&EPA
United States
Environmental Protection
Agency
Office of Chemical Safety
and Pollution Prevention
(7101)
EPA712-C-009
January 2012
        Ecological Effects
        Test Guidelines

        OCSPP 850.4300:
        Terrestrial Plants
        Field Study

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                                     NOTICE

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

     The OCSPP harmonized test guidelines serve as a compendium of accepted scientific
methodologies and protocols that are intended to provide data to inform regulatory decisions
under TSCA, FIFRA, and/or FFDCA. This document provides guidance for conducting the
test, and is also  used  by EPA, the public, and the companies that are subject to data
submission requirements under TSCA, FIFRA, and/or the FFDCA.  As a guidance
document, these guidelines are not binding on either EPA or any outside parties, and the
EPA may depart from the guidelines where circumstances warrant and without prior notice.
At places in this  guidance, the Agency uses the word "should."  In this guidance, the use of
"should" with regard to an action means that the action is recommended rather than
mandatory. The procedures contained in this guideline are strongly recommended for
generating the data that are the subject of the guideline,  but EPA recognizes that departures
may be appropriate in specific situations. You may propose alternatives to the
recommendations described in these guidelines, and the Agency will assess them for
appropriateness on a  case-by-case basis
     For additional information about these test guidelines and to access these guidelines
electronically, please go to http://www.epa.gov/ocspp and select "Test Methods &
Guidelines" on the left side navigation menu.  You may also access the guidelines in
http://www.requlations.qov grouped by Series under Docket ID #s: EPA-HQ-OPPT-2009-
0150 through EPA-HQ-OPPT-2009-0159, and EPA-HQ-OPPT-2009-0576.

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OCSPP 850.4300: Terrestrial plants field study.

(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  121-1  Target  Area Phytotoxicity  Testing  and OPP 124-1
       Terrestrial Field Testing (Pesticide  Assessment Guidelines Subdivision  J—Hazard
       Evaluation: Nontarget Plants); the Non-Target Plants:  Target  Area Testing Standard
       Evaluation Procedure; the Non-Target Plants: Terrestrial Field Testing Tier  3 Standard
       Evaluation Procedure; and ASTM E 1963-02, Standard Guide for Conducting Terrestrial
       Plant  Toxicity  Tests. This  guideline incorporates  what was formerly  Public Drafts
       OCSPP 850.4025 Target area phytotoxicity and  OCSPP 850.4300 Terrestrial  plants field
       study (April, 1996).

       (3) General. This guideline describes general procedures for performing plant  toxicity
       tests under field conditions,  whether target area or off-target area.  Thus, for pesticide
       testing guidance there are no  longer separate guidelines for target and non-target area
       tests.  This  guideline should be used  in conjunction with  OCSPP  850.4000, which
       provides general information and overall  guidance for the nontarget plants test guidelines.

(b) Purpose.  This guideline describes factors to be considered in the design and conduct of field
studies for effects of chemical substances and mixtures on terrestrial plants. Effects  considered
may include mortality, and sublethal toxic  effects  such  as decreased biomass or  other
morphological  changes, changes in population  or  community  parameters,  and  lowered
productivity such as fewer flowers, pods, fruits or seeds  or viability of seeds. The purpose of the
field study is either to provide quantification of the risk  that may occur to terrestrial plants, plant
populations or plant communities or refute the assumption that risks will occur under conditions
of actual use of the test substance (primary consideration for pesticides)  or occur under  the
pattern of production, use, disposal, or accidental release of industrial chemicals in the terrestrial
environment.

(c) Definitions.  The definitions in the  OCSPP 850.4000 guideline apply to this guideline.   In
addition, the definitions in this paragraph apply:

       Community is defined as an assemblage of populations of different species.

       Population is defined as a group of individuals of the same species.

(d) General considerations—

       (1) 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 the  variability in  the

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factors  that are  considered  in  the  design such  as chemical  mode of action,  plant
population and community dynamics, and  additionally 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 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
upland and riparian plants, and to provide a better understanding of the purpose of 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 plant population and community
dynamics  such as species composition, frequency, percent (%) cover and other indices
that describe the use and/or study area.  The general guidance in the OCSPP 850.4000
guideline applies to this guideline, except as specifically noted herein.

