United States
Environmental Protection
Office of Chemical Safety
and Pollution Prevention
January 2012
        Ecological Effects
        Test Guidelines
        OCSPP 850.2500:
        Field Testing for
        Terrestrial Wildlife


     This guideline is one of a series of test guidelines established by the United States
Environmental Protection Agency's Office of Chemical Safety and Pollution Prevention
(OCSPP) for use in testing pesticides and chemical substances to develop data for
submission to the Agency under the Toxic Substances Control Act (TSCA) (15 U.S.C. 2601,
et seq.), the Federal Insecticide, Fungicide and Rodenticide Act (FIFRA) (7 U.S.C. 136, et
seq.), and section 408 of the Federal Food, Drug and Cosmetic (FFDCA) (21 U.S.C. 346a).
Prior to April 22, 2010, OCSPP was known as the Office of Prevention, Pesticides and Toxic
Substances (OPPTS). To distinguish these guidelines from guidelines issued by other
organizations, the numbering convention adopted in 1994 specifically included OPPTS as
part of the guideline's  number.  Any test guidelines developed after April 22, 2010 will use
the new acronym (OCSPP)  in their title.

     The OCSPP harmonized test guidelines serve as a compendium of accepted scientific
methodologies and protocols that are intended to provide data to inform regulatory decisions
under TSCA, FIFRA, and/or FFDCA. This document provides guidance for conducting the
test, and is also  used  by EPA, the public, and the companies that are subject to data
submission requirements under TSCA, FIFRA, and/or the FFDCA.  As a guidance
document, these guidelines are not binding on either EPA or any outside parties, and the
EPA may depart from  the guidelines where circumstances warrant and without prior notice.
At places in this  guidance, the Agency uses the word "should."  In this guidance, the use of
"should" with regard to an action means that the action is recommended rather than
mandatory. The procedures contained in this guideline are strongly recommended for
generating the data that are the subject of the guideline,  but EPA recognizes that departures
may be appropriate in specific situations. You may propose alternatives to the
recommendations described in these guidelines, and the Agency will assess them for
appropriateness on a  case-by-case basis.

     For additional information about these test guidelines and to access these guidelines
electronically, please go to http://www.epa.gov/ocspp and select "Test Methods &
Guidelines" on the left side navigation menu.  You may also access the guidelines in
http://www.requlations.qov grouped by Series under Docket ID #s: EPA-HQ-OPPT-2009-
0150 through EPA-HQ-OPPT-2009-0159, and EPA-HQ-OPPT-2009-0576.

OCSPP 850.2500: Field testing for terrestrial wildlife.

(a) Scope—
       (1) Applicability.  This guideline is intended to be used to help develop data to submit to
       EPA under the Toxic Substances  Control Act  (TSCA) (15 U.S.C. 2601,  et seq.), the
       Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) (7 U.S.C. 136, et seq.), and
       the Federal Food, Drug, and Cosmetic Act (FFDCA) (21 U.S.C. 346a).
       (2) Background.  The source materials used in  developing this harmonized OCSPP test
       guideline include OPP 71-5 Simulated and Actual Field Testing for Mammals and Birds
       (Pesticide Assessment Guidelines  Subdivision  E—Hazard  Evaluation: Wildlife  and
       Aquatic Organisms),  and  the  Guidance Document for  Conducting Terrestrial  Field

(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

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

(d) General considerations—

       (1) Summary of the test.  The Agency uses terrestrial field studies to evaluate only those
       test substances in which significant questions on risks  to nontarget wildlife exist.  The
       test substance may be applied  in a variety of ways; the selected  method should support
       the specific study objective. The study is performed under natural conditions and  in the
       environment in which the test substance would be either applied and/or disperses to  under
       normal use practices  for pesticides or would occur under the pattern of production, use,
       disposal,  or  accidental release for industrial  chemicals.    Specific  objectives  and
       associated qualitative and quantitative  decision  statements  establishing measurement
       endpoints  and their accuracy and precision should be provided as part  of the study plan.
       Specific protocols should  be  developed as  needed and submitted to the  Agency for
       review prior to conduct of the study.

       (2) General  test guidance. In contrast to laboratory tests which  are generally amenable
       to a high degree of standardization, field study protocols are more flexible reflecting the
       case-by-case  nature of issues and decisions  a given field  study  is designed to address.
       Additionally  standardization of field studies is made difficult  by variability in  such
       factors as chemical mode-of-action, species density and  diversity, and for pesticides
       differences in use pattern, crop type, and method of application. This guideline provides
       a general outline of factors to consider in the conduct of field studies;  specific protocols
       should be developed and  submitted to the Agency for review.  Despite the variability
                                      Page 1 of 46

among field studies, several key elements common to most field studies can be identified.
This guideline was prepared to  identify and discuss these elements as they pertain to
terrestrial vertebrates, and to provide a better understanding of the purpose of such field
studies.  There are two types of field studies,  screening and definitive. The type of field
study conducted  (screening or  definitive) depends on the available data on the  test
chemical or substance in question and the terrestrial wildlife population and  community
dynamics.   Environmental  and exposure conditions under which a field study is
conducted  should resemble  the  conditions likely to be  encountered under  actual use,
production, disposal,  or  fate of the test substance  or  chemical.  Pesticides should be
applied to the site at the  rate, frequency, and  by the method specified on the label. The
general guidance  in the OCSPP  850.2000 guideline applies to this guideline,  except as
specifically noted herein.

(3) Environmental  chemistry methods.   Procedures  and  validity elements  for
independent laboratory validation of environmental chemistry methods used  to generate
data associated with this study  can be found  in OCSPP  850.6100.  Elements of the
original addendum as referenced in 40  CFR 158.630 for this purpose are now contained
in OCSPP 850.6100. These procedures, if followed, would result in data  that would
generally be of scientific  merit for the purposes described in 40 CFR 158.630.

(4) Screening field study. If the available effects data is limited to laboratory toxicity
data on  a  limited number of species,  a screening field study may be appropriate to
determine  if hazards  or risks  extrapolated to  individual  animals,  populations  and
communities from the laboratory data are occurring in the field and, if so, to what species
before conducting a definitive field study.  "Pass-fail" methods are used to determine
whether impacts occur.   These methods may  include carcass  searching, residue analysis
of species collected on study plots, residue analyses of wildlife food sources found in and
adjacent to the area of application, behavioral observations, and enzyme analysis.

(5) Definitive field study.  If a screening study indicates impacts  are  occurring, or if
other available data suggest that deleterious effects have occurred or are extremely likely,
the study design  should  be  quantitative, evaluating the magnitude of the impacts in a
definitive study.   A  quantitative  field study focuses on  the species  affected in the
screening phase.  For some test substances or  chemicals it may be appropriate to proceed
directly to a definitive study without the screening phase.  Careful consideration needs to
be given to the likelihood of impacts occurring  in order to determine which approach to
use. At the quantitative level (definitive study), the objectives should include estimating
the magnitude of acute or secondary mortality  caused  by the application, the existence
and extent of reproductive effects, and the influence of chemical use on the survival of
species of concern. Methods that can be used to address these objectives include  mark-
recapture, radio telemetry, line transect  sampling, nest monitoring, territory mapping, and
measuring young  to adult ratios.

(6) Endangered  species. Studies should not be conducted in critical habitats or areas
where endangered or threatened species could be exposed.
                                Page 2 of 46

(e) Test standards—

       (1) Test substance.   For industrial chemicals  the  substance to be tested should be
       technical grade unless the test is designed to test  a specific formulation, mixture, or end-
       use product.  For pesticides  unless  specified otherwise, data is derived  from testing
       conducted with an end-use product.  If the pesticide product is applied in a tank mixture,
       dosages  of each active ingredient (a.i.)  should be reported with identification and
       formulation for each product in the tank mix.  The OCSPP 850.2000 guideline lists the
       type of information that should be known  about the test substance before testing, and
       discusses methods for preparation for use in testing.

       (2) Residue levels.   When the test substance is applied under field condition testing,
       residues  should be determined in  selected tissues of organisms collected in and around
       the study  area  and  in  vegetation,  soil,  water,  sediments,  and other appropriate
       environmental  components.   If methods to  analyze  for the test substance are  not
       available, the submitter should consult with the Agency before beginning the test.

       (3) Sampling and experimental  design.  While examples of acceptable  experimental
       designs are given, it is beyond the purpose of this guideline to cover the fundamentals of
       this topic.  Paragraph (f) of this guideline  provides  a general outline for  a field  study
       protocol  to be submitted to the Agency for  review.  Paragraphs (e)(4) and  (e)(5) of this
       guideline discuss points to be  considered  in  designing screening and definitive field
       studies, respectively.

              (i) A study designed to refute hazard is unusual in biological research. Typically,
              an investigator  is  more  concerned about  concluding with a high degree of
              confidence that an  effect occurred, not that it failed to occur.  FIFRA  specifies
              that  a pesticide is  to  be registered only if EPA determines it will not  cause
              unreasonable  adverse  effects.   The  difference  between  an objective  of "will
              cause" and "will  not  cause"  substantially  influences  study design  and  the
              evaluation of data.

              (ii)  The adverse effects to wildlife that can result from the use of pesticides or
              under the pattern of production, use, disposal, or accidental release of industrial
              chemicals can be classified as those that affect populations of wildlife and those
              that affect individuals but not the  entire population.  Either of these effects may
              warrant regulatory  action,  including mitigation,  cancellation or suspension of  a
              pesticide use,  or controls on  production/use/disposal  of an industrial chemical.
              An  adverse effect that  results in a  reduction  in  local,  regional, or national
              populations of wildlife species is clearly  of great concern. A chemical that can
              repeatedly or frequently kill wildlife is also of concern even if these repeated kills
              may or  may  not  affect long-term populations.   The  terrestrial   field  study,
              accordingly, is designed to assess both of these types of effects.

              (iii) For pesticides, the field study should be designed to provide data that show
              whether wildlife species will not  be affected significantly by a pesticide under
              normal use practices.  For industrial chemicals produced, used, or disposed of in
              the terrestrial environment, these data are also  sought. To achieve such objectives
              fully  at the population  level, it  is necessary that detailed knowledge of  the
                                       Page 3 of 46

population dynamics and  varying environmental  conditions  for  each species
potentially at risk be available.  The theoretical aspects of population dynamics
are well documented in the literature.  However, empirical data are available for
only a few species (see paragraph (h)(8) of this guideline).  A  study designed to
provide the needed data would include information  on age  structure, age-specific
survival and reproductive rates, and the nature and form of intrinsic and extrinsic
regulatory  mechanisms.  Such a  study, when coupled with the influence of the
chemical use  (or production/use/disposal  pattern) on these parameters,  could
require several years in order to give meaningful results.

(iv) The essential question  is: How can these studies be performed in a practical,
economical manner and still provide data that can show that the chemical  under
study will not reduce or limit wildlife populations or repeatedly kill wildlife?

(v) The  question can be  answered by examining the potential  influence test
substances can have on wildlife.  These effects include:

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

       (B) Sublethal toxic  effects indirectly causing death by reducing resistance
       to other environmental  stresses such as diseases, weather, or predators.

       (C) Altered behavior such as abandonment  of nests or  young, change in
       parental care, or reduction in food consumption.

       (D) Reduced food resources or alteration of habitat.

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

(vi)  These effects can manifest themselves  in  a  population  through reduced
survival and/or lower reproductive success. However, if a field study shows that
actual use  of a chemical does not affect survival and/or reproductive success or
that only minor changes occur, it would seem reasonable to conclude that the use
of the chemical will not significantly  impact wildlife.  Further, if a field  study
provides  estimates on the  magnitude of survival and reproductive effects, it is
possible to make reasonable projections on the meaning of the effects to nontarget
populations by using available information on the  species of concern and basic
theories of population dynamics.  While less than ideal, field studies that collect
information on survival and reproductive effects and use  these data to address
population parameters should provide a reasonable  basis for evaluating potential
impacts.  This is not to imply that effects  on populations  are the only concern,
however, a study adequate to  assess these  effects will also assess the degree of
risk to individual  wildlife.

(vii) This guideline emphasizes  avian  and  mammalian wildlife.  The Agency is
also  concerned  about  other  terrestrial organisms  such  as  nontarget plants,
invertebrates, amphibians,  and reptiles.  Plants and  invertebrates  are  excluded
                         Page 4 of 46

       here from direct study, except as sources of food or pesticides to wildlife. Testing
       guidelines for nontarget insects and plants are in Groups C and D, respectively, of
       the OCSPP 850 guideline series. Established protocols, especially for acute and
       chronic toxicity testing, are available for birds and mammals, but not for reptiles
       and are limited  for amphibians.  Occasionally, however, it may be necessary to
       adapt these field techniques to apply specifically to reptiles and/or amphibians.

