
information on acute poisoning and potential sublethal effects as
suggested by enzyme, residue, or other measurements. In addition,
information has to be developed on the density and diversity of species on
the study sites as well as the sensitivity of the methods used. If no effects
are detected, assuming that the methods used were adequate to detect
levels of concern and that the species on the study site represent a good
cross-section of the nontarget species expected to be at risk, the potential
hazard indicated by lower tier tests is refuted. Unless other hazards (e.g.,
reproduction) are still of concern, additional tests would not normally be
necessary. However, if an effect is detected on one or more study sites at
rates equal to or greater than concern levels, the hazard has not been
refuted and additional tests may be necessary.
(C) In interpreting if an effect has occurred in the context of the binomial
approach, care must be employed not to assume a level of precision in
results that does not exist. Most detectable effects will exceed the concern
level for some methods used in these studies, due to the inherent
variability in the data collected to estimate the level of impact, particularly
when minimum sample sizes and areas are used. In some instances in
interpreting results it may be appropriate to use confidence limits of data
collected (or another measure of dispersion) to evaluate if concern levels
are exceeded. For example, when density estimates are used to estimate
percent mortality using the number of dead animals found during carcass
Page 21 of 46
-------
searching, the upper and lower confidence limits of the density estimate
may be more appropriate than the average, particularly when variability of
the density estimate is high. Whatever method is used, when effects are
detected that exceed concern levels they will be put into perspective in the
context of the entire study as well as other available information to
determine if or what additional data are needed. A "no-pass" result does
not necessarily mean that definitive field testing is automatically required.
(D) For example, a test may be run in an area where a species is abundant,
yet on a specific study site their numbers may be sufficiently small that a
single death exceeds the level of concern on that site. Statistically, such a
finding would indicate that the study did not pass according to the
binomial approach, and this would be the preliminary interpretation.
However, if the other sites had an adequate number of this species as well
as other species expected to be at risk and no other signs of impacts are
observed, the implications of the mortality would seem minor. On the
other hand, if diversity of species were extremely limited, it would have
greater significance. In other situations where the one dead bird is of a
species with small numbers on most sites, but density and diversity of
other species is representative of nontargets expected to be at risk, another
screening study that looks at the species in which the effect was detected
may be appropriate. Conversely, a screening study showing that there is
appreciable mortality on most study sites may be sufficient for the Agency
to consider regulatory action.
(E) In summary, the interpretation of results will go beyond the statistical
evaluation since the Agency must consider all the factors and
circumstances peculiar to each test and site. The biological interpretation
of results is, and probably always will be, a matter of scientific judgment
based upon the best available data. In general, the judgmental aspects of
biological interpretation are more important for definitive studies than for
screening studies. Nevertheless, biological considerations often will be
relevant to screening studies. Study conclusions must integrate that which
is biologically significant with that which is statistically significant.
(F) Another consideration in the interpretation of results of a field study is
the attribution of effects to the chemical being studied. A well-designed
study will include appropriate techniques to determine if an effect is
caused by a chemical. In the absence of such techniques, the Agency has
no choice but to consider that any effects were as a result of the chemical
applied. As an example, measurement of ChE levels can provide
information, since it is generally accepted that inhibition of 20% indicates
exposure and inhibition of 50% or more indicates, in birds, that mortality
is due to an inhibitor (see paragraph (h)(4) of this guideline). If the test
chemical is the only ChE inhibitor used in the vicinity of the study site, it
can be reasonably assumed that mortality associated with 60% ChE
inhibition is due to the test chemical. However, if other ChE inhibitors are
used near the site, additional information, such as residue measurements,
Page 22 of 46
-------
may be necessary to attribute death to the specific ChE inhibitor being
tested.
(5) Definitive study—
(i) Objective and scope.
(A) The definitive study is a relatively detailed investigation designed to
quantify the magnitude of impacts identified in a screening study or from
other information. In contrast to the screening study, which monitors
mainly the proportion of the local population that is expected to be
exposed, the definitive field study examines a sample of the entire local
population in the treated area. Although a definitive study may be
performed when laboratory studies indicate a high potential for field
mortality, it is more likely to be requested when there is evidence that
actual field mortality has occurred, as in a screening study, or where
reproductive effects are being investigated. The objectives of the
definitive study are:
(1) To quantify the magnitude of acute mortality caused by the
application.
(2) To determine the existence and extent of reproductive
impairment in nontarget species from the application.
(3) To determine the extent to which survival is influenced.
(B) Due to the intense effort and time required to estimate these
parameters, the definitive study should be limited to one or a few species
believed to be at the highest risk. If it can be shown that minimal (as
defined at the onset of the study) or no changes occur in study parameters
to high risk species, there is likely to be minimal potential for adversely
affecting other presumably low risk species from use of the chemical in
question.
(C) The definitive study, in addition to estimating the magnitude of effects
of acute toxicants, also can be applied to estimating the magnitude of
chronic or reproductive effects. Although the discussion has emphasized
chemicals that are acutely toxic, with few exceptions it is applicable to
chemicals that cause chronic effects.
(D) In general, the definitive study will provide limited insight into
whether or not effects are within the limits of compensation for the species
of concern. However, using the data collected in these studies coupled
with available information on the species of concern and basic theories of
population dynamics, the meaning of the observed effects on the species
can be evaluated.
Page 23 of 46
-------
(ii) Sampling and experimental design.
(A) The principles of statistical design of studies are well documented and
it is beyond the scope of this guideline to cover the fundamentals of this
topic. However, there are a few points on this topic that warrant
discussion relative to the definitive study.
(B) In the design of field studies, it is necessary to consider carefully what
constitutes a sampling unit. Eberhardt, under paragraph (h)(10) of this
guideline, points out that special problems are faced in designing
experiments on wild animal populations. Study sites must be large in
order to limit the influence of boundary effects, such as movements into
and out of the area. Large study sites can be very expensive both in terms
of actually applying the experimental treatment and in the assessment of
results. Eberhardt also states that numerous observations, even a full year
of data, on a single study site may result in very sound values for that site,
but do not provide a basis for inferences to other sites. Hurlbert (see
paragraph (h)(19) of this guideline) has discussed the problems associated
with field studies where there was no replication or replicates were not
statistically independent, which he terms pseudoreplication. Of the field
studies he evaluated, 48% of those applying inferential statistics had
pseudoreplication.
