&EPA
United States
Environmental Protection
Agency
Office of Chemical Safety
and Pollution Prevention
(7101)
EPA712-C-007
January 2012
        Ecological Effects
        Test Guidelines

        OCSPP 850.4450:
        Aquatic 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 850.4450: Aquatic 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 material used in developing  this harmonized OCSPP test
       guideline  is the OPP  124-2 Aquatic Field Testing, Pesticide Assessment Guideline
       Subdivision J.

(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 aquatic plants.  Effects considered
may include mortality, and sublethal toxic effects such as decreased biomass or changes in
population  or community parameters.   The purpose of the  field study is either to provide
quantification of the  risk that may  occur to  aquatic  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 environment.  This guideline
should be  used  in conjunction with  OCSPP  guideline  850.4000,  which provides general
information and overall guidance for the nontarget plants test guidelines. Additional guidance on
aquatic field studies can be found in the OCSPP 850.1950 guideline.

(c) Definitions.  The definitions in the OCSPP 850.4000 guideline apply to this guideline.  In
addition, the following definitions 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
       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 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 as needed
       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 aquatic 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

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the aquatic  plant population and  community dynamics such as species composition,
frequency 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.   Additional  guidance on  aquatic  field  studies  can be found  in  the OCSPP
850.1950 guideline.

(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 OCSPP
850.4000 and  paragraph  (i)(20)  of  this  guideline).   Specific protocols should be
developed as needed 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.

(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
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


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       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 populations  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.

              (i) 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  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 non-target plant population.

              (ii) Test species may include but not be limited to algae, mosses, ferns and allies,
             floating, submersed and  emergent aquatic vascular plants. Test plants should be
             in a  stage of development under which exposure to the test  substance would
             normally  occur.  Justification should be provided if surrogate species are used  to
             represent those of the natural habitat.
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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,  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.  For pesticides, the  single
              exposure  level is equivalent to the maximum label  rate (pounds a.i.  per
              acre (Ib a.i./A) directly  applied to a one acre pool of water that is 6 inches
              deep (21,280 cubic feet (ft3) or 602,581 liters). For example, a 1 Ib a.i./A
              (453,592 milligrams (mg) per acre) label application rate and  assuming a
              water density of 1 gram per milliliter is equal to 0.75 milligrams per liter
              (mg/L). Adjustments for water being less dense at warmer temperature
              (i.e.,  1 milliliter < 1 g) can be made (see in paragraph (i)(17)).

              (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
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              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.

       (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.  Studies should not be performed at a time or season when there is a
       period of natural senescence.   This dieback may contribute to the lessening of the
       test substance's effect on the  aquatic plant test species.  For pesticides, the test
       substance is  to be applied over a  period  of  time  or season according to label
       instructions.
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(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), (i)(4), (i)(6), (i)(7),  (i)(9), (i)(16) and (i)(20) of this guideline
       provide resources for 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) 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.
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(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
       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 require 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.

(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 required, 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)(20)  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
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              likely to be desired.  (Under no circumstances should field studies on chemicals
              be conducted in areas where endangered species could be exposed.)

              (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 an  effect should one
              occur.  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 or one or more performance criteria in Table 1
were not met. 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 aquatic plants field study
1.  The population of test plants and/or replicates was of an insufficient size to characterize the  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
       OCSPP 850.4000. Due to the variability among tests and test objectives, this list should
       not be considered comprehensive.

       (2) Test substance.

              (i) Identification of the chemical or end-use product (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.
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       (ii) Storage conditions of the test substance.

       (iii) Methods of preparation of test substance for application into a surface water
       or adjacent land  area  and foliage,  the maximum  label  rate, and  the actual
       application rate  (Ib a.i./A) with the finished spray volume per acre for flowable
       applications.   The volume  of surface water at the time the test substance was
       applied.

       (iv) If residue analysis is performed on plants  or portions of plants, 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 aquatic field  testing study as to  the type of  systems
       (enclosed, controlled areas of a lake, pond,  swamp, or stream, or artificial water
       systems such as aquaria, or large tubs).

       (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) Type of aquatic site  (such as swamp, bog, freshwater marsh, lake,
              pond, reservoir, stream, coastal wetlands, or irrigation  ditch)  and flow,
              turnover, flooding regime  or tidal action, as appropriate.

              (B) Size  of each treatment site, including:  area (length and width  profile)
              and depth profile, and volume.

              (C) Water quality characteristics and profile,  including  pH, temperature,
              hardness,  alkalinity  or   salinity,   if  applicable,   turbidity  (visual),
              conductivity  (if possible), and dissolved oxygen (for submerged plants
              only).

              (D)  Substrate  characteristics  of  the  treated  and   control  sites:
              name/designation  of sediment  types  and  its  physical  and chemical
              properties, including texture, organic carbon content, pH, and Eh.

              (E) Habitat structure of adjacent terrestrial,  riparian, or wetland systems.

                     (1) Location and distribution of soil types and texture, with percent
                     organic matter in relationship to treated sites and controls.
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                     (2) Location and distribution of vegetation including type, density,
                     and diversity in relationship to treated sites and controls.

                     (3) Canopy and ground cover.

                     (4) Degree and direction of slope.

              (F) Map or diagram  showing location of treated sites and  controls and
              adjacent terrestrial, riparian, and wetland systems.

       (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.

(4) Species at test site.

       (i) For investigation of a crop species (e.g., rice) the following information should
       be reported.

              (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,  if
              appropriate.

