
United States
Environmental Protection Agency
   Office of Chemical Safety
   and Pollution Prevention
   (7101)
EPA 712-C-013
January 2012


Ecological Effects
Test Guidelines



OCSPP 850.4000:

Background and Special Considerations-Tests with Terrestrial and Aquatic Plants, Cyanobacteria, and Terrestrial Soil-Core Microcosms

                                       
                                       

  
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  NOTICE
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  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.
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  	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.  
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  	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 850.4000: Background and special considerations: tests with terrestrial and aquatic plants, cyanobacteria, and terrestrial soil-core microcosms.
(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.  This guideline provides general information applicable to conducting OCSPP Series 850, Group D toxicity tests with terrestrial and aquatic plants and cyanobacteria (formerly referred to as blue-green algae).  The source materials used in developing this harmonized OCSPP test guideline include OPP 120-1 Overview, 120-2 Definitions, 120-3 Basic Test Standards, and 120-4 General Evaluation and Reporting Requirements, Pesticide Assessment Guidelines Sub-division J--Hazard Evaluation: Nontarget Plants); ASTM E 1963-02, Standard Guide for Conducting Terrestrial Plant Toxicity Tests, the Pesticides Reregistration Rejection Rate Analysis: Ecological Effects report; and the background materials in the OCSPP Series 850, Group D specific guidelines.
	(3) General.  
            (i) The OCSPP Series 850, Group D provides guidelines applicable to conducting laboratory and field toxicity tests with terrestrial and aquatic plants, cyanobacteria, and terrestrial (soil-core) microcosms.  Where appropriate, individual guidelines in the OCSPP Series 850, Group D are harmonized with OECD test guidelines.  The guidelines in the OCSPP Series, Group D are applicable to evaluating the hazards and risks of industrial chemicals and pesticides to various terrestrial and aquatic plant species and cyanobacteria resulting from direct or indirect exposure.  Data concerning the determination of outdoor pesticidal effects on plants and cyanobacteria are used in evaluating risks to nontarget plants in ecological risk assessment of pesticides (40 CFR part 158, paragraph (k)(28) of this guideline).  These data are also of use in assessments of potential off-target injury to endangered and threatened plant species listed by the Fish and Wildlife Service, Department of Interior, and when phytotoxicity concerns arise from incidents or during Special Review.  Phytotoxicity data are also occasionally requested in order to assess the potential hazard of certain pesticides to plants within the pesticide treatment area (target area testing).
            (ii) Information is provided on the design and conduct of tests with terrestrial and aquatic plants, cyanobacteria, and terrestrial plant microcosms, emphasizing the importance of adequately characterizing the test substance, use of suitable experimental designs, and establishing the physical and chemical conditions of the test system in order to provide a scientifically sound understanding of how the test substance behaves under test conditions.  Also considered are an identification of the factors that can affect the test outcome and interpretation of test results.  This general information is primarily applicable to the guidelines for laboratory toxicity tests and microcosms, since field tests are designed on a case-by-case basis.  However, the OCSPP 850.4000 guideline lists critical elements of quality assurance and reporting standards common to all the guidelines in OCSPP Series 850, Group D guidelines.
            (iii) The OCSPP Series 850, Group D guidelines have generally been validated in formal round-robin tests or through repeated use.
      	(iv) Each submitted study should meet the data quality objectives for which the test is designed.  Test validity elements critical to determining the scientific soundness and acceptability of the study have been listed for each guideline in the OCSPP Series 850, Group D.
      	(v) Certain aspects of the OCSPP 850.1000 guideline are also relevant to toxicity studies with aquatic plants and microorganisms (the OCSPP 850.4400 guideline, Aquatic plant toxicity test using Lemna spp.; the OCSPP 850.4450 guideline, Aquatic plants field study; and the OCSPP 850.4500 guideline, Algal toxicity; and the OCSPP 850.4550 guideline, Cyanobacteria toxicity).
      	(vi) The guidelines contained in the OCSPP Series 850, Group D recommend specific procedures to be used in almost all circumstances in order to result in a satisfactory study result, but also provide general guidance that allows for some latitude, based upon study-specific circumstances.  It is recognized that certain problems, some of which are unavoidable, may arise both before and during testing and provisions have thus been made in the guidelines for dealing with those that are commonly encountered.  These guidelines provide for exceptions, while at the same time maintaining a high level of scientifically sound, state-of-the-art guidance so that following this guidance will provide ecological effect information that is scientifically defensible for its intended use, while also taking into consideration the chemistry and environmental fate of the test substance.  For a satisfactory test, the experimental design, execution of the experiments, classification of the organism, sampling, measurement, and data analysis should be accomplished by use of sound scientific techniques recognized by the scientific community.  Uniformity of procedures, materials, and reporting should be maintained throughout the toxicity evaluation process.  Refinements of the procedures to increase their accuracy and effectiveness are encouraged.  When such refinements include major modifications of any test procedure, the Agency should be consulted before implementation.  Also when in doubt, users of these guidelines should consult with the appropriate regulatory authorities for clarification or additional information before proceeding.  All references supplied with respect to protocols or other test standards are provided as recommendations.
      	(vii) For pesticides, a tiered testing approach given in 40 CFR 158.540 for plant protection data requirements provides for greater efficiency of testing resources while assuring data development as warranted for hazard or risk assessment.  Tests in the lower tiers (Tier I and Tier II) are designed to screen test substances to determine their potential to cause adverse affects on seedling emergence (the OCSPP 850.4100 guideline), vegetative vigor (the OCSPP 850.4150 guideline), and aquatic plant and cyanobacteria growth and reproduction (the OCSPP 850.4500 and OCSPP 850.4550 guidelines).  For pesticides, a Tier I test, referred to as a limit test in these Group D guidelines, tests a single concentration and compares effects observed with appropriate controls.  Tier II testing for pesticides (multiple-concentration definitive test in these Group D guidelines provides for generation of dose-response curves for test substances which are known phytotoxicants or which in Tier I testing demonstrated phytotoxicity.  Tier III plant tests currently include the Terrestrial Plants Field Study (the OCSPP 850.4300 guideline); Terrestrial Soil-Core Microcosm Test (the OCSPP 850.4900 guideline); and Aquatic Plants Field Study (the OCSPP 850.4450 guideline) and are designed on a case-by-case basis to address specific objectives concerning detrimental effects on nontarget plants.  Progression to Tier III would occur on a case-by-case basis to further refine and characterize the estimate of phytotoxicity risk.  For the evaluation of pesticides, the criteria to proceed from one tier to the next are given in 40 CFR 158.540.  Testing of pesticides in any of these tiers can be expanded by conducting additional tests that are normally specific to industrial chemicals (e.g., the OCSPP 850. 4800 Plant Uptake and Translocation Test; the Rhizobium-Legume Toxicity Test (the OCSPP 850.4600 guideline) and adapting them to pesticide use as needed to address specific exposure and effect concerns.
      	(viii) Phytotoxicity data may also be used to evaluate the potential hazard and risk of industrial chemicals.  Phytotoxicity data are requested when there are verified terrestrial exposures or aquatic exposures and are also occasionally requested for industrial chemicals to assess their potential hazard to crop plant test species.  This testing is part of the Tier I (base set) suite of tests in the OPPT testing scheme developed for determining environmental effects (see the references in paragraphs (k)(10), (k)(11), (k)(16), (k)(17), (k)(30), and (k)(31)of this guideline for further details).  This testing scheme is deterministic for the most part, flexible, sequential, consistent, iterative, transparent, discriminatory of the extent of toxicity, and applicable to all types of chemicals.