(C) According to Eberhardt, under paragraph (h)(9) of this guideline, lack
of replication seems to be based on the mistaken assumption that variances
based on subsampling of sites (intrasite variability) are suitable bases for
comparing treatment effects (intersite variability). This, he believes, is not
a valid basis for a statistical test, because it is the variance of sites that are
treated alike that is relevant to a test of treatment differences. Although
subsampling of sites may be necessary to collect the data, it is the
difference between sites that is important for analysis.
(D) An important point to consider in designing a definitive study is to be
sure that the study will detect a substantial impact when, in fact, it occurs.
In statistical terms this concept is referred to as the power of the test (1- P).
Experience with classical experimental designs with random assignment
of experimental treatment and controls, has shown that the probability of a
Type II error (P, false acceptance error rate) is generally high (unless very
large numbers of replicates are available). Eberhardt, under paragraph
(h)(9) of this guideline, indicates that, all too often in field studies on
impacts to wildlife, either by default or lack of understanding, there is only
a 50% chance of detecting an effect, which he likens to settling the issue
by flipping a coin and doing no field study whatsoever. Since a definitive
study is carried out under the assumption that effects will occur, the
Agency believes minimizing Type II errors is extremely important.
(E) As suggested, the more generally used experimental designs require
inordinately large sample sizes to obtain small Type II errors. For
Page 24 of 46
-------
example, based on a coefficient of variation (cv) of 50% (this is a
relatively homogeneous sample for the kinds of data collected in field
studies), a 20% minimum detectable difference between means, a Type II
error (P) of 0.2 and a Type I error (a) of 0.05, the number of replications
required can be estimated using the formula in Equation 10 (in Eberhardt
(see paragraph (h)(8) of this guideline)).
Equation 10
where:
n = number of replications;
zp = pth percentile of the unit normal distribution (z\.a and zi_p are
critical values of the unit normal distribution);
cv = coefficient of variation;
5 = detectable mean difference expressed as the proportion of the
control group mean (i.e., 5 = (|ii - |i2)/(m)).
(F) Thus, for the example in paragraph (e)(5)(ii)(E), n = 63.6 replicates
which rounds to 64 replicates for both control and treatment groups, or
128 total study plots, are required to detect a 20% difference between
treatments and controls with an 80% chance of being sure to detect a real
difference (zi_o.2o = 0.84) at a 0.05 level of significance (zi_o.o5 = 1.645).
.65 . ,. ,. _ ,„ Equation 11
(G) With more sophisticated designs, the number of replicates can be
reduced under some circumstances and still meet the Agency's aim to limit
the probability of a Type II error to 0.2 with a detectable difference of 20
to 25%. For example, a paired plot design can be used, substantially
reducing the number of replicates required. Pairing serves to reduce the
effective coefficient of variation by reducing the variation attributable to
experimental error. The lower coefficient of variation reduces the number
of replicates. A quantitative difference or, preferably, a ratio of treated to
the total of treated and control responses, can be analyzed statistically to
test for a treatment effect on the measured response variables (see
paragraph (h)(31) of this guideline). The logic of using paired plots is
that, while no two areas are ever exactly alike, two areas that are not
widely separated in space are ordinarily subjected to much the same
climatic factors, have populations with about the same genetic makeup,
and generally the two populations can be expected to follow much the
same trend over time, apart from a chemical effect (see paragraph (h)(8) of
this guideline). If all plots are approximately equal in area and habitat and
Page 25 of 46
-------
population densities between pairs are similar, it is postulated that when
no chemical impacts occur, the mean ratio of treatment to treatment plus
control will equal one-half. Then a t-test or an exact randomization test
(see paragraph (h)(l 1) of this guideline) may be applied to test whether the
average number of survivors on the treated plots is equal to the average
number of survivors on controls.
(H) The number of pairs required can be estimated using the formula in
Equation 12.
n = (zi-a + zi-p ) r-7; - \ Equation 1 2
where,
n = number of paired plots;
zp = pth percentile of the unit normal distribution (zi_a and zi_p are
critical z-scores);
q = survival ratio;
p = mortality ratio; and
c = mean number of survivors on control plots.
(I) Thus, 10 pairs of plots (20 total) with a mean of 28 individuals per plot
would be needed at an 80% assurance of detecting a treatment-induced
impact of 20% or greater at a 0.05 level of significance if c = 28.
Increasing the mean number of individuals per plot (c) causes a reduction
in n.
n = (1.645 + 0.84)2 7 — ,* ' ., . = 9.8 Equation 13
2
(J) In some field situations, pairing may not be feasible. In these
situations, other designs would be more appropriate or a less rigorous
design may have to be used. However, in planning field studies, the
power of the study design must be considered to determine the limitations
of the study. Studies with adequate replication are highly preferred to
support registration — the use of less replication will not necessarily render
the study inadequate. However, it is wrong to use a study with low power
to imply no biological damage, when the study is not capable of detecting
the damage if it occurred. In cases where large numbers of replicates are
impractical, subjective and biological knowledge should be used in a
decision process to decide if there was a treatment effect. In most
instances, it is highly advisable to involve statisticians or biometricians
Page 26 of 46
-------
who are familiar with this kind of field study in the planning and analysis
phase of the field work to avoid costly technical errors.
(iii) Study area and site selection. Selection of geographical areas and study
sites within the areas for the definitive test generally requires the same
considerations as for a screening study. For the definitive study, however, the
selected areas and study sites should have adequate populations of the species of
concern. For pesticides, the crop of concern should be grown on a representative
portion of the area and consideration needs to be given to whether the target pest
species will be present. If it is not, it is necessary to consider what influence its
absence may have on potential results. For example, if the pest is a major food
source for nontarget species, its absence could significantly influence results.
Finally, the potential variation in populations of concern over the geographical
areas selected should be considered. It may be difficult to find sites that are
sufficiently similar to provide paired plots, which limits the coefficient of
variation so that the desired sensitivity can be achieved.
(iv) Number and size of sites. As suggested under paragraph (e)(5)(ii) of this
guideline, the number of sites will depend upon the species density on sites and
the sensitivity required. Ideally, sample size should be large enough so there will
be an 80% probability of being sure to detect a 20% difference when it exists.
The size of the study site must be large enough to provide adequate samples. The
size depends on the survey methods used, sensitivity required, and the density and
range of the species of concern. For a paired-plot design, the number of sites
required is a function of the average density of the species. In general, the
breeding density of the species of concern can be used to provide a rough estimate
of the size of area needed to provide adequate samples. However, preliminary
sampling most likely will be required to verify the estimates.
