EPA 540/09-88-109
September 1988
GUIDANCE DOCUMENT FOR CONDUCTING
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Prepared by Edward C. Fite, Larry W. Turner, Norman J. Cook and Clayton Stunkard
Ecological Effects Branch, Hazard Evaluation Division, Office of Pesticide Programs
United States Environmental Protection Agency
Washington, D.C.
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GUIDANCE DOC JM T FOR ctt rn T RESTBIAL FIELD STUDIES
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doc itient is a techj ical paper intended to provide gu o
to design and perform terrestrial field studies relative to data require nents
under the Federal Insecticide, mgicide arid I deriticide Act as nded (FIF1 A,
P. L. 92-516). The paper discusses when the Agency requires these tests, their
objective and suggests a general approach and sc*ue experinentaj. designs which
cx,uld be used to address Agency’s ncerns. A variety of basic wildlife investi-
gative ITethods which have been found useful in these type of studies are briefly
reviewed along with adequate references to assist scientists planning to undertake
a study to support a Federal Pesticide Registration.
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ACKNOWLEDGEMENTS
The authors wish to thank those individuals or groups who reviewed and commented
on the various drafts of this document. The comments and recommendations received
proved helpful and were pertinent to the techniques and methodologies presented. We are
especially grateful to the members of the Subpanel of the Science Advisory Panel. Harold
Bergman (Chairman), John M. Thomas, Robert K. Ringer, Douglas Robson. Barbara
Walton, Stanley Temple and Jerry Schnoor, for providing invaluable comments on the
issues presented by Agency staff. With their recommendation we were able to expand
and clarify many of the document’s sections. We owe a special thanks to Douglas
Robson, Professor of Biological Sciences, Biometrics Unit, Cornell University and
member of the Subpanel, for providing extensive input and guidance on the statistical
portions of the document Also, we are grateful to Al Vaughn. Richard Lee. Pierre
Mineau, Brian Collins. Bill Williams, John M. Emlen, Rick Bennett, Anne Fairbrother,
Linda Lyon, Bill Jacobs, Robert Whitrnore, Louis Best, John McCarthy, James Gilford,
Richard Tucker, Robert McLaughlin. William Gross, Phil Ross, Les Touart, Dan Rieder,
James Goodyear, Doug Urban, Henry Craven. Ann Stavola and Margaret Rostker for their
helpful comments. We also would like to credit the ASTM Subcommittee of Upland
Field Studies which undoubtedly influenced us through discussions at their meetings we
attended and Mike Slimak, Chief, Ecological Effects Branch, during the initial
development of this document.
We extend a special thanks to Ms Anne Barton, Acting Director of the Hazard
Evaluation Division, for her continuing support and direction on this project.
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11
PREFACE
This document is a technical paper intended to provide guidance on how to perform
terrestrial field studies, those studies designed to address the potential adverse effects of
proposed pesticide use(s) to nontarget iJ4I1fc These studies are presented as outlined in
§ 71-5 of the Pesticide Assessment Guidelines, Subdivision E - Hazard Evaluation:
Wildlife and Aquatic Organisms. EPA-54019-82-024. October 1982. Such studies
represent Tier IV, the most complex of the terrestrial tests presented in Subdivision E.
They are required to support those pesticide uses the Agency determines are likely to
result in adverse effects to nontarget terrestrial wildlife. Such studies consist of testing
performed in the field under actual pesticide use conditions and, generally, they address
the potential acute, subacute and/or chronic adverse effects of pesticide residues to
nontarget mammals and birds. The effects to birds and mammals axe emphasized because
the lower-tier Subdivision E tests usually employ these organisms, but effects to other
terr strial organisms, such as amphibians and reptiles are also examined and considered.
Terrestrial field studies, as discussed in this paper, are typically mulriyear/multisite
studies and consist of two levels of tests: a level 1 or screening study, which essentially
determines if adverse impacts occur to nontarget wildlife under actual pesticide use
conditions and a level 2 or definitive study, that quantifies those adverse effects identified
in the screening study or from other information. Also, the Agency requires that these
tests be performed only with nonendangered organisms and only in areas where impacts
to endangered or threatened species will not occur.
As an amplification of § 71-5 Subdivision E, this paper discusses a variety of basic
biological research techniques and wildlife investigative methods for use in assessing the
effects of pesticides in the field. These n thods and techniques axe not new, for the
majority of them have been used by wildlife biologists, fisheries biologists and game
managers for decades. They axe presented here, along with adequate references, in order
to assist scientists planning to undertake terrestrial field studies. This document is
intended to provide guidance (it is not a cookbook or checklist) and will be updated by
the Agency as the state of the art for performing these studies advances.
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111
TABLE OF CONTENTS
ACKNOWLEDGEMENTS i
PREFACE ii
TABLE OF CONTENTS iii
I14TRODUCT ION
When Required 1
Objective of Field Studies 2
General Approach 4
Sampling and Experimental Design 4
SCREENING STUDY 5
Objective and Scope 5
Geographic Area Selection 5
Study Site Selection 6
Number of Sites 7
Size of Study Sites 10
Chemical Application 10
Methods 11
Carcass Searches 11
Radio telemetry 12
Tests of Cholinesterase Inhibition 12
Residue Analysis 14
Behavioral Observations 15
Density and Diversity Estimates 15
Interpretation of Results 16
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iv
DEFINITIVE STUDY 19
Objective and Scope 19
Sampling and Experimental Design 19
Study Area and Site Selection 22
Number and Size of Sites 22
Methods 22
Mortality and Survival 23
Mark-Recapture 23
Territory Mapping Method 24
Radio Telemetry 25
Other Methods for Mortality and Survival 25
Reproduction and Survival of Dependent Young 25
Nest Monitoring 25
Behavioral Observations 25
Age Structure of Populations 26
Ancillary Methods 26
Interpretation of Results 27
LITERATURE cm D 31
Appendix A - Selected References 35
Appendix B - Suggested Components of a Field Study 39
Protocol for Subnhirr2l to EEB for Review
Appendix C - Carcass Searches 41
Appendix D - Example of Methods Available for 45
Investigating Particular. Identified Effects
Appendix E - Terrestrial Field Studies 51
When Required
Appendix F - Paired Plot Design 65
Basis for Formula for n Pairs
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iNTRODUCTION
Data from full scale terrestrial field studies are required by 40 CFR 158.145 on a case-
by-case basis to support the registration of an end-use product intended for outdoor
application. Because these studies are complex and costly, the Agency requires these tests
to evaluate only those products that appear to pose significant risks to nontarget wildlife.
Laboratory tests generally are amenable to a high degree of standardization. In
contrast, field study protocols must retain a high degree of flexibility. Variables such as
chemical mode of action, use pattern, crop type, method of application and species density
and diversity make standardization difficult in field studies. Therefore, Subdivision E,
Hazard Evaluation: Wildlife and Aquatic Organisms, of the Pesticide Assessment
Guidelines (EPA. 1982) provides only a general outline for field studies. Specific
protocols must be developed as needed and submitted to the Agency for review.
Regardless of the variability among field studies, several key elements common to most
field studies can be identified. This guidance document was prepared to identify and
discuss these elements as they pertain to terrestrial vertebrates, and to provide a better
understanding of the purpose of field studies.
WHEN REQUIRED
The Federal Insecticide, Fungicide, and Rodendcide Act, as amended (FIFRA, P.L
92-516), specifies that for a product to be registered or for continued registration, EPA
must determine that it will not cause unreasonable adverse effects on the environment.
The law further stares that the Agency must specify what data are necessary to make this
determination, but acquiring that data is the responsibility of those requesting registration
or continued registration.
For nontarger wildlife species (i.e.. terrestrial v rtebrates with emphasis on birds and
mammals), the Agency requires a series ofl tsIhat are arranged in a hierarchical or tier
system, progressing from basic laboratory tests to applied field studies. This tier system,
detailed in Subdivision £ of the Pesticide Assessment Guidelines (EPA, 1982), provides a
means to identify materials that may pose an inordinate risk, and at the same time ensures
that the process does not irresponsibly limit use of safe materials. Typically, the initial
screen consists of a comparison of results from three avian laboratory toxicity tests (an
acute oral LD and two dietary LC , studies) and one mammalian toxicity test (an acute
rat oral LD O) with estimated environmental concentrations (EECs). In addition, when
labeling contains directions for using the material under conditions where wild
vertebrates may be subject to repeated or continuous exposure to the pesticide, when the
material is stable in the environment, or when the material is stored or accumulated in
plant or animal tissues, data on avian reproductive effects are required and mammalian
reproduction data (from rodent or other mammalian test species) are examined. If
environmental concentrations on wildlife food items are at or below the threshold levels
eliciting a biological response in the avian or mammalian laboratory studies, usually it is
assumed that the probability of seriously impacting nontarget species is low. However,
for those materials where environmental concentrations exceed the Io’wesr-effec: level
eliciting a biological response, field studies usually are considered.
In assessing the need for field studies for those chemicals whose EECs exceed the
lowest-effect level (LEL). a great deal of judgment is required. Several factors, or
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INTRODUCtION
appropriate combinations of these factors, need to be considered in addition to the basic
laboratory data and EECs. These include:
- The chemical properties of the pesticide (e.g., persistence, conversion to
toxic metabolites, retention on food, repellency);
- Intended use pattern (e.g., treated habitats, expected presence of species,
including endangered species, extent of use areas, number of applications
and treatment intervals);
- Margin between EEC and the LEL
- Dose/response relationships noted in labomtory tests, including slope of
dose-response line, dine of mortality or reproductive effects, signs of
intoxication and abnormal behavior, and gross pathological changes as
noted in gross necropsies.
When the margin between the exposure level and the lowest-effect level is small the
likelihood of a decision by the Agency to require an actual field study is small. The
other factors mentioned above are seriously considered in this situation where more
judgment is needed. Conversely, when the margin between the exposure level and the
toxicological effect level is great and begins to approach, for example, the LC 51 , then the
likelihood of a decision by the Agency to require an actual field study is great. However,
the final determination of whether a field study will be iecpiired is based on the weight of
evidence, factoring in all pemnent information. An in-depth discussion of how the
Agency determines when terrestrial field studies are required is in Appendix E.
OBJECTIVE OF FIELD STUDIES
The purpose of the field study is to either refute the assumption that risks to wildlife
will occur under conditions of actual use of the pesticide and/or to provide some quantifi-
cation of the risk that may occur. The purpose is twofold because the FIFRA requirement
to determine unreasonable adveise effects implies the need for a risk-benefit analysis.
Thus if the assumption of risk cannot be refuted, and in order to refine the risk-benefit
analysis, field studies should quantify the adverse effects that would occur from actual use
of the pesticide.
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 regis-
tered only if EPA determines it will no: cause unreasonable adverse effects. While the
difference between an objective of “will cause” and “will not cause” may seem trivial, it
substantially influences study design and the evaluation of data.
The adverse effects to wildlife that can result from the use of pesticides 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
cancellation or suspension. An adverse effect that results in a reduction in local, regional,
or national populations of wildlife species is clearly of great concern. A pesticide 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,
must be designed to adequately assess both of these types of effects. In most cases,
however, the assessment of population effects presents the greatest difficulties, and a
study adequate to assess this effect will also assess the degree of risk to individual
wildlife. Consequently, throughout this Guidance Document the primary emphasis is on
designing a study to assess the risk of a population effect the study, however, must also
be adequate to address the risks to individual organisms.
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TERRESTRIAL FIELD STUDIES Fite. Turner. Cook ancLStunkard
The field study must be designed to provide data that show whether wildlife species
Will no: be affected significantly by a pesticide under normal pesticide use practices. To
fully achieve this objective at the population Level, one must have detailed knowledge of
the population dynamics and varying environmental conditions for each species potentially
at risk. The theoretical aspects of population dynamics axe well documented in the litera-
ture. However, empirical data aie available for only a few species (Eberhardt, 1985). 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
pesticide application on these parameters, would require several. if not many years in
order to begin to give meaningful results. The cost of obtaining such data could make
these studies impractical, if not impossible.
The essential question then is: How can these studies be performed in a practical,
economical manner and still provide data that can show that the pesticide under study will
not reduce or lintit wildlife populations or repeatedly kill wildlife?
One can begin to answer the question by examining the potential influence pesticides
can have on wildlife. These effects include:
- Direct poisoning and death by ingestion, dermal exposure, and/or
inhalation;
- Sublethal toxic effects indirectly causing death by reducing resistance to
other environmental stresses such as diseases, weather, or predators;
- Altered behavior such as abandonment of nests or young, change in
parental care, or reduction in food consumption;
- Reduced food resources or alteration of habitat; or
- Lowered productivity through fewer eggs laid, reduced litter size, or
reduced fertility.
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 pesticide
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, one can 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, as indicated
previously, a study adequate to assess these effect will also assess the degree of risk to
individual wildlife.
This document emphasizes avian and mammalian wildlife. The Agency is also
concerned about ocher terrestrial organisms such as noruarget plants, invertebrates,
amphibians, and reptiles. Plants and invertebrates are excluded here from direct study,
except as sources of food or pesticides to wildlife. Testing guidelines for nonrarget plants
and insects axe in Subdivisions J arid L. respectively. Established protocols. especially for
acute and chronic toxicity testing, are available for birds and mammals, but not for
reptiles and amphibians. Further, the Agency assumes that “protection” for reptiles and
amphibians is provided through the risk assessment process for birds and mammals.
Occasionally, however, it may be necessary to adapt these field techniques to apply
specifically to reptiles and/or amphibians.