      	(ix) For industrial chemicals, dose-response testing occurs at Tier I to determine as quickly and as efficiently as possible, the potential phytotoxicity of these chemicals during early seedling emergence and growth of terrestrial plants (the OCSPP 850.4230 guideline) and to aquatic plants (the OCSPP 850.4500 guideline).  In contrast to pesticides, the potential phytotoxicity of industrial chemicals is often not characterized.  Thus, range-finding tests, a preliminary step to define dose-response testing, are more commonly conducted than limit or maximum challenge tests that use only one test concentration.  If phytotoxicity is noted at Tier I multiple-concentration definitive testing, additional seedling emergence tests with more plant species are conducted at Tier II to better delimit this toxicity to sensitive plant groups or under specific exposure scenarios.  For example, if monocot species exhibit the greatest sensitivity, additional monocots are tested; if a wetland monocot is found to be the most sensitive, additional wetland monocots (and also dicots) are then tested.  Chronic (reproductive effect) tests may sometimes be part of Tier II (or more commonly part of Tier III).  Tier II plant tests for industrial chemicals are designed to investigate potential chronic cumulative toxicity and bioconcentration potential of industrial chemicals.  Tier III plant tests currently are the Plant Uptake and Translocation Test (the OCSPP 850.4800 guideline), possibly the Terrestrial Soil-Core Microcosm Test (the OCSPP 850.4900 guideline), and the Rhizobium-Legume Toxicity Test (the OCSPP 850.4600 guideline).  Field testing is conducted at Tier IV, if necessary for widely distributed chemicals with high exposure, which are toxic to plants.  Testing of industrial chemicals in any of these tiers can be expanded by conducting additional tests that are normally specific to pesticides (e.g., the OCSPP 850.4100, 850.4150, and 850.4300 guidelines) and adapting them to industrial chemicals as needed.  These pesticide-specific tests should be viewed as supplements to the TSCA plant tests, not as substitutes.  For industrial chemicals, the base set Tier I tests and requirement to proceed from one tier to the next are referenced in paragraphs (k)(10), (k)(11), (k)(16), (k)(17), (k)(30), and (k)(31)of this guideline.
      	(x) While performing field tests, all necessary measures should be taken to ensure that nontarget plants and animals, especially endangered or threatened species, will not be adversely affected either by direct hazard or by impact on food supply or food chain.
(b) Definitions.  Terms used in the OCSPP Series 850, Group D guidelines have the meanings set forth in Section 3 FIFRA regulations at 40 CFR 152.3 (Pesticide Registration and Classification Procedures); 40 CFR 158.300 (Product Chemistry Definitions); 40 CFR part 160 (Good Laboratory Practice Standards); the OCSPP 850.1000 guideline (background for aquatic organism testing); and in TSCA Section 3 regulations 40 CFR part 792 (Good Laboratory Practice Standards); and the Agency's "Terms of Environment, Glossary, Abbreviations and Acronyms" (see paragraph (k)(22) of this guideline).  The definitions below apply to the OCSPP Series 850 Group D test guidelines and where applicable, the individual test guidelines contain additional or test-specific definitions.
	Active ingredient (a.i.) is any substance (or group of structurally similar substances if specified by the Agency) that will prevent, destroy, repel or mitigate any pest, or that functions as a plant regulator, desiccant, or defoliant within the meaning of FIFRA (40 CFR152.3).
	Adjuvant is a subsidiary ingredient or additive in a mixture which modifies, enhances or prolongs by physical action the activity of the active ingredient(s).  Examples of agricultural chemical adjuvants include but are not limited to surfactants, crop oils, anti-foaming agents, buffering compounds, drift control agents, compatibility agents, stickers and spreaders.
	Algae includes the green algae (Chlorophyta), golden algae and diatoms (Chrysophyta), brown algae (Phaeophyta), and red algae (Rhodophyta).  Organisms formerly classified as blue-green algae (Cyanophyta) are now classified as Cyanobacteria.
	Aquatic plants includes those plants that are totally aquatic (free-floating or attached, submersed or emerged) that may inhabit still or flowing water bodies.
	Axenic is a culture of one organism free from other organisms.
	Concentration-response curve is the graphical and mathematical relationship between the concentration of a substance and a specific biological response produced from toxicity tests when percent response (e.g., growth) values are plotted against concentration of test substance for a given exposure duration.  This is also referred to as the dose-response curve or concentration-effect curve.
	Control refers to test organisms exposed to test conditions and test matrix (water, sediment, medium, etc.) in the absence of any introduced test substance as part of the test design for the purpose of establishing a basis of comparison with a test substance for known chemical or biological measurements.
	Culture (noun) refers to the organisms which are raised on-site or maintained under controlled conditions to produce test organisms through reproduction.
	Culture (verb) is to grow, raise, or maintain organisms under controlled conditions to produce test organisms through reproduction.
	Desirable plants are those plants that are not to be detrimentally affected during pesticide application.  They may include crops, ornamentals, or native plants inside or outside of the area of intended application.
	Direct exposure refers to the direct application of a pesticide or industrial chemical to a plant or the location where the plant would reside.
	Effect concentration (ECx) is the experimentally derived concentration of a test substance in test matrix (e.g., water, growth medium, soil, sediment) that would be expected to cause a specified effect in x percent (x%) of a group of test organisms under specified exposure conditions.
	Effect concentration, median (EC50) is the experimentally derived concentration of a test substance in a test matrix (e.g., water, growth medium, soil, sediment) that would be expected to cause a defined effect in 50% of a group of test organisms under specified exposure conditions.
	Formulation, as used within these guidelines, is a packaged end use product (e.g., dust, wettable powder, emulsifiable concentrate, ultra low volume, etc.) of the test substance and may contain one or more active ingredients and one or more inert ingredients.
	Germination refers to the physiological events associated with re-initiation of embryo growth and mobilization of reserve nutrients in seeds.  The emergence of the seedling radicle from the seed coat defines the end of germination and the beginning of early seedling growth.
	Hormesis refers to a stimulatory effect on a given response variable, occurring at a low exposure concentration of an otherwise toxic test substance (e.g., where the higher exposure concentrations produce adverse effects).
	Indirect exposure refers to exposure of plants or cyanobacteria resulting from movement of the pesticide or industrial chemical through the environment by runoff, soil erosion, spray drift, etc.
	Inert ingredient is any substance (or group of structurally similar substances if designated by the Agency), other than an active ingredient, which is intentionally included in a pesticide product (40 CFR 152.3).
	Inhibition concentration (ICx) is the experimentally derived concentration of a test substance in a test matrix (e.g., water, growth medium, soil, sediment) that would be expected to cause a given percent, x, inhibition or reduction in a non-quantal response from the smoothed mean control response.  For example, the IC25 for growth is the concentration of test substance that would cause a 25% reduction in growth in a test population from the control response and the IC50 is the concentration of test substance that would cause a 50% reduction in growth from the control response.
	Limit of detection (LOD) is the analytic level below which the qualitative presence of the material is uncertain.  This is typically defined by the lowest concentration producing a signal two standard deviations above the background noise from a matrix blank sample.