(v) Methods.
(A) Essentially, the methods used in a definitive study are a means to
quantitate reproductive and mortality rates of animals on treatment and
control areas. There are many texts and monographs available on methods
of sampling to estimate these parameters. Anyone not familiar with the
theory and principles of the various techniques should review these
references in depth. The objective of this portion of the guideline is to
provide a general guide to the various methods that could be used in a
definitive field study. In addition, these methods can be applicable to some
screening studies.
(B) The methods to be used in an individual field study will depend on the
nature of the identified concerns. Some methods are useful for
investigating several types of concerns, and most types of concerns can be
studied by several methods. When the concern becomes more specific
(e.g., secondary hazards to raptors), the use pattern or pattern of
production, use, disposal, or accidental release is limited, and/or habitat
type is limited, the range of applicable methods tends to become narrower.
Page 27 of 46
-------
(C) Methods described below are divided into three categories: Methods
for assessing mortality and survival of adults and independent juveniles,
methods for assessing reproduction and survival of dependent juveniles,
and ancillary methods. The intent of this guideline is to present methods
that are likely to be useful in many situations, rather than an exhaustive list
of all available methods. The Agency encourages the use of other
methods when they are scientifically valid and have a high probability of
detecting an effect.
(D) While it is absolutely essential to have a detailed plan that describes
the selected actions (with contingencies) for achieving the study
objectives, investigators must remain flexible because unanticipated
problems always come up in long term studies. Even with highly
experienced and resourceful field biologists, the most carefully planned
studies can be compromised due to the unpredictability of wild animals
and natural events. When a natural disaster occurs early in the study, it
may be wise to reinitiate the study. If the event occurs after substantial
data already have been collected (e.g., early in the second year of a
multiyear study), it may be more appropriate to extend the study an
additional year or more to help provide for the additional needs. If the
study is to be terminated, the report should describe thoroughly the nature
of the events and the consequences if they affect the study results.
(vi) Mortality and survival. It is very important to understand the autecology of
the species being studied in order to select the most appropriate methods for
investigating them. In addition, the choice of particular methods must consider
the applicability of the method based on the chemical use pattern and study site
characteristics.
(A) Mark-Recapture.
(1) There are several mark-recapture methods available, each
based on the same basic premise. A sample of animals is captured,
marked, released, and another sample is collected where some of
the animals are captured again. The characteristics of this
identifiable sample are used to estimate population parameters.
Mark-recapture studies can provide information on:
(a) Size of the population.
(b) Age-specific fecundity rates.
(c) Age-specific mortality rates.
(d) Combined rates of birth and immigration.
(e) Combined rates of death and emigration.
Page 28 of 46
-------
(2) Seber (see paragraph (h)(32) of this guideline) reviewed the
various mark-recapture methods and subsequent statistical
analyses. Less detailed but still very useful reviews are provided
in paragraphs (h)(4) and (h)(15) of this guideline. Nichols and
Pollock provide a valuable comparison of methods under
paragraph (h)(26) of this guideline. Table 2 provides a brief
summary of some of the various mark-recapture methods discussed
in these references.
Table 2.—Mark-Recapture Techniques
Method
Peterson Method (Lincoln Index)
Schumacher's Method
Bailey's Triple Catch
Jolly-Seber Method
Applications/Requirements/Assumptions
Estimation of population size. Usually only two sampling periods.
Closed population.
Estimation of population size. More than two sampling periods.
Marking continues throughout sampling. Closed population.
Estimate of birth rate and death rate in addition to population size.
Requires data from two marking occasions and two recapturing
occasions. Open population.
Estimates mortality and recruitment in addition to population size.
Requires more than two sampling periods and that each animal's
history of recapture be known. Open population.
(3) When considering the use of one of these mark-recapture
models, one must carefully evaluate the applicability of the method
to the circumstances under consideration. While in theory mark-
recapture techniques should be an excellent method for evaluating
effects of chemicals on wildlife populations, some mark-recapture
analyses are not particularly robust; small deviations from their
implicit assumptions can produce large errors in the results (see
paragraph (h)(4) of this guideline). However, some of the more
recent and sophisticated analytical methods are robust and can deal
with deviations from assumptions in closed populations (see
paragraph (h)(28) of this guideline).
(4) Mark-recapture methods are particularly useful for small
mammals because these animals are seldom amenable to the visual
and auditory observations necessary for using transect, territory
mapping, or similar methods. However, mark-recapture also may
be useful for birds provided a sufficient number of birds can be
captured and marked. In some situations, birds may be
"recaptured" with use of binoculars via visual observations of
marked individuals. Animals must remain marked for the duration
of the study. Typically, mammals are toe-clipped or ear-marked
and birds are banded. Marking should not make the animals more
susceptible to the effects of the chemical (e.g., anticoagulants with
toe clipping). Dyes may be useful unless they are lost by wear or
molting.
Page 29 of 46
-------
(B) Territory mapping method.
(1) A common spatial census method is territory mapping, wherein
the territories of individuals are mapped before and after treatment,
on both treated and untreated plots. The method is usually
applicable when birds are defending territories. It involves a series
of census visits to the study sites during which birds located by
sight or song are recorded on a map. The information from all the
visits is plotted for each species. Birds exhibiting territorial
behavior appear on the map as clusters of individual contacts. The
clusters are used to estimate both the size and number of territories.
The pre- and post-treatment censuses for treated sites are compared
with the pre- and post-treatment censuses for control sites to
determine changes in populations of territorial individuals that may
be attributed to the chemical (see paragraph (h)(12) of this
guideline). Further details of this method are given by the
International Bird Census Committee under paragraph (h)(21) of
this guideline, and its application to evaluating impact caused by
pesticides is reviewed by Edwards et al. (see paragraph (h)(12) of
this guideline).
(2) Problems with this method can occur. Under some
circumstances, replacement from outside the area can be so rapid
that territories are refilled before the census is completed. There
usually is a floating population of silent, nonterritorial birds who
may quickly reoccupy empty territories (see paragraph (h)(35) of
this guideline). The effects of replacement can be overcome for
some species by capturing and marking the territorial individuals
prior to treatment, so they can be distinguished from the floaters.
Also, replacement may not be a problem when the study areas are
in the center of a relatively large treated area.