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INTRODUCTION
GENERAL APPROACH
Field studies required to support registration have evolved into two types, screening
and definitive. The type(s) of study(ies) required depends on the available data on the
chemical in question. If the available information is limited to laboratory toxicity data on
a limited number of species, coupled with EECs, a screening field study may be
appropriate, with the objectives of determining if impacts are occurring and, if so, to what
species. 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. For
some 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. In some instances, where there is
insufficient information to indicate which species axe at risk in the field but available data
strongly suggest adverse effects will occur, it may be appropiiate for a field study to
begin with the general approach of a screening study, followed by a quantitative phase
that focuses on the species affected in the screening phase. In certain instances there may
be sufficient data and information for the Agency to decide additional testing including
field testing is not necessary prior to a regulatory action.
The design of field studies differs between the screening study and the definitive study.
If the objective of the study is to determine if impacts are occurring, “pass-fail” methods
can evaluate whether or not animals are being stressed by the application. 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. 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 pesticide 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.
SAMPLING AND EXPERIMENTAL DESIGN
While examples of acceptable experimental designs are given, it is beyond the purpose
of this paper to cover the fundamentals of this topic. Appendix A lists several general
and specific references that can provide an in-depth review of this subject. Appendix B
provides a general outline for a field study protocol to be submitted to the Agency for
review. The following sections generally outline points to be considered in designing
screening and definitive field studies. As stated in the introduction, specific protocols
must be developed on a case-by-case basis and submitted to the Agency for review.
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SCREENING STUDY
OBJECTIVE AND SCOPE
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. The
interpretations of screening study results. in most cases, are limited to “effect’ versus “no
effect” determinations. If the study indicates that the pesticide has caused little or no
detectable adverse effect, it may be reasonable to conclude that potential adverse effects
are minor. However, when effects are demonstrated, it may be necessary to determine the
magnitude of the effects, thus requiring additional testing if pesticide registration or
continued registration is still desired. Therefore, when information already available
shows that a product has caused adverse effects under normal use conditions, the
screening study may be of limited value. In addition, where analysis of laboratory or
other data strongly suggest that adverse effects are likely to occur, and are unlikely to be
attenuated by field use conditions, it may be appropriate to proceed directly to a definitive
field study.
In general, the screening study is 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, the screening study would not address
chronic effects, such as reduced reproduction, or effects such as changes in density or
diversity of populations.
In addition, 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 proposed use
pattern may indicate which species are more susceptible to the pesticide, allowing the
study to be designed to monitor those species in greater depth as well as to provide
insight into field results that show some species were affected more than others. Under
such circumstances, additional laboratory studies may be unavoidable. If residue concen-
trations in resident species are being used to indicate potential problems, the relationship
between tissue levels and the dose(s) that cause(s) adverse effects must be estimated. If
secondary poisoning is of concern, feeding secondary consumers (held in captivity) prey
items collected in the field following the application can be useful to evaluate this
potential exposure route. Also, 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.
GEOGRAPHIC AREA SELECTION
The selection of geographical areas for evaluating pesticide impacts on wildlife can be
difficult particularly for pesticides to be used on crops grown over large and diverse areas.
Ideally, studies should be performed in each biogeographic area where the pesticide could
be used. While this approach may be practical for uses restricted to localized areas and
conditions, many uses (e. g., corn, soybeans, alfalfa) would require an inordinate number
of studies In different geographic areas, due to 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 be biased toward situations likely to present the greatest risk. If hazards appear to
be low under these conditions, it can be reasonably concluded that impacts under less
severe conditions would be minor.
A careful review of the species and habitats in the various geographical areas where
the pesticide could be used is necessary to identify the areas of highest concern. A sound
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SCREENING STUDY
understanding of the biology of the species that are found in association with the potential
use 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 face (e.g., degradation) pa-
rameters vary geographically, study a.rea selection also should be biased towards
maximizing residues available to wildlife. In some circumstances preliminary monitoring
of candidate areas may be necessary to determine which should be selected for detailed
study.
STUDY SITE SELECTION
Selection of study sites within each geographic area also is extremely important in
designing field studies. Ideally, study sites should be randomly selected throughout the
study area. This approach may be practical for some areas such as rangeland or large
contiguous crops. However, due to the diversity and variability in wildlife species and
habitats in most areas, random selection would require a large number of sites to provide
a representative sample. The cost and time requirements of such studies would be
unreasonable. To maximize the hazard, the sites selected should have associated species
that would be at highest risk from the application, as well as a good diversity of species
to serve as indicators for other species not present at that specific location. In addition,
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.
Under some circumstances, it may be difficult to decide beforehand which species are
likely to be at highest risk. In most cases, field surveys of a number of sites may be
needed 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.
In general, study sites should be selected from what is considered to be a “typical”
application area, 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, as
indicated above, preliminary sampling.
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
(1933), “The potential density of game of low radius requiring two or more type& is,
within ordinary limits, proportional to the sum of the type peripheries.” 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” (Giles, 1978) 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 sires chosen to maximize the ratio of edge to
core may serve to indicate sites with higher densities and diversities of wildlife species.
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 wild-
life species are also influenced by the variety in the composition and arrangement of the
edge component cover types and its width. Also, the 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
1 Type . Th. vailous segmenb of an azi maI’s eiwtrowtent used for food, ver. or oth vequlremenb.
6
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RESThIAL FIELD STUDLES _ Fite. Turner. Cook and Stunkard
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 1 preliminary sampling of prospective study sites will be needed to identify study
sites with adequate density and diversity of wildlife species.
NUMBER OF SITES
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, one
must first define the expected probabilities that birds or mammals on a site arc affected or
not affected. Then the probability of the discrete binomial random variable x for n
replications can be used to determine the minimum number of sires at a certain level of
significance.
As an example for discussion purposes, we will define that a problem exists if some
specific mortality rare or level of some other variable occurs on more than 20 percent 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 .2n, it can be concluded that potential impacts will be below
the stated level of concern.
To calculate the minimum number of Sites necessary to show a significant difference
between the observed and expected, the following formula for the probability of the
binomial random variable x can be used (Walpole and Myers, 1972):
P(x) = ( ) p 2 q
Where,
x = number of sites showing effects
n = number of sites
p = probability of a site showing an effect
q = probability of a site not showing an effect
Then, solving for n, when x = 0, i.e.,
P(x=0) =( )q
Let P(x=0) a, then
a = q”
log a=n log q
n=logU log q
Using this formula, the minimum number of sites can be determined. Continuing with
the discussion example of 20 percent occurrence of an effect as a level of concern (i.e., a
7
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SCREENING STUDY
0.2 probability of an affected site, a 0.8 probability of a noneffected site, and a 0.05 level
of significance), n would be:
n = log 0.05 ÷ log 0.8
n = 13.43
Therefore, 14 Is the minimum number of sites needed such that the probability is not
greater than .05 that all sites surveyed would be unaffected. Or, in other words, if 20
percent of the application sites are actually affected, there is only a 5 percent chance of
finding all 14 sites unaffected when n = 14. Moreover, if 20 percent 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.
Under many circumstances, conducting this number of replications may not be
practical. However, as indicated previously, if site selection is biased toward hazard, the
number of sites can be reduced. 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 percent
of the application sites; or there is less than a 20 percent chance that all eight sites will
be judged unaffected when n = 8 sizes. 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 are occurring on less than
30 percent of the application sites, and at the 0.05 level of significance, effects are
occurring on less than 40 percent 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 percent of the application sites. Also, because worst-case study sites were
used the Agency could have additional confidence that adverse effects would occur on
less than 20 percent of all normal application sites.
However, 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. 2
The above calculations are for when x is equal to zero, no effects are observed on any
site. A similar approach can be used to estimate the number of sites necessary to show a
significant result for a critical value of x greater than zero. Again the formula for the
probability of the binomial random variable can be used summing the probabilities of x
and all outcomes less than x. Then by using increasing values of it, the number of
replications required to show statistical significance may be determined for a given level
of significance for individual x values. That is:
P(X r)= ( )
2 Under no carc nnstan should EieId studies on pestiades be conducted In cress where endangered spedes
could be expceed.
8
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TERRES1RIAL FIELD STUDIES Fite. Turner. Cook and Scunkard
The minimum value of n occurs when
P(X r) = a level
Continuing the previous example. Table 1 gives the results for x I and x 2 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 axe occurring below levels of concern is 14. if x 2, 21 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 sizes required to show a statistical significance becomes
inordinately large.
Table 1.
Probabilities for binomial random variable with p equal to .2 for x 1 and x 2 as a
function of the number of sites (N).
N P(x1) P(x2 )
8 0.5033 0.7969
9 0.4362 0.7382
10 0.3758 0.6778
11 0.3321 0.6174
12 0.2749 0.5583
13 0.2336 0.5017
14 0.1979 0.4481
15 0.1671 0.3980
16 0.1407 0.3518
17 0.1182 0.3096
18 0.0991 0.2713
19 0.0827 0.2369
20 0.0692 0.2061
21 0.0576 0.1787
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 percent of the
time for sampler of size 8 and 9, respectively. It also results in a declaration of “no
effect” 43.1 and 38.7 peivent 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.
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 ax risk as well as the parameter being sampled. It should be noted that the
9
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SCREENING STUDY
measure of effect is not limited to mortality. Other parameters. such as residue or
enzyme levels, can be used. Whatever parameter(s) is (axe) used, defining the criteria
level for an effect is extremely important, and when designing studies this issue should be
considered carefully.
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 pesticide. However, for most chemicals, this can be avoided by
employing methods, such as residue analysis and/or cholinesterase inhibition tests, that
can be used to indicate if the pesticide contributed to the observed effect. Further, studies
have shown that it is a relatively rare event to locate dead or sick animals in the wild
except under unusual conditions (Heinz et aL, 1979). Thus it is unlikely to find dead
animals that were killed by something other than the pesticide being tested.
Nevertheless, in some instances, particularly when reliable methods to confirm the
cause of effect are not available, controls may be necessary. In these cases the above
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. Then, as above, when
critical levels of effect and occurrence are defined, the binomial theorem can be used for
sample size determination, which gives eight 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 percent 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 in the section on experimental design for definitive studies, page 19.)
SIZE OF STUDY SITES
Study sites must 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 may be
included in a single study site to account for wide-ranging species or 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.
CHEMICAL APPLICATION
In planning field studies consideration should be given to application rates and
methods. in general, the test conditions should resemble the conditions likely to be
encountered under actual use of the product. 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. This evaluation should relate
wildlife Unhi7JrnOfl of the area to exposure. For example, if the crop is one that is used
by avian species as preferred nesting areas, feeding areas, or cover, then ground
application may be the method that maximizes exposure. However, if it is a crop with
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TERRESTRIAL FIELD STUDIES Fire. Turner. Cook and Stunkard
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 must be consistent with the label.
In addition, the equipment used may influence potential exposure of nontarget species.
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, one must evaluate the various equipment normally
used for the particular pesticide application to estimate the potential influence of equip-
ment choice on exposure. In some instances, preliminary tests may be required to
estimate which method and equipment poses the highest exposure.
METHODS
This section provides a general outline of methods appropriate for use in a screening
field study and indicates some of their limitations. The methods described have been
found to be most useful. However, we emphasize that 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, 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.
Essentially, the methods used in a screening study address exposure by monitoring
overt signs of toxicity such as mortality or behavioral modifications, or through 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 following methods can be useful for
screening studies.
Carcass Searches
Searching for dead or moribund wildlife has been a basic method used in field studies
to evaluate the impact of pesticides on nontarger species. Carcass searches can roughly
indicate the magnitude of kills when adequate areas axe searched and the reliability of the
search is documented. This latter point is extremely important. Rosene and Lay (1963)
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 meaning of the failure to
fmd carcasses can be assessed and the extent of the kill estimated.
Finding dead animals is seldom easy, even if every animal on a site is killecL For
example, three breeding pairs of small birds per acre is considered a large population
(Heinz e: aL 1979), 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 be even more difficult to find than birds.
Carcass searching specifically for mammals should be attempted only when cover
conditions permit a reasonable search efficiency. However, any vertebrate carcasses found
should be collected, even if the search is oriented primarily to one taxon.
Because the 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.
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SCREENiNG STUDY
There are no standard procedures for carcass searches. Appendix C outlines practices
that have been used typically and should be considered in designing searches.
Radio Telemetry
Radio telemetry has been found to be extremely useful for monitoring mortality and
other impacts caused by pesticide exposure of wildlife. Advances in miniaturizing elec-
tronic equipment over the last 15 years have made it feasible to track most vertebrate
animals. Transmitters have been developed that weigh a few grams and have been used
to track species as small as mice. Cochran’s (1980) excellent summary of this technique
provides additional details.
Radio telemetry has the advantages of providing information on the fare of individual
animals following a pesticide 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.
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 (Cochran, 1980), which could 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
(Moen, 1973).
While this technique can provide very useful information on impacts of pesticides to
wildlife, other points need to be considered in addition to cost. Capturing animals alive
and unharmed requires more time, skill, and motivation than one might expect. 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 under some conditions.
Adequate sample size is very iinportanL 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 LC,, 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 section on “number of sites”
for further details on these calculations). However, since the LC may differ between
species, 8 to 14 individuals would be required for each species, unless laboratory tests
have documented relative species sensitivity. Further complications can arise if the radio-
tagged 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.
Tests of Cholinesterafe Inhibition
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
chemical (Heinz e: a !, 1979: Hill and Fleming, 1982). 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 acetyicholinesterase (AChE), which
normally hydrolyzes the rieurotransmitter acetyicholine. Thus, ChE inhibitors permit ex-
cessive acetyicholine accumulation at synapses thereby inhibiting the normal cessation of
nerve impulses (O’Brien. 1967; Corbett. 1974).
