United States

Environmental Protection 

Agency

Office of Chemical Safety

and Pollution Prevention

(7101)

EPA 712-C-021

January 2012

Ecological Effects

Test Guidelines

OCSPP 850.2500:

Field Testing for

Terrestrial Wildlife
i

NOTICE

This guideline is one of a series of test guidelines established by the United States 

Environmental Protection Agency’s Office of Chemical Safety and Pollution Prevention 

(OCSPP) for use in testing pesticides and chemical substances to develop data for 

submission to the Agency under the Toxic Substances Control Act (TSCA) (15 U.S.C. 2601, 

et seq.), the Federal Insecticide, Fungicide and Rodenticide Act (FIFRA) (7 U.S.C. 136, et 

seq.), and section 408 of the Federal Food, Drug and Cosmetic (FFDCA) (21 U.S.C. 346a). 

Prior to April 22, 2010, OCSPP was known as the Office of Prevention, Pesticides and Toxic 

Substances (OPPTS). To distinguish these guidelines from guidelines issued by other 

organizations, the numbering convention adopted in 1994 specifically included OPPTS as 

part of the guideline’s number. Any test guidelines developed after April 22, 2010 will use 

the new acronym (OCSPP) in their title.

The OCSPP harmonized test guidelines serve as a compendium of accepted scientific 

methodologies and protocols that are intended to provide data to inform regulatory decisions 

under TSCA, FIFRA, and/or FFDCA. This document provides guidance for conducting the 

test, and is also used by EPA, the public, and the companies that are subject to data 

submission requirements under TSCA, FIFRA, and/or the FFDCA. As a guidance 

document, these guidelines are not binding on either EPA or any outside parties, and the 

EPA may depart from the guidelines where circumstances warrant and without prior notice. 

At places in this guidance, the Agency uses the word “should.” In this guidance, the use of 

“should” with regard to an action means that the action is recommended rather than 

mandatory. The procedures contained in this guideline are strongly recommended for 

generating the data that are the subject of the guideline, but EPA recognizes that departures 

may be appropriate in specific situations. You may propose alternatives to the 

recommendations described in these guidelines, and the Agency will assess them for 

appropriateness on a case-by-case basis. 

For additional information about these test guidelines and to access these guidelines 

electronically, please go to http://www.epa.gov/ocspp and select “Test Methods & 

Guidelines” on the left side navigation menu. You may also access the guidelines in 

http://www.regulations.gov grouped by Series under Docket ID #s: EPA-HQ-OPPT-2009-

0150 through EPA-HQ-OPPT-2009-0159, and EPA-HQ-OPPT-2009-0576.
Page 1 of 46

OCSPP 850.2500: Field testing for terrestrial wildlife.

(a) Scope―

(1) Applicability. This guideline is intended to be used to help develop data to submit to 

EPA under the Toxic Substances Control Act (TSCA) (15 U.S.C. 2601, et seq.), the 

Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) (7 U.S.C. 136, et seq.), and 

the Federal Food, Drug, and Cosmetic Act (FFDCA) (21 U.S.C. 346a).

(2) Background. The source materials used in developing this harmonized OCSPP test 

guideline include OPP 71-5 Simulated and Actual Field Testing for Mammals and Birds 

(Pesticide Assessment Guidelines Subdivision E―Hazard Evaluation: Wildlife and 

Aquatic Organisms), and the Guidance Document for Conducting Terrestrial Field 

Studies.

(b) Purpose. This guideline describes factors to be considered in the design and conduct of field 

studies to develop data on the effects to terrestrial wildlife species from chemical substances and 

mixtures (“test chemicals” or “test substances”) subject to environmental effects test regulations. 

The Agency will use these and other data to assess the risk to terrestrial wildlife that these 

chemicals may present through environmental exposure. The purpose of the field study is either 

to provide quantification of the effects that would occur to individuals, populations, or 

communities of terrestrial wildlife or refute the assumption that risks will occur under conditions 

of actual use of the test substance (primary consideration for pesticides) or occur under the 

pattern of production, use, disposal, or accidental release of industrial chemicals in the terrestrial 

environment.

(c) Definitions. The definitions in OCSPP 850.2000 apply to this guideline. 

(d) General considerations―

(1) Summary of the test. The Agency uses terrestrial field studies to evaluate only those 

test substances in which significant questions on risks to nontarget wildlife exist. The 

test substance may be applied in a variety of ways; the selected method should support 

the specific study objective. The study is performed under natural conditions and in the 

environment in which the test substance would be either applied and/or disperses to under 

normal use practices for pesticides or would occur under the pattern of production, use, 

disposal, or accidental release for industrial chemicals. Specific objectives and 

associated qualitative and quantitative decision statements establishing measurement 

endpoints and their accuracy and precision should be provided as part of the study plan. 

Specific protocols should be developed as needed and submitted to the Agency for 

review prior to conduct of the study.

(2) General test guidance. In contrast to laboratory tests which are generally amenable 

to a high degree of standardization, field study protocols are more flexible reflecting the 

case-by-case nature of issues and decisions a given field study is designed to address. 

Additionally standardization of field studies is made difficult by variability in such 

factors as chemical mode-of-action, species density and diversity, and for pesticides 

differences in use pattern, crop type, and method of application. This guideline provides 

a general outline of factors to consider in the conduct of field studies; specific protocols 

should be developed and submitted to the Agency for review. Despite the variability
Page 2 of 46

among field studies, several key elements common to most field studies can be identified. 

This guideline was prepared to identify and discuss these elements as they pertain to 

terrestrial vertebrates, and to provide a better understanding of the purpose of such field 

studies. There are two types of field studies, screening and definitive. The type of field 

study conducted (screening or definitive) depends on the available data on the test 

chemical or substance in question and the terrestrial wildlife population and community

dynamics. Environmental and exposure conditions under which a field study is 

conducted should resemble the conditions likely to be encountered under actual use, 

production, disposal, or fate of the test substance or chemical. Pesticides should be 

applied to the site at the rate, frequency, and by the method specified on the label. The 

general guidance in the OCSPP 850.2000 guideline applies to this guideline, except as 

specifically noted herein.

(3) Environmental chemistry methods. Procedures and validity elements for 

independent laboratory validation of environmental chemistry methods used to generate 

data associated with this study can be found in OCSPP 850.6100. Elements of the 

original addendum as referenced in 40 CFR 158.630 for this purpose are now contained 

in OCSPP 850.6100. These procedures, if followed, would result in data that would 

generally be of scientific merit for the purposes described in 40 CFR 158.630.

(4) Screening field study. If the available effects data is limited to laboratory toxicity

data on a limited number of species, a screening field study may be appropriate to 

determine if hazards or risks extrapolated to individual animals, populations and 

communities from the laboratory data are occurring in the field and, if so, to what species 

before conducting a definitive field study. “Pass-fail” methods are used to determine 

whether impacts occur. These methods may include carcass searching, residue analysis 

of species collected on study plots, residue analyses of wildlife food sources found in and 

adjacent to the area of application, behavioral observations, and enzyme analysis.

(5) Definitive field study. If a screening study indicates impacts are occurring, or if 

other available data suggest that deleterious effects have occurred or are extremely likely, 

the study design should be quantitative, evaluating the magnitude of the impacts in a 

definitive study. A quantitative field study focuses on the species affected in the 

screening phase. For some test substances or chemicals it may be appropriate to proceed 

directly to a definitive study without the screening phase. Careful consideration needs to 

be given to the likelihood of impacts occurring in order to determine which approach to 

use. At the quantitative level (definitive study), the objectives should include estimating 

the magnitude of acute or secondary mortality caused by the application, the existence 

and extent of reproductive effects, and the influence of chemical use on the survival of 

species of concern. Methods that can be used to address these objectives include markrecapture, radio telemetry, line transect sampling, nest monitoring, territory mapping, and 

measuring young to adult ratios.

(6) Endangered species. Studies should not be conducted in critical habitats or areas 

where endangered or threatened species could be exposed.
Page 3 of 46

(e) Test standards―

(1) Test substance. For industrial chemicals the substance to be tested should be 

technical grade unless the test is designed to test a specific formulation, mixture, or enduse product. For pesticides unless specified otherwise, data is derived from testing 

conducted with an end-use product. If the pesticide product is applied in a tank mixture, 

dosages of each active ingredient (a.i.) should be reported with identification and 

formulation for each product in the tank mix. The OCSPP 850.2000 guideline lists the 

type of information that should be known about the test substance before testing, and 

discusses methods for preparation for use in testing.

(2) Residue levels. When the test substance is applied under field condition testing, 

residues should be determined in selected tissues of organisms collected in and around 

the study area and in vegetation, soil, water, sediments, and other appropriate 

environmental components. If methods to analyze for the test substance are not 

available, the submitter should consult with the Agency before beginning the test.

(3) Sampling and experimental design. While examples of acceptable experimental 

designs are given, it is beyond the purpose of this guideline to cover the fundamentals of 

this topic. Paragraph (f) of this guideline provides a general outline for a field study

protocol to be submitted to the Agency for review. Paragraphs (e)(4) and (e)(5) of this 

guideline discuss points to be considered in designing screening and definitive field 

studies, respectively.

(i) A study designed to refute hazard is unusual in biological research. Typically, 

an investigator is more concerned about concluding with a high degree of 

confidence that an effect occurred, not that it failed to occur. FIFRA specifies 

that a pesticide is to be registered only if EPA determines it will not cause 

unreasonable adverse effects. The difference between an objective of “will 

cause” and “will not cause” substantially influences study design and the 

evaluation of data.

(ii) The adverse effects to wildlife that can result from the use of pesticides or 

under the pattern of production, use, disposal, or accidental release of industrial 

chemicals can be classified as those that affect populations of wildlife and those 

that affect individuals but not the entire population. Either of these effects may 

warrant regulatory action, including mitigation, cancellation or suspension of a 

pesticide use, or controls on production/use/disposal of an industrial chemical. 

An adverse effect that results in a reduction in local, regional, or national 

populations of wildlife species is clearly of great concern. A chemical that can 

repeatedly or frequently kill wildlife is also of concern even if these repeated kills 

may or may not affect long-term populations. The terrestrial field study, 

accordingly, is designed to assess both of these types of effects.

(iii) For pesticides, the field study should be designed to provide data that show 

whether wildlife species will not be affected significantly by a pesticide under 

normal use practices. For industrial chemicals produced, used, or disposed of in 

the terrestrial environment, these data are also sought. To achieve such objectives 

fully at the population level, it is necessary that detailed knowledge of the 
Page 4 of 46

population dynamics and varying environmental conditions for each species 

potentially at risk be available. The theoretical aspects of population dynamics 

are well documented in the literature. However, empirical data are available for 

only a few species (see paragraph (h)(8) of this guideline). A study designed to 

provide the needed data would include information on age structure, age-specific 

survival and reproductive rates, and the nature and form of intrinsic and extrinsic 

regulatory mechanisms. Such a study, when coupled with the influence of the 

chemical use (or production/use/disposal pattern) on these parameters, could 

require several years in order to give meaningful results.

(iv) The essential question is: How can these studies be performed in a practical, 

economical manner and still provide data that can show that the chemical under 

study will not reduce or limit wildlife populations or repeatedly kill wildlife?

(v) The question can be answered by examining the potential influence test 

substances can have on wildlife. These effects include:

(A) Direct poisoning and death by ingestion, dermal exposure, and/or 

inhalation.

(B) Sublethal toxic effects indirectly causing death by reducing resistance 

to other environmental stresses such as diseases, weather, or predators.

(C) Altered behavior such as abandonment of nests or young, change in 

parental care, or reduction in food consumption.

(D) Reduced food resources or alteration of habitat.

(E) Lowered productivity through fewer eggs laid, reduced litter size, or 

reduced fertility.

(vi) These effects can manifest themselves in a population through reduced 

survival and/or lower reproductive success. However, if a field study shows that 

actual use of a chemical does not affect survival and/or reproductive success or 

that only minor changes occur, it would seem reasonable to conclude that the use 

of the chemical will not significantly impact wildlife. Further, if a field study 

provides estimates on the magnitude of survival and reproductive effects, it is 

possible to make reasonable projections on the meaning of the effects to nontarget 

populations by using available information on the species of concern and basic 

theories of population dynamics. While less than ideal, field studies that collect 

information on survival and reproductive effects and use these data to address 

population parameters should provide a reasonable basis for evaluating potential 

impacts. This is not to imply that effects on populations are the only concern, 

however, a study adequate to assess these effects will also assess the degree of 

risk to individual wildlife.

