United States

Environmental Protection 

Agency

Office of Chemical Safety

and Pollution Prevention

(7101)

EPA 712-C-007

January 2012

Ecological Effects

Test Guidelines

OCSPP 850.4450:

Aquatic Plants

Field Study
i

NOTICE

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

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

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

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

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

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

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

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

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

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

the new acronym (OCSPP) in their title.

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

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

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

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

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

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

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

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

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

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

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

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

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

appropriateness on a case-by-case basis.

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

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

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

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

0150 through EPA-HQ-OPPT-2009-0159, and EPA-HQ-OPPT-2009-0576.
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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 enduse 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 (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). For 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 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 responserelationship 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)(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) 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.
Page 13 of 15

(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, 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. 

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.
Page 14 of 15

(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) Holst, 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., and F.J. 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, 

l987. 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.
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. 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).