(4) Screening study—

       (i) Objective and scope.

              (A) The screening study is designed primarily  to  demonstrate that  the
              hazard suggested by  lower  tier laboratory or pen studies does not exist
              under actual use conditions (primary consideration for pesticides) or occur
              under the pattern  of  production, use, disposal,  or  accidental  release of
              industrial chemicals.  The  interpretations of screening study  results, in
              most cases, are limited  to "effect" versus "no-effect" determinations.  If
              the study indicates that the chemical has caused little or  no detectable
              adverse effect, it may be reasonable to  conclude that potential adverse
              effects  are  minor.   When  effects  are  demonstrated, determining  the
              magnitude  of the  effects is done  with additional testing (see paragraph
              (e)(5) of this guideline).  Therefore, when information already available
              shows that a chemical  has caused adverse effects under normal use
              conditions  (primary consideration for pesticides) or under the pattern of
              production, use, disposal, or accidental release of industrial chemicals, the
              screening study may be  of limited value.  It may be appropriate to proceed
              directly to a definitive field  study in  cases where  analysis of laboratory or
              other data strongly suggest that adverse effects are likely to occur, and are
              unlikely to be attenuated by field conditions.

              (B) Screening studies  are limited to addressing the potential for acute toxic
              effects,  such as direct poisoning  and death,  and sublethal toxic effects
              potentially affecting  behavior and/or  survival.   In  most  instances, a
              screening study would not address reproductive effects  or effects such as
              changes in density or diversity of populations.

              (C) Further  laboratory   and/or pen  studies  may  be useful  prior to
              proceeding to the field, or may be necessary to interpret  results of the field
              study.  For example, additional toxicity data on species that are expected
              to be exposed from  the  normal use pattern (primary  consideration  for
              pesticides)  or under the pattern of production, use, disposal, or accidental
              release  of industrial  chemicals may indicate which  species are  more
              susceptible to the chemical,  allowing the  study to be designed  to monitor
              those species in greater depth as well as to provide insight into field results
              that show that some species were affected more than others, and additional
              laboratory  studies  may  be  unavoidable.   If  residue  concentrations in
              resident  species  are  being used  to indicate  potential  problems,  the
              relationship between tissue levels and the doses that cause adverse effects

                                Page 5 of 46

       is  estimated.  If secondary poisoning is of concern, feeding secondary
       consumers (held in captivity) prey items collected in the field following
       application of the test substance can be useful to evaluate this potential
       exposure  route.   Laboratory toxicity  tests  for secondary consumers
       coupled with residue analysis of prey items can indicate the potential for
       secondary poisoning of nontarget  species.  In designing field studies, the
       utility of laboratory and/or pen tests should  not be neglected, and where
       appropriate their use is encouraged.

(ii) Geographic area selection.

       (A) Studies should be performed in representative biogeographic areas
       where the chemical could be used (primary consideration for pesticides) or
       occur under the pattern of production, use, disposal, or accidental release
       of industrial chemicals taking into account the diversity and variability in
       wildlife species and habitats involved.  To keep the number of geographic
       areas at a manageable level while still accomplishing the  purpose of the
       field study, geographical area selection should emphasize situations likely
       to present the greatest risk.

       (B) A  careful review  of  the   species  and  habitats in  the  various
       geographical areas where the pesticide  product could be  used  or the
       industrial  chemical occur under the pattern of production, use, disposal, or
       accidental release is necessary to identify the areas of highest concern. A
       sound understanding  of the  biology  of the  species that are  found in
       association with the potential pesticide  or industrial  chemical  exposure
       sites is essential.  Identifying these areas  may  require an  extensive
       literature review and consultation with experts familiar with the  areas and
       species of concern. The  study area selected should be frequented by those
       species that would have high exposure, based on their feeding or other
       behavioral aspects.  If exposure and fate (e.g.,  degradation) parameters
       vary geographically, study area selection also should be biased towards
       maximizing  residues  available  to  wildlife.   In  some  circumstances
       preliminary  monitoring  of   candidate  areas  may  be  appropriate to
       determine which ones should be selected for detailed study.

(iii) Study site selection.

       (A) In general, study  sites should be  selected from what is considered to
       be a typical or representative  environment in which the chemical would be
       either applied and/or disperses to under normal production/use/disposal
       practices,  but at  the  same time,  study  sites  should  contain the  widest
       possible diversity  and density of wildlife species.   Identifying potential
       study sites may require consultation with experts familiar with the areas
       where studies are proposed, and preliminary sampling.

       (B) To  maximize the hazard, the sites  selected should have associated
       species that would be at highest risk from exposure, as well as a good
       diversity of species to serve  as indicators for other species not present at
                         Page 6 of 46

       that  specific  location.   The choice of study  sites  that are as similar as
       possible  in terms of abundance, diversity,  and associated habitat  will
       facilitate an analysis of the results.

       (C) Field surveys of a  number of sites  are used to identify which sites
       should be selected for detailed study.  Even when high risk species can be
       identified, preliminary surveys may be needed to  determine which sites
       have adequate numbers  of the high risk species as well as a good diversity
       of other species.

       (D) In the initial evaluation  of potential study sites, edge effect may
       indicate  which  sites   support  the  larger  and  more varied  wildlife
       populations.  As stated by Aldo Leopold (see  paragraph  (h)(23) of this
       guideline), "The potential density of game of low radius requiring two or
       more types is, within ordinary limits, proportional to the sum of the type
       peripheries."   (Type is defined as the various segments  of an animal's
       environment  used for food, cover, or other requirements.)  If study sites
       are selected to maximize edge  effect the potential for high density and
       diversity should be increased.  One quantitative measure of edge and edge
       effect (see paragraph (h)(15)  of this  guideline) is the distances around
       individual plant communities in relation to the unit area of the community.
       Population densities, in general, are  positively related to  the number of
       feet of edge per unit area  of community.  Study sites chosen to maximize
       the ratio of edge  to core may serve to indicate sites with higher densities
       and diversities of wildlife  species.

       (E) While this ratio can be helpful in  selecting  study sites,  the other
       characteristics of edge should not be neglected in screening potential study
       sites.  Density and diversity of wildlife species are  also influenced by the
       variety in the composition and arrangement of the edge component cover
       types and its  width.   Also,  interspersion  (the  plant types  and  their
       association with one another) influences densities of wildlife species.  The
       edge effect is the sum  of all the  characteristics of edge and hence each
       component needs to be considered.  An agricultural field  with  a relative
       high edge to core ratio may not have as high a density and diversity as one
       with a lower ratio but greater variety, width, and interspersion. In general,
       edge characteristics can be used to screen potential study sites;  however,
       preliminary sampling of prospective study sites will be needed to identify
       study sites with adequate density and diversity of wildlife species.

(iv) Number of sites.

       (A) The number of sites needed can be estimated using the  binomial
       theorem.  Briefly, the  rationale is that for each study site there are two
       possible outcomes, either effect or no-effect. Trials of this type are known
       as binomial  trials and, when  repeated,  the  results  will  approximate a
       binomial distribution.   In this case,  to  use the binomial theorem, it  is
       necessary first to define the expected probabilities that birds or mammals

                         Page 7 of 46

on a site are affected or not affected, after which the probability of the
discrete binomial random  variable x for n replications can be used to
determine the minimum number of sites at a certain level of significance.

(B) For purposes of illustration, a problem exists if some specific mortality
rate or level of some other variable occurs on more than 20 percent (20%)
of the  potential application sites.   Translated into binomial  probabilities,
there is a 0.2 probability of a site showing an effect and a 0.8 probability
of a site not showing an effect.  Therefore, if the results from the field trial
show that the number of sites  affected is significantly lower than 0.2, it
can be concluded that potential impacts will be below the stated level of

(C) To calculate the minimum  number of sites necessary to show a
significant difference between the observed and expected, Equation 1 for
the probability of  the  binomial  random variable x  can  be  used  (see
paragraph (h)(37) of this guideline) with rearrangement.
                                                          Equation 1

       x = number of sites showing effects,

       n = number of sites,

       p = probability of a site showing an effect, and

       q = probability of a site not showing an effect.

(D) For the illustration in paragraph (e)(4)(iv)(B) with the probability of x
= 0, no sites showing effects, set at the desired alpha level (i.e.., P(x = 0) =
a),  Equations 2 through 5  show the  sequential steps in rearranging and
solving Equation 1 for n.
                                                          Equation 2
                    a = q                                Equation 3

                                                          Equation 4
                  n =  	                             Equation 5
                  Page 8 of 46

(E) The minimum number of sites can be determined using Equation 5.
Continuing with the discussion example  of 20% occurrence of an effect
(i.e.., a 0.2 probability of an affected site, a 0.8 probability of a nonaffected
site) at an alpha  (a) level of concern  of 0.05  sample size would be
calculated using  Equation 5  as demonstrated in  Equations 6  with  a
resulting n = 13.4.

              n =  — -  =13.4                        Equation 6
(F) Therefore in the example provided, 14 is the minimum number of sites
needed  such that the probability is not greater than 0.05 that all sites
surveyed would be unaffected.  In other words, if 20% of the application
sites are actually affected, there is only a 5% chance of finding all  14 sites
unaffected when n = 14.  Moreover, if 20% of the application sites are
actually affected, we expect to find 1, 2, 3, and  4  sites  affected with
probabilities of 0.15, 0.25, 0.25, and 0.17, respectively, when n = 14.

(G) Under many circumstances, conducting this number of replications
may not be practical.  However, the number of sites can be reduced if site
selection is biased toward hazard. While arguable, it seems logical that if
the worst cases are sampled, a less stringent level of significance could be
accepted.  While this must be determined on a case-by-case basis, the
Agency believes a minimum acceptable level of significance under worst-
case conditions is  0.2  rather than  0.05  under  average or normal use
conditions.  At this level, eight sites showing no  effect would be required
to conclude at the 0.2 level  of significance that the effect occurred on less
than 20% of the application sites or — there is less than a 20% chance that
all eight sites will be judged unaffected when n =  8  sites.  Under some
circumstances,  this  may not seem  adequately protective.  It  should  be
noted, however, that based on this same design, it could be concluded that,
at the 0.1 level of significance, effects occur on less than 30% of the
application sites, and at the 0.05 level of significance, effects occur on less
than 40% of the  application sites.  Hence, with eight sites, it could be
concluded with a relatively high degree of confidence that  effects would
occur on less than 40% of the application sites. Because worst-case study
sites were used, the  Agency could have additional confidence that  adverse
effects would occur on less than 20% of all normal application sites.

(H) Under some circumstances, particularly if endangered  species could
be exposed from the proposed use, additional replication may be  desirable.
Under these conditions, a high degree of confidence that an effect was a
rare occurrence would be required.  (Under no circumstances should field
studies  on chemicals be conducted in  areas where endangered  species
could be exposed.)

(I) The  calculations under paragraphs (e)(4)(iv)(C)  through (e)(4)(iv)(G)
of this guideline are for when x = 0, no effects are observed on any site.  A
                  Page 9 of 46

                     similar approach can be used to estimate the number of sites necessary to
                     show a significant result for a  critical value of x > 0. Again the formula
                     for the probability of the binomial random variable can be used summing
                     the probabilities of x and all  outcomes of less than x.  Then by using
                     increasing values of n, the number of replications  required to show
                     statistical significance may be determined for a given level of significance
                     for individual x values.
                                                                               Equation 7
                     (J) The minimum value of n in Equation 7 occurs when P(X< r) is set to
                     the desired alpha level (i.e. P(X < r) = a level).  Continuing the previous
                     example, Table 1 gives the results for x < 1 and x U2 for the previously
                     defined acceptable occurrence level of effect (i.e., a 0.2 probability of an
                     affected site,  a 0.8 probability of nonaffected site).  From the table,  the
                     minimum number of sites needed when the critical value for x is set at 1,
                     to conclude that (at a 0.2 level of significance) effects are occurring below
                     levels of concern is 14. If x < 2, 21 sites  are needed  in order to have an
                     equivalent criterion.   As  can be seen, as x (the number of sites with
                     effects)  increases, the number  of sites required to  show  a statistical
                     significance becomes inordinately large.

