United States Environmental Protection Agency Office of Chemical Safety and Pollution Prevention (7101) EPA 712

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- 007 January 2012 

# Ecological Effects **Test Guidelines** OCSPP 850.4450 : Aquatic Plants Field Studyi **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 Roden ticide 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 the se 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 the ir 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 partie s, 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 propos e alternatives to the recommendations described in these guidelines, and the Agency will assess them for appropriateness on a case

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- case basis. For additional information about these test guidelines and to access these guidelines electronically, please go to http://www.epa.gov/ocspp and select “Test Methods & Guidelines” on the left side navigation menu. You may also access the guidelines in http://www.regulations.gov group ed by Series under Docket ID #s: EPA

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- OPPT

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- 0150 through EPA

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- OPPT

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- 0159, and EPA

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**O CSPP**  **850. 4** **4 5**  **0 :**  **Aq uatic plant** **s 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 cons idered in the design and conduct of field studies for effects of chemical substances and mixtures on aquatic plants. E ffects considered may include mortality, and sublethal toxic effects such as decreased biomass or changes in population or community p arameters. The purpose of the field study is either to provide quantification of the risk that may o ccur 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

c onsideration 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 thi s 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.** Inc ontrast 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 i s 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 f or 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. D espite the variab ility 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 p ertain to aquatic plants, and to provide a better understanding of the purpose of f ield studies. There are two ty pes of field studies, screening and definitive. The ty pe of field study

c onducted (screening or definitive) depends on the available data on the test chemical or substance in question andPage **2** of **15**

the aquatic plant population and community dy namics 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 b e 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. S pecific objectives and associated qualitative and quantitative decision statements establishing measurement endpoints and their accuracy and precision should be provided as part of the study p lan. D evelopment 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 f or 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 c an 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 b e 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 i s to determine whether impacts to plant populations are occurring and to which species. “Pass

- fail” methods are used to determine whether impacts occur. E ffects 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 f ocuses on the species affected in the screening phase. F or some test substance s or chemicals it may b e appropriate to proceed directly

to a definitive study

without the screening phase. C areful 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 includePage **3** of **15**

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 technic al grade unless the test is designed to test a specific formulation, mixture, or end

use product. F or pesticides the substance to be tested is usually the ty pical 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 ty pe 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

d uration prior to conduct of the field study

. S everal seasons or one or more ye ars may

b e appropriate where one of the objectives of the definitive field study includes evaluating lowered productivity d ue to effects on populations or evaluating alteration in community integrity. S easonal 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 ty

pe of species investigated should be based on the specific objectives of the field study

. The scope and scale can vary f rom 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. T est plants should be in a stage of development under which exposure to the test substance would normally o ccur. Justification should be provided if surrogate species are used to represent those of the natural habitat.Page **4** of **15**

(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 o f 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 o f application should be consistent with the label. Equipment used may influence potential exposure of nontarget species. The diversity of ty pes of application equipment, depending on the particular use pattern involved, could influence exposure. The various ty pes of equipment nor

mally 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

b e appropriate. F or 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

speci fied on the label. F or pesticides, the single exposure level is equivalent to the maximum label rate (pounds a.i. per acre (lb 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). F or example, a 1 lb 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 ofPage **5** of **15** treatment levels selected when fitting a response -relationship should be sufficient to meet the level o f precision desired and allow determination of the goodness

- of

- fit. Consult a statistician for assistance in determining the number of treatment levels. F or 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. F or 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

b ear 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) **Tim ing.**  Consideration should be given to the season(s) over which the study is conducted. Studies should not be performe d at a time or season when there is a period of natural senescence. This dieback may c ontribute to the lessening of the test substance ’s effect on the aquatic plant test species. F or pesticides, the test substance is to be applied over a period of time or season according to label instructions.Page **6** of **15**

(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 protoco l 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 hy pothesis testing approaches, the magnitude of the difference the study i s designed to detect between treated and untreated plots and the power (ability) of the design to detect this difference should be discussed. C oefficient of variation estimates from screening studies, literature, or la boratory tests that closely

a pproximates reality

c an 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 c onsideration 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 sti ll 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. As ound understanding of the biology o f the species that are found in association with the areas is essential. Identifying these areas is likely to inc lude 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

.Page **7** of **15**

(8) **Study site selection.** (i) Within a geographic area, study s ites should be selected from those considered to be ty pical application sites for pesticides, or a ty pical exposure site which occurs under the pattern of production, use, disposal, or acc idental release of industrial chemicals, but at the same time, study s ites 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 o f species to serve as indicators for other species not present at that specific location. The choice of study s ites that are as similar as possible in terms of abundance, diversity, and associated habitat will facilitate an analys is of the results. (iii) Identifying potential study s ites may