(C) Radio telemetry. Radio telemetry can be an extremely useful
technique to provide information on the effects of a chemical application
on nontarget species. As discussed for screening studies, radio telemetry
can be used to monitor for mortality as well as to provide useful
information on behavioral modification caused by the chemical
application. The points discussed previously (for screening studies)
generally are applicable to definitive studies. However, for the definitive
study, the number of radio-tagged animals needed depends upon the
variation between sites and the sensitivity required. For example, with
behavioral observation, intra- and intersite variation will influence the
number of radio-tagged animals required. In some instances, it might not
be practical to radio-tag the number of animals required to provide a
rigorously designed study. Under these conditions, the limitations should
be specified, and the maximum number of animals that can be practically
radio-tagged and monitored should be used.
Page 30 of 46
-------
(D) Other methods for mortality and survival. Other techniques for
assessing density and diversity are discussed for screening studies; most of
these, especially line-transect methods, are useful for definitive studies.
Some methods, such as catch per unit effort or counts of animal signs, do
not provide actual measures of density but may still be used to compare
effects on treated and untreated plots.
(vii) Reproduction and survival of dependent young. Some of the techniques
for assessing mortality and adult survival are also useful for assessing
reproduction and survival of young. Some, but not all, mark-recapture methods
can provide information on fecundity. Radio-tagging nestlings or suckling young
of moderate and large size animals may be used to assess survival of dependent
young. Radio telemetry and territory mapping are useful for locating dens or
nests for further study. The following methods are more specific for assessing
reproductive parameters.
(A) Nest monitoring.
(1) Nest monitoring is useful for evaluating the effect of chemicals
on breeding birds. The typical procedure is to search the study site
to find active nests and subsequently to check those nests to
determine their fate. Information collected on each nest should
include number of eggs laid, number hatched, number of young
fledged, and if and when the nest was abandoned or destroyed,
both before and after chemical application. While all definitive
studies should consider this technique, it also may be useful in
screening studies.
(2) This technique is relatively straightforward. However, it may
not be practical if nests are scarce or otherwise hard to find.
Because the breeding success of birds can be highly variable and
can be quite low, it is sometimes difficult to obtain sufficient data
on the success of the same species in enough sites to yield
satisfactory results for statistical comparison with controls (see
paragraph (h)(18) of this guideline). In some cases, artificial nest
structures can be constructed to increase nest densities. In a few
situations where sufficient numbers are available, the technique
may be applicable to mammal den monitoring.
(B) Behavioral observations. Behavioral observations associated with
reproduction can be quite useful, especially for birds. Techniques are
simple, but labor intensive. When used, such observations most likely
would be combined with nest monitoring since both techniques require
locating reproductive sites. Typically, the frequency and duration of
behaviors will be compared for treated and untreated plots. Incubation,
parental care (especially feeding for altricial birds), and following
behavior (for precocial animals) are behaviors that are particularly
amenable to such study. Courtship, mating, and nest building are other
Page 31 of 46
-------
behaviors that could be studied in some situations, but locating sufficient
numbers of animals displaying these behaviors to permit quantitative
analysis is difficult.
(C) Age structure of populations.
(1) Comparisons of young to adult ratios of selected species
between treated and untreated plots may indicate reproductive
effects. The timing of the application and of breeding of selected
species is critical. For assessing reproductive impairment or
survival of dependent young, per se, the duration of this technique
should be limited to single breeding and rearing periods, which
may be repeatedly assessed. However, longer study periods that
may even include several years can be used to assess the
combination of reproductive success and age-specific mortality,
even if the two cannot be separated.
(2) Obviously, use of this method requires that the age of
individual animals be determined. In some cases, it may be
necessary only to distinguish among adults, subadults, and
juveniles. In mammals, this may usually be accomplished by
examining pelage, development of testes or mammaries, or tooth
eruption or wear characteristics. In birds, plumage or
characteristics of particular (species-dependent) feathers may be
used. For carcasses or sacrificed animals, observation of the
ossification of bones or development of reproductive organs are
useful. In other cases, particularly where comparisons are made
among populations in different years, it may be appropriate to
distinguish age classes of adults. In mammals, tooth eruption,
wear, or enamel layers, or eye lens weights are useful. It is more
difficult to separate age classes of many adult birds, although
overall plumage or feather characteristics can provide some
indication. In some slow-maturing birds (e.g., gulls), plumage may
be used to distinguish year classes of sub-adults. Additional
details on aging birds and mammals are presented by Larson and
Taber (see paragraph (h)(22) of this guideline).
(viii) Ancillary methods.
(A) At least some ancillary methods are essential in every field study. As
used here, ancillary methods are generally of two types. Certain of these
methods are important for determining the nature or existence of effects or
for establishing causal relationships. Others of these methods do not
address effects directly, but they provide important information for
interpreting the results of the study.
(B) Many of the methods for determining effects have been discussed for
screening studies. Enzyme analysis, such as for ChE inhibition, and
observations of signs of toxicity can show that animals were exposed to or
Page 32 of 46
-------
killed by a toxicant of a particular type. Where it is possible that animals
may be exposed to other chemicals of the same type (e.g., feeding in a
nearby area treated with pesticides), residue analysis in nontarget animals
may be necessary to determine which specific chemical caused the signs
or alterations in enzymes. Even though carcass searches, per se, are not
recommended for definitive studies, it is still essential to recover and
analyze any carcasses found accidentally or obtained through radio-
tracking. Residue and/or enzyme analysis of live animals collected will
frequently be important.
(C) Among the other ancillary methods, analysis of environmental
residues is crucial and will probably be necessary in nearly every
definitive field study. As discussed for screening studies, the most
important environmental residues are those that occur on or in wildlife
food sources, which may include insects, plant parts, or even other
vertebrates, depending upon the species that are the primary focus of the
investigation. The investigator should review the literature on food habits
of the species being studied; often it will be appropriate to assess food
habits on the specific study sites, particularly where the literature is not
adequate to define food habits in the agricultural ecosystem under study.
Such an assessment should include the availability of food sources and the
number of mobile animals that spend only part of the time in and adjacent
to treated sites. The habitat should be thoroughly described to include
both the morphology and species that are relevant to wildlife. Frequently,
it will be important to locate and describe roosting, denning, or nesting
sites for mobile wildlife that use treated sites part of the time.
(ix) Interpretation of results.