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TERRESTRIAL FIELD STUDIES Fire. Turner. Cook and Stunkard
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 percent
in birds has been found sufficient to assume that death is pesticide related (Ludke et a!,
1975); depressions of more than 70 percent are often found in dead birds poisoned by
these chemicals (Bunyan er al., 1968a Bunyan et al., 1968b; Shellenberger es a!., 1970),
although some individual birds with less than 50 percent inhibition may die (Ludke et a!.,
1975; Bunyan er a!, 1968b). A 20 percent depression of brain ChE has been suggested
as an indication of exposure (Ludke et at., 1975). ChE concentrations in blood can also
be used to indicate exposure, avoiding the necessity of sacrificing the animal. However,
blood CIIE 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 (Hill and Fleming, 1982).
Although there are several colorimetric methods for determining ChE activity, the
general methods are similar. Brain tissues (or blood samples) are taken and analyzed for
ChE concentrations. Comparisons are then made between pro- and posttrearmenc and
between treated and untreated areas. It is important to ensure that “untreated” controls
have not been exposed to any ChE inhibitors. It also should be noted that, at the present
time, absolute enzyme levels in the literature are derived from various different, although
similar, methods and are reported in different ways. For example, Ludke et a!. (1975)
used a modification of the Eliman et a!. (1961) method and reported results of ChE
activity as nanomoles of acetylthiocholine iodide hydrolyzed/ minute/mg of protein,
whereas Bunyan e: at. (1968a) used their own colorimerric method (in addition to a pH
change method) and reported the results as microrrioles of acetylcholine hydrolyzed/
hour/mg 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.
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 percent 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 percent 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.
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 (Hill and Fleming, 1982).
Hill and Fleming (1982) have reviewed a technique for field monitoring and diagnosis
of acute poisoning of avian species, discussing sample collection, sample numbers,
preservation procedures, and sources of error. Their publication is recommended for
review for additional details.
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SCREENIM1 STUDY
Residue AnaLysis
Residue analyses of wildlife food sources provide information about the level and
duration of pesticide 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 the animals were affected. For this application of residue analyses though, labo-
ratory 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 pesticides. it
will be necessary to analyze also for residues of active metabolites.
For determining residues on wildlife food sources, the investigator should collect
samples of insects, seeds, leafy parts of plants, etc., immediately after pesticide 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 habi-
tats surrounding the treatment sites as well as in the treated fields. 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.
When residue analysis is used to evaluate exposure in nontarget animals, the tissues
selected for analysis differ depending on the purpose. Heinz a a!. (1979) indicated that
residues in brains of birds and mammals can be used to determine if death is pesticide
related for many chemicals. Sublethal exposure, they believe, is judged better from
residues in other tissues. Therefore, Heinz a a!. (1979) propose analyses of whole body
homogenates to quantify the body burden of a pesticide. If this is not feasible, they
suggest analyzing muscle tissue, 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, Heinz a a!. (1979) suggest 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 undependable 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
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 necessary to collect, it is necessary to
estimate the standard deviation and to set arbitrarily a 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, as presented by Snedecor and
Cochran (1967), is: n = 4a ’ + L’ for 95 percent probability, where a is the standard devi-
ation and L is the allowable limit around the mean. For example, if one wants to know
the residue concentrations on vegetation within ± 10 ppm and estimates a standard
deviation of 20 ppm, then n = 4(20)2 + (10)’ or 16 samples are required to have a 95
percent probability that the sample mean value will be within ± 10 ppm of the true mean.
In some situations, there ma f be little information useful for estimating the standard
deviation, or the standard deviation may be rather large, thus requiring a very large
14
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TERRESTRIAL FtELD STUDIES Fite. Turner. Cook and Stunkani
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 percent confidence limits
(usually the upper boundary, as in the case of residies) 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. Of course, the width of the
confidence intervals decreases with increasing sample sizes; so an investigator should take
as large a sample as feasible.
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 percent confidence limits are:
I ± (t. ) (s +
where s is the standard deviation estimated from the sample of size n.
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” at p = 0.2 in 20 percent of the
samples. This approach requires the establishment of threshold values which are
determined on a case-by-case basis. In general, residues reflecting an LC level of
exposure would seem to be a maximum acceptable effect concentration for a screening
study. Ideally, for each species analyzed for residues, an LC, 0 would be determined in
the laboratory. Then a group of animals would be exposed to an I.C 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 LC, dietary concentration would provide an
indication of threshold Levels.
The number and timing of collection periods must be considered and 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.
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 pesticide in the Laboratory. Other
abnormal behavior (e.g.. territorial males abruptly ceasing singing, birds not feeding.
reduced avoidance of humans) also may be important.
Density and Dversity Estimates
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 pesticide 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
15
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SCREENING STUDY
ate completed. Seasonal changes, such as migration, molt, or incubation, that can affect
real or apparent densities, also must be considered.
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. (Appendix A provides references on the various techniques available.)
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.
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.
In the line transect method, an observer walks a distance (L) across an area in
nonintersecting and nonoverlapping lines, counting the number of animals si hted and/or
heard (N), and recording one or more of the following statistics at the time of first
observation:
- Radial distance from observer to animal;
- 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.
Although the field procedures are simple, they must be understood adequately and
implemented well to obtain good estimates of density (Burnham et ci., 1980).
Bumham et at. (1980) provide a thorough review of the theory and design of line
transect sampling. This monograph should be reviewed for details along with other
references listed in Appendix A.
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 (1980), as well as other references listed in
Appendix A, review the various methods available, explaining their advantages and disad-
vantages.
INTERPRETATION OF RESULTS
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. There arc a few points that can be discussed
however, that may-be helpful when designing studies.
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 will have been 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
16
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TERRESTRIAL FIELD STUDIES Fire. Turner. Cook andStunkard
sites at rates equal to or greater than concern levels, the hazard has not been refuted and
additional tests may be necessary.
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. 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,
most detectable effect.s will exceed the concern level. 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 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.
For example, a test may be ron 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.
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.
Another consideration in the interpretation of results of a field study is the attribution
of effects to the pesticide being studied. A well-designed study will include appropriate
t chniques to determine if an effect is caused by a pesticide. In the absence of such
techniques. the Agency has no choice but to consider that any effects were as a result of
the pesticide use. As an example, measurement of ChE levels can provide information.
since it is generally accepted that inhibition of 20 percent indicates exposure and
inhibition of 50 percent or more indicates. in birds, that mortality is due to an inhibitor
(Ludke et aL, 1975). If the test chemical is the only cholinesterase inhibitor used in the
Vicinity of the study site, it can be reasonably assumed that a mortality associated with 60
percent CitE inhibition is due to the test chemical. However, if other CitE inhibitors are
used near the site, additional information, such as residue measurements, may be
necessary to atiribute death to the specific CItE inhibitor being tested.
Appendix D provides further discussion and examples of what is involved in planning
and conducting a screening study.
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DEFINITIVE STUDY
OBJECTIVE AND SCOPE
The definitive study is a relatively detailed investigation designed to quantify the
magnitude of impacts identified in a screening study or from other information. T n
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 done
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:
- To quantify the magnitude of acute mortality caused by the application;
To determine the existence and extent of reproductive impairment in
nontarget species from the application; and
- To determine the extent to which survival is influenced.
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 pesticide in question.
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 we have emphasized chemicals that are acutely toxic, with few excep-
tions the discussion is applicable to chemicals that cause chronic effects.
In general, the definitive study will provide limited insight into whether or not effects
axe 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.
SAMPLING AND EXPERIMENTAL DESIGN
As indicated previously, the principles of statistical design of studies are well
documented and it is beyond the scope of this document to cover the fundamentals of this
topic. However, there axe a few points on this topic that warrant discussion relative to
the definitive study.
itt the design of field studies, one must carefully consider what constitutes a sampling
unit. Eberhardt (1978) 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. Huribert (1984) 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
Preceding page blank
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DEFINITIVE STUDY
evaluated, 48 percent of those applying inferential statistics had pseudoreplication.
According to acrhardt (1978), 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.
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. Experience with “classical” experimental
designs with random assignment of experimental “treatment” and “controls,” has shown
that the probability of a Type U error is generally high (unless very large numbers of
replicates are available). Eberhardt (1978) indicates that, all too often in field studies on
impacts to wildlife, either by default or lack of understanding, there is only a 50 percent
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 U errors is
extremely important
As suggested above, the more generally used experimental designs require inordinately
large sample sizes to obtain small Type I! errors. For example, based on a coefficient of
variation of 50 percent (a relatively homogeneous sample for the kinds of data collected
in field studies; Eberhardt, 1976), a 20 percent minimum detectable difference between
means, a Type II error of 0.2 and a Type I error of 0.05, the number of replications
required can be estimated as:
( Z 1 . + Z J (CV)’ [ 1÷(1.8yJ
&
where
n = number of replications
Z, and a le critical values of the unit normal distribution
CV = coefficient of variation
6 = detectable nean difference expressed as the proportion of the
control group mean, i.e.,
For the above example:
(1.65 + 0.84)’ (0.5)2 [ 1 +(l..2)h]
11=
(.2)’
n = 63.6 replicates
Thus, for the above parameter, 64 replicates for both control and treatment groups, or
128 total study plots. are required to detect a 20 percent difference between treatments
20
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TERRESTRIAL FIELD STUDIES Fite. Turner. Cook and Stunkard
and controls with an 80 percent chance of being sure to detect a real difference at a .05
level of significance.
With more sophisticated designs, the number of replicates can be reduced under some
circumstances and still meet the Agency’s aspiration to limit the probability of a Type II
error to 0.2 with a detectable difference of 20 to 25 percent. Por 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 zeducing the variation attributable
to experimental error. The lower coefficient of variation reduces the number of replicates.
Then 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 (SAP, 1987).
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
pesticide effect (Eberhardt, 1976). Then, if all plots arc approximately equal in area and
habitat and population densities between pairs are similar, we are postulating that when no
pesticide impacts occur, the mean ratio of treatment to treatment plus control will equal
one-half. Then a t-test or an exact randomization test (Edgington, 1980) 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.
The number of pairs required can be estimated using the following formula’:
4q 1
= (Z + Z ,.j
p ’ (1+q 1 ) ‘
where,
n = number of paired plots
ZI.a and Z are critical Z scores
= survival ratio
p 1 = mortality ratio
= mean number of survivors on control plots
Therefore, at an 80 percent assurance of detecting a treatment-induced impact of 20
percent or greater at a 0.05 level of significance if = 28,
4(.8)
n = (1.65 + 0.84)’
(.2)’ (1 + .8) 28
n = 9.84
Thus, 10 pairs of plots (20 total) with a mean of 28 individuals per plot would be needed.
Increasing the mean number of individuals per plot ( ). causes a reduction in n.
In some field situations, pairing may not be feasible. in these situations, other designs
would be more appropriate or less rigorous design may have to be used. However, in
See Appendbi F r devc4cpinent of thu formulL
21
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DEFINITIVE STUDY
planning field studies, one must be careful to consider the power of the study design to
determine the limitations of the study. Studies with adequate replication axe highly
preferred to support registration; the use of less replication will not necessarily render the
study inadequate. However, what is objectionable is to use a study with low power to
imply no biological damage, when the study was not capable of detecting it 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 who are
familiar with this ldnd of field study in the planning and analysis phase of the field work
to avoid costly technical errors.
STUDY AREA AND SHE 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 must have adequate populations of the
species of concern. Obviously, the crop of concern must be grown on a representative
portion of the area. Also, consideration needs to be given to whether the target pest
species will be present. If it is not, one must consider what influence its absence may
have on potential results. For example, if the pest is a major food souive for nontarget
species, its absence could significantly influence results. Finally, the potential variation in
populations of concern over the geographical area(s) 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.
NUMBER AND SIZE OF SITES
As suggested in the section on study design for the definitive test, 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 percent probability of being sure to
detect a 20 percent difference when it exists. The size of the study sire must be large
enough to provide adequate samples. The size depends on the survey methods used, sen-
sitivity 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.
METHODS
Essentially, the methods used in a definitive study are a means to quarnitate
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 para-
meters (see Appendix A). Anyone not familiar with the theory and principles of the
various techniques should review these references in depth. The objective of this section
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.
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) or the use pattern and/or
22
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TERRESTRIAL FIELD STUDIES Pita. Turner. Cook and Stunlcani
habitat type is limited, the range of applicable methods tends to become more narrow.
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 guidance document is to present methods that are likely to be useful in many
situations, rather than an exhaustive list of all av iil ib1e methods. The Agency encourages
the use of other methods when they are scientifically valid, and have a high probability of
detecting an effect.
While it is absolutely essential to have a detailed investigational plan that describes the
selected actions (with contingencies) for achieving the study objectives. investigators must
remain flexible because anticipated 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 initiate the
study again. If the event occurs after substantial data already have been collected (e.g.,
early in the second year of a multiyeai 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 event(s) and its
(their) consequences if they (it) affect the study results.
MORTALITY AND SURVIVAL
It is very important to understand the auzecology of the species being studied in order
to select the most appropriate methods for investigating those species. In addition, the
choice of particular methods must consider the applicability of the method based on the
pesticide use pattern and study site characteristics.
Mark-Recapture
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 then are used to estimate population parameters. Mark-recapture
studies can provide information on:
- Size of the population;
- Age-specific fecundity rates;
- Age-specific mortality razes;
- Combined rates of birth and immigration; and
- Combined razes of death and emigration.
Seber (1982) reviewed the various mark-recapture methods and subsequent statistical
analyses. Less detailed, but still very useful, reviews are provided by Caughley (1977)
and Hanson (1967). Nichols and Pollock (1983) provide a valuable comparison of
methods. Table 2 provides a brief summary of some of the various mark-recapture
methods discussed in these references.