(2) Summary of test.   The test substance may be applied in  a variety of ways; the
selected method should support the specific study objective. Application methods range
from a single application, at a single dose or at a wide range of anticipated test substance
doses (or concentrations), as may be found  in the environment to multiple applications at
a single dose  or over a wide range  of anticipated  test substance levels.   The test  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. Development of data quality objectives for
generating environmental effects data  for decision  making  include:  development  of
decision rules, specifying limits on  decision  errors,  and  optimizing  design  (see the
OCSPP 850.4000 guideline and  paragraph  (i)(ll) of this guideline).   Specific protocols
should be developed  and submitted  to  the Agency for review prior to conduct of the
study.

(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  850.6100.  Elements of the original
addendum  as  referenced in  40  CFR 158.660  for this purpose are  now contained  in
850.6100. These procedures, if followed, would result in data that would generally be  of
scientific merit for the purposes described in 40 CFR 158.660.

(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  populations  and  communities from the
laboratory data are occurring in the field and, if so, to what species before conducting a
definitive field study.  The objective of the screening field study  is to determine whether
impacts to plant populations are occurring and to which species.  "Pass-fail" methods are
used to determine whether impacts occur.  Effects considered include measurements  of
survival, biomass, density, frequency or other appropriate indicators.


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       (5) Definitive field study.  If a screening study indicates impacts are occurring, or if
       other available data suggest or document 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
       should 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 the effects caused by the  application, the existence and
       extent  of reproductive effects,  and the  influence of chemical use on  the  survival and
       ecological function of species of concern.

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

(e)  Test standards.   Environmental and  exposure conditions under which a  field test  is
conducted should resemble the conditions likely to be encountered under actual production, use,
disposal or fate of the test substance.

       (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 the substance to be tested  is usually the typical end-use
       product (TEP). In addition, if an adjuvant is recommended for use on a TEP label, the
       adjuvant is added with the TEP at the label rate to constitute the test substance.  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 850.4000 guideline lists the type information  that should be known about the
       test substance before testing.

       (2) Test duration. Due to the highly variable nature of objectives for field studies, no
       single  test  duration can be established for  the  screening  or  definitive  field studies.
       Investigators are  encouraged  to  consult with  the Agency to reach agreement  on
       acceptable  study duration prior to conduct of the field study.  Several seasons or one or
       more years may be appropriate where one of the  objectives of the definitive field study
       includes evaluating lowered productivity due to effects on seed viability or evaluating
       alteration in community integrity.   Seasonal and annual  variation in plant species,
       population  and community  attributes should be considered when selecting the study
       duration. The field study duration should be selected to meet the stated study objectives.

       (3) Study species.  The number and type of species investigated should be based on the
       specific objectives  of the field study. The scope and scale can vary from investigation of
       effects to a specific crop species to one or more plant communities which are comprised
       of a wide-range of species.  For a community the test may investigate a selected cross-
       section of the nontarget plant population.
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(4) Administration of test substance—

       (i) Test substance application

              (A) The choice of method for test substance application is dependent upon
              the properties of the test substance, expected exposure pathways for plants
              in the environment from purposeful application, when the test substance is
              a  pesticide,  or expected exposure pathways  based on  the pattern  of
              production, use, disposal, or accidental release,  when the test substance is
              an industrial chemical,  and the anticipated range and distribution of test
              substance quantities likely to be found in the environment.

              (B) For pesticides, consideration should also  be given to proposed  or
              registered application rates  and application methods.   Where the  study
              objective is to directly  measure effects or "lack of effects" from labeled
              use, the method of application used  and the frequency  of application
              should  be consistent with the  label.   Equipment used  may influence
              potential  exposure of  nontarget  species.    The  diversity of types  of
              application  equipment  that,  depending on  the  particular  use  pattern
              involved, could influence exposure.  The  various types  of equipment
              normally used for the particular pesticide application should be evaluated
              to  estimate  the  potential influence of equipment used  on exposure.  In
              some instances, preliminary  tests  may  be required to estimate which
              method and equipment  poses the highest exposure.  The use of small site
              field equipment that may mimic the application equipment may be  useful.

       (ii) Treatment levels.

              (A) For a screening field  study,  where the objective is to  determine
              whether impacts occur  or not (i.e.,  "pass-fail"), a single treatment level
              plus a control may be appropriate.  For a pesticide screening field study,
              where the study objective is to directly measure effects  from labeled use,
              the treatment level  should be  applied at a minimum at the  maximum use
              rate and frequency specified on the label.