	Limit of quantitation (LOQ) is the analytic level below which the quantitative amount of the material is uncertain.  This is typically defined by the lowest concentration of fortified matrix successfully analyzed.
	Limit test is a toxicity test performed with a single test substance concentration and a control to establish that the value for the measurement endpoint of concern (e.g., EC50) is greater than the test substance concentration (limit concentration).
	Lowest observed effect concentration (LOEC) is the lowest concentration of a test substance to which organisms are exposed under specified exposure conditions that causes a statistically significant adverse effect as compared to the control(s).  Throughout these guidelines, the terms LOEC and lowest observed adverse effect concentration (LOAEC) have the same meaning in these guidelines.
	Maximum acceptable toxicant concentration (MATC) is the highest concentration at which a test substance can be present and not be toxic to the test organism.  The MATC lies within the range between the LOEC and no observed effect concentration (NOEC).  Operationally, for industrial chemicals, the MATC is defined as the geometric mean of these values.  The MATC is also referred to (in the Pre-Manufacture Notification (PMN) program of OPPT) as the chronic value or chronic no-effect-concentration (NEC).
	Measured concentration is an analytically derived measure above the method detection limit.
	Measurement endpoint is a quantitative measurable response to a stressor that is used to infer a measure of protection or evaluate risk to valued environmental entities.  Examples of measurement endpoints include, but are not limited to, number of organisms that survive, visual phytotoxicity, growth measurements (e.g., algal population density, plant height, plant dry weight, etc.).  Each test-specific guideline identifies the measurement endpoint(s) to be determined by the proscribed study.  The term "measurement endpoint" is used synonymously with the term "measures of effect."
	Medium is the chemically-defined culture solution used in microcosms and in culturing and testing certain organisms such as aquatic plants.
	Method detection limit (MDL) is operationally defined as the concentration of constituent that, when processed through the complete method, produces a signal with 99% probability that it is different from the blank.  It is computed as the standard deviation multiplied by the Student's t constant corresponding to the appropriate degrees of freedom (n-1).  Thus, for seven spiked samples prepared at the hypothetical LOQ, the MDL is 3.143 times the standard deviation of the mean of the seven replicate measurements.
	Nominal concentration is, for aquatic tests, the calculated concentration of active ingredient that would exist if all test substance added to the test solution was completely dissolved and did not dissipate in any way.  For terrestrial tests, it is the target concentration intended for application to test plants.
	Nontarget microorganism is any microorganism species to which the pesticide is not directly applied.  These species are not intended to be controlled, injured, killed, or detrimentally-affected in any way by a pesticide.
	No observed effect concentration (NOEC) is the highest concentration of a test substance to which organisms are exposed under specified exposure conditions that does not cause a statistically significant adverse effect as compared to the control(s).  The NOEC is the test concentration immediately below the LOEC and can only be defined in the presence of the LOEC.  Throughout these guidelines, the terms NOEC and no observed adverse effect concentration (NOAEC) have the same meaning.
	Nontarget plant is any plant species to which the pesticide is not directly applied.  These species are not intended to be controlled, injured, killed, or detrimentally-affected in any way by a pesticide.  Nontarget plants include desirable or undesirable plants outside of the target area.
	Pest-free refers to being as free of pests as reasonably possible.  For all phytotoxicity tests, damaging insects, pathogens, and surrounding weeds should be controlled so that healthy desirable plants are available for testing.  With this action detrimental effects can be attributed to the test substance in question, not to another pesticide, or to weeds, damaging insects, or pathogens.
	Phytotoxicity or plant toxicity refers to unwanted detrimental deviations from the normal pattern of appearance, growth, and function of plants in response to the test substance.  The phytotoxic response may occur during germination, growth, differentiation, maturation, and reproduction of plants, and may be of a temporary or long-term nature.  Phytotoxic responses include adverse effects on growth habit, yield, and quality of plants or their commodities to the extent that a relationship between cause and effect can be established.
	Plants comprise vascular and nonvascular plants (including algae and cyanobacteria). 
	Reagent water is water that has been prepared by deionization, glass distillation, or reverse osmosis.
	Replicate is the experimental unit within a toxicity test.  It is the smallest physical entity to which treatments can be independently assigned.
	Solubility is the amount of chemical dissolvable in test water and is operationally defined as the amount of test substance retained in the supernatant of a conventionally centrifuged sample of test medium or dilution water.  This amount of test substance is considered to represent a conservative measure of the most bioavailable fraction which may include some colloidal material not removed by centrifugation in addition to the truly dissolved fraction. 
	Static renewal system is a static system in which the test solution is renewed at specified intervals during the test.
	Stock solution is the concentrated solution of the test substance which is dissolved and introduced into the dilution water or test medium.
	Support medium is the matrix used to support the plant during growth.  There are three types of support media: natural, formulated, and artificial media.  Natural support medium is derived entirely from a combination of natural soils found in the environment.  Formulated support medium is derived from a combination of both natural soils and artificial media (including components such as sphagnum moss).  Artificial support medium is derived entirely from washed quartz sand or glass beads.
	Target area is the area intentionally treated with a pesticide when label use directions are followed.
	Target area plants are all plants located within the target area, and includes both desirable and undesirable species.
	Test chamber is the container in which the test organisms are maintained during the test period.
	Test substance refers to the specific form of a chemical substance or mixture being evaluated (e.g., pesticide active ingredient or formulation, industrial chemical).
	Test solution refers to the test substance and the dilution water or growth medium in which the test substance is dissolved or suspended.
	Terrestrial plants are plants that do not require an aquatic medium for growth and may include plants that inhabit semi-aquatic areas such as swamps or wetlands.
	Treatment group refers to the set of replicates that receive the same amount (if any) of the test substance; controls are treatment groups that receive none of the test substance.
	Typical end-use product (TEP) is a term used to convey direction to a data producer to use a commonly used end-use product, a pesticide formulation for field or other end use (excludes products with labeling that allows use of the product to formulate other pesticide products), as the test substance.  The term includes any physical apparatus used to deliver or apply the pesticide if distributed or sold with the pesticide.
	Vehicle is any agent which facilitates the mixture, dispersion, or solubilization of a test substance with a carrier (e.g., water, growth medium) used to expose the test organisms (40 CFR 160.3, 40 CFR 792.3).
(c) Apparatus, facilities, and equipment--
      (1) Laboratory facilities and equipment.  The type of facilities and equipment for conducting the toxicity tests with the organisms in this group of guidelines varies depending upon the nature of the test and the organism.  In general, these toxicity tests use normal laboratory glassware, supplies and equipment as well as equipment for maintaining the organisms under the test conditions and controlling the test conditions (e.g., temperature, humidity, and lighting).  See specific OCSPP Series 850, Group D guidelines for identification of any atypical facility, equipment, or supplies used in the test.  Construction materials and equipment that are toxic, may affect toxicity, or that may sorb test substances should not be used.
      (2) Maintenance and reliability.  All equipment used in conducting the test, including equipment used to prepare and administer the test substance, and equipment to maintain and record environmental conditions, should be of such design and capacity that tests involving this equipment can be conducted in a reliable and scientific manner.  Equipment should be inspected, cleaned, and maintained regularly, and be properly calibrated.  All materials that will come in contact with the test organisms and test substance should be cleaned before use.  Cleaning procedures should be appropriate to remove known or suspected contaminants.