       Table 1.—Probabilities for Binomial Random Variable with p = 0.2 for x < 1 and x <
2 as a Function of the Number of Sites (n).
                     (K) When the probability of an affected site is 0.2, application of the rule
                     of zero-observed-affected-sites results in a declaration of "no-effect"  16.8
                     and 13.4% of the time for samples  of size 8  and 9, respectively.  It also
                     results  in  a declaration of "no-effect" 43.1  and 38.7% of the  time for
                     samples of size 8 and 9, respectively, when the probability of an affected
                     site is 0.1,  a value less than the criterion probability.  Application of the
                                      Page 10 of 46

       rule of zero-observed-affected-sites for a declaration of no-effect means
       that a study is considered to be negative (shows no effect) if, and only if,
       none of the sites show effects.

       (L) Under any  condition,  it is extremely important with  the  binomial
       approach to define the critical or threshold level for an effect, and to be
       sure that the methods used are sensitive enough to detect an effect should
       one occur.  These assessments depend upon the species potentially at risk
       as well  as the parameter  being  sampled.  It  should be noted that the
       measure of effect is not limited to mortality.   Other parameters, such as
       residue or  enzyme levels,  can be used.   Whatever parameters are used,
       defining the criteria level for an effect is extremely important, and when
       designing studies this issue should be considered carefully.

       (M) Using this  approach,  control (reference)  sites are  not an absolute
       necessity.  While  the  Agency  encourages their use, in some cases the
       additional information  gained from the control  sites for a screening study
       may not justify the additional effort required.   In most instances, control
       sites would serve to protect from erroneously attributing effects  due to
       other  causes to the chemical.  However, for most chemicals,  this  can be
       avoided  by employing  methods,   such as  residue   analysis  and/or
       cholinesterase (ChE) inhibition tests, that can  be used to indicate if the
       chemical contributed to the observed effect. Further, because studies have
       shown that it is a relatively rare event to locate dead or sick animals in the
       wild  except under unusual  conditions  (see  paragraph (h)(18) of this
       guideline),   it  is unlikely  to find  dead animals  that  were  killed  by
       something other than the chemical being tested.

       (N) Nevertheless,  controls may be necessary  when reliable methods to
       confirm the cause of effect are not available.  In these cases the binomial
       design can be modified to a paired-plot binomial design,  with  a treatment
       plot and a comparable control plot for each study site within an  area.
       When critical  levels of effect and occurrence  are  defined,  the  binomial
       theorem can be used for sample size determination, which gives  8 site
       pairs  (16 paired plots) showing  less than a  defined difference between
       plots to conclude at the 0.2  level of significance that the effect occurred on
       less than  20%  of  the application sites.  Alternatively, a quantitative
       difference or,  preferably, ratio of treated to control  responses  could be
       used  to test for a  treatment effect  on  each  of the  measured  response
       variables.   (This is  discussed further under paragraph  (e)(5)(ii)  of this

(v) Size of study sites.  For a satisfactory field study, study sites should be  large
enough to provide adequate samples. The size is dependent on the methods used,
the sensitivity required, and the  density and diversity of species and their ranges.
In some cases, particularly with  slow-acting poisons or where species  at high risk
have relatively  large home ranges, areas several times larger than  the treatment
area may need to be examined.  In some circumstances, several  fields in an area

                         Page 11 of 46

may be included in a single study site to account for wide-ranging species of
lower densities.  Except in the unusual circumstance where fields are extremely
large (e.g., forested and range areas), the study site should never be less than an
individual field and the surrounding area.  The nature of the surrounding area is
discussed further under individual methods. Another consideration is the distance
between  study sites—in general, sites should be separated adequately to ensure
independence, which is dependent mainly on the range of the  species that could
be exposed.

(vi) Chemical application.

       (A) In general, the study conditions should  resemble the conditions likely
       to be  encountered under actual use of the product  (primary consideration
       for  pesticides) or occur under the pattern of production, use, disposal, or
       accidental release of industrial  chemicals.  For pesticides, consideration
       should be given to application rates and methods and in  most instances the
       pesticide should  be  applied  at  maximum use  rates and  frequencies
       specified on the label. If more than one application method is specified on
       the  label, the method that maximizes exposure of nontarget species should
       be used.

       (B) This  evaluation  should relate wildlife utilization  of the area to
       exposure. For example, for pesticides if the crop is one that is used by
       avian  species as preferred nesting  areas, feeding areas, or cover,  ground
       application may be the method that maximizes exposure. However, if it is
       a crop with low utilization by wildlife species, but with high utilization of
       its edges, aerial application where drift could increase exposure may be
       more  appropriate.  In any case, the method of application used should be
       consistent with the label.

       (C) Equipment  used  may influence  potential exposure of  nontarget
       species.  In some instances, preliminary tests may be required to estimate
       which method and equipment poses the highest exposure.  For pesticides
       there  is a diversity of types of farming equipment  that, depending on the
       particular use pattern involved, could influence exposure.  For example,
       for  pesticides applied in-furrow at planting there are several types of
       covering devices employed on seeders, such as drag  chains,  drag bars,
       scraper blades,  steel presswheels, etc., in which the efficiency may vary
       for  covering  the pesticide.   In general, the various equipment normally
       used for the particular pesticide application has to be evaluated to estimate
       the  potential influence of equipment choice on exposure.

(vii) Methods.  This section provides a general outline of methods appropriate for
use in a screening  field study and indicates some of their limitations.   While
methods  described have been found to be most useful, a  screening study is not
limited to  these methods.  If other methods are more appropriate, their  use  is
encouraged.  Because procedures  should  be adapted to specific situations, the
outlines  presented  should  not  be interpreted as strict  protocols.   Normally,

                        Page 12  of 46

different methods will be combined to evaluate potential impacts.  Due to the
indefinite number of variables and  the unpredictability of wild animals, even
normally reliable procedures can sometimes prove inadequate.  The methods used
in a screening study address exposure by monitoring overt signs of toxicity such
as mortality or behavioral modifications, or by evaluating parameters that indicate
animals are under stress, such as residue  concentrations in  tissues or degree of
enzyme inhibition.  Measurements of density and diversity of species are needed
to aid in evaluating the results.  The  methods in paragraph (e)(4)(vii)(A) through
(e)(4)(vii)(F)  of this guideline can be  useful for screening studies.

       (A) Carcass searches.

              (1) Searching for dead or moribund wildlife is a basic method used
              in field studies to  evaluate  the impact of chemicals on nontarget
              species.  Carcass  searches can roughly indicate the magnitude of
              kills  when adequate areas are searched and the reliability of the
              search is documented.  This  latter point is extremely important.
              Rosene and  Lay  (under paragraph  (h)(30)  of this guideline)
              indicated that finding  even a few dead animals suggests that there
              has been considerable mortality; failure to find carcasses  is poor
              evidence  that no  mortality has  occurred.  The  reliability  of the
              search is based upon the percentage of carcasses recovered by
              searchers  and the rate of  disappearance.    By  knowing  the
              reliability, the significance of failure to find  carcasses  can be
              assessed and the extent of the kill estimated.

              (2) Finding dead animals is seldom easy, even if every animal on a
              site is killed.  For example, three breeding pairs of small birds per
              acre is considered a large population (see paragraph (h)(18) of this
              guideline), and  under average cover  conditions, a small  bird  is
              difficult to detect.  Small mammals may be more abundant but, due
              to their typically secretive habits, they are more likely to die under
              cover and can be  even more difficult  to find  than birds.  Carcass
              searching specifically for mammals should be attempted only when
              cover conditions permit reasonable search efficiency.  However,
              any vertebrate carcasses found  should be collected,  even if the
              search is oriented primarily to one taxon.

              (3) Because  results may be biased by scavenging and failure to
              find   carcasses,  the   sensitivity  of   this procedure  should  be
              determined.   Under  conditions of  heavy  cover  and/or high
              scavenger removal, other methods may be more appropriate.

              (4)  There  are  no standard  procedures for  carcass  searches.
              Paragraph (e)(6) of this guideline outlines practices that have been
              used typically and should be considered in designing searches.
                        Page 13 of 46

(B) Radio telemetry.

       (1) Radio telemetry has been found  to be extremely useful  for
       monitoring  mortality and other  impacts caused  by  chemical
       exposure  of wildlife.   Advances  in miniaturizing electronic
       equipment have made it feasible to track most vertebrate animals.
       Transmitters weighing a few grams have been used to track species
       as small as mice.  Cochran's excellent summary of this technique
       provides additional details (see paragraph (h)(5) of this guideline).

       (2) Radio telemetry has the advantages of providing  information
       on the fate of individual animals following a chemical application
       and of facilitating carcass recovery for determining the  cause of
       death. Although the initial cost of this technique may be more than
       for other methods, the increase in information obtained under some
       circumstances can more than justify  the  cost.   The method  is
       particularly useful with less common or wide-ranging species.

       (3) In addition to mortality, radio telemetry can be used to monitor
       behavioral   modification  as  well  as  physiological   changes.
       Automatic radio-tracking systems permit continual surveillance of
       the location of animals (see  paragraph (h)(5) of this guideline),
       which can be used to provide insight into behavioral changes such
       as nest abandonment, desertion of young, or decreases in activities
       such as flying or feeding.   Radio telemetry equipment is also
       available for the transmission  of physiological data such as heart
       rates or breathing rates (see paragraph (h)(25) of this guideline).

       (4) While this technique can provide very useful information on
       impacts  of   chemicals  to wildlife,  other points  need  to  be
       considered in addition  to cost.   Capturing  animals alive and
       unharmed requires time, skill,  and  motivation. For the method to
       be consistently  successful, the investigator must be thoroughly
       familiar with the habits of the species under  study and  with the
       various capture  methods that can  be  used.   Even  for the most
       experienced  investigator, adequate  sample  sizes can  be difficult to

       (5) Adequate sample size is very important. The binomial theorem
       can  be used to  estimate minimum  sample size per site, if the
       question  is limited to mortality.   Briefly stated, to be sure that
       nontarget  species  are  not  being  affected  by  environmental
       concentrations greater than, for example, an  LC20, the  expected
       binomial  probabilities would  be  0.2  for  mortality and  0.8  for
       nonmortality.  Depending on the level of significance, 8 (a = 0.2)
       to 14 (a = 0.05) individuals would need to be monitored per site
       (see  paragraph (e)(4)(iv) of this guideline for further details on
       these calculations).  However, since the LC20 may differ between

                 Page 14 of 46

       species, 8 to 14 individuals would be  required for each species,
       unless   laboratory  tests   have  documented  relative  species
       sensitivity.  Further complications can arise if radiotagged animals
       leave  the area or if the  movements  of individuals limit  their
       exposure. If these complications occur at relatively low rates, a
       few additional radio-tagged animals may be sufficient to overcome
       these problems.

(C) Tests of Cholinesterase inhibition.

       (1)  Measuring cholinesterase  (ChE)  concentrations in  animal
       tissues has been  found to be a very  useful field technique for
       evaluating  exposure  of nontarget  animals to  ChE-inhibiting-
       chemicals (see paragraphs  (h)(18) and  (h)(19) of this guideline).
       These  chemicals,  including  organophosphates and carbamates,
       affect the synaptic  transmission in the cholinergic  parts of the
       nervous system by binding to the active  site of acetylcholinesterase
       (AChE),   which   normally  hydrolyzes  the  neuro-transmitter
       acetylcholine.  Thus, ChE inhibitors permit excessive acetylcholine
       accumulation at synapses,  thereby inhibiting the normal  cessation
       of nerve impulses  (see paragraphs (h)(6)  and  (h)(27) of this

       (2)  The depression  of AChE  activity, when  measured  and
       compared to controls, can indicate the degree to which an animal is
       affected.  Brain ChE depression of >50% in  birds has been found
       sufficient to assume that death is  pesticide-related (see paragraph
       (h)(24) of this guideline). Depressions of more than 70% are  often
       found in dead birds poisoned by these  chemicals  (see paragraphs
       (h)(l),  (h)(2), and (h)(33) of this guideline),  although   some
       individual birds with less  than  50%  inhibition may die.  A 20%
       depression of brain ChE has been suggested as an indication of
       exposure  (see paragraph  (h)(24)   of this  guideline).    ChE
       concentrations in blood can  also be used to indicate exposure,
       avoiding the necessity of sacrificing the animal.  However, blood
       ChE concentrations  are influenced more by  environmental and
       physiological factors than are brain ChE concentrations.  Because
       ChE activity varies among  species, the  degree of depression must
       be based on an estimated normal value  for concurrently tested
       controls  of  the  species potentially at risk.   Because of this
       difference between species, each case must be considered unique
       (see paragraph (h)(19) of this guideline).