              (B) For a definitive field study, where the objective is to evaluate the
              magnitude of effects  across  a  range of environmental  concentrations,
              multiple treatment levels plus a control would be appropriate. The range
              of treatment levels selected  should bracket the range  of  environmental
              concentrations for which conclusions  are to be drawn.  The number  of
              treatment levels selected when fitting a response-relationship  should be
              sufficient to meet the level of precision desired and allow determination of
              the goodness-of-fit. Consult a statistician for assistance in determining the
              number of treatment levels.  For a pesticide  definitive field study where
              the study objective  is to directly measure effects from labeled use, one  of
              the treatment levels should at a minimum include the maximum  use rate
              and frequency specified on the label.
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       (iii) Application timing.  When the test substance, particularly a herbicide, plant
       regulator, desiccant, or defoliant, is  applied  to  any desirable  nontarget plants
       within or adjacent to the target area,  the stage of growth or development of the
       plants at application should be observed and recorded. Field studies should not be
       done during the period of seasonal  senescence of the foliage in which the leaves
       die back in  the  late  summer.   For serial  applications, record  the  times  of
       application (or application interval) for each product or tank mix involved in the
       serial application.

(5) Test conditions.  The test conditions for  conducting  a field test  should resemble the
conditions likely to be encountered under  actual use, disposal, or accidental spill of an
industrial  test substance  or  under actual application  conditions for a pesticide.  While
each field study is unique, some elements may be common among many field studies.

       (i) Review of pertinent literature.  A well-designed  protocol should include a
       restatement of the concerns to be addressed to ensure  that there is an adequate
       understanding  of the  Agency's   position.    Literature  and  other  available
       information  that may bear upon the  problem  should be reviewed  and pertinent
       information  summarized in the  protocol.  It  is possible that the  literature may
       contain a valid answer to the questions raised by the Agency. At a minimum, the
       literature may orient the investigator to address the concerns in a particular way.

       (ii) Site characteristics.   All  protocols  should contain a description  of the
       characteristics to be used, or that were used, in selecting sites within a given area.
       If sites were selected a description of the study  sites should also in the protocol.

       (iii) Methods. All protocols  should  contain a description of the methods to  be
       used in conducting the  study.  The  protocol should  provide the reasons why
       particular methods are being used,  including,  at least qualitatively, the meaning
       that different results might have based on choice of methods.

       (iv) Timing.  Consideration should be given to the season(s) over which the study
       is  conducted.  For certain species (ferns, shrubs, trees, etc) tests should not  be
       performed at a time or season where the timing of the study outdoors (August and
       September) is during a period of senescence of the foliage in which the leaves die
       back in late summer.  This dieback may contribute to the lessening of the test
       substance's effect on the terrestrial plant test species.  If effects to a crop species
       are under consideration, plants should be grown under crop/cultivar agronomic or
       horticultural practices. For pesticides, the test substance is to be applied over a
       period of time or season according to label instructions.

(6) Sampling and experimental design.

       (i) While examples of acceptable experimental designs are given, it is  beyond the
       purpose of this guideline to cover the fundamentals  of this topic.  References in
       paragraphs (i)(2) through (i)(7), and (i)(l 1) of this guideline provide resources for
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       guidance regarding sampling and experimental design, especially for measuring
       effects on plants in natural habitats.

       (ii) A well-designed protocol  will  contain  an experimental  design that will
       indicate how the results are to be assessed quantitatively and a section on how
       results will be interpreted.

              (A) As part of the description of the experimental design for hypothesis
              testing approaches, the magnitude of the difference the study is designed
              to detect between treated and untreated plots and the power (ability) of the
              design to detect this  difference  should be  discussed.   Coefficient  of
              variation estimates from  screening studies, literature, or laboratory  tests
              that  closely approximates reality can be used to design the study and
              determine the number of replicates.

              (B) As part of the description of the  experimental design for response-
              relationship field  studies,  the  environmental   range  for  which  the
              predictions  are  to  address,  the  treatment  spacing,  and  approach for
              assessing fit should be discussed.

(7) Geographic area selection.