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 pesticides on wildlife populations, some mark-recapture analyses are not particularly
robust; small deviations from their implicit assumptions can produce large vrrors in the
results (Caughley, 1977). However, some of the more recent and sophisticated analytical
23
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DEFINITIVE STUDY
methods aie robust and can deal with deviations from assumptions in closed populations
(Otis et a!, 1978).
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.
Table 2.
Mark-Recapture Techniques.
Method Applications / Requirements I Assumptions
Peterson Method Estimation of population size. Usually only two sampling
(Lincoln Index) periods. Closed Population.
Schumacher’s Estimation of population size. More than two sampling periods;
Method marking continues throughout sampling. Closed population.
Bailey’s Triple Estimate of birth rate and death rate in addition to population
Catch size. Requires data from two marking occasions and two
recapturing occasions. Open population.
Jolly-Seber Estimates mortality and recruitment in addition to population
Method size. Requires more than two sampling periods and that each
animal’s history of recapture be known. Open population.
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 pesticide (e.g., anticoagulants with toe clipping).
Dyes may be useful unless they are lost by wear or molting.
Territory Mapping Method
A common spatial census method is territory mapping, wherein the territories of mdi-
viduals are mapped before and after treatment, on both treated and untreated plots. The
method is usualiy 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 exhibi-
ting territorial behavior appear on the map as clusters of individual contacts. The clusters
axe used to estimate both the size and number of territories. The pre- and posttxeatment
censuses for treated sites are compared with the pie- and postutatment censuses for
control sites to determine changes in populations of territorial individuals that may be
attributed to tbe pesticide (Edwards e: al., 1979). Further details of this method are given
by the International Bird Census Committee (1970). and its application to evaluating
impact caused by pesticides is reviewed by Edwards e: al. (1979).
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, non-territorial birds who may quickly
reoccupy empty territories (Stewart, 1951). The effects of replacement can be oveocome
for some species by capturing and marking the territorial individuals prior to utatinent, so
they can be distinguished from the floaters. Also, replacement may not be a problem
when the study areas axe in the center of a relatively large treated area.
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TERRESTRIAL FIELD STUDIES Fite. Turner. Cook andSmnkard
Radio Telemetry
Radio telemetry can be an extremely useful technique to provide information on the
effects of a pesticide 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 pesticide 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 behav-
ioral observation, intra- and inter-site 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.
Other Methods for Mortality and Survival
Other techniques for assessing density and diversity are discussed for screening studies;
most of these, especially linetransect 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.
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 nesdings 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.
Nest Monitoring
Nest monitoring is useful for evaluating the effect of pesticides 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 pesticide application. While all
definitive studies should consider this technique, it also may be useful in screening
studies.
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 difticuk to obtain sufficient data on the
success of the same species in enough sites to yield satisfactory results for statistical
comparison with controls (Heinz a al, 1979). 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.
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 altric La!
birds), and following behavior (for precocial animals) are behaviors that are particularly
25
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DEFINiTIVE STUDY
birds), and following behavior (for precocial animals) are behaviors that are particularly
amenable to such study. Courtship, mating, and nest building are other behaviors that
could be studied in some situations, but locating sufficient numbers of animals displaying
these behaviors to permit quantitative analysis is difficult
Age Structure of Populations
Comparisons of young-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 are critical. For assessing reproductive impairment or survival of
dependent young, per Se, the duration of this technique should be limited to single
breeding-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.
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, sub-
adults, 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, the ossification of bones or development of
reproductive organs are useful. In other cases, particularly where comparisons are 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. ft 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 (1980).
ANCILLARY METHODS
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.
Many of the methods for determining effects have been discussed for screening studies.
Enzyme analysis, such as for choilnesterase inhibition, and observations of signs of
toxicity can show that animals were exposed to or killed by a toxicant of a particular
type. Where it is possible that animals may be exposed to other pesticides of the same
type (e.g., feeding in a nearby area treated with other pesticides), residue analysis in
nonrarget animals may be necessary to determine which specific pesticide 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.
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 amount
26
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TERRESTRIAL FTFLD STUDIES Fite. Turner. Cook and Sturikard
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
aie 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.
INTERPRETATION OF RESULTS
Each field study is unique, although 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.
First, the well-designed protocol wifi contain a restatement of the concerns to be
addressed to ensure that there is an adequate understaMing of the Agency’s position.
Then the investigator should review the literature and other available information that may
bear upon the problem. h is possible that the literature may contain a valid answer to the
questions raised by the A cncy. Far more likely, the literature may orient the investigator
to address the concerns in a particular way. An example is provided by Hegdal and
BIa ckiewicz (1984) who conducted a study to address the Agency’s concerns for
secondary toxicity to barn owls (specificaUy) from the use of an anticoagulant bait
proposed for use on comrnensal rodents in and around agricultural buildings. A review of
the literature by these investigators indicated to them that 1) laboratory studies suggested
a legitimate potential for secondary poisoning to exposed raptors, but 2) the food habits of
barn owls consist primarily of microtine rodents in most areas, suggesting a low potential
for actual exposiu . 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 investi-
gators were able to nanow the study while still providing sufficient information for
evaluation.. However, ii 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.
Second, the well designed protocol will contain reasons why particular methods are
being used, including, at least qualitatively, the meanin that different results might have.
For example, a protocol may include collection of residues in non-target 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 organisme through analysis of
their food, exposure in nosatarget animals as a result of eating contaminated food, or that a
particular pesticide was likely to be the cause of any observed effects. The interpretation
of results is facilitated substantially by a statement of what is intended by using a
particular technique. In the previously cited example from Hegdal and Blaskiewicz
(1984), it was clearly stated that collection of owl pellets was 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.
27
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DEFINITIVE STUDY
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 previous sections, 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. Ideally, 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. Of course, 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.
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; or
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.
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 repro-
ductive 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.
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: rare of population increase (r) =
birth rate - death rate, where r can be positive (population growth) or negative (population
reduction). Where the concern is for specific populations of a species, then immigration
and emigration are also important. These characteristics differ among species, and data
will not always be available. The application of sound scientific judgment to the best
available infonnation 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 be necessary where endangered species are of concern or where other
species cannot be studied directly.
Ideally, 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. 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
28
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TERRESTRIAL FIELD STUDIES Fite. Turner. Cook and Stunkard
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 characrcnstics. 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 cuirently practical.
29
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LITERATURE CITED
American Institute of Biological Sciences (AIBS). Aquatic Hazards of Pesticides Task
Group. 1978. Criteria and Rationale for Decision Making in Aquatic Hazard
Evaluation. Report to the Environmental Protection Agency. Contract No. 68-012457.
August, 1978. 71 pp.
Balcomb, R. 1986. Songbird carcasses disappear rapidly from agricultural fields. The
Auk 103(4): 8 17-820.
Bunyan, P.J., D.M. Jennings and A. Taylor. 1968a. Organophosphorus poisoning, some
properties of avian esterases. J. Agr. Food Chem. 16: 326-331.
Bunyan, P.J., D.M. Jennings and A. Taylor. 1968b. Organophosphorus poisoning,
diagnosis of poisoning in pheasants owing to a number of common pesticides. 1. Agr.
Food Chem. 16: 332-339.
Burnham, K.P., D.R. Anderson and J. L. I 1980. Estiniation of density from line
transect sampling of biological populations. Wildi. Monogr., No. 72.
Caughley, 0. 1977. Analysis of Vertebrate Populations. John Wiley and Sons, New
York. 234 pp.
Cochran, W.W., 1980. Wildlife telemetry. pp. 509-520, in Wildl fe Management
Techniques Manual. S.D. Schemnita, Ed. The Wildlife Society, Washington, D.C.
Corbett, J.R. 1974. The Biochemical Mode of Action of Pesticides. Academic Press, Inc.,
New York. 330 pp.
Davis, D.E. and R.L. Winstead. 1980. Estimating the numbers of wildlife populations. pp.
221-246. in Wildltfe Management Techniques Manual. S.D. Schemnitz (Ed.), The
Wildlife Society. Washington, D.C.
Eberhardt, L.L. 1985. Assessing the dynamics of wild populations. J. Wild]. Manage.
49(4): 997-1012.
Eberhardt, LL. 1978. Appraising variability in populations studies. I. Wildi. Manage.
42(2): 207-237.
Eberhardt, L.L. 1976. Quantitative ecology and impact assessment. I. of Envir. Manage.
4: 27-70.
Edgington, E. 1980. Randomization Test. Dekker, Marcel Inc., New York, New York.
287 pp.
Edwards, P1., SM. Brown, M.R. Fletcher and P.1. Stanley. 1979. The use of a bird
territory mapping method for detecting mortality following pesticide application.
Agro-Ecosystems 5: 271-282.
Eilman, G.L, K.D. Courtney, V. Andres, Jr. and R.M. Featherstone. 1961. A new and
rapid colorimeu ic determination of acetyicholinesterase activity. Biocheni. Pharmacol.
7:88-95.
Environmental Protection Agency. 1982. Pesticide Assessment Guidelines, Subdivision E -
Hazard Evaluation: Wildlife and Aquatic Organisms. Office of Pesticides and Toxic
Substances. Washington. D.C. 87 pp.
Hanson, W.R. 1967. Estimating the density of an animal population. J. Res. Lepidoptera
6: 203-247.
Heath, R.G., LW. Spann, E.F. Hill and J.F. Kreitzer. 1972. Comparative Dietary
Toxicities of Pesticides to Birds. U.S. Fish and Wildlife Service. Special Scientific
Report -- Wildlife No. 152.. February. 57 pp.
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LITERATURE Cnth
Hegdal. P.L. and R.W. BI kiewicz. 1984. Evaluation of the potential hazards to barn
owls of Talon (brodifacoum bait) used to control rats and house mice. I. Environ.
ToxicoL Chem. 3:167-179.
Heinz, G.H., E.F. Hill, W.H. Stickel and L.F. Stickel. 1979. Environmental contaminant
studies by the Paruxent 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.
Hill, E.F. and W.J. Fleming. 1982. Anticholinesterase poisoning of birds: field monitoring
and diagnosis of acute poisoning. J. Environ. Toxicol. Chem. 1: 27-38.
Hill, E.F., R.G. Heath, LW. Spann and J.D. Williams. 1975. Lethal Dietary Toxicities of
Environmental Pollutants to Birds. U.S. Fish and Wildlife Service. Special Scientific
Report -- Wildlife No. 191. 61 pp.
Hudson, R.H., R.K. Tucker and M.A. Haegele. 1984. Handbook of Toxicity of Pesticides
to Wildljfe. U.S. Department of the Interior. Fish and Wildlife Service. Resource
Publication 153. 90 pp.
Huribert, S.H. 1984. Pseudoreplication and the design of ecological field experiments.
Ecological Monographs 54:187-211.
Giles, R.H. Jr. 1978. Wildl(fe Manageme u. W.H. Freeman and Company, San Francisco,
CA. 416 pp.
International Bird Census Committee. 1970. Recommendations for an international
standard for a mapping method in bird census work. Bull. Ecol. Res. Comm. 9:
49-52.
Kendall, M. and A. Stuart. 1977. The Advanced Theory of Statistics Volume 1,
Distribution Theory, 4th Ed. MacMillan, New York. ‘172 pp.
Larson, I. S., and R. D. Taber. 1980. Criteria of sex and age. pp. 143-202, in Wildlife
Management Techniques Manual. S. D. Schernnitz (Ed.), The Wildlife Society,
Washington, D. C.
Leopold, A. 1933. Game Management. Charles Scribner’s Sons, New York. 481 pp.
Ludke, J.L., E.L Hill and M.P. Dieter. 1975. Cholinesterase (CtiE) response and related
mortality among birds fed ChE inhibitors. Arch. Environ. Contamin. Toxicol. 3: 1-21.
Moen, A.N. 1973. Wildljfe Ecology an Analytical Approach. W.H. Freeman and
Company. San Francisco. 458 pp.
Nichols, J.D. and K.H. Pollock. 1983. Estimation methodology in contemporary small
mammal capture-recapture studies. I. MammaL 64(2): 253-260.
O’Brien, R.D. 1967. Insecticides: Action and Metabolism. Academic Press, Inc., New
York. 332 pp.
Otis, DL, K.P. Burnham, G.C. White and D.R. Anderson. 1978. Statistical interference
from capture data on closed animal populations. Wildl. Monogr. 62: 1-135.
Ripley, T.H. 1980. Planning wildlife management investigations and projects. pp. 1-6. in
Wild4fe Management Techniques Manual. S.D. Schemnitz (Ed.), The Wildlife Society,
Washington, D.C.
Rosene, W. Jr. and D.W. Lay. 1963. Disappearance and visibility of quail remains. I.
Wildl. Manage. 27: 139-142.
Scientific Advisory Panel (SAP). 1987. Final Scientific Advisory Panel subpanel’s report
on the January 7-8. 1987 meeting concerning terrestrial field studies. U.S. EPA,
Washington, D.C.
32
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TERRESTRIAL FIELD STUDIES Fite. Turner. Cook and Stunkard
Seber, G.A.F. 1982. The Estimation of Animal Abundance and Related Parameters.
Macmillan Publishing Co., Inc., New York 645 pp.
Shellenberger, T.E., B.J. Gough and L.A. Escuriex. 1970. The comparative toxicity of
organophosphare pesticides in wildlife. pp. 205-210, in W.B. Diechmarin, (Ed)
Pesticide Synzposium. Halos, Miami, FL.
Snedecor, G.W. and W.G. Cochran. 1967. Statistical Methods. Sixth Edition. The Iowa
State University Press, Ames, IA. 593 pp.