require consultation with experts familiar with the areas where studies are proposed, and preliminary s ampling. Field surveys of a number of sites may b e 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 s ites in species, diversity, biomass, and selected study variables. The control sites should also be located as close as possible to selected treated study s ites but at enough of a distance and juxtaposition that cross - contamination from application or treatment will not occur. (10) **Size of**  **study sites.**  Study s ites should be large enough to provide adequate sample. The size is dependent on the methods used, the sensitivity required, and the density and diversity o f species. C onsideration should be given to the distance between study s ites. 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 b e 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 identi fied 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 p lan. P aragraph (i)(20) provides guidance on estimating number of replicates for a number o f 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 isPage **8** of **15** likely to be desired. (Under no circumstances should field studies on chemicals be conducted in areas where endangered species could be exposed.) (iii) Iti s 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 ty pes of problems to be assessed and the variety o f available investigative methods, the key to understanding and interpreting a field study lies in the development of a sound protocol. As ound 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 b ear upon the problem. It is possible that the literature may c ontain a valid answer to the questions raised b y the Agency. Far more likely, the literature may orient the investigator to address the concerns in a particular way
. B y using the available literature on both the chemical and the particular species of concern, the investigators may b e 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

o f the study

f or 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 p articular 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 p lant 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 observe d 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 pl ots and the power (ability) of the design to detect this difference. An experimental design with number of replicates based on an estimated coefficient ofPage **9** of **15**

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. A s 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 c onsistent manner. A s 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 c ombine the individual techniques in a wide variety o f 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 f or various species, depending on their biology and ecological characteristics. However, determining a result for the whole field study that would unequivocally l ead to a statement of the degree of risk, while obviously desirable, is not currently practical. (g) **Test validity**  **elements**
**.** Inthe case of fiel d 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 p lants 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 samp ling 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 )(1) 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.Page **10** of **15** (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 (lb 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) S ite description of the aquatic field testing study as to the ty

pe of systems (enclosed, controlled areas of a lake, pond, swamp, or stream, or artificial water systems such as aquaria, or large tubs). (ii) Lo cation 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) Ty pe 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 ty pes 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 ) Lo cation and distribution of soil types and texture, with percent organic matter in relationship to treated sites and controls.Page **11** of **15** (2 ) Location and distribution of vegetation including ty pe, density, and diversity

in relationship to treated sites and control s. (3 ) Canop y 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 s ystems. (iv) Climatological data during the test: records of appli cable conditions for the ty pe 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

o f 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.Page **12** of **15** (C) Stage of development and condition of plants at test initiation. (5) **Study conditions and experimental design.** Description of the study c onditions 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 ty pe and frequency

o f 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 site s 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 ty pe and design (nozzles, orifices, pressures, flow rates, volumes, etc .)) and method for calibrating the application equipment), information about any s olvent 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 o f water quality monitoring performed during the screening or definitive study

. (viii) Methods and frequency o f characterizing adjacent terrestrial, riparian, or wetland s ystem vegetation and soils or sediments. (ix) Methods and frequency o f climatological monitoring performed during the screening or definitive study f or air temperature, thermoperiod, humidity, rainfall and watering regime, light intensity, and wind speed. (x) The photoperiod and light quality

. (xi) Methods and frequency o f monitoring of other ancillary nontreatment related factors that may

influence the measures of effect at the study s ite should be reported. F or 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.Page **13** of **15** (xii) For the screening and definitive studi es, 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, yi eld of seeds or fruit, germination rate of second generation, phy

totoxicity rating, diversity, abundance) by f ield site and treatment (provide the raw data), and summary statistics. I f phy

totoxic ity rating measures are made a description of the rating s ystem 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 rea sons 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, S.J. 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. E nvironmental Toxicology and Chemistry 11: 1

-4. (3) Davis, J.A., 1981. C omparison of static

-replacement and flow

-through bioassays using duckweed, Lemna gibba G

- 3. EPA Report No. EPA 560/6

- 81

- 003.Page **14** of **15**

(4) DeNo ye lles, 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. MPPEAL75: 1

- 88. (5) Fairchild, J.F., et al., 1992. P opulation- , community

- , and ecos ystem

- level responses of aquatic mesocosms to pulsed doses of a py rethroid 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) Holst, R.W., et al., 1982. Effect of several pesticides on the growth and nitrogen assimilation of Azolla

-

Anabaena sy

mbiosis. Weed Science 30:54

- 58. (9) Little, T.M., and F.J. Hills, 1978. Agricultural Experimentation

--Design and Analys is

. Wiley, NY. (10) Sculthorpe, C.D., 1967. The Biology o f 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: E ffects of Chlorpy rifos on a Natural System. Miscellaneous Publication No. 75, 34th Annual Meeting of the Entomological Society o f America, Nov. 29

--Dec. 3, l987. MPPEAL75: 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.Page **15** of **15**

(16) Truelove, D., 1977. Research Methods in Weed Science , 2nd Ed., Southern Weed Science Society, Auburn Printing Inc., Auburn, AL. (17) Urban, D.J. 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. P esticide Assessment Guidelines Subdivision J, Hazard Evaluation: Non

-target plants. O ffice 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. O ffice 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 o f America, Nov. 29

-Dec. 3, 1987MPPEAL7 5: 1

- 88 (1987).