       (i) Studies  should be performed in representative biogeographic areas where the
       test substance 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.  To keep the number of
       geographic areas at a manageable level while still accomplishing the purpose of
       the field study,  area selection should emphasize situations likely to present the
       greatest  risk taking into account the diversity and variability in  ecosystems
       involved.

       (ii) A careful review of the species and habitats in the geographical areas involved
       should be performed to aid in identifying the  areas of highest concern. A sound
       understanding of the biology of the species that are found in association with the
       areas  is essential.  Identifying these areas  is likely to include a literature review
       and consultation with experts familiar with the areas and species of concern.  The
       study area selected  should be appropriate for the species of concern.  If exposure
       and fate (e.g., degradation, transformation) parameters vary geographically,  study
       area  selection should also focus on those areas  with factors which maximize
       residues available for exposure.  In some circumstances preliminary monitoring of
       candidate areas may be appropriate  to determine which ones should be selected
       for detailed study.

(8) Study site selection.

       (i) Within a geographic area, study sites should be selected from those considered
       to be typical  application sites for pesticides,  or a  typical exposure site  which
       occurs under  the pattern of production, use,  disposal,  or accidental release  of

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       industrial  chemicals, but at the same time, study sites should contain the widest
       possible diversity and density of plant species for the geographic area.

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

       (iii)  Identifying potential  study  sites may  call for consultation with experts
       familiar with  the areas where  studies are  proposed,  and preliminary sampling.
       Field surveys of a number of sites may be used to identify which  sites contain
       species likely to be at highest risk.  Preliminary surveys may also be used to
       determine which sites have adequate numbers of the high risk species as well as a
       good diversity of other species.

       (iv) If crop  species are the selected study  species,  then the  selection of the site
       should be representative of major production sites for that particular crop. In such
       situations, diversity and density of non-crop plant species may not be necessary to
       answer the questions posed by the study.

(9) Control sites.  Controls sites should be selected to be as comparable  with treated
study sites in species, diversity, biomass, and selected study variables. The control sites
should also be located as close as possible to selected treated study sites but  at enough of
a distance and juxtaposition that  cross-contamination from application or treatment will
not occur.

(10) Size of study sites.  Study sites should be large enough to  provide adequate sample.
The size is dependent on the  methods used, the sensitivity needed,  and the density and
diversity of species.  Consideration should be given to the distance between study sites.
Sites should be separated adequately to ensure independence.

(11) Number of sites.

       (i) The number of sites (or replicates) to include in the study  may be estimated in
       many ways, but the number should be sufficient to detect the size of difference
       with a given level  of  power identified as  part of the data quality objectives or
       estimate  the  parameter(s) of  interest with the level of  desired confidence
       identified as part of the data quality objectives.   The methodology and rationale
       for selecting the number of sites  should be clearly  outlined and described in the
       study plan.  Paragraph (i)(ll) of this guideline provides guidance on estimating
       number of replicates for a number of statistical methods. Recommend consulting
       a statistician when estimating the number of replicates which should be used.

       (ii) Under  some  circumstances,  particularly if endangered  species could be
       exposed from the proposed use, additional replication may be desirable because
       under these  conditions, a high degree of confidence that effects are  negligible is
       likely to be  desired. (Under no circumstances should field studies on chemicals

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              be  conducted  in  areas  where endangered species could  be  exposed  unless
              approval is provided by the FWS authority for that particular listed species.)

              (iii) It is important to define the critical or threshold level for an effect, and to be
              sure that the  methods used are  sensitive  enough  to  detect the effect  size of
              concern. Whatever parameters are used, defining the criteria level for an effect is
              extremely important, and when designing studies this issue should be considered
              carefully.

              (iv)  Careful  consideration  should be  given to the controls  having  sufficient
              number of replicate sites so that a statistical  analysis can  provide meaningful
              insight regarding the study objective.

(f) Interpretation of results.  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.  A sound protocol  should
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, a well-designed protocol should 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 concerns in a particular way.  By using the available literature on both the
       chemical and the particular species of concern, the investigators  may be able to narrow
       the  study  while still  providing sufficient information for evaluation.   However, in
       narrowing the focus of the study (e.g., to a single  species or a  single geographic area) it
       may limit the adequacy of the study for evaluating effects to other  species, or for other
       use patterns that may result in exposure to different species or geographic areas.