Sokal, R.R. and FJ. Rohlf. 1969. Biometry. The Principles and Practice of Statistics in
Biological Research. W.H. Freeman and Company, San Francisco. 776 pp.
Stewart, R.E. and LW. Alrich. 1951. Removal and repopulation of breeding birds in a
spruce-fir forest community. The Auk 68: 471-482.
Urban. D.J. and NJ. Cook. 1986. Hazard Evaluation Divisions Standard Evaluation
Procedure, Ecological Risk Assessment. U.S. Environmental Protection Agency.
Office of Pesticide Programs, Washington, D.C. 96 pp.
Walpole, R.E. and R.H. Myers. 1972. Probability and Statistics for Engineers and
Scientists. The Macmillan Company, New York. 506 pp.
33
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APPENDIX A
SELECTED REFERENCES
There are many publications available, ranging from journal articles to textbooks, that
are pertinent to evaluating the impacts of pesticides to wildlife. The following list,
although not exhaustive, is presented as a starting point for those not familiar with the
subject area.
EXPERIMENTAL DESIGN AND STATISTICS
Cochran, W.G. 1977. Sampling Techniques. Third ed. John Wiley and Sons, Inc., New
York. 611 pp.
Cochran, W.G. and G.M. Cox. 1957. Experimental Design. John Wiley and Sons. Inc.,
New York. 611 pp.
Green, R.H. 1979. Sampling Design and Statistical Methods for Environmental Biologists.
John Wiley and Sons. New York 25711.
Johnson, D.H. 1979. Estimating nest success: The Mayfield method and an alternative.
The Auk 96:651-661.
Otis, D.L., K.P. Bumham, G.C. White and D.R. Anderson. 1978. Statistical Inference
from Capture Data on Closed Animal Populations. Wildi. Monogr. No. 62. The
Wildlife Society, Washington, D.C. 135 pp.
Sokal, R.R. and F.J. Rohlf. 1981. Biomary: The Principles and Practice of Statistics in
Biological Research. Wit Freeman and Company, San Francisco. 859 pp.
Steel, R.G.D. and J.H. Tome. 1960. Principles and Procedures of Statistics. McGraw-
Hill, New York. 481 pp.
Wa]pole, RE. and RH. Myers. 1972. Probability and Statistics for Engineers and
Scientists. The Macmillan Company, New York. pp. 506.
METHODS, GENERAL
Best, R.G. 1982. Handbook of Remote Sensing in Fish and Wildlife Management. Remote
Sensing Institute, South Dakota State University, Brooking. South Dakota. 125 pp.
Blower, 1G., LM. Cook and IA. Bishop. 1981. Estimating the Sire of Animal
Popularion.c. George Allen and Unwin Limited, Boston. 128 pp.
Caughicy. G. 1977. Analysis of Vertebrate Populations. John Wiley and Sons, New
York. 234 pp.
Copen, D.E 1981. The Use of Multi van ate Statistics in Studies of Wildl fe Habitat.
USDA Forest Service. General Technical Report RM-87. 249 pp. Rocky Mountain
Forest and Range Experiment Station, Fort Collins, CO.
Davis, D.E. 1982. Handbook Cf Census Methods for Terrestrial Vertebrates. CRC Press,
Inc.
Eberhardt, L.L 1985. Assessing the dynamics of wild populations. I. WildL Manage.
49(4):997-1012.
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APPENDIX A SELEcii y REFERENCES
Eberhardt, L.L. 1978. Appraising variability in population studies. I. WildL Manage.
42(2): 207-237.
Erickson, PA. 1979. Environmental impact Assessmera: Principles and Application.c.
Academic Press, New York. 395 pp.
Gilmer, D.S., L.M. Cowardin, R i. Duval, L.M Mechlin, C.W. Shaiffer and V.B.
Kuechie. Procedures for the Use of Aircraft in Wildlife Biotelemetry Studies.
U.S.D.I. Fish and Wildlife Service Resource Publication 140. Washington, D.C.
19 pp.
Miller, H.W. and D.H. Johnson. 1978. Interpreting the results of nesting studies. I.
Wildi. Manage. 42(3): 471-476.
Pettingill, OS. 1970. Ornithology in Laboratory and Field. Burgess Publishing Company.
Minneapolis. 524 pp.
Ralph, C.J. and J.M. Scott. 1980. Estimating Number of Terrestrial Birds. Proceedings of
an International Symposium held at Asiomar, California, October 26-3 1, 1980. The
Cooper Ornithological Society. 630 pp.
Schemnirz, S.D. 1980. WiIdi(fe Techniques Manual. The Wildlife Society, Washington,
D.C. 686 pp.
Southwood, T.R.E. 1978. Ecological Methodr: With Particular Reftrence to the Study of
Insect Populations. Chapman and Hall, New York. 524 pp.
Young, E. 1975. The Capture and Care of Wild Animals. Ralph Curtis Books,
Hollywood, Florida. 224 pp.
MARK-RECAPTURE
Brownie, C., D.R. Anderson, K.P. Burnhani and D.S. Robson. 1985. Statistical Inftrence
from Band Recovery Data-A Handbook. USD1, Fish and Wildlife Service Resource
Publication No. 156. Washington, D.C. 305 pp.
Hanson, W.R. 1967. Estimating the density of an animal population. I. Res. Lepidoptera
6:203-247.
Nichols, ID. and K.H. Pollock. 1983. Estimation methodology in contemporary small
mammal capture-recapture studies. J. Mamm. 64(2):253-260.
Otis, D.L., K.P. Bumham, G.C. White and D.R. Anderson. 1978. Statistical inference
from capture data on closed animal populations. Wild!. Monogr. 62: 1-135.
Seber, G.A.F. 1982. The Estimation of Animal Abundance and Related Parameters.
Macmillan Publishing Co., Inc., New York. 654 pp.
White, G.C., DR. Anderson, K.P. Burnharn and DL. Otis. 1982. Capture-Recapture and
Removal Methodr for Sampling Closed Populations. Los Alamos National Laboratory,
Pubi. LA- 8787-1-NERP, Los Alamos, NM. 235 pp.
LINE TRANSECT
Best, L.B. 1981. Seasonal changes in detection of individual bird species. Studies in
Avian Biology. 6:252-261.
Burnham, K.P., D.R. Anderson and J.L Luke. 1985. Efficiency and bias in strip and
line transect sampling. J. Wild!. Manage. 49(4): 1012-1018.
36
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TERREST UAL FI LD STUDIES File. Turner. cook and Stunkard
Burnham, KP., DR. Anderson and IL Laake. 1980. Estimation of Density from Line
Transect Sampling of Biological Populations. Wildi. Monogr. No. 72.
Cram, B.R., K.P. Burnharn, D.R. Anderson and IL. Ti ik . 1978. A Fourier Series
Estimator of Population Density for Line Transect Sampling. Utah State University
Press. 25 pp.
Eberhardt. L.L. 1978. Transect methods for population studies. I. Wildi. Manage. 42(1):
1-31.
Mikol, S.A. 1980. Field Guidelines for Using Transects to Sample Non-Game Bird
Populations. U.S.D.I, Fish and Wildlife Service Pub. 80/58. Washington, D.C.
26 pp.
TERRITORY MAPPING
Best, L.B. 1975. Interpretational errors in the ‘mapping method” as a census technique.
The Auk. 92(3):452-460.
Edwards, P.J., S.M. Brown, M.R. Fletcher and P.L Stanley. 1979. The use of a bird
temtory mapping method for detecting mortality following pesticide application. Agru-
Ecosystems. 5:271-282.
International Bird Census Committee. 1970. Recommendations for an international
standard for a mapping method in bird census work. Bull. EcoL Res. Comnt, 9:49-52.
ANTICHOLINESTERASE
Grue, C.E., G.V.N. Powell and C.H. Corsuch. 1982. Assessing effects of
organophosphates on songbirds: comparison of a captive and a free-living population.
J. WildI. Manage. 46(3);766-768.
Hill, E.F. and WI. Fleming. 1982. Anticholinesterase poisoning of birds: field monitoring
and diagnosis of acute poisoning. I. Environ. Toxicol. Chem. 1:27-38.
37
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APPENDIX B
SUGGESTED COMPONENTS OF A FIELD STUDY PROTOCOL
for Submittal to EEB for Review.
Adapted from Ripley (1980)
I. Title
II. Problem Definition
A. A review and summary of the available information on the pesticide in
relation to nontarget hazard, including use information.
B. A precise statement of the goals and purpose of the study(ies)
(objective(s)).
C. A brief statement of the problem and the context in which it exists,
specifying the limits of the proposed work (Scope).
D. Precise statements of the major hypotheses to be tested.
L II. Methods and Materials
A. 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.
B. Descriptions
1. Identify the study area(s) selected and their general suitability for
achieving the objectives of the study or what criteria will be used to select
study areas.
2. Identify the species present or expected to be present on the study
area(s), discussing characteristics pertinent to the problem being evaluated.
3. State the research procedures, designs and sampling plans to be used.
a. Specify the kind and amount of data needed and to be sought.
b. Describe in detail how all data are to be obtained, including details
of application, instrumentation, equipment, sampling procedures. etc.
4. Describe how the data are to be treated, including specifying what
stad tics are to be calculated, what models will be used, what tests of data
will be used, etc.
. Describe in detail the methods to be used to check the sensitivity and
accuracy of the procedures used.
6. DescrIbe Quality Assurance procedures for application, instrumentation,
equipment and records.
7. Briefly describe the resources (people, facilities, etc.) to be. applied to
the study.
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APPENDIX C
CARCASS SEARCHES
DESIGN
In designing carcass searches, the following factors need to be known or determined:
- 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;
- Probability of finding (a) dead animal(s) 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);
- Size of the search area; and
- Number of carcasses found.
These factors can be combined in the following formula:
N=DREAP
where
N = number of carcasses found
D = density in animals/acre
R = proportion of carcasses remaining (nonremoval)
E = search efficiency
A = acres searched
P proportion of population killed
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.
The sensitivity of the carcass search approach is equivalent to the percent detectable
kill of the population. To determine the sensitivity, the formula is adjusted:
N
- bREA
Since P is a proportion:
percent detectable kill = P X 100 = D RE A X 100
Obviously, if any 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
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APPENDIX C CARCASS SEARCHES
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 denomini tor meaning that mortality can be detected only when a
high percentage of the population is killed. For example, in 5 acre fields with only 2
birds/acre and R and E estimated at a moderate 0.5, only an 80 percent 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.
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
searchers’ attention 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.
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.
ESTIMATING EFFICIENCY OF CARCASS SEARCH
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 species 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.
The number of caz asses placed should be approximately equal to 20 percent 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 pesticide-related if found. One
potential 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.
42
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TERRESTRIAL HELD STUDIES Fit& Turner. Cook and Stunkard
ESTIMATING CARCASS REMOVAL RATE
Carcass removal should be monitored to determine local variability in scavenger
activity. The density of both carcasses and scavengers can influence the rare 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 sca-
vengers. 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.
Carcasses planted in monitoring trials should simulate mortalities actually occurring
from the pesticide. in most cases, small to moderate sized species such as starlings or
blackbirds, or laboratozy 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 percent have been removed. Ideally, the number used should approx-
imate densities resulting from effects of the pesticide under study; however, in most
instances, this will not be known. Therefore, a density of approximately 20 percent of the
population of nontarget species on the area is recommended.
Timing of carcass removal trials should be such that they do not affect scavenger
removal of pesticide-killed birds or the feather-spots of the removed carcass could be
erroneously classified as a pesticide 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.
43
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APPENDIX D
EXAMPLES OF METHODS AVAILABLE FOR_INVESTIGATING
PARTICULAR, IDENTIFIED EFFECTS
Every field study must address specific concerns for a specific chemical in a specific
use pattern. lust as each chemical differs at least slightly from other chemicals, each field
study is likely to differ at least slightly from other field studies. It is impossible to
provide thorough directions or methodology that will apply to all pesticides, including
those yet to be developed. However, some of the kinds of information required in field
studies can be related to the types of’ concerns or effects that have been identified from
lower tier testing or other information. Table D-l provides a general list of methods that
axe most likely to be appropriate for addressing typical concerns. Following the table is a
discussion of methods for two chemicals. For some pesticides and use patterns, there
may be more than one kind of identified concern; in such situations, the field study
methodology should be able to address all of the identified concerns.
One critical aspect of field studies is not considered in the following discussions. The
kinds of techniques used for investigating effects are less important than the validity of
and within the methods for elucidating effects. including lower tier data. The use of
every conceivable technique is ineffectual, not to mention very costly, if sites are
inappropriate, application rates axe low, sampling design will allow only a low probability
of detecting an effect, exposure (or lack thereof) is not documented, etc.
A second critical aspect is subjective. How accurately can the investigator piedict the
results of the field study or its various aspects? Using acute mortality as an example, if
the investigator is nearly certain that field mortality will not occur (for whatever reasons),
then a screening study would be not only appropriate, but also cost-effective. Conversely,
if the investigator believes that there is a likelihood that mortality will occur above
concern levels, then a screening study may be a waste of time and money, except that it
might have utility as a baseline study for the forthcoming definitive study. Similarly, the
requirement for, or nature of, a field study may depend on the environmental
concentrations, especially in or on wildlife food resources. Although residue estimation
techniques have frequently been shown to be reasonably accurate, there are some situa-
tions where estimations are far from measured residues. If the investigator is genuinely
confident that actual residues axe far less than estimated, to the extent that a requirement
would be removed, then actual residue data should be obtained to provide a more cost-
effective measure of likely effects. But, as in the previous example, there is little point in
obtaining such data prior to a field study if the investigator predicts that actual residues
will be similar so estimated residues.