(vii) This guideline emphasizes avian and mammalian wildlife. The Agency is 

also concerned about other terrestrial organisms such as nontarget plants, 

invertebrates, amphibians, and reptiles. Plants and invertebrates are excluded 
Page 5 of 46

here from direct study, except as sources of food or pesticides to wildlife. Testing 

guidelines for nontarget insects and plants are in Groups C and D, respectively, of 

the OCSPP 850 guideline series. Established protocols, especially for acute and 

chronic toxicity testing, are available for birds and mammals, but not for reptiles 

and are limited for amphibians. Occasionally, however, it may be necessary to 

adapt these field techniques to apply specifically to reptiles and/or amphibians.

(4) Screening study―

(i) Objective and scope. 

(A) The screening study is designed primarily to demonstrate that the 

hazard suggested by lower tier laboratory or pen studies does not exist 

under actual use conditions (primary consideration for pesticides) or occur 

under the pattern of production, use, disposal, or accidental release of 

industrial chemicals. The interpretations of screening study results, in 

most cases, are limited to “effect” versus “no-effect” determinations. If 

the study indicates that the chemical has caused little or no detectable 

adverse effect, it may be reasonable to conclude that potential adverse 

effects are minor. When effects are demonstrated, determining the 

magnitude of the effects is done with additional testing (see paragraph 

(e)(5) of this guideline). Therefore, when information already available 

shows that a chemical has caused adverse effects under normal use 

conditions (primary consideration for pesticides) or under the pattern of 

production, use, disposal, or accidental release of industrial chemicals, the 

screening study may be of limited value. It may be appropriate to proceed 

directly to a definitive field study in cases where analysis of laboratory or 

other data strongly suggest that adverse effects are likely to occur, and are 

unlikely to be attenuated by field conditions.

(B) Screening studies are limited to addressing the potential for acute toxic 

effects, such as direct poisoning and death, and sublethal toxic effects 

potentially affecting behavior and/or survival. In most instances, a 

screening study would not address reproductive effects or effects such as 

changes in density or diversity of populations.

(C) Further laboratory and/or pen studies may be useful prior to 

proceeding to the field, or may be necessary to interpret results of the field 

study. For example, additional toxicity data on species that are expected 

to be exposed from the normal use pattern (primary consideration for 

pesticides) or under the pattern of production, use, disposal, or accidental 

release of industrial chemicals may indicate which species are more 

susceptible to the chemical, allowing the study to be designed to monitor 

those species in greater depth as well as to provide insight into field results 

that show that some species were affected more than others, and additional 

laboratory studies may be unavoidable. If residue concentrations in 

resident species are being used to indicate potential problems, the 

relationship between tissue levels and the doses that cause adverse effects 
Page 6 of 46

is estimated. If secondary poisoning is of concern, feeding secondary 

consumers (held in captivity) prey items collected in the field following 

application of the test substance can be useful to evaluate this potential 

exposure route. Laboratory toxicity tests for secondary consumers 

coupled with residue analysis of prey items can indicate the potential for 

secondary poisoning of nontarget species. In designing field studies, the 

utility of laboratory and/or pen tests should not be neglected, and where 

appropriate their use is encouraged.

(ii) Geographic area selection. 

(A) Studies should be performed in representative biogeographic areas 

where the chemical could be used (primary consideration for pesticides) or 

occur under the pattern of production, use, disposal, or accidental release 

of industrial chemicals taking into account the diversity and variability in 

wildlife species and habitats involved. To keep the number of geographic 

areas at a manageable level while still accomplishing the purpose of the 

field study, geographical area selection should emphasize situations likely 

to present the greatest risk.

(B) A careful review of the species and habitats in the various 

geographical areas where the pesticide product could be used or the 

industrial chemical occur under the pattern of production, use, disposal, or 

accidental release is necessary to identify the areas of highest concern. A

sound understanding of the biology of the species that are found in 

association with the potential pesticide or industrial chemical exposure 

sites is essential. Identifying these areas may require an extensive 

literature review and consultation with experts familiar with the areas and 

species of concern. The study area selected should be frequented by those 

species that would have high exposure, based on their feeding or other 

behavioral aspects. If exposure and fate (e.g., degradation) parameters 

vary geographically, study area selection also should be biased towards 

maximizing residues available to wildlife. In some circumstances 

preliminary monitoring of candidate areas may be appropriate to 

determine which ones should be selected for detailed study.

(iii) Study site selection. 

(A) In general, study sites should be selected from what is considered to 

be a typical or representative environment in which the chemical would be 

either applied and/or disperses to under normal production/use/disposal 

practices, but at the same time, study sites should contain the widest 

possible diversity and density of wildlife species. Identifying potential 

study sites may require consultation with experts familiar with the areas 

where studies are proposed, and preliminary sampling. 

(B) To maximize the hazard, the sites selected should have associated 

species that would be at highest risk from exposure, as well as a good 

diversity of species to serve as indicators for other species not present at 
Page 7 of 46

that specific location. The choice of study sites that are as similar as 

possible in terms of abundance, diversity, and associated habitat will 

facilitate an analysis of the results.

(C) Field surveys of a number of sites are used to identify which sites 

should be selected for detailed study. Even when high risk species can be 

identified, preliminary surveys may be needed to determine which sites 

have adequate numbers of the high risk species as well as a good diversity 

of other species.

(D) In the initial evaluation of potential study sites, edge effect may

indicate which sites support the larger and more varied wildlife 

populations. As stated by Aldo Leopold (see paragraph (h)(23) of this 

guideline), “The potential density of game of low radius requiring two or 

more types is, within ordinary limits, proportional to the sum of the type 

peripheries.” (Type is defined as the various segments of an animal's 

environment used for food, cover, or other requirements.) If study sites 

are selected to maximize edge effect the potential for high density and 

diversity should be increased. One quantitative measure of edge and edge 

effect (see paragraph (h)(15) of this guideline) is the distances around 

individual plant communities in relation to the unit area of the community. 

Population densities, in general, are positively related to the number of 

feet of edge per unit area of community. Study sites chosen to maximize 

the ratio of edge to core may serve to indicate sites with higher densities 

and diversities of wildlife species.

(E) While this ratio can be helpful in selecting study sites, the other 

characteristics of edge should not be neglected in screening potential study 

sites. Density and diversity of wildlife species are also influenced by the 

variety in the composition and arrangement of the edge component cover 

types and its width. Also, interspersion (the plant types and their 

association with one another) influences densities of wildlife species. The 

edge effect is the sum of all the characteristics of edge and hence each 

component needs to be considered. An agricultural field with a relative 

high edge to core ratio may not have as high a density and diversity as one 

with a lower ratio but greater variety, width, and interspersion. In general, 

edge characteristics can be used to screen potential study sites; however, 

preliminary sampling of prospective study sites will be needed to identify

study sites with adequate density and diversity of wildlife species.

(iv) Number of sites. 

(A) The number of sites needed can be estimated using the binomial 

theorem. Briefly, the rationale is that for each study site there are two 

possible outcomes, either effect or no-effect. Trials of this type are known 

as binomial trials and, when repeated, the results will approximate a 

binomial distribution. In this case, to use the binomial theorem, it is 

necessary first to define the expected probabilities that birds or mammals 
Page 8 of 46

on a site are affected or not affected, after which the probability of the 

discrete binomial random variable x for n replications can be used to 

determine the minimum number of sites at a certain level of significance.

(B) For purposes of illustration, a problem exists if some specific mortality 

rate or level of some other variable occurs on more than 20 percent (20%) 

of the potential application sites. Translated into binomial probabilities, 

there is a 0.2 probability of a site showing an effect and a 0.8 probability 

of a site not showing an effect. Therefore, if the results from the field trial 

show that the number of sites affected is significantly lower than 0.2, it 

can be concluded that potential impacts will be below the stated level of 

concern.

(C) To calculate the minimum number of sites necessary to show a 

significant difference between the observed and expected, Equation 1 for 

the probability of the binomial random variable x can be used (see 

paragraph (h)(37) of this guideline) with rearrangement.

x n x

p q

x

n P x













( )  Equation 1

where:

x = number of sites showing effects,

n = number of sites, 

p = probability of a site showing an effect, and

q = probability of a site not showing an effect.

(D) For the illustration in paragraph (e)(4)(iv)(B) with the probability of x

= 0, no sites showing effects, set at the desired alpha level (i.e., P(x = 0) = 

α), Equations 2 through 5 show the sequential steps in rearranging and 

solving Equation 1 for n.

n

q

n P x 









  

0

( 0) Equation 2

n   q Equation 3

log  nlog q Equation 4



















q

n

log

log

Equation 5
Page 9 of 46

(E) The minimum number of sites can be determined using Equation 5. 

Continuing with the discussion example of 20% occurrence of an effect 

(i.e., a 0.2 probability of an affected site, a 0.8 probability of a nonaffected 

site) at an alpha (α) level of concern of 0.05 sample size would be 

calculated using Equation 5 as demonstrated in Equations 6 with a 

resulting n = 13.4.

13.4

log0.80

log 0.05

















n  Equation 6

(F) Therefore in the example provided, 14 is the minimum number of sites 

needed such that the probability is not greater than 0.05 that all sites 

surveyed would be unaffected. In other words, if 20% of the application 

sites are actually affected, there is only a 5% chance of finding all 14 sites 

unaffected when n = 14. Moreover, if 20% of the application sites are 

actually affected, we expect to find 1, 2, 3, and 4 sites affected with 

probabilities of 0.15, 0.25, 0.25, and 0.17, respectively, when n = 14.

(G) Under many circumstances, conducting this number of replications 

may not be practical. However, the number of sites can be reduced if site 

selection is biased toward hazard. While arguable, it seems logical that if 

the worst cases are sampled, a less stringent level of significance could be 

accepted. While this must be determined on a case-by-case basis, the 

Agency believes a minimum acceptable level of significance under worstcase conditions is 0.2 rather than 0.05 under average or normal use 

conditions. At this level, eight sites showing no effect would be required 

to conclude at the 0.2 level of significance that the effect occurred on less 

than 20% of the application sites or ― there is less than a 20% chance that 

all eight sites will be judged unaffected when n = 8 sites. Under some 

circumstances, this may not seem adequately protective. It should be 

noted, however, that based on this same design, it could be concluded that, 

at the 0.1 level of significance, effects occur on less than 30% of the 

application sites, and at the 0.05 level of significance, effects occur on less 

than 40% of the application sites. Hence, with eight sites, it could be 

concluded with a relatively high degree of confidence that effects would 

occur on less than 40% of the application sites. Because worst-case study 

sites were used, the Agency could have additional confidence that adverse 

effects would occur on less than 20% of all normal application sites.

(H) Under some circumstances, particularly if endangered species could 

be exposed from the proposed use, additional replication may be desirable. 

Under these conditions, a high degree of confidence that an effect was a 

rare occurrence would be required. (Under no circumstances should field 

studies on chemicals be conducted in areas where endangered species 

could be exposed.)

(I) The calculations under paragraphs (e)(4)(iv)(C) through (e)(4)(iv)(G) 

of this guideline are for when x = 0, no effects are observed on any site. A 
Page 10 of 46

similar approach can be used to estimate the number of sites necessary to 

show a significant result for a critical value of x > 0. Again the formula 

for the probability of the binomial random variable can be used summing 

the probabilities of x and all outcomes of less than x. Then by using 

increasing values of n, the number of replications required to show 

statistical significance may be determined for a given level of significance 

for individual x values.

 

x n x

r

x

p q

x

n P X r



 









  

0

Equation 7

(J) The minimum value of n in Equation 7 occurs when P(X < r) is set to 

the desired alpha level (i.e. P(X < r) = α level). Continuing the previous 

example, Table 1 gives the results for x < 1 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 are occurring below 

levels of concern is 14. If x < 2, 21 sites are needed in order to have an 

equivalent criterion. As can be seen, as x (the number of sites with 

effects) increases, the number of sites required to show a statistical 

significance becomes inordinately large.

Table 1.—Probabilities for Binomial Random Variable with p = 0.2 for x ≤ 1 and x ≤ 

2 as a Function of the Number of Sites (n).

n P(x < 1) P(x < 2)

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

(K) When the probability of an affected site is 0.2, application of the rule 

of zero-observed-affected-sites results in a declaration of “no-effect” 16.8 

and 13.4% of the time for samples of size 8 and 9, respectively. It also 

results in a declaration of “no-effect” 43.1 and 38.7% of the time for 

samples of size 8 and 9, respectively, when the probability of an affected 

site is 0.1, a value less than the criterion probability. Application of the 
Page 11 of 46

rule of zero-observed-affected-sites for a declaration of no-effect means 

that a study is considered to be negative (shows no effect) if, and only if, 

none of the sites show effects.