      (3) Permits.  Experimental use permits may be required for the terrestrial testing of pesticides under field conditions involving more than 10 acres such as in studies described in the OCSPP 850.4300 (terrestrial plant field testing) guideline.  Permits may be required for aquatic field testing of pesticides of more than 1 acre.
      (4) Field equipment.  If relevant, the application equipment used in testing products in small field plot studies should be designed to simulate conventional application equipment.  This can be accomplished by using the basic components of commercial application equipment in the design of the small-plot equipment.  For example, nozzle types, sizes, and arrangements on small plot sprayers can be identical to those used by growers on commercial ground sprayers.  Single-row commercial granular application equipment mounted on a garden tractor for small plot trials should produce results comparable to those from a multiple of such units on a large tractor.  For large-scale field trials, commercial application equipment should be used.  Specific details as to descriptions of equipment design, adjustment, and operation should be provided in test reports.
(d) Experimental design and data analysis--
      (1) Design elements.  Elements of experimental design such as the number of test treatments, progression factor between treatment levels, number of replicates, and number of organisms per replicate and per treatment are based upon the purpose of the test, variability expected in response measurements, and the type of statistical procedures that will be used to evaluate the results.  See the test-specific guidelines for specific information relating to these aspects of test design.  General principles of test design are set forth in this guideline.  General guidance on the statistical analysis of ecotoxicity tests can be found in the references in paragraphs (k)(1), (k)(4), (k)(13), (k)(14), (k)(15), (k)(25), (k)(26), and (k)(27) of this guideline.
      (2) Calculation of endpoints--
      	(i) Background--
                  (A) Qualitative data.  Some of the data generated in phytotoxicity tests are qualitative, such as ratings based upon visual symptoms of phytotoxicity.  Qualitative data such as ratings are not statistically analyzed for these tests but may be used to report qualitative no-effect levels.
                  (B) Quantitative data.  
                        (1) Quantitative data generated in phytotoxicity tests may be of three types: 
                              (a) Quantal (dichotomous), where the variable has only two mutually exclusive outcomes, (e.g., dead or alive) -- note that quantal data are a special case of discrete data; 
                              (b) Discrete, where there is a finite number of values possible or there is a space on the number line between two possible values (e.g., number of fronds, fruits or seeds produced); or 
                              (c) Continuous, where the variable can assume a continuum of possible outcomes (e.g., height, weight).
                        (2) These data may be analyzed using regression-based techniques or hypothesis-testing procedures depending on the objectives and endpoints of a specific test guideline.  For information on the advantages and disadvantages of these approaches, see the references in paragraphs (k)(7), (k)(8), (k)(14), (k)(15), and (k)(18) of this guideline.  Specific test guideline objectives, either point estimate or hypothesis-based endpoints or both, are identified in each specific test guideline.
            (ii) Point estimates and concentration-response or dose-response tests.  This type of toxicity test is designed to allow calculation of a concentration- or dose-response curve (mathematical model) and to estimate one or more specific points (point estimates) on the curve, such as an EC25 or EC50.  Regarding terminology, the term ICx is more appropriately used for continuous endpoints, rather than ECx.  Because of the normal variation in sensitivity of individuals within a group of test organisms, a measure of the degree of certainty in the model parameters and the point estimate value(s) should be determined.
                  (A) No single statistical technique is appropriate for all data sets, and the assumptions and requirements of each method should be known before using (see paragraphs (k)(1), (k)(9), (k)(12), (k)(18), and (k)(29) of this guideline).  Not all methods suitable for continuous data are appropriate for quantal data (see paragraphs (k)(6) and (k)(12) of this guideline).  For point estimate tests, regression-based methods that model the full concentration- or dose-response relationship and provide error estimates of the model parameters and point estimate(s) are desired.  The regression model used to fit data should be recorded, and the error estimates of the model parameters (e.g., standard error of slope and intercept), and goodness-of-fit should be calculated and recorded.  For a point estimate (e.g., IC50) the 95% confidence interval and standard error are calculated and recorded.  If data do not fit a regression-based model, other point estimator methods (e.g., binominal, moving average, trimmed Spearman-Karber, linear interpolation (e.g., Bootstrap ICp)) are available (see paragraphs (k)(23), (k)(26) and (k)(27) of this guideline).  The method used to estimate the endpoint and, if applicable, 95% confidence interval for the point estimate, should be recorded.
                  (B) To account for experimental variability, select a statistical method that retains data for the individual replicates through the regression calculations as opposed to pooling the replicates.  Where hormesis occurs, it is preferable to select a statistical method that provides for analysis of the impact of stimulation on the calculated endpoints as opposed to discarding stimulatory data.
                  (C) Concentration-effect models are good estimating tools only for the range of concentrations used to fit them; therefore, endpoints that are extrapolated beyond the range of the concentrations tested would be considered to be of lower confidence or potentially, of such low confidence that they would not be appropriate to estimate.
            (iii) Hypothesis-based methods--
                  (A) Multiple-concentration or multiple-dose definitive tests.  In this type of test, the purpose is to determine if the biological response to a treatment level differs from the response of the control.  Hypothesis testing-based endpoints, expressed as the NOEC and LOEC (or NOEL and LOEL), are calculated by determining statistically significant differences from the control.  The null hypothesis is that no difference exists among the mean (or median if nonparametric), control and treatment responses.  The alternative hypothesis is that the treatment(s) result in an adverse biological effect relative to the control sample.  Parametric and nonparametric analysis of variance (ANOVA) tests and multiple-comparison tests are often appropriate for continuous data and may be appropriate for some categorical data (rank, order, score).  Contingency table tests are usually appropriate for categorical data.  Parametric tests are based on normal distribution theory and assume that the data within treatments are a random sample from an approximately normal distribution and that the error variance is constant among treatments.  These assumptions should be examined using appropriate tests, and data transformations (see paragraph (d)(2)(v)(A)) of this guideline or nonparametric techniques should be used where the assumptions are not met.  Where possible multiple comparison tests that restrict the number of comparisons made should be used.  Generally, the more powerful multiple-comparison tests are those which assume a dose-response relationship in the data.  When the assumption of a monotonic dose-response holds, Williams' and Jonckheere's test, respectively, are examples of parametric and nonparametric tests that can be used.  When the assumption of a monotonic dose-response fails, Dunnett's t-test and either Steel's many-one rank test or the Wilcoxon rank sum test with Bonferroni adjustment, respectively, are examples of parametric and nonparametric multiple comparison tests requiring no assumption about the dose-response but which restrict comparisons of the treatments to a control.  A measure of the sensitivity of the test, such as the minimum significant difference (parametric tests), should be calculated.  Alternatively, a calculation of the number of replicates necessary to achieve data quality objectives given the actual measured test responses and variability should be made.  At a minimum, the percent reduction from the control for each treatment should be calculated.
                  (B) Types of decisions and errors.  
                        (1) Table 1 presents the two possible outcomes and decisions that can be reached in the statistical hypothesis tests discussed in paragraph (d)(3)(ii)(A) of this guideline: 
                              (a) There is no difference among the mean control and treatment responses; or 
                              (b) There is a difference among the mean control and treatment responses (concerned with direction, where response is adverse relative to the control).  
                        (2) Statistical tests of hypothesis can be designed to control for the chances of making incorrect decisions.  The types of incorrect and correct decisions that can be made in a hypothesis-based test and the probability of making these decisions are represented in Table 1.  For multiple comparison tests the Type I error rate is controlled to account for multiple test comparisons.