EXAMPLE 1
A cholinesterase inhibiting (“irreversible”) compound with the potential for causing
mortality quickly after ingestion of environmentally relevant amounts. Avian reproductive
tests show reduced productivity of young apparently as a result of parental toxicity, but
no evidence of impairment of other reproductive processes.
Discussion- If the reproductive effect levels are above the environmental levels, this
investigation would focus on acute mortality. Unless there are several documented kills
from normal use, a screening study would be the likely approach. Such a study would
be oriented towards both birds and mammals unless acute toxicity data indicate one of
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Table D-1.
Priority (1=highest priority) of field study methods for addressing different types of
concerns or effects.
Lethality 1
Sublethal 2
Method
Fast
Chronic
or
Delayed
Acute
Chronic
Resource
Loss 3
(Food or
Cover)
Environmental Residues 4 1
Nontarget Residues 4 2
Density and Diversity 3 1
1
1
2
1
1
2
1
1
3
1
1
Enzyme Analysis 2
Behavioral Observations 2
Nest and Nest Box Monitoring 3
1
1
2
1
1
1
1
1
1
2
1
Carcass Searching 1
Radio Telemetz 7 ° 2
Mark-recaptum 3
3
1
1
2
2
2
2
2
2
Adult: Young Ratios 3
Gross Histopathology
Resource Survey
2
2
2
2
2
2
1
Footnotes
‘Lethal responses may be fad (e.g., onset of’ mortality is less than I hour an labosatoty suulies) or delayed (onset 12 ins. or
more) or iermadiaie. The same techniques are useful, an general, for any type of lethal response arcept thai caitass
seaidung is most useful with fast-acting compounds Cc 1 boor onset) and decreases in utility with slower acting com-
pounds. Carcass marches am of questionable validity (with exception for unique situations), when the onset c i moctality is
greater mli ii 12 hours. Conversely, although radio telemetry and Inset-recapture techniques may be usefUl for a fast-amng
compound, they become increasingly useful whui lire time-to-toxicity is intermediate and essential [ or delayed or decmnc
lethal responses.
Defined as a resp to a magic or repeated application ci a pesticide that 1 .rbw a die fimma of a nomarget organism to
survive. mr imudiou or t young. Acote effects may render an orgnusmn more suscept i ble to predation, cause list
abandonmei* or spontarreous abortion. lmpafr die ability of edialts to feed their young, etc. Cheonic effects may be similar
an nature or Involve mote subtle ects on in eedsolve pcterulsl. but dwy are medfested more slowly.
Loss of aesoulcas, audi as nontargat di i provide food or cover for wildlife, typically does riot provide a basis
for ccsxtoodng field studir ’ However, in ansi cizoumatariom, paitioulady where large omtiguou emeaga Is treated, effects
on food or cover may be xcu,iiwi. .d mel may wanarat a field study.
As used an this table. asivhomn l mean residues in soil. water, wildhfe food resources. etc. N arget residues
are thom residues fcmmd In wildlife (either found deed or collected live) that may Indicate the toxic again onasing effears.
In some cases, mach as wheiethcse Is a for both primary arid secondary toxicity, the same alma! (e.g.. a mouse or
sparrow) may be pest of tire nontarget reskbe collection for armitthig cause of effects and also pen of the aitvucmmaiflal
residue collection as a food source for a predator. If existing chemical fate data are inadequate to amese changes in envi.
zunmerulal residues, sampling at several intervals may be vary usefuL
All field studies must provide some description ci the species. mimbera and nature of utilizedon of nontarget wildlife asso-
ciated with the study site(s). Such a descnption is essential duling the sate selection process to mau i. that the dudy can
provide useful Information. This entry on the table raises to density mad versuy estimates that am made during the al
study and may be fairly general (e.g.. [ or the purpose of determining the size of the carcass sesath ama) or fatty detailed
(e.g., when the estimates are used to compare changes an populations pie- and post-application or between treated areas end
controls).
‘Typically, as the usefulness of carcass searches foe ‘ag lethality decreases with longer time-to-toxicity for a
pesticide, the utility of radio telemneny and/or mark-recapture methods Increases. Mark-recapture end radio telemetry
normally will not both be used in the mane study. Mark-recapture is most useful with moderately common species with
low mobibty (e.g., small mammals). wbezma rariso telemehy u most useful for less common or mote mobile species (e.g.,
rapioca especially, bait also birds in esanz1 and mo ate size mammals).
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TERRESTRIAL FIELD STUDIES Fite. Turner. Cook and Stunkard
these taxa to be much less sensitive than the other. After sites are selected where there
is an adequate abundance and diversity of wildlife, appropriate techniques would include:
- Carcass searching for birds and mammals, cover permitting. If carcass
searching is not feasible for mammals or birds, mark-recapture or radio
telemetry are useful alternatives.
- Collection of environmental residues.
- Cholinesterase assays to assess cause of mortality, supplemented by
residue analysis if other cholinesterase inhibitors are used in the area.
- Density and diversity estimates for use in calculating search area size and
probability of detecting dead wildlife.
If there is a question about the environmental levels, relative to reproductive effect levels,
the collection of environmental residues during a screening study may permit an
assessment of the potential reproductive effects in the field. If reproductive effect levels
axe lower than environmental concentrations as determined either by data collection during
a screening study or through acceptable estimation techniques, then a definitive field study
would be appropriate for assessing such effects. However, different approaches would be
used depending, first, upon whether or not a screening study had been conducted and,
second, the results of the screening study.
EXAMPLE 2
A valid screening study showed no acute mortality. Concerns would be focused on
potential reproductive impairment in the field. Appropriate techniques should include:
- Additional environmental residues.
- Nontarget residues in live-collected wildlife.
- Oiolinesterase assay in collected wildlife.
- Behavioral observations, particularly related to reproductive and
nesting/parental behavior.
- Monitoring of nest/dens or artificial structures to evaluate productivity
relative to control sites.
- For mammals, evaluation of young-adult ratios relative to control sites
and/or pretreatment.
- Depending on the use pattern and nature of the test plots, radio telemetry
and/or mark-recapture techniques may be useful alternatives.
A valid screening study indicated greater than the concern level for mortality occurred
over a stated percent/sites. Concerns and techniques would be as above plus additional
techniques shbuld be employed to determine the extent and importance of acute mortality.
EXAMPLE 3
- Quantitative density and diversity methods for treated (including nearby
habitat) and control sites.
- Mark-recapture methods may be particularly useful for mammals.
47
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APPENDIX D Ecamp]es of Methods Available
- Radio telemetry has some disadvantages (primarily the number of
organisms required) for quantitative acute effects, but could be useful for
this purpose if already being used to investigate productivity parameters.
EXAMPLE 4
No screening study has been done. Environmental residues, either from actual data or
from acceptable estimation techniques. exceed both acute and reproductive effect levels.
If the actual existence of acute effects and the estimation of environmental residues are
questionable, a screening study may be useful, but, unless both residues are lower than
reproductive effect levels and no mortality is found, the screening study would have to be
followed by a definitive study. Unless the investigator was quite confident that a
screening study would be “clean” on both counts. it would be quicker and more cost-
effective to proceed directly to a definitive study. Since a definitive study for assessing
reproductive effects is nearly always a multi-year study, the assessment of acute effects in
the first year could be of the screening type. If effects above concern Levels axe found, a
more thorough assessment of acute effects may be made in the second and/or subsequent
years. Appropriate techniques for both acute and reproductive concerns have been listed
above. However, because both concerns would be investigated at the same time, the
investigator should consider carefully how these techniques can be combined in the most
efficient manner.
EXAMPLE 5
An anticoagulant rodenticide causes mortality after a delay of one to several days
(regardless of whether due to one or several feedings). In addition to nontarget mortality
from primary exposure, there is a concern for secondary toxicity to predators or
scavengers feeding on either dead or live target rodents.
Discussion- Anticoagulants frequently have quite different toxicity to different taxa of
wildlife. Concerns for secondary toxicity may be based on reasonable scenarios or on
known incidents of secondary poisoning and the concerns may be for a broad or narrow
array of secondary consumers. If concerns axe for one taxon (e.g., buteonid raptors or
wild canids) and are based upon potential, rather than known effects, laboratory studies on
secondary toxicity would be strongly recommended and should provide accurate
information on residues in primary consumers as well as toxicity to the secondary
consumer. Assuming that this laboratory study supports the potential for field effects and
provides dose-response information (or a NOEL), the residues in primary consumers
(equals secondary exposure levels) are important in interpreting any field results. It is
essential that any secondary toxicity field study include considerations of food habits of
the secondary consumer.
With regard to ptimary nontarget toxicity, it can be assumed that a rodenricide will
kill nontarget rodaits. and probably other nontarger mammals that ingest the toxicant.
Birds, as primary consumers, may or may not be particularly sensitive, but if extended
laboratory studies indicate they are, they should be Included in the field study design.
Except where the sensitivity of birds is equivocal, with respect to exposure, there is little
point in screening studies for a vertebrate toxicant; this example compound is designed to
kill rodents. Appropriate techniques for this example compound include:
- Behavioral observations, especially regarding food habits of consumers.
- Residues in target and nontarget primary consumers and possibly
secondary consumer.
48
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TERRES1RIAL FIELD STUDIES Fite.. Turner. Cook and Stunkard
- Mark-recapture for small nontarget mammals.
- Radio telemetry for secondary consumers, larger nontarget mammals and
birds; effects on birds could also be studied through density and diversity
(i.e.. census) methods or mark-recapture.
- Insofar as possible, target and nontarget carcasses should be collected for
analysis, but systematic carcass searches are of little use when mortality is
delayed.
49
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APPENDIX E
TERRESTRIAL FIELD STTJDIES,
WHEN ARE THEY REQUIRED?
GENERAL
The Agency utilizes a tiered system of ecological effects (usually toxicity) testing to
determine the potential risks of proposed pesticide uses to nontarget aquatic and terrestrial
organisms. These tests are outlined in various subdivisions of the Guidelines with
Subdivision E addressing the pesticide’s effects to birds, mammals and aquatic vertebrates
and invertebrates; Subdivision J addressing nontarget plant effects; and Subdivision L
addressing nontarget insect effects. However, the terresthal toxicity or adverse effects,
data usually available for risk assessments are as follows:
Tier 1
Mammalian Toxicity Data
Submitted in support of (human) toxicology data requirements (e.g., rat acute oral
W , acute dermal toxicity; 90-day t eding studies — rodent and nonrodent *‘s 81-I
through -7; 82-1 through -5; 83-1 through -4; 84-2 through -4; and § s 85-1, 85-2 and
86-1).
Avian Toxicity Data
Avian acute oral LD , (upland gamebird or waterfowl species) (*71-1); Avian dietary
LC , (upland gamebird) (*71-2); and Aviari dietary LC (waterfowl species) (*71-2).
Tier 2
Wild Mammal Toxicity Data
Generally, a dietary LCm or acute oral LD , study with a non-endangered
representative species that is likely to be exposed (*71-3).
Avian Reproductive Studies
Studies using upland gamebird and waterfowl species (*71-4).
Special Studies
Studies with specified avian or mammalian species such as nontarget mammalian
reproduction studies, avian acute dermal LI),, avian cholinesterase test. avian or
mammalian secondary toxicity (*70-1).
Tiers 3 and 4
Field Tests
Simulated and/or actual field testing with avian and/or mammalian species (*71-5).
Preceding page blank
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p ijrx E WHEN ARE STUDIES REOUIRED?
Test Species
The typical mammalian and avian indicator species used in the toxicity tests above are
the domestic rat, bobwhite quail, ring-necked pheasant and mallard duck. Other
organisms such as cottontail rabbits, voles and songbirds are sometimes used on a case-
by-case basis to address specific risks. Generally, those organisms, representative of areas
where pesticide applications are likely to occur, axe utilized (excepting endangered
species).
ECOLOGICALITERRESTRIAL RISK ASSESSMENT
in order to determine when terrestrial field studies are required to support a pesticide
use proposed for registration, the Agency must perform an ecological risk assessment.
This process is composed of two major areas: an aquatic risk assessment and a terrestrial
risk assessment. The Agency aLso assesses the risks to nontargec plants and to nonrarget
invertebrates (primarily, to beneficial insects such as honey bees). However, since the
aquatic and, especially, the terrestrial assessments are the major determinants of when
terrestrial field studies axe required, they will be discussed in detail here.
Components of Ecological Risk
The components of both the terrestrial and aquatic risk assessments can be presented as
follows:
Toxicological Environmental Ecological
Hazards X Exposure = Risks
or
Effects (Toxicity) Exposure Estimates of
Data X Data = Ecological Risks
Table E-l breaks this relationship down further to show the data and/or information
utilized.
As required by k1LKA, when the Agency performs an ecological risk assessment, it
performs the terrestrial and aquatic risk segments together. The terrestrial assessment has
the greatest impact on determining when terrestrial field studies are required, but the
aquatic segment is an important element that could show a need for such studies. For
example, if a pesticide use provided for adverse impacts on aquatic food sources and the
Agency estimated that such impacts could adversely affect nontarget terrestrial organisms,
then a terrestrial field tudy might be required.
A similar discussion, relative to adverse effects on wildlife habitat and terrestrial food
items (e.g., invertebrates such as earthworms, insects, slugs), can be presented. However,
although the Agency tries to address these areas in its ecological risk assessment, the EPA
focuses on the acute, subacute and/or chronic risks to mammals, birds and aquatic
vertebrates arid invertebrates via ingestion, dermal exposure, inhalation and/or aquatic
exposure. It does not usually address adverse effects via loss of habitat or from loss of
terrestrial food items unless endangered species are involved or catastrophic losses appear
52
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TERRESTRIAL FIELD STUDIES Fite. Turner. Cook and Stunkard
Table E-1.