(L) Under any condition, it is extremely important with the binomial 

approach to define the critical or threshold level for an effect, and to be 

sure that the methods used are sensitive enough to detect an effect should 

one occur. These assessments depend upon the species potentially at risk 

as well as the parameter being sampled. It should be noted that the 

measure of effect is not limited to mortality. Other parameters, such as 

residue or enzyme levels, can be used. Whatever parameters are used, 

defining the criteria level for an effect is extremely important, and when 

designing studies this issue should be considered carefully.

(M) Using this approach, control (reference) sites are not an absolute 

necessity. While the Agency encourages their use, in some cases the 

additional information gained from the control sites for a screening study 

may not justify the additional effort required. In most instances, control 

sites would serve to protect from erroneously attributing effects due to

other causes to the chemical. However, for most chemicals, this can be 

avoided by employing methods, such as residue analysis and/or 

cholinesterase (ChE) inhibition tests, that can be used to indicate if the 

chemical contributed to the observed effect. Further, because studies have 

shown that it is a relatively rare event to locate dead or sick animals in the 

wild except under unusual conditions (see paragraph (h)(18) of this 

guideline), it is unlikely to find dead animals that were killed by

something other than the chemical being tested.

(N) Nevertheless, controls may be necessary when reliable methods to

confirm the cause of effect are not available. In these cases the binomial 

design can be modified to a paired-plot binomial design, with a treatment 

plot and a comparable control plot for each study site within an area. 

When critical levels of effect and occurrence are defined, the binomial 

theorem can be used for sample size determination, which gives 8 site 

pairs (16 paired plots) showing less than a defined difference between 

plots to conclude at the 0.2 level of significance that the effect occurred on 

less than 20% of the application sites. Alternatively, a quantitative 

difference or, preferably, ratio of treated to control responses could be 

used to test for a treatment effect on each of the measured response 

variables. (This is discussed further under paragraph (e)(5)(ii) of this 

guideline.)

(v) Size of study sites. For a satisfactory field study, study sites should be large 

enough to provide adequate samples. The size is dependent on the methods used, 

the sensitivity required, and the density and diversity of species and their ranges. 

In some cases, particularly with slow-acting poisons or where species at high risk 

have relatively large home ranges, areas several times larger than the treatment 

area may need to be examined. In some circumstances, several fields in an area 
Page 12 of 46

may be included in a single study site to account for wide-ranging species of 

lower densities. Except in the unusual circumstance where fields are extremely 

large (e.g., forested and range areas), the study site should never be less than an 

individual field and the surrounding area. The nature of the surrounding area is 

discussed further under individual methods. Another consideration is the distance 

between study sites--in general, sites should be separated adequately to ensure 

independence, which is dependent mainly on the range of the species that could 

be exposed.

(vi) Chemical application. 

(A) In general, the study conditions should resemble the conditions likely

to be encountered under actual use of the product (primary consideration 

for pesticides) or occur under the pattern of production, use, disposal, or 

accidental release of industrial chemicals. For pesticides, consideration 

should be given to application rates and methods and in most instances the 

pesticide should be applied at maximum use rates and frequencies 

specified on the label. If more than one application method is specified on 

the label, the method that maximizes exposure of nontarget species should 

be used.

(B) This evaluation should relate wildlife utilization of the area to 

exposure. For example, for pesticides if the crop is one that is used by

avian species as preferred nesting areas, feeding areas, or cover, ground 

application may be the method that maximizes exposure. However, if it is 

a crop with low utilization by wildlife species, but with high utilization of 

its edges, aerial application where drift could increase exposure may be 

more appropriate. In any case, the method of application used should be 

consistent with the label.

(C) Equipment used may influence potential exposure of nontarget 

species. In some instances, preliminary tests may be required to estimate 

which method and equipment poses the highest exposure. For pesticides 

there is a diversity of types of farming equipment that, depending on the 

particular use pattern involved, could influence exposure. For example, 

for pesticides applied in-furrow at planting there are several types of 

covering devices employed on seeders, such as drag chains, drag bars, 

scraper blades, steel presswheels, etc., in which the efficiency may vary 

for covering the pesticide. In general, the various equipment normally 

used for the particular pesticide application has to be evaluated to estimate 

the potential influence of equipment choice on exposure.

(vii) Methods. This section provides a general outline of methods appropriate for 

use in a screening field study and indicates some of their limitations. While 

methods described have been found to be most useful, a screening study is not 

limited to these methods. If other methods are more appropriate, their use is 

encouraged. Because procedures should be adapted to specific situations, the 

outlines presented should not be interpreted as strict protocols. Normally, 
Page 13 of 46

different methods will be combined to evaluate potential impacts. Due to the 

indefinite number of variables and the unpredictability of wild animals, even 

normally reliable procedures can sometimes prove inadequate. The methods used 

in a screening study address exposure by monitoring overt signs of toxicity such 

as mortality or behavioral modifications, or by evaluating parameters that indicate 

animals are under stress, such as residue concentrations in tissues or degree of 

enzyme inhibition. Measurements of density and diversity of species are needed 

to aid in evaluating the results. The methods in paragraph (e)(4)(vii)(A) through 

(e)(4)(vii)(F) of this guideline can be useful for screening studies.

(A) Carcass searches. 

(1) Searching for dead or moribund wildlife is a basic method used 

in field studies to evaluate the impact of chemicals on nontarget 

species. Carcass searches can roughly indicate the magnitude of 

kills when adequate areas are searched and the reliability of the 

search is documented. This latter point is extremely important. 

Rosene and Lay (under paragraph (h)(30) of this guideline) 

indicated that finding even a few dead animals suggests that there 

has been considerable mortality; failure to find carcasses is poor 

evidence that no mortality has occurred. The reliability of the 

search is based upon the percentage of carcasses recovered by 

searchers and the rate of disappearance. By knowing the 

reliability, the significance of failure to find carcasses can be 

assessed and the extent of the kill estimated.

(2) Finding dead animals is seldom easy, even if every animal on a 

site is killed. For example, three breeding pairs of small birds per 

acre is considered a large population (see paragraph (h)(18) of this 

guideline), and under average cover conditions, a small bird is 

difficult to detect. Small mammals may be more abundant but, due 

to their typically secretive habits, they are more likely to die under 

cover and can be even more difficult to find than birds. Carcass 

searching specifically for mammals should be attempted only when 

cover conditions permit reasonable search efficiency. However, 

any vertebrate carcasses found should be collected, even if the 

search is oriented primarily to one taxon.

(3) Because results may be biased by scavenging and failure to

find carcasses, the sensitivity of this procedure should be 

determined. Under conditions of heavy cover and/or high 

scavenger removal, other methods may be more appropriate.

(4) There are no standard procedures for carcass searches. 

Paragraph (e)(6) of this guideline outlines practices that have been 

used typically and should be considered in designing searches.
Page 14 of 46

(B) Radio telemetry. 

(1) Radio telemetry has been found to be extremely useful for 

monitoring mortality and other impacts caused by chemical 

exposure of wildlife. Advances in miniaturizing electronic 

equipment have made it feasible to track most vertebrate animals. 

Transmitters weighing a few grams have been used to track species 

as small as mice. Cochran's excellent summary of this technique 

provides additional details (see paragraph (h)(5) of this guideline).

(2) Radio telemetry has the advantages of providing information 

on the fate of individual animals following a chemical application 

and of facilitating carcass recovery for determining the cause of 

death. Although the initial cost of this technique may be more than 

for other methods, the increase in information obtained under some 

circumstances can more than justify the cost. The method is 

particularly useful with less common or wide-ranging species.

(3) In addition to mortality, radio telemetry can be used to monitor 

behavioral modification as well as physiological changes. 

Automatic radio-tracking systems permit continual surveillance of 

the location of animals (see paragraph (h)(5) of this guideline), 

which can be used to provide insight into behavioral changes such 

as nest abandonment, desertion of young, or decreases in activities 

such as flying or feeding. Radio telemetry equipment is also 

available for the transmission of physiological data such as heart 

rates or breathing rates (see paragraph (h)(25) of this guideline).

(4) While this technique can provide very useful information on 

impacts of chemicals to wildlife, other points need to be 

considered in addition to cost. Capturing animals alive and 

unharmed requires time, skill, and motivation. For the method to 

be consistently successful, the investigator must be thoroughly 

familiar with the habits of the species under study and with the 

various capture methods that can be used. Even for the most 

experienced investigator, adequate sample sizes can be difficult to 

obtain.

(5) Adequate sample size is very important. The binomial theorem 

can be used to estimate minimum sample size per site, if the 

question is limited to mortality. Briefly stated, to be sure that 

nontarget species are not being affected by environmental 

concentrations greater than, for example, an LC20, the expected 

binomial probabilities would be 0.2 for mortality and 0.8 for 

nonmortality. Depending on the level of significance, 8 (α = 0.2) 

to 14 (α = 0.05) individuals would need to be monitored per site 

(see paragraph (e)(4)(iv) of this guideline for further details on 

these calculations). However, since the LC20 may differ between 
Page 15 of 46

species, 8 to 14 individuals would be required for each species, 

unless laboratory tests have documented relative species 

sensitivity. Further complications can arise if radiotagged animals 

leave the area or if the movements of individuals limit their 

exposure. If these complications occur at relatively low rates, a 

few additional radio-tagged animals may be sufficient to overcome

these problems.

(C) Tests of Cholinesterase inhibition. 

(1) Measuring cholinesterase (ChE) concentrations in animal 

tissues has been found to be a very useful field technique for 

evaluating exposure of nontarget animals to ChE-inhibitingchemicals (see paragraphs (h)(18) and (h)(19) of this guideline). 

These chemicals, including organophosphates and carbamates, 

affect the synaptic transmission in the cholinergic parts of the 

nervous system by binding to the active site of acetylcholinesterase 

(AChE), which normally hydrolyzes the neuro-transmitter 

acetylcholine. Thus, ChE inhibitors permit excessive acetylcholine 

accumulation at synapses, thereby inhibiting the normal cessation 

of nerve impulses (see paragraphs (h)(6) and (h)(27) of this 

guideline).

(2) The depression of AChE activity, when measured and 

compared to controls, can indicate the degree to which an animal is 

affected. Brain ChE depression of >50% in birds has been found 

sufficient to assume that death is pesticide-related (see paragraph 

(h)(24) of this guideline). Depressions of more than 70% are often 

found in dead birds poisoned by these chemicals (see paragraphs 

(h)(1), (h)(2), and (h)(33) of this guideline), although some 

individual birds with less than 50% inhibition may die. A 20% 

depression of brain ChE has been suggested as an indication of 

exposure (see paragraph (h)(24) of this guideline). ChE 

concentrations in blood can also be used to indicate exposure, 

avoiding the necessity of sacrificing the animal. However, blood 

ChE concentrations are influenced more by environmental and 

physiological factors than are brain ChE concentrations. Because 

ChE activity varies among species, the degree of depression must 

be based on an estimated normal value for concurrently tested 

controls of the species potentially at risk. Because of this 

difference between species, each case must be considered unique 

(see paragraph (h)(19) of this guideline).

(3) Although there are several calorimetric methods for 

determining ChE activity, the general methods are similar. Brain 

tissues (or blood samples) are taken and analyzed for ChE 

concentrations. Comparisons are made between pre- and posttreatment and between treated and untreated areas. It is important 
Page 16 of 46

to ensure that untreated controls have not been exposed to any ChE 

inhibitors. It also should be noted that absolute enzyme levels in 

the literature are derived from various different, although similar, 

methods and are reported in different ways. For example, Ludke et 

al. (see paragraph (h)(24) of this guideline) used a modification of 

the method reported in paragraph (h)(13) of this guideline and 

reported results of ChE activity as nanomoles of acetylthiocholine 

iodide hydrolyzed per minute per milligram of protein, whereas 

Bunyan et al. (see paragraph (h)(1) of this guideline) used their 

own colorimetric method (in addition to a pH change method) and 

reported the results as micromoles of acetylcholine hydrolyzed per 

hour per milligram of protein. Therefore, without a tightly

standardized method, it is necessary to use concurrent controls of 

the same species obtained from the general vicinity (but untreated) 

of the exposed birds, rather than literature values. Because of the 

greater variation in plasma ChE levels than for brain, more 

controls are necessary to evaluate blood samples.