	Table 1.--Types of Errors and the Probabilities of Making Correct and Incorrect Decisions Based on the Results of Testing

                          Actual (or True) Condition:
Test Decision Outcome:
                     Treatment Response = Control Response
                   Treatment Response < Control Response
Treatment Response = Control Response
                               Correct Decision
                          probability = 1- alpha (α)
                        Type II error (False negative)
                            probability = beta (β)
Treatment Response < Control Response
                         Type I error (False positive)
                               probability = α
                               Correct Decision
                      probability (Power of test) = 1-β
                  (C) Power of the test.  Power of the test versus percent reduction in treatment response relative to the control mean at various coefficients of variation is provided in the reference in paragraph (k)(23) of this guideline.  Examples are specifically given for 5 and 8 replicates for a one-tailed test alpha (α) of 0.05 and 0.10.  Effects on the number of replicates at various coefficients of variation are also provided in the reference in paragraph (k)(23) of this guideline for various low α and beta (β) values (i.e., α + β = 0.25).  See also the references in paragraphs (k)(9) and (k)(24) of this guideline.
                  (D) Limit test.  In a limit test it is only necessary to ascertain that: a fixed point standard (such as the EC25 or IC25 for terrestrial plants or EC50 or IC50 for aquatic plants) is greater than a given "limit" concentration (Tier I test for pesticides); and the response at a given "limit" concentration does not differ from the control response.  Only one treatment, the "limit" concentration, and the appropriate control(s) are tested.  This is referred to as a limit test or maximum challenge concentration test.
                        (1) Fixed point standard.  For a fixed point limit test, the null hypothesis is that the limit treatment mean response as compared to the control response is greater than or equal to a fixed point response of concern (e.g., 25% for terrestrial plants).  The alternative hypothesis is that the limit treatment mean response as compared to the control response is less than the fixed point response of concern. (Concerned with direction, where response is inhibition relative to the control switch hypotheses around.)
                        (2) Difference between two means (or medians).  For testing if the treatment level affects the test organism, the null hypothesis is that the treatment mean (or median) response is equal to the control response mean (or median) level and the alternative hypothesis is that the treatment mean response differs from the control response.  The direction of the alternative hypothesis depends on what is considered an adverse direction for the specific response being evaluated, such as decreased survival, shoot height and biomass or increased mortality as compared to the control response.  Examples of parametric and nonparametric two-group comparison tests are Student's t-test and Wilcoxon rank sum test, respectively.
            (iv) Transformations, outliers, and non-detects--
                  (A) Transformations.  Transforma-tion of data (e.g., square root, log, arcsine-square root) may be useful for a number of statistical analysis purposes.  The two main reasons are to satisfy assumptions for statistical testing and to derive a linear relationship between two variables, so that linear regression analysis can be applied.  Added benefits include consolidating data that may be spread out or that have several extreme values (see reference in paragraph (k)(24) of this guideline).  Once the data have been transformed, all statistical analyses are performed on the transformed data.
                  (B) Outliers.  Outliers are measurements that are extremely large or small relative to the rest of the data and, therefore, are suspected of misrepresenting the population from which they were collected.  Unless there is a known documented reason for the outlier(s), such as measurement system problems or instrument breakdown, the statistical analyses performed should at a minimum include results using the full data set (i.e., the suspected outlier(s) are not discarded).  Outliers should not be discarded based on a statistical outlier test (see reference in paragraph (k)(24) of this guideline).  The analyst may conduct all statistical analysis of the data with both a full and truncated (presumed outliers are discarded) data set, however, so that the effect of the presumed outlier(s) on the conclusion may be assessed.
                  (C) Nondetects.  Data generated from chemical analysis that falls below the LOD of the analytical procedure are generally described as not detected, or nondetects, (rather than as zero or not present) and the appropriate LOD should be reported.  There are a variety of ways to evaluate data that include both detected and non-detected values (see reference in paragraph (k)(24) of this guideline).  However, for a satisfactory test in a number of the Group D guidelines, test substance concentrations should not be below the LOD (see specific OCSPP Series 850, Group D guidelines), except in controls.
      (3) Selection of test treatments--
            (i) Point estimate and concentration-response or dose-response test.  Toxicity tests where the objective is the concentration-response curve and a specific point on the curve (e.g., IC50) usually consist of one or more control treatments and at least five test treatments which should bracket the specific point(s) of concern for the test.  To obtain a reasonably precise estimate of the IC50 using probit analysis for example, one or more treatments should be between, but not including, 0 and 50% and one or more treatments should be between, but not including, 50 and 100%.  Where the objective is to derive the concentration-response curve and to determine more than one specific point response on the curve (e.g., IC25 and IC50), the use of additional treatment levels to ensure that both point response values are each bracketed is encouraged.  The spacing between test treatments depends upon the expected slope of the concentration- or dose-response curve, information about which can be gained during a range-finding test.  The minimum geometric ratio between doses is 2.  The maximum geometric ratio between doses should be 4, with adequate justification documented for using a ratio of 4.  When the objective of the test is to determine a regression-based estimate and sample size constraints apply, the use of more treatment levels is preferable to the use of more replicates.  The inclusion of additional treatment levels rather than additional replicates results in better characterization of the overall concentration- or dose-response relationship.
            (ii) Hypothesis-based test--
                  (A) Multiple-concentration or -dose definitive test.  Each test usually consists of one or more control treatments and at least five test treatments which span the expected environmental concentrations and where at least the lowest treatment level is the NOEC (or NOEL).  The test treatments are usually selected in a geometric series in which the ratio is minimally 2 to 4.  A key assumption is that the response data are monotonic with increasing concentration or dose (i.e., the degree of biological effect increases as treatment concentration increases) or that there is a threshold response such that a NOEC (or NOEL) for a given biological response should not occur at a treatment concentration higher than one found to be statistically different from the control for the given biological response.  Where these assumptions do not hold it is recommended that additional concentrations be included to better characterize the relationship of the biological response with exposure concentration.  If the failure is suspected to be due to high variability in a given response measurement, the number of replicates should be increased.
                  (B) Limit test.  A limit test consists of a single treatment level and the appropriate control(s).  Individual OCSPP Series 850 Group D guidelines identify the concentration or dose that satisfies the limit treatment level test for that guideline.
      (4) Randomization.  For test results to be satisfactory test treatments should be randomly assigned to individual test chambers or the group of test chambers constituting a replicate and the test chambers or replicates randomly assigned to locations.  When practically feasible, the locations should be randomly reassigned during the test.  Randomized block designs may be desirable for terrestrial plant tests, completely randomized designs may also be used.  For test results to be satisfactory, test organisms (for example, seeds or seedlings) should ideally be randomly assigned to the test containers; where this is not practical impartial assignment can be used.  (Note: random assignment as used here implies a mathematically-based unbiased assignment method and impartial assignment implies a non mathematically-based unbiased assignment procedure.)  All test chambers should be treated as similarly as possible to eliminate potential bias in test results.  The methods used to randomize treatments among test chambers and test chambers among locations should be recorded, as well as methods of impartial organism assignment to test chambers.