The relationship between the
the type information utilized:
components of ecological risk can be broken down to show
Toxicological Hazard Data
X Exposure Data = Ecological Risks
- Laboratory eco-toxicity
data (e.g., LD ,, LC 1 ,
and NELs)
- Physical and - Integration of data into
chemical Agency statement of risk
properties for both endangered and
- Human Toxicity data (e.g.,
NELs, Chronic effects)
non-endangered species
- Chemical fate and
transport data
- Field data (sometimes
available) or, small
- Nontarget organism and
habitat information for both
pen avian and mammalian
endangered and non-endangered species
species
- Pesticide use information
- Pesticide incidents data
(e.g., avian field
- Pesticide residues (estimated and/or actual)
kills)
likely, based on Agency estimates or a body of data that shows that such losses are pos-
sible.
The Agency does this because the largest and often times best, effects data base
available is the toxicity/effects data for mammals, birds and aquatic vertebrates and
invertebrates. As the state of the an improves, however, EPA will perform more
ecosystem-level risk assessments utilizing the effects data for all ecosystem components.
The process that usually generates the requirement for a terrestrial field study is the
Agency s ecological risk assessment, especially its two major components the aquatic
assessment and the terrestrial assessment. Figure E-l is a schematic presentation of how
the Agency moves from this assessment process to the field study requirement.
The terrestrial risk segment of the ecological risk assessment is usually the major area
that “triggers” the requirement for a terrestrial field study. The terrestrial risk assessment
process examines the potential risks of pesticide uses to non-human, nontarget terrestrial
organisms; primarily, to nontargec mammals and avian species. Reptiles and amphibians
are not necessarily ignored. but it is assumed that when birds and mammals are “pro-
tected,” via Agency risk procedures and criteria, that some “protection” is afforded reptiles
and amphibians. Further, as the state of the art of toxicity testing develops, other
organisms, such as reptiles and amphibians. can be considered more accurately in the risk
assessment process (Urban and Cook, 1986).
53
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Figure E-1.
A Schematic Presentation of How EPA Moves from the Risk Assessment Process to Field
Study Requirements.
Terrestrial Risk Aquatic Risk
Assessment Assessment
Ecological Risk Assessment
Agency Statement Assessing Ecological Risk From
Uses Of Pesticide
Agency’s Regulatory Outputs:
- Require Additional Data
Terrestrial Field Study
- Require Restricted Use
C12csificanon to Reduce
Risk
- Require Pesticide Use Restrictions
On Label to Reduce Risk
- Initiate Special Review Based On
Risk Criteria
- Recommend For Registration
- Recommend Against Registration
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TERRESThIAL FIELD STUDIES Fite. Turner. Cook and Stu ajxj
EXPOSURE DATA
Under the exposure portion of the relationship, Toxicological Hazards X Environmental
Exposure = Ecological Risks, the Agency examines five areas:
Physical/Chemical Properties
The EPA requires that this information be submitted for all pesticides arid, generally.
the information of most value in the ecological risk process includes: color, physical stare,
melting point, vapor pressure, density or specific gravity, solubility, dissociation constant,
octanol/water partition coefficient. pH, molecular weight and chemical structure (Urban
and Cook, 1986). These data give the Agency an indication whether the pesticide is
highly soluble, volatilizes readily, is related in chemical structure to other pesticide
compounds, etc.
Chemical Fate/Transport Data
The data submitted to the Agency and typically utilized in the risk process Includes:
hydrolysis, photodegradation in water or on soil, metabolism studies, leaching potential.
field dissipation (residue decline curves, metabolites) and bioaccumulazion. Usmg these
data the EPA estimates which potential exposures are likely: acute, subacute, chronic
(reproductive) and/or secondary or, possibly, tertiary because of build-up in the food chain
(Urban and Cook, 1986).
Pesticide Use Information
Generally, much of the pesticide use information is submitted by the pesticide
applicant. However, the Agency also examines public literature that may provide
pertinent data (e.g.. the U.S. Depaitment of Agriculture’s Agricultural Statistics handbook)
on the proposed use. Information that is factored into the terrestrial risk assessment
process includes: the type of formulation (granular, wettable powder. flowable,
microencapsulated), type of application equipment (helicopter, plane, ground), crop
acreages to be treated, amount of pesticide to be applied (amount per acre, low volume,
high volume, ultralow volume (ULV)). timing of application (time of day, time of year),
number of applications per season, intervals between applications, use site(s), target
pest(s), inerts in the formulation and diluents, surfactants, adjuvants or stickera used, if
any.
Nontarget Organism/Habitat Information
The Agency, primarily through the use of its staff expertise, the public literature and
contacts with the U.S. Fish and Wildlife Service (USFWS), the Office of Endangered
Species (OES), State fish and game agencies, academicians and other experts in the field,
determines the nonrarger avian and mammalian species, including reptiles and amphibians,
when possible, are likely to be exposed. Both non-endangered and endangered organisms
are considered including: what species/habitats are exposed; what life stages are exposed;
for how long does exposure occur, whether exposure is acute, intermittent or chronic; and
what food sources may be contaminated. For federally listed endangered species the OES
or the National Marine Fisheries Service (NMFS) is contacted via informal and formal
consultation procedures. Documents, information and data are forwarded to OES (or
55
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ppENDIX E WHEN ARE STUDIES REOUIRED?
NMFS) during the formal consultation and the OES responds with a formal Biological
Opinion identifying those endanpred species likely to be affected by the pesticide use
pattern(s). This Opinion is reviewed by the EPA and recommendations, based on the
opinion and relative to the pesticide use(s). are developed by the Agency. It should be
noted that every attempt is made by the EPA to protect federally listed (and, when
possible, State listed) endangered species.
Actual Pesticide Residues
The Agency has access to up to four data bases for actual pesticide residues. These are:
Chemical FateiTrans port Data
Pesticide residues in the form of half-life estimates, actual measured residues and
residue decline curves are generally available in this data package. Such data are
submitted by the pesticide registrant and are reviewed and validated by the Agency. Said
data are heavily utilized in the ecological risk assessment process.
Residue Tolerance Data
Actual residue data are required by the Agency for pesticides used on crops that may
be consumed by humans and/or domestic animals (such as cattle) or on crops that may be
processed into human and/or cattle food or feed items. Also, residues in/on fish and
shellfish are required to support pesticides used in aquatic sites. These residue data,
however, are usually of limited utility to the ecological risk assessment process because
such data generally consist of residues determined at the time of crop harvest and for crop
items consumed by humans, but not necessarily by nontarget wildlife. These data are
developed for use in the human risk assessment process; however, when possible, the
Agency utilizes said information in its ecological risk assessment.
Residues In/On Wildlife Food Items
Occasionally, but rarely, data for pesticide residues in/on wildlife food items such as
insects, other invertebrates, seeds, pods, forage or nuts are submitted by the registrant to
the Agency prior to the determination that a terrestrial field study is required. However.
the Agency normally does not request such data until it determines that a terrestrial field
study is needed to assess the risks.
Public Literature
Whenever possible, the Agency nrili,-es actual pesticide residue data found in the
literature. Often, however, such data are lacking, particularly residues in/on pertinent
wildlife food items or they are not readily available because of time constraints in the
pesticide review process. Further, the previous three areas concerned residue data
collected and submitted by the pesticide registrant. For this area the registrant may or
may not, review the literature; it is not a requirement, but it Is recommended.
Summary
The ecological risk assessment usually utilizes the residue data generated with the
chemical fate and transport data since these residues are the most readily available. Such
data are used to determine the fate and transport of the pesticide. Unfortunately, said data
56
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TERRES1RIALFIELD STUDIES Fite. Turner. Cook and Sturikard
are of limited value relative to wildlife food items. To address residues on wildlife food,
the Agency develops estimates of residue levels. A discussion of how this is done is
presented below.
Estimated Residues
The estimated acute terrestrial residues utilized by the Agency are those shown in
Table E-2 and they are based primarily upon the works of Floerger and Kenaga (1972)
and Kenaga (1973). This residue profile provides a worst case scenario, that is, the
maximum expected residues likely to be found in or on vegetation and/or invertebrate
(Insect) surfaces immediately after application. This approach maximizes acute hazard
determinations because day-zero (the day of application) residues are utilized.
Table E-2.
Maximum Expected Residues and Typical Residues of Pesticides on Differing Categories
of Vegetation Types (from Hoerger and Kenaga,. 1972).
Residues (in ppm) for a Pesticide Dosage of 1 Lb/Acre
Plant Category
Immediately
Application
After
Six Weeks
Application
After
Upper
Limit
Typical
Limit
Upper
Limit
Typical
Limit
Range Grass
240
125
30
5
Grass
110
92
20
1-5
Leaves and Leafy Crops
125
35
20
<1
Forage Crops
(Small Insects)
58
33
1
<1
Pods Containing Seeds
12
3
1.5
<1
(Large Insects)
Grain (Large Insects)
10
3
1.5
<1
Fruit (Large Insects)
7
1.5
1.5
<0.2
The Agency considers this approach reasonable because; 1) in most instances actual
residue data are lacking, 2) the data presented by Hoerger and Kenaga (1972) appear to
correlate fairly well with those of other researchers, 3) the pesticides and crops considered
by the authors cover those reviewed routinely by the Agency and 4) as mentioned earlier,
the Agency makes every attempt to correlate these estimates with actual residue data on
pertinent wildlife food items (Urban and Cook, 1986).
In using this approach, the EPA:
- Uses the extrapolation proposed by Kenaga (1973) which is that residues
on insects can be estimated from residue data for plants, or plant parts, with
the same surface area to mass ratio as the insects in question. For small
insects the values for dense foliage (forage crops) axe used; for large insects
the values for pods, grain and even fruit can be utilized;
57
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APPENDIX E WHEN ARE STUDIES REQUIRED?
- Utilizes a quantity of pesticide per square foot approach for assessing the
risks of baits, seeds or granules to nontarget organisms (e.g., mg product/sq
ft or number of seeds, baits or granules/sq ft); and
- Recognizes that the upper limit residue values presented by Hoerger and
Kenaga (1972) are in terms of wet weight whereas most bird consumption
values for the avian dietary LC, 0 studies are presented in terms of dry
weight. (When appropriate and logical, the EPA uses a factor of three for
adjusting a dry-weight diet to an estimated wet-weight diet (e.g., LC , + 3)
as suggested by various authors.)
For chronic residues, the EPA would correlate the available chemical fate and transport
data with the acute terrestrial EECs in an attempt to obtain decline curves for appropriate
wildlife food items. Whenever possible, however, actual pesticide residue data would be
utilized.
TOXICOLOGICAL HAZARD DATA
General
The toxicological or effects data utilized in the hazard portion of the terrestrial risk
equation consist not only of the terrestrial toxicity data outlined previously, but also other
data that prove to be pertinent to the Agency’s terrestrial risk assessment. Such other data
include freshwater (and, depending on the pesticide use pattern, marine/estuanne)
vertebrate and Invertebrate toxicity nontarget beneficial insect effects data and
noncarget plant effects data. These data can be acute, subacute and/or long term or
chronic in nature; in most cases the data are developed in laboratory studies. For a full
listing of the types of toxicity or effects data that can be required by the Agency the
following should be consulted:
- The various subdivisions of the Pesticide Assessment Guidelines; and
- 40 CFR (158). Data Requirements for Pesticide Registration; Final Rule;
Wednesday, October 24, 1984.
It should be noted that the majority, if not almost all, of the toxicity/effects data
utilized by the EPA are developed and submitted by the pesticide registrant.
Consequently. the registrant has certain statutory tights concerning citation of these studies
and they cannot be used by other applicants without permission from or offering
compensation to the owner.
Study Reliability/Statistics
Without going into the actual study design of the different toxicity/effects studies, it
can be said that each study, whether short term (some are performed in 48 hours) or long
term and highly complex (some are performed over weeks, months or even years), is
critically reviewed by the Agency’s scientific staff. The study’s acceptability, relative to
good scientific practice and its ability to support the pesticide submission under Agency
consideration, are determined. Further, each study receives a statistical evaluation and,
typically, only those data with the best statistical reliability axe used in the ecological risk
assessment process. (Note that, if studies are determined to be totally unacceptable, they
are not used. Marginal studies may be used, but they are identified as such.) Also, the
Agency has developed and continues to develop, Standard Evaluation Procedures (SEPs)
for each kind of data that is required to support a pesticide submission. These SEPs
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TERRESTRIAL FIELD STUDIES Fite. Turner. Cook and Stunkard
present the procedures used to evaluate the toxicityfeffects data submitted and ensure that
comprehensive and consistent treatment of the science in the data reviews is maintained
by Agency staff (Urban and Cook, 1986).
INTEGRATION OF EXPOSURE AND TOXICOLOGICAL HAZARD
DATA
General
Obviously, the critical step in any risk assessment is the integration of exposure and
toxicological hazard data into a statement or conclusion concerning the risks to those
organisms of concern when exposed to the items under study (in this case, pesticides).
Generally, the reliability of the risk assessment is greater when the statistical reliability
and scientific accuracy of the available data is high. For non-human, nontarget organisms
the Agency makes every attempt to achieve such a desirable scenario. Unfortunately this
situation is often not obtained because, typically, the Agency only has available:
- A roxicityleffects data set that does not contain all of the required
terrestrial studies. These studies might include: one avian acute oral LD
study, two avian dietary LC studies and acute, subacute and/or chronic
studies with domestic mammals. Two avian reproduction studies may be
required in some use situations.