       (3)  Although  there  are  several  calorimetric  methods  for
       determining  ChE activity, the general methods are similar.  Brain
       tissues (or  blood samples)  are  taken and analyzed  for  ChE
       concentrations.  Comparisons are made between pre- and  post-
       treatment and between treated and untreated  areas.  It is important

                 Page 15 of 46

to ensure that untreated controls have not been exposed to any ChE
inhibitors.  It also should be noted that absolute enzyme levels in
the  literature are derived from various different, although similar,
methods and are reported in different ways. For example, Ludke et
al. (see paragraph (h)(24) of this guideline) used a modification of
the  method reported  in  paragraph  (h)(13) of this  guideline and
reported results of ChE activity as nanomoles of acetylthiocholine
iodide hydrolyzed per minute per milligram of protein, whereas
Bunyan et al. (see paragraph  (h)(l) of this guideline) used  their
own colorimetric method (in addition to a pH change method) and
reported the results as micromoles of acetylcholine hydrolyzed per
hour  per  milligram  of  protein.   Therefore,  without a  tightly
standardized method,  it is necessary to use concurrent controls of
the  same species obtained from the general vicinity (but untreated)
of the exposed birds, rather than literature values.  Because of the
greater  variation  in  plasma  ChE  levels  than for brain, more
controls are necessary to evaluate blood samples.

(4)  Tests for ChE activity can be used to help confirm cause of
death and monitor levels of exposure.   In the latter case, 5 to  10
individuals  of each species  are collected  before treatment and at
periodic intervals following treatment.  Mean inhibition of 20% or
more is considered an indication of exposure to  a ChE inhibitor.
Confirmation of cause of death  may be determined by analyzing
brain tissue from wildlife  found dead following  treatment and
comparing the activity with controls. Inhibition of 50% or more is
considered  strongly  presumptive evidence  that mortality  was
caused  by a  ChE-inhibiting  compound.    The  cause-effect
relationship can be further supported by chemical analysis of the
contents of the digestive tract  or other tissues for the chemical in

(5)  For this technique to provide  accurate  information, prompt
collection and proper preservation of specimens are essential. ChE
concentrations  in  tissues are influenced  by  time since  death,
ambient temperatures, and whether or not reversible ChE inhibitors
are  being investigated.   Therefore, the response of postmortem
brain ChE  to ambient conditions can seriously affect diagnosis of
antiChE poisoning. Samples must be collected shortly after death
and  frozen immediately to halt changes in tissue  or  enzyme-
inhibitor complexes.    A  technique  for  field  monitoring and
diagnosis of acute poisoning of  avian species has been reviewed,
discussing   sample  collection,   sample  numbers,  preservation
procedures, and sources of error (see  paragraph (h)(19)  of this
           Page 16 of 46

(D) Residue analysis.

       (1) Residue analyses of wildlife food sources provide information
       about the  level and  duration of chemical  exposure.   Residue
       analysis of animal tissues also can indicate actual exposure levels.
       If the relationship between tissue concentrations and toxic effects
       is known for the species in question, residue analyses can provide a
       measure  of the  degree  to which animals  are affected.   For this
       application of residue analyses, laboratory trials  are necessary to
       establish the relationship between residue  levels  and toxicity.  In
       addition to death, these  laboratory trials should include such  signs
       as anorexia, asthenia, asynergy, or ataxia.  For chemicals that are
       readily metabolized by vertebrates,  residue analysis may not be
       appropriate for diagnostic purposes.  With many chemicals, it will
       be necessary to analyze for residues of active metabolites also.

       (2)  For  determining  residues on  wildlife  food  sources, the
       investigator should collect samples of insects, seeds, leafy parts of
       plants, etc.,  immediately after chemical application and at periods
       thereafter.   Samples should  be  analyzed for the chemical to
       determine potential exposure rate and duration.  The  application
       method needs to  be considered  in  determining where  to take
       samples.  If drift is likely, samples should be taken from habitats
       surrounding the treatment sites  as  well  as  in the treated  sites.
       Because  analysis can be  costly, the investigator should  consider
       carefully the number of  samples necessary  to provide adequate
       data.  Where feasible, samples from different  locations within a
       site should  not  be pooled.   Separate analysis  of samples can
       provide data on the range and variability  of exposure  as well as
       mean levels.

       (3) When residue analysis is used to evaluate exposure in nontarget
       animals,  the tissues selected for analysis differ depending on the
       purpose.   Heinz et al  (see paragraph  (h)(17)  of this guideline)
       indicated that for many chemicals, residues in brains of birds and
       mammals can be used to determine if death is chemical-related.
       The authors believe that sublethal exposure is judged  better from
       residues in other tissues.  Therefore, they proposed that analyses of
       whole  body homogenates should be used to  quantify the  body
       burden of a chemical.   If this  is not feasible, analysis of muscle
       tissue is  suggested, because muscle residues reflect body burden
       more  nearly than  those of any other tissue, and the  amount of
       muscle tissue is  not unduly large.  For persistent chemicals, it has
       been suggested  that residues  in liver and fat tissues could be
       misleading  for  determining  acute  body  burdens.    Liver  is  a
       processing organ and its  residue level largely represents current
       availability of the chemical.  Residues in fat are greatly affected by
       changes  in  the  amount  of  body  fat, and  are  not  dependable

                  Page 17 of 46

indicators of body burden of the chemical; however, for some
chemicals,  liver,  fat, or other tissues may be  good qualitative
indicators that exposure did occur. In general, laboratory trials or
data gathered in metabolism or other studies may be necessary to
determine which tissues can provide the most useful information.
Residue analysis of eggs taken from nests in treatment areas can
indicate the degree of contamination that a treatment has caused, as
well as possible reproductive effects of the treatment.

(4)  Two approaches may  be  used to determine the number of
samples  to  be collected.  Frequently, residue  samples  will  be
collected to establish a mean value and confidence limits.  To
determine the number of  samples  to  collect, it is  necessary to
estimate the standard deviation and to set an arbitrary limit from
the  mean value that is acceptable.  Although the mean value does
not need to be estimated, it is also necessary to have  some idea of
the  mean so that the standard  deviation can be estimated and the
limit can be set. The formula  for the number of samples  for 95%
probability,  from  the  reference in  paragraph  (h)(34)  of  this
guideline, is presented in Equation 8.

                .  2 /
            /? = 4cr /2                              Equations
                  / -/-/


       G = the standard deviation, and

       L = the allowable limit around the mean.

(5)  For  example,  to  calculate  the  residue  concentrations  on
vegetation  within  ±10 ppm,  with  an estimate  of  the  standard
deviation of 20 ppm, then n = 4(20)2/(10)2 means 16 samples are
required to have a 95% probability that the sample mean value will
be within ±10 ppm of the true mean.

(6)  In some situations, there may be little information useful for
estimating the standard deviation, or the standard deviation may be
rather large, thus requiring a very large  sample  size.  For some
types of samples,  such as residues in nontarget wildlife carcasses,
the  sample size cannot be increased to permit more precision. The
mean value of a parameter certainly has utility, but it also is very
important to establish confidence  limits around the mean.   In
general, the Agency will use the 95% confidence limits  (usually
the  upper boundary, as in the case of residues) in the assessment of
the  data.  This approach will  substantially reduce the impact of
outliers but will still incorporate the range  of reasonable values
into the  assessment.  In  addition,  the use  of confidence limits
reduces the necessity  for taking  a  large  number  of samples.
           Page 18 of 46

       Because  the  width of the  confidence  intervals decreases with
       increasing sample sizes, investigators should take samples that are
       as large as feasible.

       (7) Since the sample size will nearly always be less than  30, the
       calculation of confidence limits should be based on  Student's  t-
       distribution. The t values are derived from tables available in most
       statistics books, and the 95% confidence limits are:
                                                          Equation 9

              s  = the  standard deviation estimated from the sample of
              size n.

       (8)  Alternatively, the  binomial  approach  may  be used for
       determining if residues, typically in collection of live nontarget
       animals,  exceed  a particular  threshold value  that  indicates  an
       effect.  The required sample size is the same as presented for the
       binomial  approach  in determining the number  of study  sites.
       Specifically, in the preceding  example a minimum of 8  samples
       with none exceeding the threshold value or 14 samples with one or
       none exceeding the threshold value indicates no effect atp = 0.2 in
       20% of the samples.   This approach requires the establishment of
       threshold values which are determined on a case-by-case basis.  In
       general, residues reflecting a 20% lethal concentration (LC2o) level
       of exposure would  seem to  be a maximum  acceptable effect
       concentration for a screening study.  A median lethal concentration
       (LCso)  should be determined  in the laboratory for  each  species
       analyzed  for residues, after which a group of animals would be
       exposed to an LC20 concentration to determine the mean threshold
       concentration of residues.  Since this approach is impractical for a
       screening study, it is suggested that the mean residue concentration
       in  bobwhite  and/or  mallards  exposed   to   an   LC20   dietary
       concentration would provide an indication of threshold levels.

       (9)  The number and timing of collection periods should be based
       on the  persistence of the specific chemical under study.  Where
       persistence in the  field has not  been adequately determined, it may
       be necessary to sample at regular intervals (e.g., days 0, 1, 3, 7, 14,
       28,  56) to provide data on degradation rates.

(E) Behavioral observations.  Observations  of behavior sometimes can
be  an  extremely  important indicator of  treatment effects.     Such
observations might include characteristic signs of toxicity or behavioral
changes  seen in test animals exposed to the  chemical  in the laboratory.
                  Page 19 of 46

Other abnormal behavior (e.g., territorial males abruptly ceasing singing,
birds not feeding, reduced avoidance of humans) also may be important.

(F) Density and diversity estimates.

       (1) It is necessary to know the number of individuals and variety of
       species on and  around a study site in  order to indicate which
       species could have been exposed  and to aid in evaluating the
       significance  of  mortalities  or  other findings.    In  addition,
       preliminary information on density and diversity is necessary for
       site selection and to determine the size of study sites.  Under some
       circumstances,   comparisons   of  density  estimates  between
       treatment and control sites, or between before and after treatments,
       may be  used to indicate chemical  impacts.   In general,  the
       usefulness of these comparisons is limited in a screening study due
       to the relatively small acreage involved.   If mortality occurs,
       replacement from outside is likely  to  be so  rapid that losses are
       replaced before censuses are completed. In addition it is necessary
       to  consider  seasonal   changes,  such as  migration,  molt,  or
       incubation, which can affect real or apparent densities.

       (2)  Several techniques may be used to estimate the density and
       diversity of wildlife species,  including  counts of animal signs,
       catch-per-unit effort, mark-recapture,  and  line-transect sampling.
       Although the methods  selected depend on the species  of concern,
       for the screening field test line transect methods are likely to be the
       most useful for birds.

       (3)  The major advantage of  line-transect  sampling  is that  it is
       relatively easy to use in the field once a proper sample of lines has
       been chosen.  However, line-transect-sampling is not applicable to
       all  species,  particularly  those that  are  not  easily observed.
       Individuals using line-transects must be extremely competent in
       species identification.

       (4)  In  the line transect method an  observer walks  a  distance (L)
       across  an  area  in nonintersecting and  non-overlapping  lines,
       counting the number of animals sighted  and/or heard  (N), and
       recording one or more of the following statistics at the time of first

              (a) Radial distance from observer to animal.

              (b) Right-angle distance from the animal  sighted or heard
              to the path of the observer  or angle  of sighting  from the
              observer's path  to the point at  which the  animal  was first
              sighted or heard.
                  Page 20 of 46

              (5)  Although  the field  procedures are  simple,  they must be
              understood adequately  and implemented  well to  obtain  good
              estimates of density (see paragraph (h)(3) of this guideline).  The
              authors provide a thorough review of the theory and design of line-
              transect-sampling and  this monograph should be reviewed  for

              (6) For  mammals, density and diversity  estimates from  capture
              data may be the most practical for a screening study.  There are
              several ways of estimating the populations from capture data, some
              relatively  simple, that  may provide adequate  information  for a
              screening study. Davis and Winstead (see paragraph (h)(7) of this
              guideline), review the various  methods available,  explaining their
              advantages and disadvantages.

(viii) Interpretation of results.

       (A) The numerous variables involved  in field studies makes a meaningful
       discussion of the  interpretation of results  somewhat tenuous, particularly
       with the almost inexhaustible array of results that could  occur. Each study
       must be considered unique and therefore will  require a case-by-case
       analysis that incorporates not only the actual study but other relevant
       information that is available.