      (5) Number of replicates.  The number of replicate test chambers for a given treatment is dependent upon the objective of the specific guideline test.  Except for field tests which are designed on a case-by-case basis, the minimum number of replicates for a given test is described in each individual OCSPP Series 850 Group D guideline.  The number of replicates selected should yield results that are statistically significant at the 90 to 95% level of confidence with a significance level of less than 0.10.  The sample size and number of replicates for each tested plant species in the tiered test scheme should be sufficient to calculate the IC25 or IC50 (progression criteria for terrestrial and aquatic plants, respectively) and to detect differences of concern when treatments are compared to controls.  For terrestrial plant tests, a replicate or experimental unit will usually consist of more than one test chamber (pot or flat) because, for a satisfactory test, plants should not be crowded in the test chambers.  For a satisfactory test, the group of pots or flats constituting an experimental unit or replicate should be kept together throughout the test duration.  For tests with algae, cyanobacteria, and aquatic vascular plants, the test chamber constitutes the experimental unit or replicate.  All test chambers within a replicate and all replicates within a treatment should be treated as similarly as possible.  Except for field tests which are designed on a case-by-case basis, the minimum number of replicates for a given test described in each individual OCSPP Series 850 Group D guidelines.  
            (i) Regression-based test.  When the objective of the test is to determine a regression-based estimate and sample size constraints apply, the inclusion of additional concentrations rather than additional replicates results in better characterization of the overall concentration-response relationship.  For pesticides the objective of some OCSPP Group D guideline tests includes determination of both a regression-based point estimate (e.g., IC25) and a hypothesis-based endpoint (e.g., NOEC) in which case the minimum number of replicates will be determined by the hypothesis-based method.
            (ii) Hypothesis-based test.  For hypothesis-based tests, the determination of the test-specific number of replicates depends upon the objectives of the test, the statistical method(s) that may be used, the coefficient of variation, the size of effect to be detected, and the acceptable error rate.  (Note: several of the non-parameteric multiple-comparison tests recommended cannot be performed without at least a minimum of 4 replicates).  Individual testing facilities should consider variability observed in their laboratory and adjust the number of replicates upward where the minimum replication number identified in the test specific guideline is not sufficient to provide the statistical power to detect adverse effects to the test organisms or, if appropriate, identify and correct any environmental, handling, and culturing conditions, etc. that are resulting in the high variability.
      (6) Controls.  Control groups are used to ensure that effects observed are associated with or attributed only to the test substance exposure.  In phytotoxicity evaluations, all treated plots, plants, and commodities should be compared directly to untreated control plots, plants, and commodities in order for the study to be considered satisfactory.  The appropriate control group should be similar in every respect to the test group except for exposure to the test substance. Within a given study, all test organisms including the controls should be from the same source. To prevent bias, a system of random assignment of the test plants to test and control groups should be used for an acceptable test.  (In field trials, it is useful to have one set of replicates assigned, in adjacent plots, to a control and in order from lowest concentration to highest concentration for visual comparison, with the remaining replicates randomly assigned).  Where a carrier, vehicle, or adjuvant other than water is used, appropriate experiments and controls should be included to distinguish the possible action of the carrier, vehicle, or adjuvant.  Untreated control (check) plots should be treated and evaluated in the same manner as the treatment plots with respect to other pesticides or chemical (fertilizers, etc.) and cultural practices.  A vehicle control (solvent control) is also tested if a vehicle was used to prepare the test substance.  To demonstrate satisfactorily that the vehicle has no unacceptable effect, the highest concentration of the vehicle that was added to any of the test chambers is used in the vehicle control.  It is recommended that the vehicle concentration be the same at each treatment level.  If either the control or vehicle control results are not satisfactory for the test, indicating problems with test organisms or test procedures, the test results should be considered unacceptable.  If both the control and the vehicle control results verify test organism health and status, the control and vehicle control results are compared using an appropriate statistical method to determine if there is an effect of the vehicle on the test organisms.  If there is a statistically significant difference between the control and the vehicle control, indicating either a positive or negative vehicle effect for any of the measured response variables using an α-level of 0.05, the study may be considered unacceptable.
(e) Test substance characterization--
      (1) Background information on the test substance.  The information in paragraphs (e)(1)(i) through (e)(1)(vi) of this guideline should be known about the test substance prior to testing:
            (i) Chemical name; CAS number; molecular structure; source; lot or batch number; purity and/or percent a.i.; identities and concentrations of major ingredients and major impurities; radiolabeling if any, location of label(s), and radiopurity; date of most recent assay and expiration date for sample.
      	(ii) Appropriate storage and handling conditions for the test substance to protect the integrity of the test substance.  (Note: health and safety precautions should also be known.  These considerations are beyond the scope of these guidelines and depend upon the characteristics of the test substance).
      	(iii) Physical and chemical properties of the test substance, including solubility in water and various solvents; vapor pressure; hydrolysis at various pH; pKa; soil sorption behavior, etc.  Of particular relevance are rates for processes such as hydrolysis, photolysis, and volatilization.
      	(iv) Stability, and if relevant, solubility, under the test conditions (refer to OCSPP 850.1000 guideline).
      	(v) Physical and chemical properties and stability information for the analytical standard (if applicable).
      	(vi) Analytical method for quantification of the test substance in the test solutions, test matrix (support medium) or in the dosing solutions.  Analyses are conducted with the specific media which will be used during the test; i.e., under test conditions.
      (2) Preliminary analyses.  
            (i) The Agency recommends preliminary testing of the test substance.  The information about stability and solubility of the test substance should be developed under actual test conditions.  This information can be gained while doing the range-finding studies.
            (ii) Information on the behavior of a test substance should be based on experiments conducted under the same conditions as those occurring during the test.  These include but are not limited to:
                  (A) Test matrix characteristics (e.g., support medium, culture medium, soil, etc.).
			(B) Temperature, humidity, lighting, etc.
			(C) With test organisms in place (when practical).
			(D) Use of the same test containers.
            (iii) The tests in paragraphs (e)(2)(iii)(A) through (e)(2)(iii)(D) of this guideline should be performed:
			(A) Stability trials conducted under actual test conditions. 
			(B) If relevant, solubility trials conducted under test conditions. 
                  (C) Chemical analysis methods as detailed in paragraph (g) of this guideline.
                  (D) Determination of storage stability of the test substance in the samples to be collected for chemical analyses should be determined.  This includes determining whether and how samples can be stored for future analysis.
      (3) Sample storage.  If samples of the treatment solutions or other exposure matrices collected for chemical analysis cannot be analyzed immediately, they should be handled and stored appropriately to minimize loss of the test substance.  Loss could be caused by such processes as microbial degradation, hydrolysis, oxidation, photolysis, reduction, sorption, or volatilization.  Stability determination under storage conditions, whether it refers to storing the test substance before testing or storing samples awaiting analysis, is required by GLP regulation. Test substance stability under storage conditions should be documented.
      
      (4) Analytical test substance determinations.  
            (i) For terrestrial tests, solutions to be applied should be measured at test initiation for a satisfactory test.  See specific guidelines for any additional testing.
		(ii) For aquatic tests see the OCSPP 850.1000 guideline for guidance.
            (iii) For field tests, media and frequency of testing depends on the objective of the study and is determined on a case-by-case basis.
      (5) Application of test substance.  For a satisfactory study, application from equipment should be made from lowest concentration to highest concentration so as to minimize residue carryover.
      (6) Mode of action.  When they have been determined, the primary and secondary modes of action with respect to plant morphogenic and biochemical levels should be reported.
(f) Preparation of test substances.  