- A limited number of test species used in the laboratory studies: e.g.,
mallard duck, bobwhite quail, ring-necked pheasant, rat, mouse, dog, guinea
pig and rabbit.
- A limited number of data points. Generally, only five or six
dose/concentration levels are used in the acute studies to develop the LD 54 ,
or LC .
- Laboratory results from lower-tiered terrestrial effects studies that are
difficult to extrapolate to many field situations.
- Estimated Environmental Concentrations (EECs) rather than actual field
residue data for pesticides.
From this it can be seen that the Agency is often extrapolating from a situation of
limited information to a “real world” that has multiple species, animal populations and
endangered species that are sensitive to ecological perturbations. To perform such
extrapolations, a link is needed between the observed laboratory effects (or
pharmacological vulnerability) and the estimated field effects (or ecological vulnerability)
(Hudson et al., 1984). Terrestrial field studies serve as the link. They are studies
designed to derermirz what effects, if any, occur under actual pesticide use conditions. Jn
essence, the results of such studies either support or refute the Agency’s estimates of field
effects.
Specific Extrapolative Techniques
The actual integration process requires the Agency to carefully correlate the exposure
and toxicological hazard data discussed above. Terrestrial EECs, actual pesticide residue
data, the pesticide’s physical and chemical properties, pesticide use information, chemical
fate and transport data, and nontarget organism/habitat information are integrated with the
available laboratory (and, possibly, field or pesticide incidents data) mammalian, avian
59
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APPENDIX E WHEN ARE STUDIES REOUTRED?
and aquatic effects/toxicity data. Determinations on potential acute, subacute, secondary
and/or chronic risks axe developed for both nonendangered and endangered nontarget fish
and wildlife. Further, conclusions concerning: labeling, use restrictions, classification of
uses (e.g.. Restricted Use), the need for a Special Review, whether the product should be
registered or not and the need for further data (e.g.. terrestrial field study) are made. A
complete discussion of the Agency’s extrapolative techniques for determining what field
effects are likely based on effects observed in laboratory studies and using aquatic and
telTestlial EECs is presented in the EPA’s SEP. Ecological Risk Assessment. EPA54O/ 9-
85-001, June. 1986. Specifically, the terrestrial risk assessment procedures are presented
on pages 29 through 52. For convenience and brevity those techniques will not be
repeated here, but interested parties should consult that document.
Dose-Response Curves
An especially critical pan of the toxicological hazard data set the Agency uses in its
ecological risk assessment and for determining when terrestrial field studies axe required is
the dose-response curves developed for LD /LC% studies. In utilizing these curves, the
EPA critically examines the study design of each LD 0 /I C, study and performs a variety
of functions including: 1) recalculating and verifying the statistical results (using, for
example, Finney Probit), 2) examining closely the variability of the test results, particu-
larly the 95 percent confidence liniits for the LDJOILCSO values, 3) examining the observed
and expected results closely at the 100 percent, 50 percent and 0 percent response levels
and at the lowest effect level (LEL), 4) checking the slope of the dose-response curve and
5) noting the toxic symptoms and any sublethal responses that occur during the study
(AIBS. 1978). LD/LC values other than the LDSLC O may be developed, but with the
knowledge that: the most statistically precise value is the LD C/LCSO value; such extreme
values as LDII LC 10 or LD, ,/LC may not be accurate due to curvature of the dose-
response line; and specially designed studies are actually needed to determine accurately
such extreme values (Heath et al, 1972; Hill et al, 1975; Hudson et al., 1984).
The Agency also critically examines longer term dose-response curves in a similar
manner. At most, however, only three data points are available: a no effect level (NEL),
a low effect level (LEL) and a high effect level (HEL).
“TRIGGERS” THAT REQUIRE FIELD STUDIES
There are several specific conditions or criteria that “trigger” terrestrial field studies
requirement. Considering the above discussion, it can be seen that a flexible, weight-of-
evidence approach is used by the Agency to perform an ecological risk assessment and to
determine when terrestrial field studies are required. Many factors must be considered
and integrated in the process. However, it is still possible to identify those conditions or
criteria that must be met in order for a terrestrial field study to be required for a pesticide
proposed for a particular use pattern.
Toxicity
The pesticide is acutely or chronically toxic to birds or mammals as shown by
laboratory effects/toxicity studies. “Acutely” and “chronically” toxic are obviously relative
terms to be used with discretion. Generally speaking, certain classes of compounds are
considered to be highly toxic to groups of organisms whereas others are not. For
example. many organophosphates and carbamates are considered to be acutely toxic to
avian organisms. The Agency recognizes these chemical characteristics and, therefore,
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TERRE.SIRIAL FIELD STUDIES Fite. Turner. Cook and Stunkard
uses tbe tennis “acutely” and “chronicaliy” in a flexible manner with the belief that
specific criteria to distinguish between “acute” and “chronic” are inappropriate.
Exposure
Nontarget terrestrial organisms are likely to be exposed acutely and/or chronically to
pesticide residues under actual field conditions. Specifically, nontarget organism,
primarily avian and manrinalian species, must be present in or adjacent to the treated
areas. In essence, the likelihood of exposure for these organisms must be high.
EECs
Actual or estimated (terresthal EECs) pesticide residues are present in the nontarget
terrestrial organism’s environxuent and arc available to terrestrial organisms at levels equal
to or greater than the acute and/or chronic lowest effect levels (LELs) observed in the
laboratory effects/toxicity studies for birds and mammals.
Again, the Agency recognizes the limitations of using estimated residues, but does so
only when pertinent actual residue data are lacking. Relative to use of the LEL, the EPA
notes that other criteria (e.g., 1/5th or 1/10th of the LD or LC ) have been used in the
past in ecological risk assessiuents. However, in an attempt to be flexible and hopefully
to include chemicals that might be of potential concern, the Agency has chosen to use the
LEL. An extreme example, but one that supports this approach, would be a pesticide that
caused blindness in test birds or mammals at the LEL Obviously, the Agency would be
seriously concerned with the potential risks to birds and maznnrals in the wild.
Residues
When the amount or duration of pesticide residues (as described above) increases
relative to the acute and/or chronic effect levels observed in the laboratory effects/toxicity
studies for mammals and birds, the Agency’s ecological concerns increase and the
likelihood that a terrestrial field study is required increases also.
This criterion is more open-ended, but it correlates exposure data on actual or estimated
residues with toxicological hazard data.
Acute Risks or Concerns- Although the EPA has no specific “cut-off’ point (for an EEC,
actual residue value or toxicological effect) that can be presented here, the following can
be stated:
Is Terrestrial Field
Residue/Effect Level Study Required ?
Residue < NEL No
NEL
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APPENDTX F
WHEN ARE ST1TflTP PFnTrr rn
Generally speaking, the Agency has minimal concerns when actual or estimated
pesticide residues (that axe acute or of short duration) are below the LEL (as determined
in laboratory studies). As these residues increase relative to the LD , or LC values
determined in laboratory studies, the Agency’s ecological concerns increase and the
likelihood of requiring terrestrial field studies increases.
In utilizing these ratios of residues to effect levels, the Agency must closely examine
the acute dose-response curves developed in the laboratory acute effects/toxicity studies
for mammals and birds. As an example, the following three hypothetical acute dose-
response curves for A, B and C are presented to clarify the above and, particularly, the
situation when: LEL < Residue
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TERRESThIAL FIELD STUDIES Fite. Turner. Cook and Stunkard
Chronic Risks or Concerns
For chronic risks or concerns a terrestrial field study is generally required when the
pesticide residues (actual or estimated) equal or exceed the LEL. Because the LEL is
usually an effect on a reproductive parameter and the potential for adverse population
effects can be greater for chronic risks than for acute risks, the Agency is more conserva-
tive and requires the teriesulal field study in order to address the potential chronic or
reproductive risks. Also, mitigation of chronic risks by label use restrictions and/or
restricted use classification may not be as readily achieved as for acute risks.
Is Terrestrial Field
Residue / Effect Level Study Required?
Residue < NEL No
NEL
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APPENDIX F
PAIRED PLOT DESIGN
BASIS FOR FORMULA FOR n PAIRS
This section presents, in detail, the basis for the formula to determine the number of
blocks (pairs of plots) necessary to test the hypothesis that a pesticide has no short-term
effect on wildlife mortality.
Suppose we have n blocks, each with a treated plot and an untreated (control) plot.
Assume that the “true” number of “individuals” on the i control plot is C,, and that the
“true” number of “individuals” on the i treatment plot is T , , for i = 1, 2, ... , n. Also,
assume that T , = q C, for each block, but that C, need not be constant for all blocks.
The parameter q is the survival ratio, with its complement, p = l-q. being the short-term
mortality ratio. For the totality of blocks, define parameters:
=( C ,)+n and =(ZT,)+n,
the means of the abundance (density) parameters C, and T , respectively. Clearly,
T=qC.
Next, postulate that an observed count of the number of individuals on any plot is
subject to measurement error. That is, if c, and t, are the observations for the i block,
each is a single value from an infinite number of repeated, independent attempts to count
the number of individuals. Assume that possible values of c and t, follow independent
Poisson distributions such that the probability of observing a specific value for c (or t,)
is a function of the abundance parameter C ,(orT ,) for the plot’. We may say that the
variation of count for a plot is “locally Poisson.” The mean and variance of a Poisson
distribution are equal in value. For the totality of blocks, define the following statistics:
c=Zc, t=Lt ,, =c+n, and i=t+n.
From distribution theory, it is known that the distribution of a sum of independent
observations from different Poisson distributions is also a Poisson distribution with a
parameter equal to the sum of the parameters of the distributions whose observations are
summed. Proof of this assertion is available in Kendall and Stuart (1977, pp. 280-281),
and need not be presented here. Hence, c has a Poisson distribution with parameter n , r
hasonewithaparamcrofnq ,andw=c+thaSonewithaparametern (1+q).
At this point , we examine the conditional distribution of t, and also c, given a value for
w = c + t. Note that c, t, and w each have discrete distributions. Denote the following
probabilities:
P (C) - the probability of the event (value of) c,
P (t) - the probability of the event (value of) r,
‘If y has a Poiaaon dist 1butIon wub a parameter t, then tha probability thaty Is equal a value r may be pr ed as
P(y r)—i t’— -- r!
In this formula 1 a the base of the natural Iogsnthrns, and ri the facturial of r.
Preceding page blank
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APPENDIX F NUMBER OF BLOCKS NECESSARY
P (w) - the probability of the event (value of) w,
P (c,t) - the probability of the event c and:, and
P (tlw) - the probability of event t. given event w.
P (c.t) is known as a joint probability, while P (tlw) is a conditional probability.
Because of the assumption of independence of c 1 and t , events c and t are also
independent. Hence, P (c.t) = P (c)• P (t). By definition of a conditional probability,
P (tiw) = P (t,w) + p (w).
Since P (t,w) = P (c,t),
then P (tlw) = P (c,t) + P (w).
By substitution, we arrive at an intermediate result
w!
P(tlw)=
(1+q)w c! t!
This may be rewritten as
I w4
w!(q\/1 \
P(tlw)= 1— )(— )
c!t! \l+qJ\1+qJ
an expression that is readily recognized as the probability function for a binomial
distribution with parameters w and P = q + (1+q). We rewrite the equation as
fw\
P(tlw)=( )pt(lpy.s
\t /
and note that w-t is c.
Therefore, the conditional distribution of t, given w is a binomial distribution. The
limiting form of the binomial distribution with P near .5 and large w is a normal
distribution with an equal to wP, and variance equal to wP(l-P). This result suggests
that,bydeflningP=t+w,wemayobtainanestimatorofPthazisnormallydistributed
with mean P and variance equal to P(l-P) + w. This merely represents a simple linear
transformation of the conditional binomial distribution of t.
Suppose we test the null hypothesis H 0 : q=q, with a level of significance equal to a,
against an alternative hypothesis H 1 : q=q 1 , with power equal to 1-B, for q 1 < q 0 .
Equivalent hypotheses are j! P=P 0 , and H 1 : P=P 1 , with P 0 = q 1 -‘- (l+q 0 ) and P 1 = q 1 +
(1+q 1 ). A critical value of F, may be designated as to repi ent the point on the scale
of P that divides the scale into two d ecision regions; values of P < P correspond to a
decision to reject H 0 , those where > Pd to nonrejection of H 0 .
Now, = ( d-PO) + JP 0 (l-1’ ) + w and
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TERRESTRIAL FIELD STUDIES Fite. Turner. Cool and Stunkard
Z 14 = ( ,-P 1 ) ÷ /P 1 (l-P) + w
axe values of transformed to Z-scoies with respect to the distribution of 1’ under the
null and alternative hypotheses. In order to simplify our algebra, we replace P 0 (1-P 0 ) by
P 1 (l-P ). This replacement makes little difference for null and alternative values of P
relatively close to one another, and/or when w is relatively large with respect to P 0 (l-P 0 ).
Solving each of these equations for P , and setting the results equal to each other,
produces an equation for w, written as
P 1 (l-P 1 )
w = (Z 1 . + Z 1 .
(P 0 -P 1 ) 2
Since w = c + t = n ( + I), and on the average, = , andt= T = q , we replace w
by n (1+q 1 ). The choice of q 1 results in a slightly larger value of n, than if q 0 is
selected. Also, we substitute the following quantities:
P 0 = 112 (when q 0 =I), P 1 (1-P 1 ) = q 1 + (l+q 1 ) 3 , and
Al
(P P =
4 (l+q )’
Finally,
4q 1
n = + Z)’
Pi 2 (1+q 1 ) c
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