       (B) In  general, the results of the screening  field  study should  provide
       information  on  acute  poisoning  and  potential  sublethal  effects as
       suggested by  enzyme, residue, or other measurements.   In addition,
       information has to be developed on the density and diversity of species on
       the study sites as well as the sensitivity of the methods used. If no effects
       are detected, assuming that the methods used were adequate to detect
       levels of concern and that the  species on  the study site represent a good
       cross-section of the nontarget species  expected to be at risk, the potential
       hazard  indicated by lower tier tests is  refuted.  Unless other hazards (e.g.,
       reproduction) are  still of concern, additional tests would not normally be
       necessary. However, if an effect is detected on one or more study sites at
       rates equal to or greater than  concern  levels,  the hazard has not been
       refuted and additional tests may be necessary.

       (C) In interpreting if an effect has occurred in the context of the binomial
       approach, care  must be employed not to  assume a level of precision in
       results that does not exist. Most detectable effects will exceed the concern
       level for  some methods used  in these  studies,  due to  the  inherent
       variability in the data collected to estimate the level of impact, particularly
       when minimum sample sizes and  areas are used.  In  some instances in
       interpreting results it may be appropriate to use confidence limits of data
       collected (or another measure of dispersion) to  evaluate if concern levels
       are exceeded.  For example, when density estimates are used to estimate
       percent mortality using the  number of dead animals found during carcass

                        Page 21 of 46

searching, the upper and lower confidence limits of the density estimate
may be more appropriate than the average, particularly when variability of
the density estimate is high.  Whatever method is used, when effects  are
detected that exceed concern levels they will be put into perspective in  the
context of the entire  study  as well  as other  available information to
determine if or what additional data are needed.  A "no-pass" result does
not necessarily mean that definitive field testing is automatically required.

(D) For example, a test may be run in an area where a species is abundant,
yet on a specific study site their numbers may be sufficiently small that a
single death exceeds the level of concern on that  site. Statistically, such a
finding would indicate  that the  study  did not pass  according to  the
binomial  approach,  and this would  be the preliminary interpretation.
However, if the other sites had an  adequate number of this species as well
as other species expected to be at risk and no other signs of impacts  are
observed, the  implications of the  mortality would seem minor. On  the
other  hand,  if diversity of species were extremely limited, it would have
greater significance. In  other situations where the one dead bird is of a
species with small numbers  on most  sites, but  density and diversity of
other  species is representative of nontargets expected to  be at risk, another
screening study that looks at the species in which the effect was detected
may be appropriate. Conversely,  a screening study  showing that there is
appreciable mortality on most study sites may be sufficient for the Agency
to consider regulatory action.

(E) In summary, the interpretation of results will go beyond the  statistical
evaluation  since  the  Agency   must consider all  the  factors  and
circumstances peculiar to each test and site.  The biological interpretation
of results is, and probably always  will be, a matter of scientific judgment
based upon the best available data.  In general, the judgmental aspects of
biological interpretation are more important for definitive studies than  for
screening studies.   Nevertheless, biological  considerations  often will be
relevant to screening studies.  Study conclusions must integrate that which
is biologically significant with that which is statistically significant.

(F) Another consideration in the interpretation of results of a field study is
the attribution of effects  to the chemical being studied.  A well-designed
study will include appropriate techniques  to  determine if an effect is
caused by a chemical. In the absence of such techniques, the Agency has
no choice but to consider that any  effects were as a result of the chemical
applied.   As an  example,  measurement  of  ChE  levels can provide
information, since it is generally accepted that inhibition of 20% indicates
exposure and inhibition of 50% or more indicates, in birds, that mortality
is due to an inhibitor (see paragraph (h)(4)  of this guideline).  If the test
chemical is the only ChE inhibitor used in the vicinity of the study site, it
can be reasonably assumed that mortality associated with 60%  ChE
inhibition is due to the test chemical. However, if other ChE inhibitors  are
used near the site, additional information, such as residue measurements,

                  Page 22 of 46

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

                     (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

              (C) The definitive study, in addition to estimating the magnitude of effects
              of acute  toxicants,  also can  be  applied  to estimating the magnitude of
              chronic or reproductive  effects.  Although the discussion has emphasized
              chemicals that are acutely toxic,  with few exceptions it is applicable to
              chemicals that cause chronic effects.

              (D) In general,  the definitive study  will provide  limited insight into
              whether or not effects  are within the limits of compensation for the species
              of concern.  However, using the data  collected  in these studies coupled
              with available information on the species of concern and  basic theories of
              population  dynamics,  the meaning of the observed effects on  the species
              can be evaluated.
                               Page 23 of 46

(ii) Sampling and experimental design.

       (A) The principles of statistical design of studies are well documented and
       it is beyond the scope  of this guideline to cover the fundamentals of this
       topic.   However, there are a few points  on this topic that warrant
       discussion relative to the definitive study.

       (B) In the design of field studies, it is necessary to consider carefully what
       constitutes  a sampling unit.  Eberhardt, under paragraph (h)(10) of this
       guideline,  points  out  that special problems  are  faced  in  designing
       experiments on wild animal populations.  Study  sites must be large in
       order to limit the influence of boundary effects, such as movements  into
       and out of the area. Large study sites can be very expensive both in terms
       of actually applying the experimental treatment and in the assessment of
       results.  Eberhardt also states that numerous observations, even a full year
       of data, on  a single study site may  result in very sound values for that  site,
       but do not provide a basis for inferences to other  sites.   Hurlbert  (see
       paragraph (h)(19) of this guideline) has discussed the problems associated
       with field studies  where there  was no  replication or replicates were not
       statistically independent, which he terms pseudoreplication.  Of the field
       studies he  evaluated,  48% of  those  applying  inferential  statistics  had

       (C) According to Eberhardt, under paragraph (h)(9) of this guideline,  lack
       of replication seems to be based on the mistaken assumption that variances
       based on subsampling of sites (intrasite variability) are suitable bases for
       comparing treatment effects (intersite variability). This, he believes, is not
       a valid basis for a statistical test, because it is the variance of sites that are
       treated alike that is relevant to a test of treatment differences.  Although
       subsampling of sites may  be  necessary to  collect the data,  it is the
       difference between sites that is important for analysis.

       (D) An important point to consider in designing a definitive study is to be
       sure that the study will detect a substantial impact when, in fact, it occurs.
       In  statistical terms this concept is referred to as the power of the test (1- P).
       Experience with classical experimental designs  with random  assignment
       of experimental treatment and controls, has shown that the probability of a
       Type  II error (P, false acceptance error rate) is generally high (unless very
       large  numbers  of replicates are available).   Eberhardt, under paragraph
       (h)(9) of this guideline, indicates that, all too often in field  studies on
       impacts to wildlife, either by default or lack of understanding, there is only
       a 50% chance of detecting an  effect, which he likens to settling the issue
       by flipping a coin and doing no field study whatsoever.  Since a definitive
       study is  carried out under the assumption  that effects will  occur, the
       Agency believes minimizing Type  II errors is  extremely important.

       (E) As suggested, the more generally  used experimental designs require
       inordinately large sample sizes to obtain small Type II errors.   For

                         Page 24 of 46

example,  based on  a coefficient of  variation (cv)  of 50%  (this is  a
relatively homogeneous  sample for the kinds of data  collected in field
studies), a 20% minimum detectable difference between means, a Type II
error (P) of 0.2 and a Type I error (a)  of 0.05, the number of replications
required can be estimated using the formula in Equation 10 (in Eberhardt
(see paragraph (h)(8) of this guideline)).
                                                         Equation 10

       n = number of replications;

       zp = pth percentile of the unit normal distribution (z\.a and zi_p are
       critical values of the unit normal distribution);

       cv = coefficient of variation;

       5 = detectable mean difference expressed as the proportion  of the
       control group mean (i.e., 5 = (|ii - |i2)/(m)).

(F) Thus, for the example in paragraph  (e)(5)(ii)(E), n = 63.6 replicates
which rounds to 64 replicates  for both control  and  treatment groups, or
128 total study plots,  are  required to detect  a  20% difference between
treatments  and controls with an 80% chance of being sure to detect a real
difference (zi_o.2o = 0.84) at a 0.05 level of significance (zi_o.o5 = 1.645).
         .65  .	 ,.   ,.   _  ,„                Equation 11
(G) With more sophisticated designs, the number  of replicates  can be
reduced under some circumstances and still meet the Agency's aim to limit
the probability of a Type II error to 0.2 with a detectable difference of 20
to 25%.   For example, a  paired plot design can be  used, substantially
reducing the number of replicates required. Pairing serves to reduce the
effective coefficient of variation by reducing the variation attributable to
experimental error.  The lower coefficient of variation reduces the number
of replicates.  A quantitative difference or, preferably, a ratio of treated to
the total  of treated and control responses, can be analyzed statistically to
test  for  a treatment  effect  on the  measured response  variables (see
paragraph (h)(31) of this  guideline).   The logic of using  paired plots is
that, while no two areas  are  ever exactly alike, two areas that are not
widely separated in space are  ordinarily subjected to much the  same
climatic  factors, have  populations with about the same genetic makeup,
and generally the two populations can be expected to follow much the
same trend over time, apart from a chemical effect (see paragraph (h)(8) of
this guideline). If all plots are approximately equal in area and habitat and
                 Page 25 of 46

population densities between pairs are similar, it is postulated that when
no chemical impacts occur, the mean ratio of treatment to treatment plus
control will equal one-half.  Then a t-test or an  exact randomization test
(see paragraph (h)(l 1) of this guideline) may be applied to test whether the
average number of survivors on the treated plots is equal to the average
number of survivors on controls.

(H) The number of pairs required can be estimated using the formula in
Equation 12.
        n = (zi-a + zi-p )   r-7; - \                    Equation 1 2

       n = number of paired plots;

       zp = pth percentile of the unit normal distribution (zi_a and zi_p are
       critical z-scores);

       q = survival ratio;

       p = mortality ratio; and

       c = mean number of survivors on control plots.

(I) Thus, 10 pairs of plots (20 total) with a mean of 28 individuals per plot
would be needed at  an 80% assurance of detecting a  treatment-induced
impact  of 20%  or greater  at  a  0.05 level  of significance if c = 28.
Increasing the mean number of individuals per plot (c)  causes a reduction
in n.
   n = (1.645 + 0.84)2  7 — ,*  '  .,   .  = 9.8           Equation 13
(J)  In some field  situations, pairing may not be feasible.   In these
situations, other designs would  be more appropriate or a less rigorous
design may have to be used.  However, in planning field studies, the
power of the study design must be considered to determine the limitations
of the study.  Studies with adequate replication are highly preferred to
support registration — the use of less replication will not necessarily render
the study inadequate.  However, it is wrong to use a study with low power
to imply no  biological damage, when the study is not capable of detecting
the damage  if it occurred. In cases where large numbers of replicates are
impractical,  subjective and biological knowledge should be  used in a
decision  process to decide if there  was a treatment effect.   In  most
instances, it is highly advisable  to involve statisticians or biometricians

                  Page 26 of 46

       who are familiar with this kind of field study in the planning and analysis
       phase of the field work to avoid costly technical errors.

(iii) Study area and site selection.  Selection of geographical areas and  study
sites  within the  areas  for  the definitive test generally requires the  same
considerations as for a  screening study.  For the definitive study, however,  the
selected areas and study sites should have adequate populations of the species of
concern.  For pesticides, the crop of concern should be grown on a representative
portion of the area and consideration needs to be given to whether the target pest
species will be  present.  If it is not, it is necessary to  consider what influence its
absence may have on potential results.  For example, if the pest is a major food
source for nontarget species, its absence  could significantly influence  results.
Finally, the potential variation in populations of concern over the geographical
areas  selected should be considered.  It may be difficult to  find sites that  are
sufficiently  similar  to  provide paired plots, which limits the coefficient  of
variation so that the desired sensitivity can be achieved.

(iv) Number and size of sites.  As suggested under  paragraph (e)(5)(ii) of this
guideline, the number of sites will depend  upon the species density on sites and
the sensitivity required.  Ideally, sample size should be large enough so there will
be an 80%  probability of being sure to detect a 20% difference when it exists.
The size of the study site must be large enough to provide adequate samples. The
size depends on the survey methods used, sensitivity required, and the density and
range of the species of concern.  For a paired-plot design, the number  of sites
required  is  a function  of the  average  density  of the  species. In general,  the
breeding density of the species of concern can be used  to provide a rough estimate
of the size of area needed to provide adequate samples.  However,  preliminary
sampling most likely will be required to verify the estimates.

(v) Methods.