              (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, if appropriate.

              (D) Seed size class,  if appropriate.

              (E) Description of handling and processing of plants 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.

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              (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., foliar  exposure,
       surface water contact), application or delivery methods (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.

       (v) Number of applications and dates applied.

       (vi) Study duration.

       (vii) Methods and frequency of measuring flow, turnover, flooding or tidal regime
       during the study.

       (viii) Methods and frequency  of water quality monitoring performed during the
       screening or definitive study.

       (viii) Methods  and frequency of characterizing  adjacent terrestrial,  riparian, or
       wetland system  vegetation and soils or sediments.

       (ix) 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.

       (x) The photoperiod and light quality.

       (xi) 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.
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              (xii) 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,  water  quality)  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
              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 following references should be consulted for additional background
material on this test guideline.

       (1) Campbell, P.J., D. Arnold, T. Brock, N. Grandy, W. Heger, F. Heimbach,  SJ. Maund
       and M. Streloke, 1999.  Guidance document on higher-tier aquatic risk assessment for
       pesticides (HARAP),  Report of a  SETAC-Europe/OECD/EC workshop, 19-22  April
       1998.

       (2)  Crossland, N.O. and T.W.  LaPoint,  1992.  The design of mesocosm experiments.
       Environmental Toxicology and Chemistry 11: 1-4.

       (3)  Davis, J.A.,  1981.   Comparison of static-replacement  and flow-through bioassays
       using duckweed, Lemna gibba G-3. EPA Report  No. EPA 560/6-81-003.
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(4) DeNoyelles, F. Jr. et al., 1987. Use of Experimental Ponds to Assess the Effects of a
Pesticide on the Aquatic Environment. Miscellaneous Publication No. 75, 34th Annual
Meeting of the Entomological Society of America, Nov. 29 - Dec. 3, 1987.  MPPEAL 75:
1-88.

(5) Fairchild, J.F., et al., 1992. Population-, community-, and ecosystem-level responses
of aquatic  mesocosms to pulsed doses  of a pyrethroid  insecticide. Environmental
Toxicology and Chemistry 11: 115-129.

(6)  Graney,  R.L.  et al. (Eds.),  1994. Aquatic Mesocosm Studies  in Ecological Risk
Assessment Lewis, Boca Raton, FL.

(7)  Hill,  I.R. et al. (Eds), 1994.  Freshwater Field Tests for Hazard Assessment of
Chemicals, Lewis, Boca Raton, FL.

(8) Hoist, R.W., et al., 1982. Effect of several pesticides on  the growth and nitrogen
assimilation of Azolla-Anabaena symbiosis. Weed Science 30:54-58.

(9) Little, T.M., andFJ. Hills, 1978. Agricultural Experimentation—Design and Analysis.
Wiley, NY.

(10) Sculthorpe, C.D., 1967.  The Biology of Aquatic Vascular Plants. London. Arnold
Publishers.

(11) SETAC Europe, 1991. Guidance Document on Testing Procedures for Pesticides in
Freshwater Mesocosms, report from the workshop "A meeting of experts on guidelines
for  static field mesocosm tests"  held at Monks Wood Experimental Station, Abbots
Ripton, Huntingdon, UK, July 3-4, 1991.

(12) SETAC-RESOLVE,  1992.  Proceedings of a workshop on aquatic microcosms for
ecological assessment  of pesticides, held  at Wintergreen, Virginia, October  1991.
SETAC Foundation for Environmental Education and the RESOLVE Program  of the
World Wildlife Fund, 56 pp.

(13)  Siefert, R.  E., et al,  1987.  Littoral  Enclosures for Aquatic Field Testing of
Pesticides:  Effects of Chlorpyrifos on a Natural System. Miscellaneous Publication No.
75,  34th  Annual Meeting of the Entomological Society of America, Nov. 29—Dec. 3,
1987. MPPEAL 75: 1-88.

(14) Touart, L.W., 1988.  Aquatic Mesocosm Tests to Support Pesticide  Registrations.
U.S. Environmental Protection Agency, Hazard Evaluation Division; Technical Guidance
Document. National Technical Information Service, Springfield, VA.

(15) Touart, L.  W. and M. W. Slimak, 1987. Mesocosm Approach for  Assessing the
Ecological Risk of Pesticides. Miscellaneous Publication No. 75, 34th Annual Meeting of
the Entomological Society of America, Nov. 29-Dec. 3, 1987. MPPEAL 75: 1-88.
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(16) Truelove, D., 1977. Research Methods in Weed Science, 2nd Ed.,  Southern Weed
Science Society, Auburn Printing Inc., Auburn, AL.

(17) Urban, DJ. and N.J.  Cook, 1986.  Hazard Evaluation Division Standard Evaluation
Procedure: Ecological Risk Assessment. EPA-540/9-86/167, Washington, DC.

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

(19) U.S. Environmental Protection Agency, 1986. Hazard Evaluation Division Standard
Evaluation Procedure, Non-target Plants: Aquatic Field Testing - Tier 3.   Office  of
Pesticides Program, Washington, D.C. EPA 540/9-86-136, June 1986.

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

(21) Voshell,  J.R.  Jr. Using  Mesocosms to Assess the  Aquatic Ecological Risk  of
Pesticides: Theory and Practice. Miscellaneous Publication No. 75, 34th Annual Meeting
of the Entomological  Society  of America, Nov. 29-Dec.  3, 1987MPPEAL  75:  1-88
(1987).
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