(A) While each field study is unique, some elements may be common
among many field studies. When a definitive field study is required, the
requirement is based on one or more specific concerns that pertain to a
specific chemical and one or several use patterns. Because of the
substantial diversity in the types of problems to be assessed and the variety
of available investigative methods, the key to understanding and
interpreting a field study lies in the development of a sound protocol. All
protocols will contain a description of the study sites, or the characteristics
to be used in selecting sites within a given area, and the methods to be
used in conducting the study. However, a well designed protocol will go
beyond this descriptive approach in three ways.
(1) First, the well-designed protocol will contain a restatement of
the concerns to be addressed to ensure that there is an adequate
understanding of the Agency's position. The investigator should
review the literature and other available information that may bear
upon the problem. It is possible that the literature may contain a
valid answer to the questions raised by the Agency. Far more
likely, the literature may orient the investigator to address the
Page 33 of 46
-------
concerns in a particular way. An example is provided by Hegdal
and Blaskiewicz (under paragraph (h)(16) of this guideline), who
conducted a study to address the Agency's concerns for secondary
toxicity to barn owls (specifically) from the use of an anticoagulant
bait proposed for use on commensal rodents in and around
agricultural buildings. A review of the literature by these
investigators indicated to them that laboratory studies suggested a
legitimate potential for secondary poisoning to exposed raptors,
but that the food habits of barn owls consist primarily of microtine
rodents in most areas, suggesting a low potential for actual
exposure. Consequently, they designed their study to focus on
barn owl food habits and movements, and included an additive to
the bait formulation that would permit an identification of whether
or not the barn owls ate rodents that had fed on the bait. The study
adequately demonstrated that actual exposure of barn owls was
quite limited, and the proposed registration for this use was
subsequently approved. By using the available literature on both
the chemical and the particular species of concern, the
investigators were able to narrow the study while still providing
sufficient information for evaluation. However, it should be noted
that this study was not adequate for evaluating the potential for
secondary toxicity in the field to other predators that may have
different food habits, or for other use patterns that may result in
exposure to different predators or scavengers.
(2) Second, the well designed protocol will provide the reasons
why particular methods are being used, including, at least
qualitatively, the meaning that different results might have. For
example, a protocol may include collection of residues in nontarget
animals, but it also should include a statement of purpose and
meaning for such collection. Residues may be used to indicate
potential exposure to nontarget organisms through analysis of their
food, exposure in nontarget animals as a result of eating
contaminated food, or that a particular chemical was likely to be
the cause of any observed effects. Interpretation of data is
facilitated substantially by a statement of what results were
intended by using a particular technique. In the previously cited
example (see paragraph (h)(16) of this guideline), it was clearly
stated that collection of owl pellets was used to assess general food
habits and that use of a fluorescing dye in the bait was for the
purpose of ascertaining whether or not the owls fed on commensal
rodents that specifically had fed on the bait. The interpretation of
the data collected, once the purpose was stated, naturally led to the
conclusion of no-significant-exposure to the barn owls.
(3) Third, the well designed protocol will contain an experimental
design that will indicate how the results can be assessed
quantitatively. The experimental design has been discussed in
Page 34 of 46
-------
previous sections of this guideline, but there are two facets that
relate closely to the interpretation of results: The difference that
can be detected between treated and untreated plots and the power
(ability) of the design to detect this difference. An experimental
design with number of replicates based on an estimated coefficient
of variation that closely approximates reality will allow the study
to detect a stated concern level some prescribed number of times
during the study time. The actual difference between treated and
control units is measured during the field study, but the design can
form an initial basis for interpretation when combined with the
available information on the species of concern. As a result, the
well-designed protocol should include a section on interpretation.
(B) Study methods for investigating acute mortality are more
straightforward than for other kinds of effects. Nevertheless, there are
sufficient differences in the use of the data to preclude a constant
interpretation. The study may focus directly on the species of concern and
may involve little or no extrapolation, depending on such factors as the
type and the extent of use, the available toxicity data base, and home range
of the species. Extrapolation to other populations, regions, or uses might
be necessary. If the species of concern cannot be studied directly, it may
be necessary to extrapolate between species, involving interspecies
differences both in toxicological sensitivity and in ecological and
population parameters.
(C) The same kinds of considerations apply to reproductive impairment
and chronic toxicity, even though different, and often more laborious and
costly, investigative methods are involved. Where reproductive success is
impaired, information on species-specific variation in reproductive
ecology is necessary to understand how a particular degree of impairment
may relate to effects among various species. Such reproductive
considerations can include whether an avian species is a determinate or
indeterminate layer, the number of nestings per season for different
geographic areas in the use pattern, the length of the refractory period, as
well as the specific effect which can range from destruction of
reproductive organs to behavioral deficits such as nest abandonment.
Considerations of reproductive ecology among different species of
mammals include delayed fertilization or implantation, resorption of
embryos or parental infanticide due to stress, number of young per
breeding cycle, etc. All of these factors, and many others, are relevant to
determining for different species the extent of effects that could result in
population reductions or lack of ability to recover.
(D) An analysis of whether or not a particular level of effect is going to
affect wildlife populations is species-specific. For any species (or
subspecies), the changes in population can be described very simplistically
by the equation: rate of population increase (r) = birth rate minus death
rate, where values of r can be positive (population growth) or negative
Page 35 of 46
-------
(population reduction). Immigration and emigration are also important
when the concern is for specific populations of a species. These
characteristics differ among species, and data will not always be available.
The application of sound scientific judgment to the best available
information will be the basis for interpreting the results of a study. It may
be necessary to compare the results of the field study to laboratory data,
especially where laboratory data are available on a variety of species
and/or effects and the field study has focused on species other than those
of direct concern. The use of extrapolation techniques will he necessary
where endangered species are of concern or where other species cannot be
studied directly.
(E) The Agency would like to be able to obtain a standardized result from
a field study so that the result could be applied in a very consistent
manner. As discussed in previous sections of this guideline, the different
effects and species of concern will vary and will require the development
of specific protocols to address these factors. Although most of the
various techniques have some degree of standardization, the field study
may combine the individual techniques in a wide variety of ways to
address specific concerns. A standardized result might be attainable for
the individual techniques, although that result would still have to be
applied differently for various species, depending on their biology and
ecological characteristics. However, determining a result for the whole
field study that would unequivocally lead to a statement of the degree of
risk, while obviously desirable, is not currently practical.
(6) Carcass searches—
(i) Design.
(A) In designing carcass searches, the following factors need to be known
or determined:
(1) Density of the species that are likely to be exposed. For
example, granular products are most likely to result in exposure to
ground-feeding animals; therefore, birds such as warblers or
swallows should not be included in density counts for such
products.