       (2) Second, a 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 plant tissues, but it also
       should include a statement of purpose and meaning for such collection. Residues may be
       used to confirm exposure to nontarget plants by spray  drift or runoff, 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.

       (3) Third, a well designed protocol will contain an experimental design that will indicate
       how the results  can  be assessed quantitatively.   The experimental design  has been
       discussed in previous sections 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
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       variation that closely approximates reality will allow the study to detect a stated concern
       level.  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.

       (4) 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
       specific study protocols should be developed 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.

(g) Test validity elements.  In the case of  field studies, validity elements will vary with the
purpose and design of the study, and should be developed in cooperation with the Agency prior
to the implementation of the study.  Generally, studies would be considered to be unacceptable if
one or more of the  conditions in Table 1 occurred.  This list should  not be  misconstrued as
limiting the reason(s) that a test could be found unacceptable.

       Table 1.—Some test validity elements for the terrestrial plants field  study
1.  The population of test plants and/or replicates was of an insufficient size to characterize or detect
effects with an acceptable degree of certainty.

2.  The controls were contaminated with the test substance or there was insufficient sampling or study
conditions to document that controls were not contaminated.

3.  Control plants were not maintained under the same test conditions as the test substance plants.	

(h) Reporting—

       (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 850.4000 guideline.   Due to the  variability among tests and test objectives,
       this list should not be considered comprehensive.

       (2) Test substance.

              (i) Identification of the test substance  (name, state or form, source), its purity (for
              pesticides, the identity (common name, IUPAC and CAS names, CAS number)
              and concentration of active ingredient(s)), and known physical and  chemical
              properties that are pertinent to the test.

              (ii) Storage conditions of the test substance.


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       (iii) Methods  of preparation of test  substance  for application  onto foliage, the
       maximum label rate,  and the actual application rate (Ib a.i./A)  with the finished
       spray volume per acre.

       (iv) If residue analysis is performed  on foliage, describe the stability of the test
       substance under storage conditions.

       (v) Data on storage of the plant material, if applicable.

(3) Site of the test.

       (i) Site description  of the terrestrial field  testing study  such as a grassland,
       forested area, fallow field, tilled field, etc.

       (ii) Location of the test sites that represent the general regional  areas of potential
       usage  such  as  Northeastern  temperate  deciduous;  Southeastern temperate
       deciduous; Northern grassland (cool  prairie); Southern grassland (warm prairie);
       Northwestern  (and  Alaskan)  conifer  forest  and high  desert;  Southwestern
       chaparral  Mediterranean  and  low  desert;   and  Hawaiian   and  Caribbean
       semitropical and tropical regions.

       (iii) Physiographic conditions including:

              (A) The edaphic conditions and characterization including soil type  and
              texture, approximate pH and temperature, Kd and Kow values.

              (B) Where the presence of a fragipan  or shallow bedrock may lead to
              restricted leaching or soil waterflow, the  depth of that restriction.

              (C) The degree  and direction  of slope and its orientation to  the row
              direction if the slope will lead  to excessive runoff.

              (D) Map or diagram showing location of treated plants and controls.

       (iv) Climatological data during the test: 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 sites: name/designation of  soil type and its
       physical and chemical properties, including pH and percent organic matter.

(4) Species at test site.

       (i) For investigation of a crop species the information in paragraphs (h)(4)(l)(A)
       through (h)(4)(l)(H) of this guideline should be reported.
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              (A) Scientific and  common name,  plant  family  and variety including
              species/variety and cultivar if appropriate.

              (B) Test date of germination rating and germination percentage.

              (C) History  of the seed: Source, name of supplier,  seed year or growing
              season collected, batch or  lot number, seed treatment(s), and storage
              conditions.

              (D) Seed size class.

              (E) Description of handling and processing of seeds before use in test.

              (F) Planting  dates.

              (G) Stage of development, height and condition of plants that are treated.

              (H) Population density of seeds or plants.

       (ii)  For nontarget plant  species the study  design objectives and  protocols will
       impact the scale (i.e., all species, cross-section, selected species) of reporting of
       the information on nontarget species.

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

              (B) Scientific and common name, plant family and variety.

              (C) Stage of development and condition of plants at test initiation.

(5) Study conditions and experimental design. Description of the study conditions and
experimental design used in the screening or definitive tests, and any preliminary testing.