       (A) Essentially,  the  methods used  in a definitive study are  a means to
       quantitate reproductive  and  mortality rates of animals on treatment and
       control areas.  There are many texts  and monographs available on methods
       of sampling to estimate these parameters.  Anyone not familiar with  the
       theory and  principles of the various techniques  should review  these
       references in depth.   The objective of this portion of the guideline is to
       provide  a general guide to the various methods that could be used in a
       definitive field study. In addition, these methods can be applicable to some
       screening studies.

       (B) The methods to be used in an individual field study will depend on  the
       nature of  the  identified  concerns.    Some methods  are  useful  for
       investigating  several types of concerns, and most types of concerns can be
       studied by several methods.  When the  concern becomes more specific
       (e.g., secondary hazards to  raptors),  the use  pattern  or pattern   of
       production, use,  disposal, or accidental  release is  limited, and/or habitat
       type is limited, the range of applicable methods tends to become narrower.

                        Page 27 of 46

       (C) Methods described below are divided into three categories:  Methods
       for assessing mortality and survival of adults and independent juveniles,
       methods for assessing reproduction and  survival of dependent juveniles,
       and ancillary methods.  The intent of this guideline is to present methods
       that are likely to be useful in many situations, rather than an exhaustive list
       of all  available methods.    The Agency  encourages the use of other
       methods when they are scientifically valid and have a high probability of
       detecting an effect.

       (D) While it is absolutely essential to have a detailed plan that describes
       the  selected  actions  (with  contingencies)  for achieving  the  study
       objectives,   investigators  must  remain  flexible  because unanticipated
       problems always  come up  in  long term studies.   Even with  highly
       experienced and resourceful  field biologists, the most carefully planned
       studies  can be compromised due to the  unpredictability of wild animals
       and natural events. When a natural disaster occurs early in the study,  it
       may be wise to reinitiate the study. If the event occurs after substantial
       data already have been collected  (e.g., early in the second  year of a
       multiyear study),  it may be  more appropriate to extend the  study  an
       additional year or more to help provide for the additional needs.  If the
       study is to be terminated, the report should describe thoroughly the nature
       of the events and the consequences if they affect the study results.

(vi) Mortality and  survival.  It is very important to understand the autecology of
the species  being  studied in order to select the  most appropriate methods for
investigating them.   In addition, the choice of particular methods must consider
the applicability of the method based on the chemical use pattern and study site

       (A) Mark-Recapture.

              (1)  There  are  several  mark-recapture methods  available,  each
              based on the same basic premise.  A sample of animals is captured,
              marked, released, and another sample is collected where some of
              the  animals  are captured  again.    The  characteristics  of this
              identifiable sample are used to  estimate population parameters.
              Mark-recapture studies can provide information on:

                     (a) Size of the population.

                     (b) Age-specific fecundity rates.

                     (c) Age-specific mortality rates.

                     (d) Combined rates of birth and immigration.

                     (e) Combined rates of death and emigration.
                         Page 28 of 46

  (2)  Seber  (see paragraph (h)(32)  of this guideline) reviewed the
  various  mark-recapture   methods  and  subsequent  statistical
  analyses.  Less detailed but still very useful reviews are provided
  in paragraphs  (h)(4) and (h)(15)  of this guideline.  Nichols  and
  Pollock  provide  a valuable  comparison  of  methods  under
  paragraph  (h)(26) of this guideline.   Table  2 provides a brief
  summary of some of the various mark-recapture methods discussed
  in these references.

Table 2.—Mark-Recapture Techniques
Peterson Method (Lincoln Index)
Schumacher's Method
Bailey's Triple Catch
Jolly-Seber Method
Estimation of population size. Usually only two sampling periods.
Closed population.
Estimation of population size. More than two sampling periods.
Marking continues throughout sampling. Closed population.
Estimate of birth rate and death rate in addition to population size.
Requires data from two marking occasions and two recapturing
occasions. Open population.
Estimates mortality and recruitment in addition to population size.
Requires more than two sampling periods and that each animal's
history of recapture be known. Open population.
  (3)  When considering the use of  one of these mark-recapture
  models, one must carefully evaluate the applicability of the method
  to the circumstances under consideration.  While in theory mark-
  recapture techniques should be an excellent method for evaluating
  effects of chemicals on wildlife populations, some mark-recapture
  analyses  are not particularly  robust; small deviations  from  their
  implicit assumptions can produce large errors in the results (see
  paragraph (h)(4) of this guideline).  However, some of the more
  recent and sophisticated analytical methods are robust and can deal
  with deviations from  assumptions  in closed populations  (see
  paragraph (h)(28) of this guideline).

  (4)  Mark-recapture methods  are  particularly  useful  for small
  mammals because these animals are  seldom amenable to the visual
  and auditory  observations necessary for using transect, territory
  mapping, or similar methods.  However, mark-recapture also may
  be useful for  birds  provided a sufficient number of birds  can be
  captured   and  marked.  In  some  situations,  birds  may  be
  "recaptured"  with  use  of binoculars  via visual observations of
  marked individuals. Animals must remain marked for the duration
  of the study.  Typically,  mammals are toe-clipped or  ear-marked
  and birds are banded.  Marking should not make the animals more
  susceptible to the effects of the chemical (e.g., anticoagulants with
  toe  clipping).  Dyes  may be useful unless they are lost by wear or
            Page 29 of 46

(B) Territory mapping method.

       (1) A common spatial census method is territory mapping, wherein
       the territories of individuals are mapped before and after treatment,
       on both treated  and untreated plots.   The  method is usually
       applicable when birds are defending territories. It involves a series
       of census visits to the study sites during which birds located by
       sight or song are recorded on a map.  The information from all the
       visits is plotted  for  each species.   Birds exhibiting territorial
       behavior appear on the map as clusters of individual contacts. The
       clusters are used to estimate both the size  and number of territories.
       The pre- and post-treatment censuses for treated sites are compared
       with the pre-  and  post-treatment censuses for  control  sites  to
       determine changes in populations of territorial individuals that may
       be attributed  to the  chemical  (see paragraph  (h)(12) of this
       guideline).    Further  details of this method are  given by  the
       International Bird Census Committee under paragraph (h)(21) of
       this guideline, and its application to evaluating impact caused by
       pesticides is reviewed by Edwards et al.  (see paragraph (h)(12) of
       this guideline).

       (2)  Problems  with  this  method  can  occur.    Under  some
       circumstances, replacement from outside the area can be so rapid
       that territories are refilled before the census is completed.   There
       usually is a floating population of silent, nonterritorial birds who
       may quickly reoccupy empty territories (see paragraph (h)(35) of
       this guideline).  The effects of replacement can be overcome for
       some species by capturing and marking  the territorial  individuals
       prior to treatment, so they can be distinguished from the floaters.
       Also, replacement may not be a problem  when the study areas are
       in the center of a relatively large treated area.

(C) Radio  telemetry.   Radio telemetry  can  be an  extremely useful
technique to provide information on the effects of a chemical application
on nontarget species. As discussed for  screening studies, radio telemetry
can be used  to monitor  for  mortality as  well  as to  provide useful
information  on  behavioral  modification   caused  by   the  chemical
application.    The  points  discussed  previously  (for screening  studies)
generally are applicable to  definitive studies. However, for the definitive
study,  the  number of radio-tagged animals needed depends upon  the
variation between sites and the sensitivity required.  For  example, with
behavioral  observation, intra-  and  intersite variation will influence the
number of radio-tagged animals required.  In some instances, it might not
be practical to radio-tag the  number of  animals required to provide  a
rigorously designed study.  Under these conditions, the limitations should
be specified, and the maximum number of animals that can be practically
radio-tagged and monitored should be used.

                  Page 30 of 46

       (D) Other methods for mortality and  survival.  Other techniques for
       assessing density and diversity are discussed for screening studies; most of
       these, especially line-transect methods, are useful for definitive studies.
       Some methods, such as catch per unit effort or counts of animal  signs, do
       not provide actual measures of density but may still be used to compare
       effects on treated and untreated plots.

(vii) Reproduction and survival of dependent  young.  Some of the techniques
for  assessing  mortality  and  adult survival   are  also useful  for  assessing
reproduction and survival of young.   Some, but  not all, mark-recapture methods
can provide information on fecundity. Radio-tagging nestlings or suckling young
of moderate and large size animals may be used to assess survival of dependent
young.  Radio telemetry  and territory mapping are useful for locating dens  or
nests for further study. The following methods are more specific for assessing
reproductive parameters.

       (A) Nest monitoring.

              (1) Nest monitoring is useful for evaluating the effect of chemicals
              on breeding birds.  The typical procedure is to search the study site
              to find active nests  and subsequently to check those  nests  to
              determine  their fate.  Information collected on each  nest  should
              include number of eggs  laid,  number hatched, number of young
              fledged, and if and when the nest  was abandoned or destroyed,
              both before and after chemical application.   While all  definitive
              studies should consider this technique, it also may  be useful  in
              screening studies.

              (2) This technique is relatively straightforward.  However,  it may
              not be practical if nests are  scarce  or otherwise  hard to find.
              Because the breeding  success  of birds can be highly variable and
              can be quite low, it is sometimes  difficult  to obtain sufficient data
              on the  success  of the same species in enough sites  to yield
              satisfactory results for  statistical comparison with controls (see
              paragraph  (h)(18) of this guideline).  In some cases, artificial nest
              structures can be constructed to increase nest densities.   In a few
              situations where sufficient numbers are available, the technique
              may be applicable to mammal den monitoring.

       (B) Behavioral observations.   Behavioral observations associated with
       reproduction  can  be quite useful, especially for birds.  Techniques are
       simple,  but labor intensive.  When used, such observations most likely
       would be combined with  nest monitoring  since both techniques require
       locating  reproductive sites.  Typically,  the  frequency and  duration  of
       behaviors will be compared for treated and untreated plots.  Incubation,
       parental  care  (especially  feeding  for  altricial  birds), and following
       behavior  (for  precocial  animals)  are behaviors that are  particularly
       amenable to such  study.   Courtship, mating, and nest building are other

                         Page 31 of 46

       behaviors that could be studied in some situations, but locating sufficient
       numbers of animals displaying these behaviors to permit quantitative
       analysis is difficult.

       (C) Age structure of populations.

              (1)  Comparisons  of young to adult ratios  of  selected  species
              between treated  and untreated plots may indicate  reproductive
              effects.  The timing of the application and of breeding of selected
              species  is critical.   For assessing  reproductive impairment or
              survival of dependent young, per se, the duration of this technique
              should be limited to single breeding and rearing periods, which
              may be repeatedly assessed.  However, longer study periods that
              may even  include  several  years  can  be  used  to assess the
              combination of reproductive  success and age-specific mortality,
              even if the two cannot be separated.

              (2)  Obviously, use  of this  method requires that the  age of
              individual animals be  determined.    In  some cases, it may be
              necessary  only  to  distinguish  among  adults,  subadults,  and
              juveniles.   In  mammals, this may usually  be accomplished by
              examining pelage, development of testes or mammaries, or  tooth
              eruption  or  wear  characteristics.     In  birds,  plumage  or
              characteristics  of particular (species-dependent)  feathers may be
              used.   For carcasses  or sacrificed  animals, observation of the
              ossification of bones or development of reproductive organs are
              useful.  In other cases, particularly  where comparisons are made
              among populations  in  different years, it  may be appropriate to
              distinguish age classes  of adults.   In mammals, tooth eruption,
              wear,  or enamel layers, or eye lens weights are useful.  It is  more
              difficult to separate  age classes  of many adult birds, although
              overall  plumage  or feather   characteristics  can  provide  some
              indication. In some slow-maturing birds (e.g., gulls), plumage may
              be used to distinguish  year  classes of  sub-adults.  Additional
              details on  aging birds and mammals are presented by Larson and
              Taber (see paragraph (h)(22) of this guideline).

(viii) Ancillary methods.

       (A) At least some ancillary methods are essential in every field study. As
       used here, ancillary methods are generally of two types.  Certain of these
       methods are important for determining the nature or existence of effects or
       for establishing causal relationships.   Others of these  methods do not
       address effects  directly, but  they provide important  information for
       interpreting the results of the study.

       (B) Many of the methods for determining effects have been discussed for
       screening studies.   Enzyme  analysis,  such as  for ChE inhibition, and
       observations of signs of toxicity can show that animals were exposed to or
                        Page 32 of 46

       killed by a toxicant of a particular type. Where it is possible that animals
       may be exposed to other chemicals of the same type (e.g.,  feeding in a
       nearby area treated with pesticides), residue analysis in nontarget animals
       may be necessary  to determine which  specific chemical  caused the signs
       or alterations in enzymes.  Even though carcass searches, per se, are not
       recommended for  definitive studies,  it is still  essential to  recover  and
       analyze any  carcasses  found  accidentally or  obtained through radio-
       tracking.  Residue and/or enzyme analysis of live animals collected will
       frequently be important.