(4) Tests for ChE activity can be used to help confirm cause of 

death and monitor levels of exposure. In the latter case, 5 to 10 

individuals of each species are collected before treatment and at 

periodic intervals following treatment. Mean inhibition of 20% or 

more is considered an indication of exposure to a ChE inhibitor. 

Confirmation of cause of death may be determined by analyzing 

brain tissue from wildlife found dead following treatment and 

comparing the activity with controls. Inhibition of 50% or more is 

considered strongly presumptive evidence that mortality was 

caused by a ChE-inhibiting compound. The cause-effect 

relationship can be further supported by chemical analysis of the 

contents of the digestive tract or other tissues for the chemical in 

question.

(5) For this technique to provide accurate information, prompt 

collection and proper preservation of specimens are essential. ChE 

concentrations in tissues are influenced by time since death, 

ambient temperatures, and whether or not reversible ChE inhibitors 

are being investigated. Therefore, the response of postmortem 

brain ChE to ambient conditions can seriously affect diagnosis of 

antiChE poisoning. Samples must be collected shortly after death 

and frozen immediately to halt changes in tissue or enzymeinhibitor complexes. A technique for field monitoring and 

diagnosis of acute poisoning of avian species has been reviewed, 

discussing sample collection, sample numbers, preservation 

procedures, and sources of error (see paragraph (h)(19) of this 

guideline).
Page 17 of 46

(D) Residue analysis. 

(1) Residue analyses of wildlife food sources provide information 

about the level and duration of chemical exposure. Residue 

analysis of animal tissues also can indicate actual exposure levels. 

If the relationship between tissue concentrations and toxic effects 

is known for the species in question, residue analyses can provide a 

measure of the degree to which animals are affected. For this 

application of residue analyses, laboratory trials are necessary to 

establish the relationship between residue levels and toxicity. In 

addition to death, these laboratory trials should include such signs 

as anorexia, asthenia, asynergy, or ataxia. For chemicals that are 

readily metabolized by vertebrates, residue analysis may not be 

appropriate for diagnostic purposes. With many chemicals, it will 

be necessary to analyze for residues of active metabolites also.

(2) For determining residues on wildlife food sources, the 

investigator should collect samples of insects, seeds, leafy parts of 

plants, etc., immediately after chemical application and at periods 

thereafter. Samples should be analyzed for the chemical to 

determine potential exposure rate and duration. The application 

method needs to be considered in determining where to take 

samples. If drift is likely, samples should be taken from habitats 

surrounding the treatment sites as well as in the treated sites. 

Because analysis can be costly, the investigator should consider 

carefully the number of samples necessary to provide adequate 

data. Where feasible, samples from different locations within a 

site should not be pooled. Separate analysis of samples can 

provide data on the range and variability of exposure as well as 

mean levels.

(3) When residue analysis is used to evaluate exposure in nontarget 

animals, the tissues selected for analysis differ depending on the 

purpose. Heinz et al (see paragraph (h)(17) of this guideline) 

indicated that for many chemicals, residues in brains of birds and 

mammals can be used to determine if death is chemical-related. 

The authors believe that sublethal exposure is judged better from 

residues in other tissues. Therefore, they proposed that analyses of 

whole body homogenates should be used to quantify the body

burden of a chemical. If this is not feasible, analysis of muscle 

tissue is suggested, because muscle residues reflect body burden 

more nearly than those of any other tissue, and the amount of 

muscle tissue is not unduly large. For persistent chemicals, it has 

been suggested that residues in liver and fat tissues could be 

misleading for determining acute body burdens. Liver is a 

processing organ and its residue level largely represents current 

availability of the chemical. Residues in fat are greatly affected by

changes in the amount of body fat, and are not dependable 
Page 18 of 46

indicators of body burden of the chemical; however, for some 

chemicals, liver, fat, or other tissues may be good qualitative 

indicators that exposure did occur. In general, laboratory trials or 

data gathered in metabolism or other studies may be necessary to 

determine which tissues can provide the most useful information. 

Residue analysis of eggs taken from nests in treatment areas can 

indicate the degree of contamination that a treatment has caused, as 

well as possible reproductive effects of the treatment.

(4) Two approaches may be used to determine the number of 

samples to be collected. Frequently, residue samples will be 

collected to establish a mean value and confidence limits. To 

determine the number of samples to collect, it is necessary to 

estimate the standard deviation and to set an arbitrary limit from 

the mean value that is acceptable. Although the mean value does 

not need to be estimated, it is also necessary to have some idea of 

the mean so that the standard deviation can be estimated and the 

limit can be set. The formula for the number of samples for 95% 

probability, from the reference in paragraph (h)(34) of this 

guideline, is presented in Equation 8.

2

2

4

L

n   Equation 8

where:

σ = the standard deviation, and 

L = the allowable limit around the mean.

(5) For example, to calculate the residue concentrations on 

vegetation within ±10 ppm, with an estimate of the standard 

deviation of 20 ppm, then n = 4(20)2/(10)2 means 16 samples are 

required to have a 95% probability that the sample mean value will 

be within ±10 ppm of the true mean.

(6) In some situations, there may be little information useful for 

estimating the standard deviation, or the standard deviation may be 

rather large, thus requiring a very large sample size. For some 

types of samples, such as residues in nontarget wildlife carcasses, 

the sample size cannot be increased to permit more precision. The 

mean value of a parameter certainly has utility, but it also is very

important to establish confidence limits around the mean. In 

general, the Agency will use the 95% confidence limits (usually

the upper boundary, as in the case of residues) in the assessment of 

the data. This approach will substantially reduce the impact of 

outliers but will still incorporate the range of reasonable values 

into the assessment. In addition, the use of confidence limits 

reduces the necessity for taking a large number of samples. 
Page 19 of 46

Because the width of the confidence intervals decreases with 

increasing sample sizes, investigators should take samples that are 

as large as feasible.

(7) Since the sample size will nearly always be less than 30, the 

calculation of confidence limits should be based on Student's tdistribution. The t values are derived from tables available in most 

statistics books, and the 95% confidence limits are:

  













n

s x t

0.05 Equation 9

where 

s = the standard deviation estimated from the sample of 

size n.

(8) Alternatively, the binomial approach may be used for 

determining if residues, typically in collection of live nontarget 

animals, exceed a particular threshold value that indicates an 

effect. The required sample size is the same as presented for the 

binomial approach in determining the number of study sites. 

Specifically, in the preceding example a minimum of 8 samples 

with none exceeding the threshold value or 14 samples with one or 

none exceeding the threshold value indicates no effect at p = 0.2 in 

20% of the samples. This approach requires the establishment of 

threshold values which are determined on a case-by-case basis. In 

general, residues reflecting a 20% lethal concentration (LC20) level 

of exposure would seem to be a maximum acceptable effect 

concentration for a screening study. A median lethal concentration 

(LC50) should be determined in the laboratory for each species 

analyzed for residues, after which a group of animals would be 

exposed to an LC20 concentration to determine the mean threshold 

concentration of residues. Since this approach is impractical for a 

screening study, it is suggested that the mean residue concentration 

in bobwhite and/or mallards exposed to an LC20 dietary 

concentration would provide an indication of threshold levels.

(9) The number and timing of collection periods should be based 

on the persistence of the specific chemical under study. Where 

persistence in the field has not been adequately determined, it may 

be necessary to sample at regular intervals (e.g., days 0, 1, 3, 7, 14, 

28, 56) to provide data on degradation rates.

(E) Behavioral observations. Observations of behavior sometimes can 

be an extremely important indicator of treatment effects. Such 

observations might include characteristic signs of toxicity or behavioral 

changes seen in test animals exposed to the chemical in the laboratory. 
Page 20 of 46

Other abnormal behavior (e.g., territorial males abruptly ceasing singing, 

birds not feeding, reduced avoidance of humans) also may be important.

(F) Density and diversity estimates. 

(1) It is necessary to know the number of individuals and variety of 

species on and around a study site in order to indicate which 

species could have been exposed and to aid in evaluating the 

significance of mortalities or other findings. In addition, 

preliminary information on density and diversity is necessary for 

site selection and to determine the size of study sites. Under some 

circumstances, comparisons of density estimates between 

treatment and control sites, or between before and after treatments, 

may be used to indicate chemical impacts. In general, the 

usefulness of these comparisons is limited in a screening study due 

to the relatively small acreage involved. If mortality occurs, 

replacement from outside is likely to be so rapid that losses are 

replaced before censuses are completed. In addition it is necessary 

to consider seasonal changes, such as migration, molt, or 

incubation, which can affect real or apparent densities.

(2) Several techniques may be used to estimate the density and 

diversity of wildlife species, including counts of animal signs, 

catch-per-unit effort, mark-recapture, and line-transect sampling. 

Although the methods selected depend on the species of concern, 

for the screening field test line transect methods are likely to be the 

most useful for birds.

(3) The major advantage of line-transect sampling is that it is 

relatively easy to use in the field once a proper sample of lines has 

been chosen. However, line-transect-sampling is not applicable to 

all species, particularly those that are not easily observed. 

Individuals using line-transects must be extremely competent in 

species identification.

(4) In the line transect method an observer walks a distance (L) 

across an area in nonintersecting and non-overlapping lines, 

counting the number of animals sighted and/or heard (N), and 

recording one or more of the following statistics at the time of first 

observation:

(a) Radial distance from observer to animal.

(b) Right-angle distance from the animal sighted or heard 

to the path of the observer or angle of sighting from the 

observer's path to the point at which the animal was first 

sighted or heard.
Page 21 of 46

(5) Although the field procedures are simple, they must be 

understood adequately and implemented well to obtain good

estimates of density (see paragraph (h)(3) of this guideline). The 

authors provide a thorough review of the theory and design of linetransect-sampling and this monograph should be reviewed for 

details.

(6) For mammals, density and diversity estimates from capture 

data may be the most practical for a screening study. There are 

several ways of estimating the populations from capture data, some 

relatively simple, that may provide adequate information for a 

screening study. Davis and Winstead (see paragraph (h)(7) of this 

guideline), review the various methods available, explaining their 

advantages and disadvantages.

(viii) Interpretation of results. 

(A) The numerous variables involved in field studies makes a meaningful 

discussion of the interpretation of results somewhat tenuous, particularly 

with the almost inexhaustible array of results that could occur. Each study

must be considered unique and therefore will require a case-by-case 

analysis that incorporates not only the actual study but other relevant 

information that is available.

(B) In general, the results of the screening field study should provide 

information on acute poisoning and potential sublethal effects as 

suggested by enzyme, residue, or other measurements. In addition, 

information has to be developed on the density and diversity of species on 

the study sites as well as the sensitivity of the methods used. If no effects 

are detected, assuming that the methods used were adequate to detect 

levels of concern and that the species on the study site represent a good 

cross-section of the nontarget species expected to be at risk, the potential 

hazard indicated by lower tier tests is refuted. Unless other hazards (e.g., 

reproduction) are still of concern, additional tests would not normally be 

necessary. However, if an effect is detected on one or more study sites at 

rates equal to or greater than concern levels, the hazard has not been 

refuted and additional tests may be necessary. 

(C) In interpreting if an effect has occurred in the context of the binomial 

approach, care must be employed not to assume a level of precision in 

results that does not exist. Most detectable effects will exceed the concern 

level for some methods used in these studies, due to the inherent 

variability in the data collected to estimate the level of impact, particularly 

when minimum sample sizes and areas are used. In some instances in 

interpreting results it may be appropriate to use confidence limits of data 

collected (or another measure of dispersion) to evaluate if concern levels 

are exceeded. For example, when density estimates are used to estimate 

percent mortality using the number of dead animals found during carcass 
Page 22 of 46

searching, the upper and lower confidence limits of the density estimate 

may be more appropriate than the average, particularly when variability of 

the density estimate is high. Whatever method is used, when effects are 

detected that exceed concern levels they will be put into perspective in the 

context of the entire study as well as other available information to 

determine if or what additional data are needed. A “no-pass” result does 

not necessarily mean that definitive field testing is automatically required.

(D) For example, a test may be run in an area where a species is abundant, 

yet on a specific study site their numbers may be sufficiently small that a 

single death exceeds the level of concern on that site. Statistically, such a 

finding would indicate that the study did not pass according to the 

binomial approach, and this would be the preliminary interpretation. 

However, if the other sites had an adequate number of this species as well 

as other species expected to be at risk and no other signs of impacts are 

observed, the implications of the mortality would seem minor. On the 

other hand, if diversity of species were extremely limited, it would have 

greater significance. In other situations where the one dead bird is of a 

species with small numbers on most sites, but density and diversity of 

other species is representative of nontargets expected to be at risk, another 

screening study that looks at the species in which the effect was detected 

may be appropriate. Conversely, a screening study showing that there is 

appreciable mortality on most study sites may be sufficient for the Agency 

to consider regulatory action.