      (1) Procedures for test substance preparation for studies with algae, cyanobacteria, and aquatic vascular plants are addressed in the OCSPP 850.1000 guideline.  In terrestrial plant toxicity tests, exposure to the test substance is typically accomplished through application of the test substance on soil (or other suitable growth media matrix) or to the leaves and other above-ground portions of the plants.  Test substance may be added by weight or prepared in a concentrated stock solution.  For pesticides, the testing of typical end product (TEP) in plant tests generally obviates the need for addition of a vehicle since a formulation may already contain a vehicle (see paragraph (f)(3) of this guideline).
            (i) The preferred choice for preparation of a stock solution is to use reagent water (deionized, distilled or reverse osmosis water), providing test substance can be dissolved in water.  If the test substance cannot be dissolved in reagent water, vehicles are often used.  If a vehicle, i.e. a solvent, is absolutely necessary to dissolve the test substance, the amount used should not exceed the minimum volume necessary to dissolve the test substance.  If the test substance is a mixture, formulation or commercial product, none of the ingredients is considered a vehicle unless an extra amount is used in its preparation for testing.
            (ii) If a vehicle is used to prepare the test substance, a vehicle control is also included in the test, in addition to the untreated (negative) control.  The same batch of vehicle (solvent) control used to prepare the test treatments is used in the vehicle control.  For a valid test, the selected vehicle should not affect the test plants at the concentration used. A vehicle should not interfere with the metabolism (degradation) of the test substance, alter the chemical properties of the test substance, or produce physiological or toxic effects to plants.
      (2) All techniques used in stock solution preparation (shaking, stirring, sonication, heating, solvent, etc.) should be recorded.  The appearance of the stock solution should be observed and recorded.
      (3) Generally, for terrestrial plant phytotoxicity testing of pesticides, the TEP is the actual substance tested.  The submitter should consult with the Agency if any form of the test substance other than TEP is to be tested.  Aquatic plant studies may be conducted using the TEP or technical grade of the a.i.; however, if an overspray exposure is used for emergent or floating aquatic vascular plants, the test substance should be the TEP.  If an adjuvant is recommended on the product label, representative adjuvants should be included in the test at the recommended dosage for an acceptable test.  The TEP selected for testing should be the one with the highest percentage a.i. and/or the one most widely used.  The use of TEP testing should eliminate the need for a separate vehicle control as the vehicle is part of the formulation.  An untreated (negative) control is still included in the test.
(g) Analytical methods and sampling for verification of exposure--
      1) Method validation.  	
            (i) The analytical method used to measure the test substance should be validated before beginning the definitive test by appropriate laboratory practices.  An analytical method is not acceptable if likely degradation products of the test substance give positive or negative interferences which cannot be systematically identified and mathematically corrected, unless it is shown that such degradation products are not present in the test system during the test.
            (ii) Method validation is conducted for the purpose of determining the linear range, detection limit, accuracy and precision (repeatability and reproducibility) of the method for analysis of the test substance under the conditions of the test.  Thus, quality control (fortification) samples should be prepared at concentrations spanning the range of concentrations to be used in the definitive test, using the same procedures (vehicles, etc.) and in the same matrix (water, soil, etc.) representative of what will be used in the test.
            (iii) The method validation should include a determination of linearity between detector response and test substance concentration, the LOQ, the MDL, accuracy (average percent recovery) and precision (relative standard deviation).  The method validation should establish the acceptance criteria for the quality control (QC) samples that will be prepared and analyzed during the test.
      (2) Collection of samples.  Samples should be collected in such a manner as to provide an accurate representation of matrix being sampled.  For toxicity tests with algae, cyanobacteria and aquatic vascular plants, these will be aqueous samples and the OCSPP 850.1000 guideline should be consulted.  For terrestrial plant toxicity tests, the samples will generally consist of the dosing solutions, other dosing matrix, or the support medium. Analyses, if conducted, are performed to confirm the initial concentration of the test substance applied.  Samples should be processed and analyzed immediately, or handled and stored in a manner which minimizes loss of test substance through microbial degradation, photodegradation, chemical reaction, volatilization, sorption or other processes.
      (3) Analysis of test samples.  Concurrent with each analysis of test samples, quality control (fortified) samples should be analyzed.  Quality control samples are prepared by adding known amounts of the test substance to the test matrix.  Minimally, one quality control sample should be at the low end of the test concentration range and one quality control sample at the high end.  A control (zero-level fortification) sample should also be included.  Test sample recoveries may be corrected for inherent method bias as determined from concurrent analysis of freshly fortified quality control samples.
(h) Reference toxicants.  Historically, reference toxicity testing has been thought to provide three types of information relevant to the interpretation of toxicity test data: first, an indication of the relative "health" of the organisms used in the test; second, a demonstration that the laboratory can perform the test procedure in a reproducible manner over a period of time; and third, information to indicate whether the sensitivity of a particular strain or population in use at a laboratory is comparable to that of those used in other facilities and how sensitivity varies over time.  However, performance of control organisms over time may be a better indicator of success in handling and testing of at least some organisms.  Nonetheless, periodic reference toxicant testing can provide an indication of the overall comparability of results within and among laboratories.  Although a positive control is not standard for each test, a quarterly or semiannual positive control (on a guideline-specific basis) can serve as a means of detecting possible interlaboratory or temporal variation.  A reference toxicant might also be desirable when there is any significant change in source or maintenance of test organisms or in other test conditions.
(i) Monitoring of test conditions.  Test conditions are specified in each test-specific guideline in the OCSPP Series 850 Group D.  These conditions include environmental factors such as temperature, humidity, and lighting. Methods used for monitoring test conditions should be in accordance with established methods (e.g., those published by the U.S. EPA, ASTM, APHA et al., etc. (see paragraphs (k)(1), (k)(2), (k)(3) of this guideline).  Environmental conditions of specific field sites should be recorded daily throughout the duration of the test.
      (1) Temperature.  For greenhouse, growth chamber and laboratory toxicity tests or studies it is desirable that temperature be monitored continuously (recorded at least hourly).  Alternatively, the maximum and minimum should be measured daily (which is a minimum of at least two measurements during each 24 hour period during the study).  Temperature measurements should be made in at least one representative location.  For field experiments the maximum and minimum air temperature should be measured daily.
      (2) Humidity.  Where applicable, humidity should be monitored continuously in at least one representative location.
      (3) Lighting.  Guidance for lighting in greenhouse, growth chamber and laboratory toxicity tests or studies can be found in the references given in paragraphs (k)(3), (k)(5) and (k)(19) of this guideline.  The photoperiod (day length) should be recorded and a photosynthetically active radiation (PAR) sensor (measures light energy in the 400-700 nanometer wavelength range) should be used to measure light quality in micromoles of photons per square meter per second (umol/m[2]/s).  For field experiments, the approximate light quantity (usually expressed in degree of cloudiness) and photoperiod (day length) should be observed and recorded.  Frequency of light quality monitoring for a test is detailed in each individual OCSPP Series 850 Group D guideline.
	(4) Biological observations.  
            (i) Where test substances are applied in the field, the effects of that test substance on nontarget plants in the system and along the immediate border should be observed and recorded, including plant vigor, phytotoxicity or other visible symptoms, and delay or acceleration of vegetative growth, flowering or sporulation, and maturation.
            (ii)  Monitor all variations, either inhibitory or stimulatory, between the treated test organisms and the untreated control test organisms for toxicity tests conducted in greenhouses, growth chambers or laboratories.  Such variations may be due to phytotoxicity (chlorosis, necrosis, and wilting), formative effects (leaf and stem deformation), and/or effects on growth or development rates.