       (C)  Among  the  other  ancillary methods,  analysis of  environmental
       residues  is  crucial  and  will  probably be  necessary  in nearly  every
       definitive  field  study.   As  discussed for  screening studies, the most
       important environmental residues are  those that occur on or in wildlife
       food  sources, which may  include insects,  plant  parts, or  even other
       vertebrates, depending upon the species that are the primary focus of the
       investigation.  The investigator should  review the literature on food habits
       of the species being studied; often it  will be appropriate to assess food
       habits on the specific study sites, particularly where  the literature is not
       adequate to define food habits  in the agricultural ecosystem under study.
       Such an assessment should include the  availability of food sources and the
       number of mobile animals that spend only part of the time in and adjacent
       to treated sites.  The habitat should be thoroughly described to include
       both the morphology and species that are relevant to wildlife. Frequently,
       it will be  important to locate and describe roosting,  denning, or nesting
       sites for mobile wildlife that use treated sites part of the time.

(ix) Interpretation of results.

       (A) While each field study is unique, some elements may  be common
       among many field studies. When a definitive field study is required, the
       requirement is based on one or more  specific concerns  that pertain to a
       specific chemical  and  one  or several  use patterns.   Because  of the
       substantial diversity in the types of problems to be assessed and the variety
       of  available  investigative  methods,   the  key  to  understanding   and
       interpreting a field study lies in the development of a  sound protocol.  All
       protocols will contain a description of the study sites, or the characteristics
       to be used in selecting sites within a  given area, and the methods to be
       used in conducting the study.  However, a well designed protocol will go
       beyond this descriptive approach in three ways.

              (1) First, the well-designed protocol will contain a restatement of
              the concerns to be addressed to ensure that there is  an adequate
              understanding of the Agency's  position.   The investigator should
              review the literature and other available information that may bear
              upon the problem. It is possible that the literature  may contain a
              valid  answer to  the  questions  raised by the  Agency.  Far more
              likely, the  literature may orient the investigator to address the

                         Page 33 of 46

concerns in a particular way.  An example is provided by Hegdal
and Blaskiewicz (under paragraph (h)(16) of this guideline), who
conducted a study to address the Agency's concerns for secondary
toxicity to barn owls (specifically) from the use of an anticoagulant
bait  proposed  for use  on  commensal  rodents in  and  around
agricultural  buildings.    A  review  of  the  literature by these
investigators indicated to them that laboratory studies suggested a
legitimate potential for secondary  poisoning  to exposed raptors,
but that the food habits of barn owls consist  primarily of microtine
rodents in  most  areas,  suggesting  a low potential for  actual
exposure.  Consequently, they designed their study to  focus on
barn owl food habits and movements, and included an additive to
the bait formulation that would permit an identification of whether
or not the barn owls ate rodents that had fed  on the bait.  The study
adequately demonstrated  that actual  exposure of barn owls  was
quite  limited,  and the  proposed registration for this  use  was
subsequently approved.  By using the available literature on both
the  chemical  and   the  particular   species  of  concern,  the
investigators were able to narrow the study while  still providing
sufficient information for evaluation.  However, it should be noted
that this study  was not adequate for evaluating the potential for
secondary toxicity in the field to other  predators that may have
different food habits,  or for  other use patterns that may result in
exposure to different predators or scavengers.

(2) Second, the well  designed protocol  will provide the reasons
why particular methods  are  being used, including,  at  least
qualitatively, the meaning that different  results might have.  For
example, a protocol may include collection of residues in nontarget
animals, but it also should  include  a statement of purpose  and
meaning for such collection.   Residues  may  be used to indicate
potential exposure to nontarget organisms through analysis of their
food,   exposure  in  nontarget  animals  as a  result  of  eating
contaminated food, or that a particular chemical was likely to be
the cause  of any  observed  effects.   Interpretation  of data is
facilitated  substantially  by  a statement of  what  results were
intended by using a particular  technique. In the previously cited
example (see paragraph  (h)(16) of this guideline), it was clearly
stated that collection of owl pellets was used to assess general food
habits  and that use of a fluorescing  dye in the bait was  for the
purpose of ascertaining whether or not the owls fed on commensal
rodents that specifically had fed on the bait.  The interpretation of
the data collected, once the purpose was stated, naturally led to the
conclusion of no-significant-exposure to the  barn owls.

(3) Third, the well designed protocol  will contain an experimental
design  that  will  indicate  how  the results  can  be  assessed
quantitatively.   The experimental  design has  been discussed in
           Page 34 of 46

       previous sections of this  guideline,  but there are two facets that
       relate closely to the interpretation of results:  The difference that
       can be detected between treated and  untreated plots and the power
       (ability) of the design to  detect this difference.   An experimental
       design with number of replicates based on an estimated coefficient
       of variation that closely approximates reality will allow the study
       to detect a stated concern level some prescribed number of times
       during the study time.  The actual difference between treated and
       control units is measured during the field study, but the design can
       form  an initial basis  for  interpretation when combined with the
       available information on the species of concern.  As a result, the
       well-designed protocol should include a section on interpretation.

(B)  Study  methods   for  investigating   acute   mortality  are   more
straightforward than for other kinds of effects.  Nevertheless,  there are
sufficient differences  in  the use  of  the  data to  preclude a  constant
interpretation. The study may focus directly on the species of concern and
may involve little or no extrapolation, depending on such factors as the
type and the extent of use, the available toxicity data base, and home range
of the species. Extrapolation to other  populations, regions, or uses might
be necessary.  If the species of concern cannot be studied directly, it may
be  necessary to extrapolate  between species,  involving  interspecies
differences  both in  toxicological  sensitivity and  in  ecological  and
population parameters.

(C) The same kinds of considerations apply to reproductive impairment
and chronic toxicity, even though different,  and often more laborious and
costly, investigative methods are involved.  Where reproductive success is
impaired,  information  on  species-specific  variation  in  reproductive
ecology is necessary to understand how a particular degree of impairment
may  relate  to  effects  among  various species.    Such  reproductive
considerations can include whether an avian species is a determinate or
indeterminate layer, the  number of  nestings  per  season for different
geographic areas in the use pattern, the length of the refractory period, as
well  as  the  specific  effect  which   can  range  from  destruction  of
reproductive organs to behavioral  deficits  such  as nest abandonment.
Considerations  of  reproductive  ecology   among  different  species  of
mammals  include delayed fertilization or  implantation, resorption of
embryos  or  parental infanticide due   to stress, number of young per
breeding cycle, etc.  All of these  factors, and many others, are relevant to
determining for different species  the extent of effects that could result in
population reductions or lack of ability  to recover.

(D) An analysis of whether or not  a particular level of effect is going to
affect  wildlife  populations  is  species-specific.   For  any species (or
subspecies), the changes in population  can be described very simplistically
by the equation:  rate of population increase (r) = birth rate  minus death
rate, where values of r can be positive (population growth) or negative

                 Page 35 of 46

              (population reduction).  Immigration and emigration are also important
              when the concern is for specific  populations of  a  species.   These
              characteristics differ among species, and data will not always be available.
              The  application  of sound  scientific judgment to  the  best available
              information will be the basis for interpreting the results of a study.  It may
              be necessary  to compare the results of the field study to laboratory data,
              especially where  laboratory data are available on a variety of species
              and/or effects and the field study has  focused on species other than those
              of direct concern.  The use of extrapolation techniques will he necessary
              where endangered species are of concern or where other species cannot be
              studied directly.

              (E) The Agency would like to be able to obtain a standardized result from
              a field  study  so  that the result could  be applied in a very consistent
              manner.  As discussed in previous sections of this guideline, the different
              effects and species of concern will vary and will require the development
              of specific protocols to address these  factors.  Although most  of the
              various  techniques have some  degree of standardization, the field study
              may  combine the individual techniques in a  wide variety of ways to
              address  specific concerns.  A standardized result might be attainable for
              the individual techniques, although that  result would  still have to be
              applied  differently for various  species, depending  on their biology  and
              ecological characteristics.   However,  determining  a result for the whole
              field  study that would unequivocally lead to a statement of the degree of
              risk, while obviously desirable, is not currently practical.
(6) Carcass searches—

       (i) Design.
              (A) In designing carcass searches, the following factors need to be known
              or determined:

                     (1) Density of the species  that are likely  to be exposed.  For
                     example, granular products are most likely to result in exposure to
                     ground-feeding animals;  therefore,  birds  such  as  warblers  or
                     swallows  should  not  be included in  density  counts for  such

                     (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

                     (3) Size of the search area.

                     (4) Number of carcasses found..

                                Page 36 of 46

  (B) These factors can be combined in the following formula:

                 n = dxrxexaxp                        Equation 14


         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

  (D)  The sensitivity  of the carcass search  approach  is equivalent to the
  percent detectable kill of the population.  To determine the  sensitivity,
  Equation  14 is rearranged to solve  for p, the proportion  of  population
  killed, as  shown in Equation 15.  Since p is a proportion multiplying by
  100  (Equation 16) provides the percentage of the population killed.

                  p=n/          \                        Equation 15
                 ^   /(dxrxexa)                         ^

percent detectable kill = p x 100 = f n/,         0(lOO)       Equation 16
^                       ^        [^/(dxrxexa)j^   '        ^

  (E) If any of the values of d, r, e, or a are zero, the equation  cannot be
  solved and the carcass search is not applicable (i.e.., no density of birds, no
  acres searched, no carcasses remaining, no remaining carcasses found).
  However, other combinations of d, r, e and a,  such as low density and
  small acreage or low efficiency and high scavenger removal, can result in
  a small denominator meaning that mortality can be detected only when a
  high percentage of the population  is killed.  For example, in 5 acre fields
  with only two birds per acre and r  and e estimated at a moderate 0.5, only

                    Page 37 of 46

       an 80% or greater kill could be detected. In such situations, it is necessary
       to increase one of the parameters to achieve a stated level of detectability
       or else to use methods other than carcass searching.  The same equation
       can be used to estimate the minimum search area to  detect a given
       mortality level (p) by solving for a.

(ii) Search procedure.  In general, depending on the sensitivity of the search
method relative  to the habitat involved, corridors or  plots should be selected.
These areas should be searched systematically by walking predetermined routes
until the area has all been covered. Due to the concentration required to find dead
animals, other activities that could distract the attention of the searchers should be
avoided  during  carcass searching.   In homogeneous situations,  investigators
should randomly select search areas. However, in most studies it is advisable to
stratify the sampling, concentrating efforts in areas frequented by wildlife species
such as woods edges, ditch banks, field borders,  fencerows, and other habitats
where wildlife concentrate.

(iii) Duration. Searches should begin on the day of application and continue on a
daily basis for as long as mortalities or other evidence of intoxication occur.  In
general, a week  or two following application should be adequate.  However, the
length  of time  searches are  continued should be  related to  how long lethal
concentrations are expected to be present.   Normally,  the  same areas should be
searched each day.

(iv) Estimating efficiency of carcass search.

       (A) Efficiency trials should be conducted periodically (minimum 3 times
       per study site)  during the study  to determine the proportion of carcasses
       that are  detected.   Just prior  to  the initiation of a scheduled search,
       carcasses of animals representative of species found in the area should be
       variously placed within the search area.  If the study site includes edge
       habitat, carcasses should be placed in the edges as well as in the fields.  In
       general, carcasses should be placed where animals would be most likely to
       die, depending on the nature of the chemical.   Searchers should not be
       aware that  simulated mortalities have been placed;  however, they should
       be aware that these trials will occur during any scheduled search.

       (B) The  number of carcasses placed should be approximately equal to
       20% of the estimated density of species on the search area.  All placed
       carcasses should be marked to distinguish them from actual  kills.  The
       location of placed carcasses should be mapped  so those not found can be
       easily recovered following completion of that day's search activities, since
       unrecovered  carcasses  could bias  study  results.   For  example,  if  a
       scavenger were to carry off a simulated kill and  consume it  at another
       location on the study site, the remains could be erroneously classified as
       chemical-related if found. One way to avoid this problem would be to dip
       carcasses in a nontoxic substance that fluoresces under ultraviolet light so
       that the remains could be identified.

                        Page 38 of 46

             (v) Estimating carcass removal rate.