(2) Probability of finding dead animals if any are killed. This is
dependent on the probability of a carcass remaining on the study
site (i.e., not being removed by scavengers) and the probability of
detecting a carcass if it remains on the study site (search
efficiency).
(3) Size of the search area.
(4) Number of carcasses found..
Page 36 of 46
-------
(B) These factors can be combined in the following formula:
n = dxrxexaxp Equation 14
where:
n = number of carcasses found;
d = density in animals per acre;
r = proportion of carcasses remaining (nonremoval);
e = search efficiency;
a = acres searched; and
p = proportion of population killed.
(C) Carcass searches should be used only when there is a reasonable
potential to detect mortality. If such mortality does occur, the carcass
search should be able to detect it and therefore, carcasses should be found.
It is recommended that carcass searches be designed so that at least two
carcasses (n = 2) will be found if there is appreciable mortality. In
general, preliminary sampling would be required to determine these
factors. However, information from other field studies can be used in the
planning stages to determine if carcass searching would be appropriate for
use under anticipated conditions and to assist in developing the study
design.
(D) The sensitivity of the carcass search approach is equivalent to the
percent detectable kill of the population. To determine the sensitivity,
Equation 14 is rearranged to solve for p, the proportion of population
killed, as shown in Equation 15. Since p is a proportion multiplying by
100 (Equation 16) provides the percentage of the population killed.
p=n/ \ Equation 15
^ /(dxrxexa) ^
percent detectable kill = p x 100 = f n/, 0(lOO) Equation 16
^ ^ [^/(dxrxexa)j^ ' ^
(E) If any of the values of d, r, e, or a are zero, the equation cannot be
solved and the carcass search is not applicable (i.e.., no density of birds, no
acres searched, no carcasses remaining, no remaining carcasses found).
However, other combinations of d, r, e and a, such as low density and
small acreage or low efficiency and high scavenger removal, can result in
a small denominator meaning that mortality can be detected only when a
high percentage of the population is killed. For example, in 5 acre fields
with only two birds per acre and r and e estimated at a moderate 0.5, only
Page 37 of 46
-------
an 80% or greater kill could be detected. In such situations, it is necessary
to increase one of the parameters to achieve a stated level of detectability
or else to use methods other than carcass searching. The same equation
can be used to estimate the minimum search area to detect a given
mortality level (p) by solving for a.
(ii) Search procedure. In general, depending on the sensitivity of the search
method relative to the habitat involved, corridors or plots should be selected.
These areas should be searched systematically by walking predetermined routes
until the area has all been covered. Due to the concentration required to find dead
animals, other activities that could distract the attention of the searchers should be
avoided during carcass searching. In homogeneous situations, investigators
should randomly select search areas. However, in most studies it is advisable to
stratify the sampling, concentrating efforts in areas frequented by wildlife species
such as woods edges, ditch banks, field borders, fencerows, and other habitats
where wildlife concentrate.
(iii) Duration. Searches should begin on the day of application and continue on a
daily basis for as long as mortalities or other evidence of intoxication occur. In
general, a week or two following application should be adequate. However, the
length of time searches are continued should be related to how long lethal
concentrations are expected to be present. Normally, the same areas should be
searched each day.
(iv) Estimating efficiency of carcass search.
(A) Efficiency trials should be conducted periodically (minimum 3 times
per study site) during the study to determine the proportion of carcasses
that are detected. Just prior to the initiation of a scheduled search,
carcasses of animals representative of species found in the area should be
variously placed within the search area. If the study site includes edge
habitat, carcasses should be placed in the edges as well as in the fields. In
general, carcasses should be placed where animals would be most likely to
die, depending on the nature of the chemical. Searchers should not be
aware that simulated mortalities have been placed; however, they should
be aware that these trials will occur during any scheduled search.
(B) The number of carcasses placed should be approximately equal to
20% of the estimated density of species on the search area. All placed
carcasses should be marked to distinguish them from actual kills. The
location of placed carcasses should be mapped so those not found can be
easily recovered following completion of that day's search activities, since
unrecovered carcasses could bias study results. For example, if a
scavenger were to carry off a simulated kill and consume it at another
location on the study site, the remains could be erroneously classified as
chemical-related if found. One way to avoid this problem would be to dip
carcasses in a nontoxic substance that fluoresces under ultraviolet light so
that the remains could be identified.
Page 38 of 46
-------
(v) Estimating carcass removal rate.
(A) Carcass removal should be monitored to determine local variability in
scavenger activity. The density of both carcasses and scavengers can
influence the rate of removal. Under some conditions, large numbers of
carcasses may attract scavengers. In other situations a large number of
kills may dilute removal rate due to limited number of scavengers. Where
it can be adequately documented that removal of carcasses occurs almost
exclusively either at night or during the day, the timing of carcass searches
may be adjusted to minimize the effects of removal.
(B) Carcasses planted in monitoring trials should simulate mortalities
actually occurring from the chemical. In most cases, small to moderate
sized species such as starlings or blackbirds, or laboratory bobwhite or
Japanese quail chicks may be used. Carcasses should be variously placed
within the general study areas and monitored daily for at least 5 days or
until 90% have been removed. The number used should approximate
densities resulting from effects of the chemical under study; however, in
most instances, this will not be known. Therefore, a density of
approximately 20% of the population of nontarget species on the area is
recommended.
(C) Timing of carcass removal trials should be such that they do not affect
scavenger removal of chemical-killed birds or the feather-spots of the
removed carcass could be erroneously classified as a chemical kill.
Location of placed birds should be recorded on maps and may be marked
in the field with small stakes or by other inconspicuous means, preferably
at a fixed distance and direction from the carcass.
(f) Suggested components of a field study protocol. The following protocol was adapted from
the Wildlife Managements Techniques Manual (under paragraph (h)(29) of this guideline), and is
recommended for studies submitted to the Agency for review.
(1) Title
(2) Problem definition. The following information should be provided:
(i) A review and summary of the available information on the chemical in relation
to nontarget hazard, including use information.
(ii) A precise statement of the goals and purpose of the study.
(iii) A brief statement of the problem and the context in which it exists, specifying
the limits of the proposed work.
(iv) Precise statements of the major hypotheses to be tested.
Page 39 of 46
-------
(3) Methods and materials. This section should include the following:
(i) A brief discussion of various methods and procedures that have been or could
be used to evaluate the problem. This discussion should identify the strengths and
weaknesses of each method or procedure discussed.