(E) In summary, the interpretation of results will go beyond the statistical 

evaluation since the Agency must consider all the factors and 

circumstances peculiar to each test and site. The biological interpretation 

of results is, and probably always will be, a matter of scientific judgment 

based upon the best available data. In general, the judgmental aspects of 

biological interpretation are more important for definitive studies than for 

screening studies. Nevertheless, biological considerations often will be 

relevant to screening studies. Study conclusions must integrate that which 

is biologically significant with that which is statistically significant.

(F) Another consideration in the interpretation of results of a field study is 

the attribution of effects to the chemical being studied. A well-designed 

study will include appropriate techniques to determine if an effect is 

caused by a chemical. In the absence of such techniques, the Agency has 

no choice but to consider that any effects were as a result of the chemical 

applied. As an example, measurement of ChE levels can provide 

information, since it is generally accepted that inhibition of 20% indicates 

exposure and inhibition of 50% or more indicates, in birds, that mortality

is due to an inhibitor (see paragraph (h)(4) of this guideline). If the test 

chemical is the only ChE inhibitor used in the vicinity of the study site, it 

can be reasonably assumed that mortality associated with 60% ChE 

inhibition is due to the test chemical. However, if other ChE inhibitors are 

used near the site, additional information, such as residue measurements, 
Page 23 of 46

may be necessary to attribute death to the specific ChE inhibitor being 

tested.

(5) Definitive study―

(i) Objective and scope. 

(A) The definitive study is a relatively detailed investigation designed to 

quantify the magnitude of impacts identified in a screening study or from 

other information. In contrast to the screening study, which monitors 

mainly the proportion of the local population that is expected to be 

exposed, the definitive field study examines a sample of the entire local 

population in the treated area. Although a definitive study may be 

performed when laboratory studies indicate a high potential for field 

mortality, it is more likely to be requested when there is evidence that 

actual field mortality has occurred, as in a screening study, or where 

reproductive effects are being investigated. The objectives of the 

definitive study are:

(1) To quantify the magnitude of acute mortality caused by the 

application. 

(2) To determine the existence and extent of reproductive 

impairment in nontarget species from the application.

(3) To determine the extent to which survival is influenced.

(B) Due to the intense effort and time required to estimate these 

parameters, the definitive study should be limited to one or a few species 

believed to be at the highest risk. If it can be shown that minimal (as 

defined at the onset of the study) or no changes occur in study parameters 

to high risk species, there is likely to be minimal potential for adversely 

affecting other presumably low risk species from use of the chemical in

question.

(C) The definitive study, in addition to estimating the magnitude of effects 

of acute toxicants, also can be applied to estimating the magnitude of 

chronic or reproductive effects. Although the discussion has emphasized 

chemicals that are acutely toxic, with few exceptions it is applicable to 

chemicals that cause chronic effects.

(D) In general, the definitive study will provide limited insight into 

whether or not effects are within the limits of compensation for the species 

of concern. However, using the data collected in these studies coupled 

with available information on the species of concern and basic theories of 

population dynamics, the meaning of the observed effects on the species 

can be evaluated.
Page 24 of 46

(ii) Sampling and experimental design. 

(A) The principles of statistical design of studies are well documented and 

it is beyond the scope of this guideline to cover the fundamentals of this 

topic. However, there are a few points on this topic that warrant 

discussion relative to the definitive study.

(B) In the design of field studies, it is necessary to consider carefully what 

constitutes a sampling unit. Eberhardt, under paragraph (h)(10) of this 

guideline, points out that special problems are faced in designing 

experiments on wild animal populations. Study sites must be large in 

order to limit the influence of boundary effects, such as movements into 

and out of the area. Large study sites can be very expensive both in terms 

of actually applying the experimental treatment and in the assessment of 

results. Eberhardt also states that numerous observations, even a full year 

of data, on a single study site may result in very sound values for that site, 

but do not provide a basis for inferences to other sites. Hurlbert (see 

paragraph (h)(19) of this guideline) has discussed the problems associated 

with field studies where there was no replication or replicates were not 

statistically independent, which he terms pseudoreplication. Of the field 

studies he evaluated, 48% of those applying inferential statistics had 

pseudoreplication.

(C) According to Eberhardt, under paragraph (h)(9) of this guideline, lack 

of replication seems to be based on the mistaken assumption that variances 

based on subsampling of sites (intrasite variability) are suitable bases for 

comparing treatment effects (intersite variability). This, he believes, is not 

a valid basis for a statistical test, because it is the variance of sites that are 

treated alike that is relevant to a test of treatment differences. Although 

subsampling of sites may be necessary to collect the data, it is the 

difference between sites that is important for analysis.

(D) An important point to consider in designing a definitive study is to be 

sure that the study will detect a substantial impact when, in fact, it occurs. 

In statistical terms this concept is referred to as the power of the test (1- β). 

Experience with classical experimental designs with random assignment 

of experimental treatment and controls, has shown that the probability of a 

Type II error (β, false acceptance error rate) is generally high (unless very 

large numbers of replicates are available). Eberhardt, under paragraph 

(h)(9) of this guideline, indicates that, all too often in field studies on 

impacts to wildlife, either by default or lack of understanding, there is only 

a 50% chance of detecting an effect, which he likens to settling the issue 

by flipping a coin and doing no field study whatsoever. Since a definitive 

study is carried out under the assumption that effects will occur, the 

Agency believes minimizing Type II errors is extremely important.

(E) As suggested, the more generally used experimental designs require 

inordinately large sample sizes to obtain small Type II errors. For 
Page 25 of 46

example, based on a coefficient of variation (cv) of 50% (this is a 

relatively homogeneous sample for the kinds of data collected in field 

studies), a 20% minimum detectable difference between means, a Type II 

error (β) of 0.2 and a Type I error (α) of 0.05, the number of replications 

required can be estimated using the formula in Equation 10 (in Eberhardt 

(see paragraph (h)(8) of this guideline)).

       

2

2 2 2

1 1 1 1



     



 

z z cv

n Equation 10

where:

n = number of replications;

zp = p

th percentile of the unit normal distribution (z1-α and z1-β are 

critical values of the unit normal distribution);

cv = coefficient of variation;

δ = detectable mean difference expressed as the proportion of the 

control group mean (i.e., δ = (μ1 - μ2)/(μ1)).

(F) Thus, for the example in paragraph (e)(5)(ii)(E), n = 63.6 replicates 

which rounds to 64 replicates for both control and treatment groups, or 

128 total study plots, are required to detect a 20% difference between 

treatments and controls with an 80% chance of being sure to detect a real 

difference (z1-0.20 = 0.84) at a 0.05 level of significance (z1-0.05 = 1.645).

       

 

2

2 2 2

0.2

1.65  0.84 0.5 1 1 0.2

n  Equation 11

(G) With more sophisticated designs, the number of replicates can be 

reduced under some circumstances and still meet the Agency's aim to limit 

the probability of a Type II error to 0.2 with a detectable difference of 20 

to 25%. For example, a paired plot design can be used, substantially 

reducing the number of replicates required. Pairing serves to reduce the 

effective coefficient of variation by reducing the variation attributable to 

experimental error. The lower coefficient of variation reduces the number 

of replicates. A quantitative difference or, preferably, a ratio of treated to 

the total of treated and control responses, can be analyzed statistically to 

test for a treatment effect on the measured response variables (see 

paragraph (h)(31) of this guideline). The logic of using paired plots is 

that, while no two areas are ever exactly alike, two areas that are not 

widely separated in space are ordinarily subjected to much the same 

climatic factors, have populations with about the same genetic makeup, 

and generally the two populations can be expected to follow much the 

same trend over time, apart from a chemical effect (see paragraph (h)(8) of 

this guideline). If all plots are approximately equal in area and habitat and 
Page 26 of 46

population densities between pairs are similar, it is postulated that when 

no chemical impacts occur, the mean ratio of treatment to treatment plus 

control will equal one-half. Then a t-test or an exact randomization test 

(see paragraph (h)(11) of this guideline) may be applied to test whether the 

average number of survivors on the treated plots is equal to the average 

number of survivors on controls.

(H) The number of pairs required can be estimated using the formula in 

Equation 12.

 

   



















   

p q c

q

n z z

1

4

2

2

1  1  Equation 12

where,

n = number of paired plots;

zp = p

th percentile of the unit normal distribution (z1-α and z1-β are 

critical z-scores);

q = survival ratio;

p = mortality ratio; and

c = mean number of survivors on control plots.

(I) Thus, 10 pairs of plots (20 total) with a mean of 28 individuals per plot 

would be needed at an 80% assurance of detecting a treatment-induced 

impact of 20% or greater at a 0.05 level of significance if c = 28. 

Increasing the mean number of individuals per plot (c) causes a reduction 

in n.

 

   

9.8

0.2 1 0.8 28

4 0.8

1.645 0.84 2

2





















n   Equation 13

(J) In some field situations, pairing may not be feasible. In these 

situations, other designs would be more appropriate or a less rigorous 

design may have to be used. However, in planning field studies, the 

power of the study design must be considered to determine the limitations 

of the study. Studies with adequate replication are highly preferred to 

support registration—the use of less replication will not necessarily render 

the study inadequate. However, it is wrong to use a study with low power 

to imply no biological damage, when the study is not capable of detecting 

the damage if it occurred. In cases where large numbers of replicates are 

impractical, subjective and biological knowledge should be used in a 

decision process to decide if there was a treatment effect. In most 

instances, it is highly advisable to involve statisticians or biometricians 
Page 27 of 46

who are familiar with this kind of field study in the planning and analysis 

phase of the field work to avoid costly technical errors.

(iii) Study area and site selection. Selection of geographical areas and study

sites within the areas for the definitive test generally requires the same 

considerations as for a screening study. For the definitive study, however, the 

selected areas and study sites should have adequate populations of the species of 

concern. For pesticides, the crop of concern should be grown on a representative 

portion of the area and consideration needs to be given to whether the target pest 

species will be present. If it is not, it is necessary to consider what influence its 

absence may have on potential results. For example, if the pest is a major food 

source for nontarget species, its absence could significantly influence results. 

Finally, the potential variation in populations of concern over the geographical 

areas selected should be considered. It may be difficult to find sites that are 

sufficiently similar to provide paired plots, which limits the coefficient of 

variation so that the desired sensitivity can be achieved.

(iv) Number and size of sites. As suggested under paragraph (e)(5)(ii) of this 

guideline, the number of sites will depend upon the species density on sites and 

the sensitivity required. Ideally, sample size should be large enough so there will 

be an 80% probability of being sure to detect a 20% difference when it exists. 

The size of the study site must be large enough to provide adequate samples. The 

size depends on the survey methods used, sensitivity required, and the density and 

range of the species of concern. For a paired-plot design, the number of sites 

required is a function of the average density of the species. In general, the 

breeding density of the species of concern can be used to provide a rough estimate 

of the size of area needed to provide adequate samples. However, preliminary 

sampling most likely will be required to verify the estimates.

(v) Methods. 

(A) Essentially, the methods used in a definitive study are a means to 

quantitate reproductive and mortality rates of animals on treatment and 

control areas. There are many texts and monographs available on methods 

of sampling to estimate these parameters. Anyone not familiar with the 

theory and principles of the various techniques should review these 

references in depth. The objective of this portion of the guideline is to 

provide a general guide to the various methods that could be used in a 

definitive field study. In addition, these methods can be applicable to some 

screening studies.

(B) The methods to be used in an individual field study will depend on the 

nature of the identified concerns. Some methods are useful for 

investigating several types of concerns, and most types of concerns can be 

studied by several methods. When the concern becomes more specific 

(e.g., secondary hazards to raptors), the use pattern or pattern of 

production, use, disposal, or accidental release is limited, and/or habitat 

type is limited, the range of applicable methods tends to become narrower.
Page 28 of 46

(C) Methods described below are divided into three categories: Methods 

for assessing mortality and survival of adults and independent juveniles, 

methods for assessing reproduction and survival of dependent juveniles, 

and ancillary methods. The intent of this guideline is to present methods 

that are likely to be useful in many situations, rather than an exhaustive list 

of all available methods. The Agency encourages the use of other 

methods when they are scientifically valid and have a high probability of 

detecting an effect.