            (iii) Uniform scoring procedures should be used to evaluate the observable toxic responses.
            (iv) At least two methods of evaluation (such as quantitative and qualitative determinations) should be used in the evaluation of test substance effects on growth, reproduction, and yield of plants in greenhouse and controlled growth chamber experiments.  When direct measurements cannot be made, such as in large field evaluations, a 0-to-100 or 0-to-10 rating scale should be used, where 0 indicates no injury and 100 or 10 indicates a total effect or kill produced by the test substance.  An explanation of the steps of the rating scale employed should be included with the report.
(j) Reporting--
      (1) Background information.  In addition to the reporting requirements prescribed in the Good Laboratory Practices Standards (40 CFR part 792 and 40 CFR part 160), the report should include the information in paragraphs (j)(1)(i) through (j)(1)(v) of this guideline:
            (i) Test facility (name and location), study dates, and personnel.  If conducted outside of a laboratory or greenhouse, report the geographic location and describe the relation of this location to the occurrence or culture of the test species in the surrounding area.
      	(ii) The name of the sponsor, study director, principal investigator, names of other scientists or professionals, and the names of all supervisory personnel involved in the study.
      	(iii) Raw data sufficient to allow independent confirmation of the study authors' conclusions should be presented with the study report.  Raw data includes all measurements recorded during the study including, but not limited to, effects (mortality, growth, etc.), environmental conditions (temperature, etc.) and test substance concentration or dose measured as specified and are necessary for the reconstruction and evaluation of the report of that study.  The absence of raw data may make the study incomplete and impossible to review for scientific soundness and thus can lead to rejection of the study as scientifically sound.
      	(iv) The signed and dated reports of each of the individual scientists or other professionals involved in the study, including each person who, at the request or direction of the testing facility or sponsor, conducted an analysis or evaluation of data or specimens from the study after data generation was completed.
      	(v) The locations where all raw data and the final report are stored.
      	(vi) The statement prepared and signed by the quality assurance unit identifying whether or not the study was conducted in compliance with Good Laboratory Practices Standards (40 CFR part 792 or 40 CFR part 160). Alternatively the statement can indicate it was conducted under OECD Principles of Good Laboratory Practice, in accordance with the multilateral agreement with OECD member countries.
      (2) Data elements.  The test report should provide a complete and accurate description of test procedures and evaluation of test results including but not limited to the material in paragraphs (j)(2)(i) through (j)(2)(xv) of this guideline.
      	(i) Objectives and procedures stated in the approved protocol, including any changes or deviations or occurrences which may have influenced the results of the test.
      	(ii) Identification of the test substance (including source, lot, batch number, and purity), and known physical and chemical properties that are pertinent to the test.  Provide the physical state, water solubility (if relevant), pH, stability and degradation properties under test conditions and stability under storage conditions if stored prior to use or prior to sample analysis.  Where appropriate a cross-reference to OCSPP Series 830 (Product Properties Test Guidelines) guideline study results can be used to report this data.
      	(iii) Methods of preparation of the test substance and the concentrations or doses used in limit and definitive testing.  If vehicles and adjuvants (e.g. diluents, suspending agents, and emulsifiers) are used, the name, composition, and source of the vehicle and adjuvant, the nominal concentration of the test substance in the carrier, and the vehicle concentration(s) used in the test.
      	(iv) Information about the test organisms (common name, genus, species, family, and cultivar or variety), a statement providing the rationale for selection of the particular species used in the test, source, and life stage.
      	(v) A description of the test system used in definitive, limit or any preliminary testing this includes a description of substrate source, properties and characteristics; description of the experimental unit (test container, pots, flats, field plot, etc.), including number of test organism per test container and per experimental unit; number of replicates per treatment; controls; and method of test substance application including dosage rates or spray volume per unit area, application equipment (type, nozzle, orifice, pressure), time (season, stage of growth) and number of applications, tank mixture (if applicable), adjuvants (if applicable).
      	(vi) Results of measurements of test substance.  All analytical procedures should be described and documentation provided such that a bench chemist can independently determine what equipment to use and perform the analysis.  Also include the raw data, standards, quality control samples, and chromatograms from samples taken during either definitive or range-finding tests, not of standard or samples from recovery tests.  For a satisfactory test, the accuracy of the method, LOD, MDL, and LOQ should be given.
      	(vii) Exposure may be expressed as units of test substance per unit of land area treated (e.g for terrestrial plant studies,. lb a.i./A), units of concentration (e.g., milligrams per liter for aquatic plant studies), units per flow rate, or units of test substance per unit volume applied to obtain a specified degree of foliage coverage (such as to runoff).
      	(viii) For pesticides, test substance concentrations or dosages should be recorded in units of a.i. or the acid equivalent of the a.i. as appropriate.
      	(ix) If a product is applied more than once within a year or growing season, each rate and the interval between applications should be recorded. 
      	(x) Methods, frequency, and results of environmental monitoring performed during the study (air temperature, light quality, humidity, photo- and thermoperiods, etc.) and other records of test conditions such as description of watering schedules and any cultural practices during the test (if applicable).
      	(xi) Biological observations should be reported in sufficient detail to allow complete independent evaluation of the results (see specific test guidelines in this group for specific measures of effect).  For target area applications include a description of the stage of growth or development of nontarget plants within or adjacent to the target area.
      	(xii) The stage of plant development and study dates when adverse results occurred and subsided (if plants recovered) should be recorded.  Any lack of effects by the test substance should also be recorded.
      	(xiii) All data developed during the study that is suggestive or predictive of toxic effects and all concomitant gross toxicological manifestations.
      	(xiv) Calculated endpoints and a description of all statistical methods, including: software used, handling of outlier data points, handling of non-detect or zero values, tests to validate the assumptions of the analyses, level of significance, any data transformations, for hypothesis tests a measure of the sensitivity of the test (either the minimum significant difference or the percent change from the control that this minimum difference represents).  Raw data should be reported to allow independent verification of statistical procedures.
      	(xv) Methods used for test chamber and treatment randomization as well as methods for random or impartial assignment of test organisms to test chambers.
(k) References. The references in this paragraph should be consulted for additional background material on this test guideline.
	(1) American Public Health Association, American Water Works Association, Water Environment Federation, 1998.  Standard Methods for the Examination of Water and Wastewater, 20[th] edition. Part 8010, Toxicity: Introduction.
	(2) American Society for Testing and Materials, 2002.  ASTM E 1963-02.  Standard guide for conducting terrestrial plant toxicity tests.  In Annual Book of ASTM Standards, Vol. 11.06, ASTM, West Conshohocken, PA.  Current edition approved December 10, 2002.
	(3) American Society for Testing and Materials, 2002.  ASTM E 1733-95.  Standard guide for the use of lighting in laboratory testing.  In Annual Book of ASTM Standards, Vol. 11.06, ASTM, West Conshohocken, PA. Current edition approved September 10, 1995; Reapproved 2002.
	(4) American Society for Testing and Materials, 2003.  ASTM E 1847-96.  Standard practice for statistical analysis of toxicity tests conducted under ASTM guidelines.  In Annual Book of ASTM Standards, Vol. 11.05, ASTM, West Conshohocken, PA. Current edition approved December 10, 1996; Reapproved 2003.
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