(ii) Description of the procedure to be followed:
(A) Identify the study areas selected and their general suitability for
achieving the objectives of the study or what criteria will be used to select
study areas.
(B) Identify the species present or expected to be present on the study
areas, discussing characteristics pertinent to the problem being evaluated.
(C) State the research procedures, designs and sampling plans to be used:
(1) Specify the kind and amount of data needed and to be sought.
(2) Describe in detail how all the data are to be obtained, including
details of application, instrumentation, equipment, sampling
procedures, and any other information.
(D) Describe how the data are to be treated, including specifying what
statistics are to be calculated, what models will be used, what tests of data
will be used, etc.
(E) Describe in detail the methods to be used to check the sensitivity and
accuracy of the procedures used.
(F) Describe quality assurance procedures for application, instrumentation,
equipment, and records.
(G) Briefly describe the resources (people, facilities, etc) to be applied to
the study.
(g) Reporting. In addition to the reporting provisions on background information, study
protocol deviations, and test substance in paragraphs (g)(l), (g)(2), and (g)(3) of this guideline,
respectively, the test report should include, but not necessarily be limited to the information in
paragraphs (g)(4), (g)(5), and (g)(6) of this guideline on test species, test methods and conditions,
and results, respectively.
(1) Background information. Background information to be supplied in the report
consists at a minimum of those background information items listed in paragraph (j)0) of
the OCSPP 850.2000 guideline.
(2) Study protocol deviations. Provide a copy of the final field study protocol used.
Include a description of any deviations from the study protocol originally submitted and
agreed upon with the Agency or any occurrences which may have influenced the results
Page 40 of 46
-------
of the test, the reason for these changes, and any resulting effects on test endpoints noted
and discussed.
(3) Test substance.
(i) Identification of the test substance: common name, IUPAC and CAS names,
CAS number, structural formula, source, lot or batch number, chemical state or
form of the test substance, and its purity (i.e. for pesticides, the identity and
concentration of active ingredient(s)), radiolabeling if any, location of label(s),
and radiopurity.
(ii) Storage conditions of the test chemical or test substance and stability of the
test chemical or test substance under storage conditions if stored prior to use.
(iii) Methods of preparation of the test substance for application, the application
rate(s), for pesticides the maximum label rate.
(iv) For residue analysis in wildlife, vegetation, soil, water, sediments, and other
appropriate environmental components describe the stability of the test substance
under storage conditions.
(v) Data on storage of biological and environmental samples.
(4) Site of the test.
(i) A description of the field study area(s) and sites, including the size and
characteristics of all components over time, as well as prevailing meteorological
conditions.
(ii) History of the site in terms of factors that may influence the study objectives.
(iii) Map or diagram showing location of treated sites and controls and showing
locations of observations and searches.
(iv) Climatological data during the field study: records of applicable conditions
for the type of site, i.e., temperature, thermoperiod, rainfall or watering regime,
light regime including intensity and quality, photoperiod, relative humidity, wind
speed, etc.
(v) Substrate characteristics of the study area and treated sites.
(vi) Characteristics of the flora and fauna of the study area and test sites.
(5) Study species.
(i) Identify the wildlife species present in the study area(s), and habitat.
(ii) For study species the study design objectives and protocols, and the scale (i.e.,
all species, cross-section, selected species).
Page 41 of 46
-------
(ii) Characteristics of species (e.g., age, stage of development, health status)
pertinent to the problem being evaluated.
(A) Number and type of species investigated and the scale of identification
(e.g., a single species of concern, all species of a community or a selected
cross-section).
(B) Scientific and common name.
(C) Stage of development and condition of study species and other
wildlife at test initiation.
(6) Study conditions and experimental design. Description of the study conditions and
experimental design used in the screening or definitive tests, and any preliminary testing.
(i) A statement of the concerns to be addressed and the type and frequency of
monitoring.
(A) The methods of evaluating effects on terrestrial wildlife (e.g., radio
telemetry, carcass searches, ChE inhibition). The report results should
include:
(1) Observed behavioral, biochemical, and ecological effects, such
as measures of mortality and survival, reproduction, population
density, and enzyme inhibition.
(2) Residue concentrations of the test substance (and degradation
products, if evaluated) in-wildlife and wildlife food sources over
time.
(B) Statement of the data objectives for specific measures (i.e., the critical
or threshold level for an effect, precision of a point estimate).
(ii) The field study design: size of field sites, number of control sites, the number
of experimental treatment levels and the number of experimental sites (replicates)
for each treatment, the lay-out and distance of field sites to each other and to
control sites.
(iii) Methods used for treatment randomization.
(iv) Number of applications and dates applied, treatment concentrations or
application rates, frequency and pattern of administration.
(v) Method of test substance application: application or delivery methods (e.g.,
irrigation water, soil incorporated, surface soil or foliar spray) to the site including
equipment type and design (nozzles, orifices, pressures, flow rates, volumes, etc})
and method for calibrating the application equipment), information about any
solvent used to dissolve and apply the test substance.
(vi) Study duration.
Page 42 of 46
-------
(vii) Methods and frequency of climatological monitoring performed during the
study for air temperature, thermoperiod, humidity, rainfall and watering regime,
light intensity, and wind speed.
(viii) The photoperiod and light quality.
(ix) Methods and frequency of monitoring of other ancillary nontreatment related
factors that may influence the measures of effect at the study site should be
reported. For example, if effects to wildlife from application to a crop species is
studied or if a crop is treated concurrent to the investigation of wildlife effects,
cultural practices during the tests such as cultivation, pest control, should be
monitored and reported for the study sites.
(x) All analytical procedures should be described. The accuracy of the method,
method detection limit, and limit of quantification should be given.
(7) Results.
(i) Environmental monitoring data results (air temperature, humidity and light
intensity, rainfall) in tabular form (provide raw data for measurements not made
on a continuous basis), and descriptive statistics (mean, standard deviation,
minimum, maximum).
(ii) Tabulation of the results of study-specific wildlife measures by field site and
treatment (provide the raw data), and summary statistics. If categorical rating
measures are made a description of the rating system should be included.
(iii) Description of the statistical method(s), software package(s) used, the basis
for the choice of the method(s), statements of the reasons why particular methods
are being used, including, at least qualitatively, the meaning that different results
might have.
(iv) Results of the statistical analysis including graphical and tabular summaries,
and results of goodness-of-fit tests or minimum significant differences detectable,
as appropriate.