(D) While it is absolutely essential to have a detailed plan that describes 

the selected actions (with contingencies) for achieving the study

objectives, investigators must remain flexible because unanticipated 

problems always come up in long term studies. Even with highly 

experienced and resourceful field biologists, the most carefully planned 

studies can be compromised due to the unpredictability of wild animals 

and natural events. When a natural disaster occurs early in the study, it 

may be wise to reinitiate the study. If the event occurs after substantial 

data already have been collected (e.g., early in the second year of a 

multiyear study), it may be more appropriate to extend the study an 

additional year or more to help provide for the additional needs. If the 

study is to be terminated, the report should describe thoroughly the nature 

of the events and the consequences if they affect the study results.

(vi) Mortality and survival. It is very important to understand the autecology of 

the species being studied in order to select the most appropriate methods for 

investigating them. In addition, the choice of particular methods must consider 

the applicability of the method based on the chemical use pattern and study site 

characteristics.

(A) Mark-Recapture. 

(1) There are several mark-recapture methods available, each 

based on the same basic premise. A sample of animals is captured, 

marked, released, and another sample is collected where some of 

the animals are captured again. The characteristics of this 

identifiable sample are used to estimate population parameters. 

Mark-recapture studies can provide information on:

(a) Size of the population.

(b) Age-specific fecundity rates.

(c) Age-specific mortality rates.

(d) Combined rates of birth and immigration.

(e) Combined rates of death and emigration.
Page 29 of 46

(2) Seber (see paragraph (h)(32) of this guideline) reviewed the 

various mark-recapture methods and subsequent statistical 

analyses. Less detailed but still very useful reviews are provided 

in paragraphs (h)(4) and (h)(15) of this guideline. Nichols and 

Pollock provide a valuable comparison of methods under 

paragraph (h)(26) of this guideline. Table 2 provides a brief 

summary of some of the various mark-recapture methods discussed 

in these references.

Table 2.—Mark-Recapture Techniques

Method Applications/Requirements/Assumptions

Peterson Method (Lincoln Index) Estimation of population size. Usually only two sampling periods. 

Closed population.

Schumacher's Method Estimation of population size. More than two sampling periods. 

Marking continues throughout sampling. Closed population.

Bailey's Triple Catch Estimate of birth rate and death rate in addition to population size. 

Requires data from two marking occasions and two recapturing 

occasions. Open population.

Jolly-Seber Method Estimates mortality and recruitment in addition to population size. 

Requires more than two sampling periods and that each animal's 

history of recapture be known. Open population.

(3) When considering the use of one of these mark-recapture 

models, one must carefully evaluate the applicability of the method 

to the circumstances under consideration. While in theory markrecapture techniques should be an excellent method for evaluating 

effects of chemicals on wildlife populations, some mark-recapture 

analyses are not particularly robust; small deviations from their 

implicit assumptions can produce large errors in the results (see 

paragraph (h)(4) of this guideline). However, some of the more 

recent and sophisticated analytical methods are robust and can deal 

with deviations from assumptions in closed populations (see 

paragraph (h)(28) of this guideline).

(4) Mark-recapture methods are particularly useful for small 

mammals because these animals are seldom amenable to the visual 

and auditory observations necessary for using transect, territory 

mapping, or similar methods. However, mark-recapture also may 

be useful for birds provided a sufficient number of birds can be 

captured and marked. In some situations, birds may be 

"recaptured" with use of binoculars via visual observations of 

marked individuals. Animals must remain marked for the duration 

of the study. Typically, mammals are toe-clipped or ear-marked 

and birds are banded. Marking should not make the animals more 

susceptible to the effects of the chemical (e.g., anticoagulants with 

toe clipping). Dyes may be useful unless they are lost by wear or 

molting.
Page 30 of 46

(B) Territory mapping method. 

(1) A common spatial census method is territory mapping, wherein 

the territories of individuals are mapped before and after treatment, 

on both treated and untreated plots. The method is usually 

applicable when birds are defending territories. It involves a series 

of census visits to the study sites during which birds located by

sight or song are recorded on a map. The information from all the 

visits is plotted for each species. Birds exhibiting territorial 

behavior appear on the map as clusters of individual contacts. The 

clusters are used to estimate both the size and number of territories. 

The pre- and post-treatment censuses for treated sites are compared 

with the pre- and post-treatment censuses for control sites to 

determine changes in populations of territorial individuals that may

be attributed to the chemical (see paragraph (h)(12) of this 

guideline). Further details of this method are given by the 

International Bird Census Committee under paragraph (h)(21) of 

this guideline, and its application to evaluating impact caused by

pesticides is reviewed by Edwards et al. (see paragraph (h)(12) of 

this guideline).

(2) Problems with this method can occur. Under some 

circumstances, replacement from outside the area can be so rapid 

that territories are refilled before the census is completed. There 

usually is a floating population of silent, nonterritorial birds who 

may quickly reoccupy empty territories (see paragraph (h)(35) of 

this guideline). The effects of replacement can be overcome for 

some species by capturing and marking the territorial individuals 

prior to treatment, so they can be distinguished from the floaters. 

Also, replacement may not be a problem when the study areas are 

in the center of a relatively large treated area.

(C) Radio telemetry. Radio telemetry can be an extremely useful 

technique to provide information on the effects of a chemical application 

on nontarget species. As discussed for screening studies, radio telemetry 

can be used to monitor for mortality as well as to provide useful 

information on behavioral modification caused by the chemical 

application. The points discussed previously (for screening studies) 

generally are applicable to definitive studies. However, for the definitive 

study, the number of radio-tagged animals needed depends upon the 

variation between sites and the sensitivity required. For example, with 

behavioral observation, intra- and intersite variation will influence the 

number of radio-tagged animals required. In some instances, it might not 

be practical to radio-tag the number of animals required to provide a 

rigorously designed study. Under these conditions, the limitations should 

be specified, and the maximum number of animals that can be practically 

radio-tagged and monitored should be used.
Page 31 of 46

(D) Other methods for mortality and survival. Other techniques for 

assessing density and diversity are discussed for screening studies; most of 

these, especially line-transect methods, are useful for definitive studies. 

Some methods, such as catch per unit effort or counts of animal signs, do 

not provide actual measures of density but may still be used to compare 

effects on treated and untreated plots.

(vii) Reproduction and survival of dependent young. Some of the techniques 

for assessing mortality and adult survival are also useful for assessing 

reproduction and survival of young. Some, but not all, mark-recapture methods 

can provide information on fecundity. Radio-tagging nestlings or suckling young 

of moderate and large size animals may be used to assess survival of dependent 

young. Radio telemetry and territory mapping are useful for locating dens or 

nests for further study. The following methods are more specific for assessing 

reproductive parameters.

(A) Nest monitoring. 

(1) Nest monitoring is useful for evaluating the effect of chemicals 

on breeding birds. The typical procedure is to search the study site 

to find active nests and subsequently to check those nests to 

determine their fate. Information collected on each nest should 

include number of eggs laid, number hatched, number of young 

fledged, and if and when the nest was abandoned or destroyed, 

both before and after chemical application. While all definitive 

studies should consider this technique, it also may be useful in 

screening studies.

(2) This technique is relatively straightforward. However, it may

not be practical if nests are scarce or otherwise hard to find. 

Because the breeding success of birds can be highly variable and 

can be quite low, it is sometimes difficult to obtain sufficient data 

on the success of the same species in enough sites to yield 

satisfactory results for statistical comparison with controls (see 

paragraph (h)(18) of this guideline). In some cases, artificial nest 

structures can be constructed to increase nest densities. In a few 

situations where sufficient numbers are available, the technique 

may be applicable to mammal den monitoring.

(B) Behavioral observations. Behavioral observations associated with 

reproduction can be quite useful, especially for birds. Techniques are 

simple, but labor intensive. When used, such observations most likely 

would be combined with nest monitoring since both techniques require 

locating reproductive sites. Typically, the frequency and duration of 

behaviors will be compared for treated and untreated plots. Incubation, 

parental care (especially feeding for altricial birds), and following 

behavior (for precocial animals) are behaviors that are particularly 

amenable to such study. Courtship, mating, and nest building are other 
Page 32 of 46

behaviors that could be studied in some situations, but locating sufficient 

numbers of animals displaying these behaviors to permit quantitative 

analysis is difficult.

(C) Age structure of populations. 

(1) Comparisons of young to adult ratios of selected species 

between treated and untreated plots may indicate reproductive 

effects. The timing of the application and of breeding of selected 

species is critical. For assessing reproductive impairment or 

survival of dependent young, per se, the duration of this technique 

should be limited to single breeding and rearing periods, which 

may be repeatedly assessed. However, longer study periods that 

may even include several years can be used to assess the 

combination of reproductive success and age-specific mortality, 

even if the two cannot be separated.

(2) Obviously, use of this method requires that the age of 

individual animals be determined. In some cases, it may be 

necessary only to distinguish among adults, subadults, and 

juveniles. In mammals, this may usually be accomplished by

examining pelage, development of testes or mammaries, or tooth 

eruption or wear characteristics. In birds, plumage or 

characteristics of particular (species-dependent) feathers may be 

used. For carcasses or sacrificed animals, observation of the 

ossification of bones or development of reproductive organs are 

useful. In other cases, particularly where comparisons are made 

among populations in different years, it may be appropriate to 

distinguish age classes of adults. In mammals, tooth eruption, 

wear, or enamel layers, or eye lens weights are useful. It is more 

difficult to separate age classes of many adult birds, although 

overall plumage or feather characteristics can provide some 

indication. In some slow-maturing birds (e.g., gulls), plumage may

be used to distinguish year classes of sub-adults. Additional 

details on aging birds and mammals are presented by Larson and 

Taber (see paragraph (h)(22) of this guideline).

(viii) Ancillary methods. 

(A) At least some ancillary methods are essential in every field study. As 

used here, ancillary methods are generally of two types. Certain of these 

methods are important for determining the nature or existence of effects or 

for establishing causal relationships. Others of these methods do not 

address effects directly, but they provide important information for 

interpreting the results of the study.

(B) Many of the methods for determining effects have been discussed for 

screening studies. Enzyme analysis, such as for ChE inhibition, and 

observations of signs of toxicity can show that animals were exposed to or 
Page 33 of 46

killed by a toxicant of a particular type. Where it is possible that animals 

may be exposed to other chemicals of the same type (e.g., feeding in a 

nearby area treated with pesticides), residue analysis in nontarget animals 

may be necessary to determine which specific chemical caused the signs 

or alterations in enzymes. Even though carcass searches, per se, are not 

recommended for definitive studies, it is still essential to recover and 

analyze any carcasses found accidentally or obtained through radiotracking. Residue and/or enzyme analysis of live animals collected will 

frequently be important.

(C) Among the other ancillary methods, analysis of environmental 

residues is crucial and will probably be necessary in nearly every 

definitive field study. As discussed for screening studies, the most 

important environmental residues are those that occur on or in wildlife 

food sources, which may include insects, plant parts, or even other 

vertebrates, depending upon the species that are the primary focus of the 

investigation. The investigator should review the literature on food habits 

of the species being studied; often it will be appropriate to assess food 

habits on the specific study sites, particularly where the literature is not 

adequate to define food habits in the agricultural ecosystem under study. 

Such an assessment should include the availability of food sources and the 

number of mobile animals that spend only part of the time in and adjacent 

to treated sites. The habitat should be thoroughly described to include 

both the morphology and species that are relevant to wildlife. Frequently,

it will be important to locate and describe roosting, denning, or nesting 

sites for mobile wildlife that use treated sites part of the time.

(ix) Interpretation of results. 

(A) While each field study is unique, some elements may be common 

among many field studies. When a definitive field study is required, the 

requirement is based on one or more specific concerns that pertain to a 

specific chemical and one or several use patterns. Because of the 

substantial diversity in the types of problems to be assessed and the variety 

of available investigative methods, the key to understanding and 

interpreting a field study lies in the development of a sound protocol. All 

protocols will contain a description of the study sites, or the characteristics 

to be used in selecting sites within a given area, and the methods to be 

used in conducting the study. However, a well designed protocol will go 

beyond this descriptive approach in three ways.