       (i) A statement  of the concerns to  be addressed  and the type and frequency of
       monitoring  of  vegetation  measures  (e.g.,  diversity,  abundance,  biomass,
       emergence) addressing these concerns.

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

       (v) Number of applications and dates applied.

       (vi) Study duration.

       (vii) Methods and frequency of climatological monitoring performed during the
       screening or definitive 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 a  crop species is  studied  or if  a  crop  is  treated
       concurrent to the investigation of nontarget plant effects,  cultural practices during
       the  tests such as cultivation, pest control, irrigation practices; and  any nutrient
       amendments.  Any infestations of disease  or insects should be monitored  and
       reported for the study sites.

       (x)  For the screening and  definitive studies, all analytical procedures  should be
       described.  The accuracy  of the method,  method detection limit,  and limit of
       quantification should be given. Provide the ILV report.
(6) 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  vegetative  measures (e.g.,
       emergence, height, dry weight, yield of seeds or fruit, germination rate of second
       generation, phytotoxicity rating,  diversity, abundance) by field site and treatment
       (provide the raw data), and summary statistics.  If phytotoxicity rating measures
       are made a description of the rating system should be included.

       (iii) Description (i.e., method of determination) of and tabular summary of any
       secondary vegetative measures.

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

       (v) 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
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             being used, including, at least qualitatively, the meaning that different results
             might have.

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

(i) References.  The references in this paragraph should be consulted for additional background
material on this test guideline.

       (1) American Society for Testing and Materials.  2002.  ASTM E 1963-02.  Standard
       guide for conducting terrestrial plant toxicity tests. In Annual Book of ASTM Standards,
       Vol.  11.06, ASTM, West Conshohocken, PA.  Current edition approved December 10,
       2002.

       (2) Cooperrider, A. Y., R. J. Boyd and H. R. Stuart, eds.  1986.  Inventory and monitoring
       of wildlife habitat.  U.S.  Department of Interior, Bureau  of Land Management Service
       Center, Denver, CO. sviii, 858 pp.

       (3) Little, T.M. and FJ.  Hills.  1978.  Agricultural  Experimentation - Design  and
       Analysis. Wiley, NY.

       (4) Pfleeger, T., 1991.  Impact of Airborne Pesticides on Natural Plant Communities. In
       EPA Publ. EPA/600/9-91/041, Plant Tier Testing: A Workshop to Evaluate Nontarget
       Plant Testing  in  Subdivision J Pesticide Guidelines, Nov.  29-Dec.  1,  1990,  ERL,
       Corvallis, OR. pp. 108-123.

       (5) Phillips, E.A., 1959. Methods of Vegetation Study. NY: Holt, Rhinehart, and
       Winston.

       (6) Ratsch, H. and J.S. Fletcher.  1991. Plant Reproduction and/or Life Cycle Testing. In
       EPA Publ. EPA/600/9-91/041, Plant Tier Testing: A Workshop to Evaluate Nontarget
       Plant Testing  in  Subdivision J Pesticide Guidelines, Nov.  29-Dec.  1,  1990,  ERL,
       Corvallis, OR. pp. 80-89.

       (7) Truelove, B., ed., 1977.  Research Methods in Weed Science.  Southern Weed Science
       Society, Auburn Printing, Auburn, AL.

       (8) U.S.  Environmental Protection  Agency,   1982.   Pesticide Assessment  Guidelines,
       Subdivision J—Hazard Evaluation: Non-target plants.  Office of Pesticides Programs,
       EPA 540/9-82-020, Washington, DC.

       (9) U.S. Environmental Protection Agency, 1986.  Hazard Evaluation Division Standard
       Evaluation Procedure, Non-target Plants: Terrestrial Field  Testing  Tier 3.  Office of
       Pesticides Programs, Washington, D.C. EPA 540/9-86-135, June 1986.
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(10) U.S. Environmental Protection Agency, 1986. Hazard Evaluation Division Standard
Evaluation  Procedure, Non-target Plants: Target Area Testing.   Office of Pesticides
Programs, Washington, D.C. EPA 540/9-86-130, June 1986.

(11) U.S. Environmental Protection Agency,  2000.   Guidance  for the Data  Quality
Objectives Process (QA/G-4), EPA/600/R-96/055, Office of Research and Development.
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