(h) References. The following references should be consulted for additional background
material on this test guideline.
(1) Bunyan, P.J. et al. Organophosphorus poisoning, some properties of avian esterases.
Journal of Agricultural and Food Chemistry 16: 326-331 (1968).
(2) Bunyan, P.J. et al. Organophosphorus poisoning, diagnosis of poisoning in pheasants
owing to a number of common pesticides. Journal of Agricultural and Food Chemistry
16:332-339(1968).
(3) Burnham, K.P. et al. Estimation of density from line transect sampling of biological
populations. Wildlife Monographs, No. 72 (1980).
Page 43 of 46
-------
(4) Caughley, G. Analysis of Vertebrate Populations. Wiley, NY (1977).
(5) Cochran, W.W., Wildlife telemetry, pp. 509-520, in Wildlife Management
Techniques Manual. S.D. Schemnitz, Ed. The Wildlife Society, Washington, DC (1980).
(6) Corbett, J.R. The Biochemical Mode of Action of Pesticides. Academic, NY (1974).
(7) Davis, D.E. and R.L. Winstead. 1980. Estimating the numbers of wildlife populations.
pp. 221-246, in Wildlife Management Techniques Manual. S.D. Schemnitz, Ed., The
Wildlife Society, Washington, DC (1980).
(8) Eberhardt, L.L. Quantitative ecology and impact assessment. Journal of Wildlife
Management 4: 27-70 (1976).
(9) Eberhardt, L.L. Appraising variability in populations studies. Journal of Wildlife
Management 42: 207-237 (1978).
(10) Eberhardt, L.L. Assessing the dynamics of wild populations. Journal of Wildlife
Management 49: 997-1012 (1985).
(11) Edgington, E. Randomization Test. Dekker, NY (1980).
(12) Edwards, P.J. et al. The use of a bird territory mapping method for detecting
mortality following pesticide application. Agro-Ecosystems 5: 271-282 (1979).
(13) Ellman, G.L. et al. A new and rapid calorimetric determination of
acetylcholinesterase activity. Biochemical Pharmacology 7:88-95 (1961).
(14) Fite, E., L. Turner, N. Cook, and C. Stunkard, 1988. Guidance Document for
Conducting Terrestrial Field Studies. United States Environmental Protection Agency,
Office of Pesticide Programs, Washington, D.C. EPA 540/09-88-109.
(15) Hanson, W.R. Estimating the density of an animal population. Journal of Research
Lepidoptera 6: 203-247 (1967).
(16) Hegdal, P.L. and R.W. Blaskiewicz. Evaluation of the potential hazards to barn owls
of Talon (brodifacoum bait) used to control rats and house mice. Journal of
Environmental and Toxicological Chemistry 3: 167-179 (1984).
(17) Heinz, G.H. et al. Environmental contaminant studies by the Patuxent Wildlife
Research Center, pp. 8-35. in Avian and Mammalian Wildlife Toxicology. ASTM STP
693, E.E. Kenaga (Ed), American Society for Testing and Materials, Philadelphia, PA
(1979).
(18) Hill, E.F. and W.J. Fleming. Anticholinesterase poisoning of birds: field monitoring
and diagnosis of acute poisoning. Journal of Environmental and Toxicological Chemistry
1:27-38(1982).
(19) Hurlbert, S.H. Pseudoreplication and the design of ecological field experiments.
Ecological Monographs 54:187-211 (1984).
Page 44 of 46
-------
(20) Giles, R.H. Jr. Wildlife Management. Freeman, San Francisco, CA. (1978).
(21) International Bird Census Committee. Recommendations for an international
standard for a mapping method in bird census work. Bulletin of the Ecological Research
Committee 9:49-52 (1970).
(22) Larson, J. S. and R. D. Taber. Criteria of sex and age. pp. 143-202, in Wildlife
Management Techniques Manual. S. D. Schemnitz; (Ed.), The Wildlife Society,
Washington, DC (1980).
(23) Leopold, A. Game Management.Scribner's, NY (1933).
(24) Ludke, J.L. et al. Cholinesterase (ChE) response and related mortality among birds
fed ChE inhibitors. Archives of Environmental Contaminant Toxicology 3:1-21 (1975).
(25) Moen, A.N. Wildlife Ecology: An Analytical Approach. Freeman, San Francisco,
CA(1973).
(26) Nichols, J.D. and K.H. Pollock. Estimation methodology in contemporary small
mammal capture-recapture studies. Journal of Mammal 64:253-260 (1983).
(27) O'Brien, R.D. Insecticides: Action and Metabolism. Academic Press, NY (1967).
(28) Otis, D.L. et al. Statistical interference from capture data on closed animal
populations. Wildlife Monographs 62:1-135 (1978).
(29) Ripley, T.H. Planning wildlife management investigations and projects, pp. 1-6. in
Wildlife Management Techniques Manual. S.D. Schemnitz (Ed.), The Wildlife Society,
Washington, DC (1980).
(30) Rosene, W. Jr. and D.W. Lay. Disappearance and visibility of quail remains. Journal
of Wildlife Management 27:139-142 (1963).
(31) Scientific Advisory Panel. Final Scientific Advisory Panel subpanel's report on the
January 7-8, 1987 meeting concerning terrestrial field studies. Environmental Protection
Agency, Washington, DC (1987).
(32) Seber, G.A.F. The Estimation of Animal Abundance and Related Parameters.
Macmillan,NY(1982).
(33) Shellenberger, T.E. et al The comparative toxicity of organophosphate pesticides in
wildlife, pp. 205- 210, in W.B. Diechmann, (Ed.), Pesticide Symposium, Halos, Miami,
FL (1970).
(34) Snedecor, G.W. and W.G. Cochran. Statistical Methods. Sixth Edition. The Iowa
State University Press, Ames, IA (1967).
(35) Stewart, R.E. and J.W. Alrich. Removal and repopulation of breeding birds in a
spruce-fir forest community. The Auk 68:471-482 (1951).
Page 45 of 46
-------
(36) United States Environmental Protection Agency, 1982. Pesticide Assessment
Guidelines Subdivision E, Hazard Evaluation: Wildlife and Aquatic Organisms. Office
of Pesticide and Toxic Substances, Washington, D.C. EPA 540/9-82-024.
(37) Walpole, R.E. and R.H. Myers. Probability and Statistics for Engineers and
Scientists. Macmillan, NY (1972).
Page 46 of 46
------- |