(1) First, the well-designed protocol will contain a restatement of 

the concerns to be addressed to ensure that there is an adequate 

understanding of the Agency's position. The investigator should 

review the literature and other available information that may bear 

upon the problem. It is possible that the literature may contain a 

valid answer to the questions raised by the Agency. Far more 

likely, the literature may orient the investigator to address the 
Page 34 of 46

concerns in a particular way. An example is provided by Hegdal 

and Blaskiewicz (under paragraph (h)(16) of this guideline), who 

conducted a study to address the Agency's concerns for secondary 

toxicity to barn owls (specifically) from the use of an anticoagulant 

bait proposed for use on commensal rodents in and around 

agricultural buildings. A review of the literature by these 

investigators indicated to them that laboratory studies suggested a 

legitimate potential for secondary poisoning to exposed raptors, 

but that the food habits of barn owls consist primarily of microtine 

rodents in most areas, suggesting a low potential for actual 

exposure. Consequently, they designed their study to focus on 

barn owl food habits and movements, and included an additive to 

the bait formulation that would permit an identification of whether 

or not the barn owls ate rodents that had fed on the bait. The study

adequately demonstrated that actual exposure of barn owls was 

quite limited, and the proposed registration for this use was 

subsequently approved. By using the available literature on both 

the chemical and the particular species of concern, the 

investigators were able to narrow the study while still providing 

sufficient information for evaluation. However, it should be noted 

that this study was not adequate for evaluating the potential for 

secondary toxicity in the field to other predators that may have 

different food habits, or for other use patterns that may result in 

exposure to different predators or scavengers.

(2) Second, the well designed protocol will provide the reasons 

why particular methods are being used, including, at least 

qualitatively, the meaning that different results might have. For 

example, a protocol may include collection of residues in nontarget 

animals, but it also should include a statement of purpose and 

meaning for such collection. Residues may be used to indicate 

potential exposure to nontarget organisms through analysis of their 

food, exposure in nontarget animals as a result of eating 

contaminated food, or that a particular chemical was likely to be 

the cause of any observed effects. Interpretation of data is 

facilitated substantially by a statement of what results were 

intended by using a particular technique. In the previously cited 

example (see paragraph (h)(16) of this guideline), it was clearly 

stated that collection of owl pellets was used to assess general food 

habits and that use of a fluorescing dye in the bait was for the 

purpose of ascertaining whether or not the owls fed on commensal 

rodents that specifically had fed on the bait. The interpretation of 

the data collected, once the purpose was stated, naturally led to the 

conclusion of no-significant-exposure to the barn owls.

(3) Third, the well designed protocol will contain an experimental 

design that will indicate how the results can be assessed 

quantitatively. The experimental design has been discussed in 
Page 35 of 46

previous sections of this guideline, but there are two facets that 

relate closely to the interpretation of results: The difference that 

can be detected between treated and untreated plots and the power 

(ability) of the design to detect this difference. An experimental 

design with number of replicates based on an estimated coefficient 

of variation that closely approximates reality will allow the study 

to detect a stated concern level some prescribed number of times 

during the study time. The actual difference between treated and 

control units is measured during the field study, but the design can 

form an initial basis for interpretation when combined with the 

available information on the species of concern. As a result, the 

well-designed protocol should include a section on interpretation.

(B) Study methods for investigating acute mortality are more 

straightforward than for other kinds of effects. Nevertheless, there are 

sufficient differences in the use of the data to preclude a constant 

interpretation. The study may focus directly on the species of concern and 

may involve little or no extrapolation, depending on such factors as the 

type and the extent of use, the available toxicity data base, and home range 

of the species. Extrapolation to other populations, regions, or uses might 

be necessary. If the species of concern cannot be studied directly, it may

be necessary to extrapolate between species, involving interspecies 

differences both in toxicological sensitivity and in ecological and 

population parameters.

(C) The same kinds of considerations apply to reproductive impairment 

and chronic toxicity, even though different, and often more laborious and 

costly, investigative methods are involved. Where reproductive success is 

impaired, information on species-specific variation in reproductive 

ecology is necessary to understand how a particular degree of impairment 

may relate to effects among various species. Such reproductive 

considerations can include whether an avian species is a determinate or 

indeterminate layer, the number of nestings per season for different 

geographic areas in the use pattern, the length of the refractory period, as 

well as the specific effect which can range from destruction of 

reproductive organs to behavioral deficits such as nest abandonment. 

Considerations of reproductive ecology among different species of 

mammals include delayed fertilization or implantation, resorption of 

embryos or parental infanticide due to stress, number of young per 

breeding cycle, etc. All of these factors, and many others, are relevant to 

determining for different species the extent of effects that could result in 

population reductions or lack of ability to recover.

(D) An analysis of whether or not a particular level of effect is going to 

affect wildlife populations is species-specific. For any species (or 

subspecies), the changes in population can be described very simplistically 

by the equation: rate of population increase (r) = birth rate minus death 

rate, where values of r can be positive (population growth) or negative 
Page 36 of 46

(population reduction). Immigration and emigration are also important 

when the concern is for specific populations of a species. These 

characteristics differ among species, and data will not always be available. 

The application of sound scientific judgment to the best available 

information will be the basis for interpreting the results of a study. It may 

be necessary to compare the results of the field study to laboratory data, 

especially where laboratory data are available on a variety of species 

and/or effects and the field study has focused on species other than those 

of direct concern. The use of extrapolation techniques will he necessary 

where endangered species are of concern or where other species cannot be 

studied directly.

(E) The Agency would like to be able to obtain a standardized result from 

a field study so that the result could be applied in a very consistent 

manner. As discussed in previous sections of this guideline, the different 

effects and species of concern will vary and will require the development 

of specific protocols to address these factors. Although most of the 

various techniques have some degree of standardization, the field study 

may combine the individual techniques in a wide variety of ways to 

address specific concerns. A standardized result might be attainable for 

the individual techniques, although that result would still have to be 

applied differently for various species, depending on their biology and 

ecological characteristics. However, determining a result for the whole 

field study that would unequivocally lead to a statement of the degree of 

risk, while obviously desirable, is not currently practical.

(6) Carcass searches―

(i) Design. 

(A) In designing carcass searches, the following factors need to be known 

or determined:

(1) Density of the species that are likely to be exposed. For 

example, granular products are most likely to result in exposure to 

ground-feeding animals; therefore, birds such as warblers or 

swallows should not be included in density counts for such 

products. 

(2) Probability of finding dead animals if any are killed. This is 

dependent on the probability of a carcass remaining on the study 

site (i.e., not being removed by scavengers) and the probability of 

detecting a carcass if it remains on the study site (search 

efficiency).

(3) Size of the search area.

(4) Number of carcasses found..
Page 37 of 46

(B) These factors can be combined in the following formula:

n = d × r × e × a × p Equation 14

where:

n = number of carcasses found;

d = density in animals per acre;

r = proportion of carcasses remaining (nonremoval);

e = search efficiency;

a = acres searched; and

p = proportion of population killed.

(C) Carcass searches should be used only when there is a reasonable 

potential to detect mortality. If such mortality does occur, the carcass 

search should be able to detect it and therefore, carcasses should be found. 

It is recommended that carcass searches be designed so that at least two 

carcasses (n = 2) will be found if there is appreciable mortality. In 

general, preliminary sampling would be required to determine these 

factors. However, information from other field studies can be used in the 

planning stages to determine if carcass searching would be appropriate for 

use under anticipated conditions and to assist in developing the study 

design.

(D) The sensitivity of the carcass search approach is equivalent to the 

percent detectable kill of the population. To determine the sensitivity, 

Equation 14 is rearranged to solve for p, the proportion of population 

killed, as shown in Equation 15. Since p is a proportion multiplying by

100 (Equation 16) provides the percentage of the population killed.

d r e a

n p

  

 Equation 15

 

100 100











  

  

d r e a

n percent detectable kill p Equation 16

(E) If any of the values of d, r, e, or a are zero, the equation cannot be 

solved and the carcass search is not applicable (i.e., no density of birds, no 

acres searched, no carcasses remaining, no remaining carcasses found). 

However, other combinations of d, r, e and a, such as low density and 

small acreage or low efficiency and high scavenger removal, can result in 

a small denominator meaning that mortality can be detected only when a 

high percentage of the population is killed. For example, in 5 acre fields 

with only two birds per acre and r and e estimated at a moderate 0.5, only
Page 38 of 46

an 80% or greater kill could be detected. In such situations, it is necessary 

to increase one of the parameters to achieve a stated level of detectability 

or else to use methods other than carcass searching. The same equation 

can be used to estimate the minimum search area to detect a given 

mortality level (p) by solving for a.

(ii) Search procedure. In general, depending on the sensitivity of the search 

method relative to the habitat involved, corridors or plots should be selected. 

These areas should be searched systematically by walking predetermined routes 

until the area has all been covered. Due to the concentration required to find dead 

animals, other activities that could distract the attention of the searchers should be 

avoided during carcass searching. In homogeneous situations, investigators 

should randomly select search areas. However, in most studies it is advisable to 

stratify the sampling, concentrating efforts in areas frequented by wildlife species 

such as woods edges, ditch banks, field borders, fencerows, and other habitats 

where wildlife concentrate.

(iii) Duration. Searches should begin on the day of application and continue on a 

daily basis for as long as mortalities or other evidence of intoxication occur. In 

general, a week or two following application should be adequate. However, the 

length of time searches are continued should be related to how long lethal 

concentrations are expected to be present. Normally, the same areas should be 

searched each day.

(iv) Estimating efficiency of carcass search. 

(A) Efficiency trials should be conducted periodically (minimum 3 times 

per study site) during the study to determine the proportion of carcasses 

that are detected. Just prior to the initiation of a scheduled search, 

carcasses of animals representative of species found in the area should be 

variously placed within the search area. If the study site includes edge 

habitat, carcasses should be placed in the edges as well as in the fields. In 

general, carcasses should be placed where animals would be most likely to 

die, depending on the nature of the chemical. Searchers should not be 

aware that simulated mortalities have been placed; however, they should 

be aware that these trials will occur during any scheduled search.

(B) The number of carcasses placed should be approximately equal to 

20% of the estimated density of species on the search area. All placed 

carcasses should be marked to distinguish them from actual kills. The 

location of placed carcasses should be mapped so those not found can be 

easily recovered following completion of that day's search activities, since 

unrecovered carcasses could bias study results. For example, if a 

scavenger were to carry off a simulated kill and consume it at another 

location on the study site, the remains could be erroneously classified as 

chemical-related if found. One way to avoid this problem would be to dip 

carcasses in a nontoxic substance that fluoresces under ultraviolet light so 

that the remains could be identified.
Page 39 of 46

(v) Estimating carcass removal rate. 

(A) Carcass removal should be monitored to determine local variability in 

scavenger activity. The density of both carcasses and scavengers can 

influence the rate of removal. Under some conditions, large numbers of 

carcasses may attract scavengers. In other situations a large number of 

kills may dilute removal rate due to limited number of scavengers. Where 

it can be adequately documented that removal of carcasses occurs almost 

exclusively either at night or during the day, the timing of carcass searches 

may be adjusted to minimize the effects of removal.

(B) Carcasses planted in monitoring trials should simulate mortalities 

actually occurring from the chemical. In most cases, small to moderate 

sized species such as starlings or blackbirds, or laboratory bobwhite or 

Japanese quail chicks may be used. Carcasses should be variously placed 

within the general study areas and monitored daily for at least 5 days or 

until 90% have been removed. The number used should approximate 

densities resulting from effects of the chemical under study; however, in 

most instances, this will not be known. Therefore, a density of 

approximately 20% of the population of nontarget species on the area is 

recommended.

(C) Timing of carcass removal trials should be such that they do not affect 

scavenger removal of chemical-killed birds or the feather-spots of the 

removed carcass could be erroneously classified as a chemical kill. 

Location of placed birds should be recorded on maps and may be marked 

in the field with small stakes or by other inconspicuous means, preferably 

at a fixed distance and direction from the carcass.

(f) Suggested components of a field study protocol. The following protocol was adapted from 

the Wildlife Managements Techniques Manual (under paragraph (h)(29) of this guideline), and is 

recommended for studies submitted to the Agency for review.

(1) Title.

(2) Problem definition. The following information should be provided:

(i) A review and summary of the available information on the chemical in relation 

to nontarget hazard, including use information.

(ii) A precise statement of the goals and purpose of the study.

(iii) A brief statement of the problem and the context in which it exists, specifying 

the limits of the proposed work.

(iv) Precise statements of the major hypotheses to be tested.
Page 40 of 46

(3) Methods and materials. This section should include the following:

(i) A brief discussion of various methods and procedures that have been or could 

be used to evaluate the problem. This discussion should identify the strengths and 

weaknesses of each method or procedure discussed.

(ii) Description of the procedure to be followed:

(A) Identify the study areas selected and their general suitability for 

achieving the objectives of the study or what criteria will be used to select 

study areas.

(B) Identify the species present or expected to be present on the study

areas, discussing characteristics pertinent to the problem being evaluated.