                    (A) Carcass removal should be monitored to determine local variability in
                    scavenger activity.   The density  of both carcasses and scavengers can
                    influence the rate of removal.  Under  some conditions, large numbers of
                    carcasses may attract scavengers.   In  other situations  a large number of
                    kills may dilute removal rate due to limited number of scavengers. Where
                    it can be adequately documented that removal of carcasses occurs almost
                    exclusively either at night or during the day, the timing of carcass searches
                    may be adjusted to minimize the effects of removal.

                    (B)  Carcasses planted in monitoring trials  should simulate mortalities
                    actually occurring from the  chemical.  In most cases,  small  to moderate
                    sized species such as starlings  or blackbirds, or laboratory bobwhite or
                    Japanese quail chicks may be used.  Carcasses should be variously placed
                    within the general study  areas and monitored daily for at  least 5 days or
                    until 90% have  been removed.   The number used should approximate
                    densities resulting from effects of the  chemical under study;  however, in
                    most instances,   this will  not be known.    Therefore,  a  density of
                    approximately 20% of the population  of nontarget species on the area is

                    (C) Timing of carcass removal trials should be such that they do not affect
                    scavenger removal  of chemical-killed birds or the feather-spots of the
                    removed carcass could  be  erroneously  classified  as a  chemical  kill.
                    Location of placed birds should be recorded on maps and may be marked
                    in the field with  small stakes or by other inconspicuous means, preferably
                    at a fixed distance and direction from the carcass.

(f) Suggested components of a field study protocol.  The following protocol was adapted from
the Wildlife Managements Techniques Manual (under paragraph (h)(29) of this guideline), and is
recommended for studies submitted to the Agency for review.

       (1) Title

       (2) Problem definition. The following information should be provided:

             (i) A review and summary of the available information on the chemical in relation
             to nontarget hazard, including use information.

             (ii) A precise statement of the goals and purpose of the study.

             (iii) A brief statement of the problem and the context in which it exists, specifying
             the limits of the proposed work.

             (iv) Precise statements of the major hypotheses to be tested.
                                     Page 39 of 46

       (3) Methods and materials. This section should include the following:

              (i) A brief discussion of various methods and procedures that have been or could
              be used to evaluate the problem. This discussion should identify the strengths and
              weaknesses of each method or procedure discussed.

              (ii) Description of the procedure to be followed:

                     (A)  Identify the  study  areas  selected and  their general suitability  for
                     achieving the objectives of the  study or what criteria will be used to select
                     study areas.

                     (B) Identify the  species present or expected to  be  present on  the study
                     areas, discussing characteristics pertinent to the problem being evaluated.

                     (C) State the research procedures, designs and sampling plans to be used:

                           (1) Specify the kind and amount of data needed and to be sought.

                           (2) Describe in detail how all the  data are to be obtained, including
                           details  of application,  instrumentation, equipment,  sampling
                           procedures, and any other information.

                     (D) Describe how the data  are to be treated,  including  specifying  what
                     statistics are to be calculated, what models will be used, what tests of data
                     will be used, etc.

                     (E) Describe in detail  the methods to be used to check the sensitivity and
                     accuracy of the procedures used.

                     (F) Describe quality assurance procedures for application, instrumentation,
                     equipment, and records.

                     (G) Briefly describe the resources (people, facilities, etc) to be applied to
                     the study.

(g) Reporting.   In addition to  the  reporting provisions on background information, study
protocol deviations, and test substance in paragraphs (g)(l), (g)(2), and (g)(3) of this guideline,
respectively, the test report should include, but not necessarily be limited  to the information in
paragraphs (g)(4), (g)(5), and (g)(6) of this guideline on test species, test methods and conditions,
and results, respectively.

       (1)  Background information.  Background information to be supplied in the report
       consists at a minimum of those background  information items  listed in paragraph (j)0) of
       the OCSPP 850.2000 guideline.

       (2)  Study protocol deviations.  Provide a copy of the final field  study protocol used.
       Include a  description of any deviations from the  study protocol  originally submitted and
       agreed upon with the Agency or any occurrences which may  have influenced the results

                                      Page 40 of 46

of the test, the reason for these changes, and any resulting effects on test endpoints noted
and discussed.

(3) Test substance.

       (i) Identification of the test substance: common name, IUPAC and CAS names,
       CAS number, structural formula, source, lot or batch number, chemical state or
       form of the  test substance,  and its purity (i.e. for  pesticides, the identity  and
       concentration of active ingredient(s)), radiolabeling  if any, location of label(s),
       and radiopurity.

       (ii) Storage conditions of the test chemical or test substance and stability of the
       test chemical or test substance under storage conditions if stored prior to use.

       (iii) Methods of preparation  of the  test substance for application, the application
       rate(s), for pesticides the maximum  label rate.

       (iv) For residue analysis in wildlife, vegetation, soil, water, sediments, and other
       appropriate environmental  components describe the stability of the test substance
       under storage conditions.

       (v) Data on storage of biological and environmental samples.

(4) Site of the test.

       (i) A description  of the field study area(s) and  sites, including  the  size  and
       characteristics of all components over time, as well as prevailing meteorological

       (ii) History of the site in terms of factors that may influence the study objectives.

       (iii) Map or diagram showing location of treated sites and  controls and showing
       locations of observations and searches.

       (iv) Climatological data during the field study: records of applicable conditions
       for the type of site, i.e., temperature, thermoperiod,  rainfall or watering regime,
       light regime including intensity and quality, photoperiod, relative humidity, wind
       speed, etc.

       (v) Substrate  characteristics of the study area and treated sites.

       (vi) Characteristics of the flora and  fauna of the study area and test sites.

(5) Study species.

       (i) Identify the wildlife species present in the study area(s), and habitat.

       (ii) For study species the study design objectives and protocols, and the scale (i.e.,
       all species, cross-section, selected species).

                                Page 41 of 46

       (ii) Characteristics  of species  (e.g.,  age,  stage of  development, health status)
       pertinent to the problem being evaluated.

              (A) Number and type of species investigated and the scale of identification
              (e.g., a single species of concern, all species of a community or a selected

              (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

              (A) The methods of evaluating  effects  on terrestrial wildlife  (e.g., radio
              telemetry, carcass searches, ChE inhibition).  The report results should

                     (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

              (B) Statement of the  data objectives for  specific measures (i.e., the critical
              or threshold  level for an effect, precision of a point estimate).

       (ii) The field  study design: size of field  sites, number of control sites, the number
       of experimental treatment levels and the number of experimental sites (replicates)
       for each treatment,  the lay-out and distance of field sites to each other and to
       control sites.

       (iii) Methods  used for treatment randomization.

       (iv)  Number  of  applications and dates  applied,  treatment concentrations or
       application rates, frequency and pattern of administration.

       (v) Method of test  substance application: application or delivery methods (e.g.,
       irrigation water, soil incorporated, surface soil or foliar spray) to the site including
       equipment type and design (nozzles, orifices, pressures, flow rates, volumes, etc})
       and  method for calibrating the application equipment), information  about  any
       solvent used to dissolve and  apply the test substance.

       (vi) Study duration.
                                Page 42 of 46

              (vii) Methods and frequency of climatological monitoring performed during the
              study for air temperature, thermoperiod, humidity, rainfall and watering regime,
              light intensity, and wind speed.

              (viii) The photoperiod and light quality.

              (ix) Methods and frequency of monitoring of other ancillary nontreatment related
              factors that may influence the measures  of effect at the  study site should be
              reported. For example, if effects to wildlife from application to a crop species is
              studied or if a crop is treated concurrent to the investigation of wildlife effects,
              cultural  practices during the tests  such as  cultivation, pest control, should be
              monitored and reported for the study sites.

              (x) All analytical procedures should be described. The accuracy of the method,
              method detection limit, and limit of quantification  should be given.

     (7) Results.

              (i) Environmental monitoring data results (air temperature,  humidity and light
              intensity, rainfall) in tabular form (provide raw data for measurements not made
              on  a  continuous basis),  and descriptive statistics (mean,  standard deviation,
              minimum, maximum).

              (ii) Tabulation of the results of study-specific wildlife measures by field site and
              treatment (provide the raw data),  and summary  statistics.   If categorical rating
              measures are made a description of the rating system should be included.

              (iii) Description of the statistical method(s), software package(s) used, the basis
              for the choice of the method(s), statements of the reasons why particular methods
              are being used,  including, at least qualitatively, the meaning that different results
              might have.

              (iv) Results of the statistical analysis including graphical and tabular summaries,
              and results of goodness-of-fit tests or minimum significant differences detectable,
              as appropriate.

(h) References.   The following references  should be consulted for additional  background
material on this test guideline.

       (1) Bunyan, P.J. et al. Organophosphorus  poisoning, some properties of avian esterases.
       Journal of Agricultural  and Food Chemistry 16: 326-331 (1968).

       (2) Bunyan, P.J. et al. Organophosphorus poisoning, diagnosis of poisoning in pheasants
       owing  to a number of  common  pesticides. Journal  of Agricultural and Food Chemistry

       (3) Burnham, K.P. et al. Estimation of density from line transect sampling of biological
       populations. Wildlife Monographs, No. 72 (1980).

                                       Page  43 of 46

(4) Caughley, G. Analysis of Vertebrate Populations. Wiley, NY (1977).

(5) Cochran,  W.W.,  Wildlife  telemetry,  pp.  509-520,  in  Wildlife Management
Techniques Manual. S.D. Schemnitz, Ed. The Wildlife Society, Washington, DC (1980).

(6) Corbett, J.R.  The Biochemical Mode of Action of Pesticides.  Academic, NY (1974).

(7) Davis, D.E. and R.L. Winstead. 1980. Estimating the numbers of wildlife populations.
pp. 221-246, in  Wildlife Management  Techniques Manual. S.D. Schemnitz, Ed.,  The
Wildlife Society, Washington, DC (1980).

(8) Eberhardt, L.L. Quantitative ecology and impact assessment. Journal  of Wildlife
Management 4: 27-70 (1976).

(9) Eberhardt, L.L. Appraising variability in populations studies. Journal  of Wildlife
Management 42: 207-237 (1978).

(10) Eberhardt,  L.L. Assessing the dynamics of wild populations. Journal of Wildlife
Management 49: 997-1012 (1985).

(11) Edgington,  E. Randomization Test. Dekker, NY (1980).

(12) Edwards, P.J.  et  al. The use of  a bird territory mapping  method for detecting
mortality following pesticide application. Agro-Ecosystems 5: 271-282 (1979).

(13)  Ellman,  G.L.  et al.  A  new  and  rapid   calorimetric   determination  of
acetylcholinesterase activity. Biochemical Pharmacology 7:88-95 (1961).

(14) Fite, E., L. Turner, N. Cook,  and C. Stunkard,  1988.  Guidance Document for
Conducting Terrestrial  Field Studies. United States Environmental Protection Agency,
Office of Pesticide Programs, Washington, D.C. EPA 540/09-88-109.

(15) Hanson, W.R. Estimating the density of an animal population. Journal of Research
Lepidoptera 6: 203-247 (1967).

(16) Hegdal, P.L. and R.W. Blaskiewicz. Evaluation of the potential hazards to barn owls
of  Talon (brodifacoum  bait)  used to  control  rats  and house  mice.  Journal  of
Environmental and Toxicological Chemistry 3: 167-179 (1984).

(17) Heinz,  G.H. et al. Environmental contaminant studies by the  Patuxent Wildlife
Research Center, pp. 8-35. in Avian and Mammalian Wildlife Toxicology. ASTM STP
693, E.E. Kenaga (Ed), American Society for Testing and Materials,  Philadelphia, PA

(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

(19) Hurlbert, S.H. Pseudoreplication and the design of ecological field experiments.
Ecological Monographs 54:187-211 (1984).
                               Page 44 of 46

(20) Giles, R.H. Jr. Wildlife Management. Freeman, San Francisco, CA. (1978).

(21) International Bird Census Committee.  Recommendations for an  international
standard for a mapping method in bird census work. Bulletin of the Ecological Research
Committee 9:49-52 (1970).

(22) Larson, J.  S. and R. D.  Taber. Criteria of sex and age. pp. 143-202, in Wildlife
Management Techniques  Manual.  S. D.  Schemnitz;  (Ed.),  The  Wildlife  Society,
Washington, DC (1980).

(23) Leopold, A. Game Management.Scribner's, NY (1933).

(24) Ludke, J.L. et al. Cholinesterase (ChE) response and related mortality among birds
fed ChE inhibitors. Archives of Environmental Contaminant Toxicology 3:1-21 (1975).

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