(C) State the research procedures, designs and sampling plans to be used:

(1) Specify the kind and amount of data needed and to be sought.

(2) Describe in detail how all the data are to be obtained, including 

details of application, instrumentation, equipment, sampling 

procedures, and any other information.

(D) Describe how the data are to be treated, including specifying what 

statistics are to be calculated, what models will be used, what tests of data 

will be used, etc.

(E) Describe in detail the methods to be used to check the sensitivity and 

accuracy of the procedures used.

(F) Describe quality assurance procedures for application, instrumentation, 

equipment, and records.

(G) Briefly describe the resources (people, facilities, etc.) to be applied to 

the study.

(g) Reporting. In addition to the reporting provisions on background information, study

protocol deviations, and test substance in paragraphs (g)(1), (g)(2), and (g)(3) of this guideline, 

respectively, the test report should include, but not necessarily be limited to the information in 

paragraphs (g)(4), (g)(5), and (g)(6) of this guideline on test species, test methods and conditions, 

and results, respectively.

(1) Background information. Background information to be supplied in the report 

consists at a minimum of those background information items listed in paragraph (j)(1) of 

the OCSPP 850.2000 guideline.

(2) Study protocol deviations. Provide a copy of the final field study protocol used. 

Include a description of any deviations from the study protocol originally submitted and 

agreed upon with the Agency or any occurrences which may have influenced the results 
Page 41 of 46

of the test, the reason for these changes, and any resulting effects on test endpoints noted 

and discussed.

(3) Test substance. 

(i) Identification of the test substance: common name, IUPAC and CAS names, 

CAS number, structural formula, source, lot or batch number, chemical state or 

form of the test substance, and its purity (i.e. for pesticides, the identity and 

concentration of active ingredient(s)), radiolabeling if any, location of label(s), 

and radiopurity.

(ii) Storage conditions of the test chemical or test substance and stability of the 

test chemical or test substance under storage conditions if stored prior to use.

(iii) Methods of preparation of the test substance for application, the application 

rate(s), for pesticides the maximum label rate.

(iv) For residue analysis in wildlife, vegetation, soil, water, sediments, and other 

appropriate environmental components describe the stability of the test substance

under storage conditions.

(v) Data on storage of biological and environmental samples.

(4) Site of the test. 

(i) A description of the field study area(s) and sites, including the size and 

characteristics of all components over time, as well as prevailing meteorological 

conditions.

(ii) History of the site in terms of factors that may influence the study objectives.

(iii) Map or diagram showing location of treated sites and controls and showing 

locations of observations and searches.

(iv) Climatological data during the field study: records of applicable conditions 

for the type of site, i.e., temperature, thermoperiod, rainfall or watering regime, 

light regime including intensity and quality, photoperiod, relative humidity, wind 

speed, etc.

(v) Substrate characteristics of the study area and treated sites.

(vi) Characteristics of the flora and fauna of the study area and test sites.

(5) Study species. 

(i) Identify the wildlife species present in the study area(s), and habitat.

(ii) For study species the study design objectives and protocols, and the scale (i.e., 

all species, cross-section, selected species).
Page 42 of 46

(ii) Characteristics of species (e.g., age, stage of development, health status) 

pertinent to the problem being evaluated.

(A) Number and type of species investigated and the scale of identification 

(e.g., a single species of concern, all species of a community or a selected 

cross-section).

(B) Scientific and common name.

(C) Stage of development and condition of study species and other 

wildlife at test initiation.

(6) Study conditions and experimental design. Description of the study conditions and 

experimental design used in the screening or definitive tests, and any preliminary testing.

(i) A statement of the concerns to be addressed and the type and frequency of 

monitoring.

(A) The methods of evaluating effects on terrestrial wildlife (e.g., radio 

telemetry, carcass searches, ChE inhibition). The report results should 

include:

(1) Observed behavioral, biochemical, and ecological effects, such 

as measures of mortality and survival, reproduction, population 

density, and enzyme inhibition. 

(2) Residue concentrations of the test substance (and degradation 

products, if evaluated) in wildlife and wildlife food sources over 

time.

(B) Statement of the data objectives for specific measures (i.e., the critical 

or threshold level for an effect, precision of a point estimate).

(ii) The field study design: size of field sites, number of control sites, the number 

of experimental treatment levels and the number of experimental sites (replicates) 

for each treatment, the lay-out and distance of field sites to each other and to 

control sites.

(iii) Methods used for treatment randomization.

(iv) Number of applications and dates applied, treatment concentrations or 

application rates, frequency and pattern of administration.

(v) Method of test substance application: application or delivery methods (e.g., 

irrigation water, soil incorporated, surface soil or foliar spray) to the site including 

equipment type and design (nozzles, orifices, pressures, flow rates, volumes, etc.)) 

and method for calibrating the application equipment), information about any 

solvent used to dissolve and apply the test substance.

(vi) Study duration.
Page 43 of 46

(vii) Methods and frequency of climatological monitoring performed during the 

study for air temperature, thermoperiod, humidity, rainfall and watering regime, 

light intensity, and wind speed.

(viii) The photoperiod and light quality.

(ix) Methods and frequency of monitoring of other ancillary nontreatment related 

factors that may influence the measures of effect at the study site should be 

reported. For example, if effects to wildlife from application to a crop species is 

studied or if a crop is treated concurrent to the investigation of wildlife effects, 

cultural practices during the tests such as cultivation, pest control, should be 

monitored and reported for the study sites.

(x) All analytical procedures should be described. The accuracy of the method, 

method detection limit, and limit of quantification should be given.

(7) Results. 

(i) Environmental monitoring data results (air temperature, humidity and light 

intensity, rainfall) in tabular form (provide raw data for measurements not made 

on a continuous basis), and descriptive statistics (mean, standard deviation, 

minimum, maximum).

(ii) Tabulation of the results of study-specific wildlife measures by field site and 

treatment (provide the raw data), and summary statistics. If categorical rating 

measures are made a description of the rating system should be included.

(iii) Description of the statistical method(s), software package(s) used, the basis 

for the choice of the method(s), statements of the reasons why particular methods 

are being used, including, at least qualitatively, the meaning that different results 

might have.

(iv) Results of the statistical analysis including graphical and tabular summaries, 

and results of goodness-of-fit tests or minimum significant differences detectable, 

as appropriate.

(h) References. The following references should be consulted for additional background 

material on this test guideline.

(1) Bunyan, P.J. et al. Organophosphorus poisoning, some properties of avian esterases. 

Journal of Agricultural and Food Chemistry 16: 326-331 (1968).

(2) Bunyan, P.J. et al. Organophosphorus poisoning, diagnosis of poisoning in pheasants 

owing to a number of common pesticides. Journal of Agricultural and Food Chemistry 

16: 332-339 (1968).

(3) Burnham, K.P. et al. Estimation of density from line transect sampling of biological 

populations. Wildlife Monographs, No. 72 (1980).
Page 44 of 46

(4) Caughley, G. Analysis of Vertebrate Populations. Wiley, NY (1977).

(5) Cochran, W.W., Wildlife telemetry. pp. 509-520, in Wildlife Management 

Techniques Manual. S.D. Schemnitz, Ed. The Wildlife Society, Washington, DC (1980).

(6) Corbett, J.R. The Biochemical Mode of Action of Pesticides. Academic, NY (1974).

(7) Davis, D.E. and R.L. Winstead. 1980. Estimating the numbers of wildlife populations. 

pp. 221-246, in Wildlife Management Techniques Manual. S.D. Schemnitz, Ed., The 

Wildlife Society, Washington, DC (1980).

(8) Eberhardt, L.L. Quantitative ecology and impact assessment. Journal of Wildlife 

Management 4: 27-70 (1976).

(9) Eberhardt, L.L. Appraising variability in populations studies. Journal of Wildlife 

Management 42: 207-237 (1978).

(10) Eberhardt, L.L. Assessing the dynamics of wild populations. Journal of Wildlife 

Management 49: 997-1012 (1985).

(11) Edgington, E. Randomization Test. Dekker, NY (1980).

(12) Edwards, P.J. et al. The use of a bird territory mapping method for detecting 

mortality following pesticide application. Agro-Ecosystems 5: 271-282 (1979).

(13) Ellman, G.L. et al. A new and rapid calorimetric determination of 

acetylcholinesterase activity. Biochemical Pharmacology 7:88-95 (1961).

(14) Fite, E., L. Turner, N. Cook, and C. Stunkard, 1988. Guidance Document for 

Conducting Terrestrial Field Studies. United States Environmental Protection Agency, 

Office of Pesticide Programs, Washington, D.C. EPA 540/09-88-109.

(15) Hanson, W.R. Estimating the density of an animal population. Journal of Research 

Lepidoptera 6: 203-247 (1967).

(16) Hegdal, P.L. and R.W. Blaskiewicz. Evaluation of the potential hazards to barn owls 

of Talon (brodifacoum bait) used to control rats and house mice. Journal of 

Environmental and Toxicological Chemistry 3: 167-179 (1984).

(17) Heinz, G.H. et al. Environmental contaminant studies by the Patuxent Wildlife 

Research Center, pp. 8-35. in Avian and Mammalian Wildlife Toxicology. ASTM STP 

693, E.E. Kenaga (Ed), American Society for Testing and Materials, Philadelphia, PA 

(1979).

(18) Hill, E.F. and W.J. Fleming. Anticholinesterase poisoning of birds: field monitoring 

and diagnosis of acute poisoning. Journal of Environmental and Toxicological Chemistry

1: 27-38 (1982).

(19) Hurlbert, S.H. Pseudoreplication and the design of ecological field experiments. 

Ecological Monographs 54:187-211 (1984).
Page 45 of 46

(20) Giles, R.H. Jr. Wildlife Management. Freeman, San Francisco, CA. (1978).

(21) International Bird Census Committee. Recommendations for an international 

standard for a mapping method in bird census work. Bulletin of the Ecological Research 

Committee 9:49-52 (1970).

(22) Larson, J. S. and R. D. Taber. Criteria of sex and age. pp. 143-202, in Wildlife 

Management Techniques Manual. S. D. Schemnitz; (Ed.), The Wildlife Society, 

Washington, DC (1980).

(23) Leopold, A. Game Management.Scribner's, NY (1933).

(24) Ludke, J.L. et al. Cholinesterase (ChE) response and related mortality among birds 

fed ChE inhibitors. Archives of Environmental Contaminant Toxicology 3:1-21 (1975).

(25) Moen, A.N. Wildlife Ecology: An Analytical Approach. Freeman, San Francisco, 

CA (1973).

(26) Nichols, J.D. and K.H. Pollock. Estimation methodology in contemporary small 

mammal capture-recapture studies. Journal of Mammal 64:253-260 (1983).

(27) O'Brien, R.D. Insecticides: Action and Metabolism.Academic Press, NY (1967).

(28) Otis, D.L. et al. Statistical interference from capture data on closed animal

populations. Wildlife Monographs 62:1-135 (1978).

(29) Ripley, T.H. Planning wildlife management investigations and projects, pp. 1-6. in 

Wildlife Management Techniques Manual. S.D. Schemnitz (Ed.), The Wildlife Society, 

Washington, DC (1980).

(30) Rosene, W. Jr. and D.W. Lay. Disappearance and visibility of quail remains. Journal 

of Wildlife Management 27:139-142 (1963).

(31) Scientific Advisory Panel. Final Scientific Advisory Panel subpanel's report on the 

January 7-8, 1987 meeting concerning terrestrial field studies. Environmental Protection 

Agency, Washington, DC (1987).

(32) Seber, G.A.F. The Estimation of Animal Abundance and Related Parameters. 

Macmillan, NY (1982).

(33) Shellenberger, T.E. et al The comparative toxicity of organophosphate pesticides in 

wildlife. pp. 205- 210, in W.B. Diechmann, (Ed.), Pesticide Symposium, Halos, Miami, 

FL (1970).

(34) Snedecor, G.W. and W.G. Cochran. Statistical Methods. Sixth Edition. The Iowa 

State University Press, Ames, IA (1967).

(35) Stewart, R.E. and J.W. Alrich. Removal and repopulation of breeding birds in a 

spruce-fir forest community. The Auk 68:471-482 (1951).
Page 46 of 46

(36) United States Environmental Protection Agency, 1982. Pesticide Assessment 

Guidelines Subdivision E, Hazard Evaluation: Wildlife and Aquatic Organisms. Office 

of Pesticide and Toxic Substances, Washington, D.C. EPA 540/9-82-024.

(37) Walpole, R.E. and R.H. Myers. Probability and Statistics for Engineers and 

Scientists. Macmillan, NY (1972).