
Office of Chemical Safety and Pollution Prevention (7101)
                                                               EPA 712-C-16-014
                                                                   October 2016


Ecological Effects Test Guidelines



OCSPP 850.1000

Background and Special Considerations-Tests with Aquatic and Sediment-Dwelling Fauna and Aquatic 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 Act (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 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.



OCSPP 850.1000: Background and special considerations - tests with aquatic and sediment-dwelling fauna and aquatic microcosms
(a) Scope.
      (1) Applicability. This guideline is intended for use in meeting testing requirements of the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) (7 U.S.C. 136, et seq.), the Toxic Substances Control Act (TSCA) (15 U.S.C. 2601, et seq.), and the Federal Food, Drug, and Cosmetic Act (FFDCA) (21 U.S.C. 346a). It describes test procedures that, if followed, in conjunction with an OCSPP Series 850, Group A test-specific guideline would result in data that would generally be of scientific merit for the purposes described in a test-specific Series 850, Group A test guideline.
      (2) Background. This guideline provides general information applicable to conducting OCSPP Series 850, Group A toxicity tests with aquatic species, including vertebrates and invertebrates. In addition, certain aspects of the guidance provided herein are relevant to Group D toxicity studies conducted with aquatic plants and microorganisms (OCSPP 850.4400, Aquatic plant toxicity test using Lemna spp.; OCSPP 850.4500, Algal toxicity; and OCSPP 850.4550, Cyanobacteria toxicity). The source materials used in developing this harmonized OCSPP guideline are OPP 70-1 General Information, OPP 70-2 Definitions, OPP 70-3 General Test Standards, OPP 70-4 Reporting and Evaluation of Data (Pesticide Assessment Guidelines Subdivision E--Hazard Evaluation: Wildlife and Aquatic Organisms; see paragraph (l)(38) of this guideline); OECD 23 Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (see paragraph (l)(20) of this guideline); the Pesticide Reregistration Rejection Rate Analysis Ecological Effects (see paragraph (l)(31) of this guideline); and the background materials in the OCSPP Series 850, Group A specific guidelines.
      (3) General. 
            (i) The OCSPP Series 850, Group A provides guidelines applicable to conducting laboratory toxicity tests with aquatic and sediment-dwelling vertebrates and invertebrates, studies of effects in aquatic microcosms or the field, and bioaccumulation in aquatic and sediment-dwelling organisms. Aquatic microcosm and field tests are designed on a case-by-case basis. The guidelines in the OCSPP Series 850, Group A are applicable to evaluating the hazards and risks of industrial chemicals and pesticides to various aquatic organisms exposed directly or indirectly. Data concerning the effects of pesticides on aquatic organisms and bioaccumulation in aquatic organisms are used in ecological risk assessment of pesticides (40 CFR 158, see paragraph (l)(38) of this guideline). These data are also of use in assessments of potential injury to endangered and threatened aquatic organisms listed by the Fish and Wildlife Service, Department of Interior and the National Marine Fisheries Service, National Oceanic and Atmospheric Administration, Department of Commerce and when toxicity concerns arise from incidents or during Special Review. Data concerning the effects and/or bioaccumulation of industrial chemicals on aquatic organisms may be used in ecological risk assessment of new and existing industrial chemicals. These data are used for both deterministic and probabilistic risk assessments.
            (ii) Information is provided on the design and conduct of tests with aquatic and sediment-dwelling fauna, emphasizing the importance of adequately characterizing the test substance, use of suitable experimental design, 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 the factors that can affect the test outcome and interpretation of test results. In addition, emphasis is placed on providing guidance on modifications or additional steps that need to be considered when conducting toxicity tests with test substances that have poor solubility or stability under test conditions. This general information is primarily applicable to the guidelines for laboratory toxicity tests since aquatic microcosm and field tests are designed on a case-by-case basis. However, the OCSPP 850.1000 guideline lists critical quality assurance and reporting standards common to all of the guidelines in OCSPP Series 850, Group A. 
            (iii) 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; the OCSPP 850.4500 guideline, Algal toxicity; and the OCSPP 850.4550 guideline, Cyanobacteria (Anabaena flos-aquae) toxicity).
            (iv) The OCSPP Series 850, Group A guidelines have generally been validated in formal round-robin tests or through repeated use.
            (v) 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 in each guideline in the OCSPP Series 850, Group A.
            (vi) The guidelines contained in OCSPP Series 850, Group A recommend specific procedures to be used in almost all circumstances to result in a satisfactory test result, but also provide general guidance that allows for some latitude, based upon test-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 and taking into consideration the chemistry and environmental fate of the test substance so that following this guidance will provide ecological effect information that is scientifically defensible for its intended use. 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 158.243 and 40 CFR 158.630 for nontarget aquatic organisms provides for greater efficiency of testing resources while assuring data development as warranted to meet the objectives of a hazard and risk assessment. Test requirements in the first stage depend upon the type of registration (e.g., experimental use permit, manufacturing use product, end-use product). Within a registration type, specific test requirements further depend upon the pesticide use pattern and potential exposure pattern to freshwater or saltwater aquatic and sediment-dwelling fauna. Within these Group A guidelines, a limit test, which tests a single concentration and compares effects observed with appropriate control(s), provides for screening of and use of fewer test animals for low toxicity test substances or screening of test substances that are not toxic at solubility limits. In these Group A guidelines, higher tier testing for pesticides (multiple-concentration definitive test) provides for generation of concentration-response relationships for test substances that are known toxicants or that demonstrated toxicity in limit testing.
            (xiii) Data on toxicity to aquatic and sediment-dwelling fauna and effects in aquatic microcosms may also be used to evaluate the potential hazard and risk of industrial chemicals. When the pattern of production, use, or disposal indicates exposure to aquatic organisms, these tests are recommended. This testing is part of the OPPT tiered testing scheme developed for determining environmental effects (see the references in paragraphs (l)(16), (l)(17), (l)(26), (l)(27), (l)(40), and (l)(41) 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.
            
            (xiv) For industrial chemicals, concentration-response testing occurs with an aquatic vertebrate and an aquatic invertebrate at Tier I. (See 850.4000 for a discussion of aquatic plant testing requirements). This is because OPPT is interested in determining the potential toxicity of these chemicals to aquatic organisms. In contrast to pesticides, the toxicity of industrial chemicals to aquatic organisms is generally uncharacterized. Thus, range-finding tests, a preliminary step to define concentration-response testing, are more commonly conducted than limit or maximum challenge tests that use only one test concentration. If toxicity is noted at Tier I, additional testing with more test species may be conducted at Tier II and beyond to better characterize the toxicity to sensitive groups or under specific exposure scenarios. Chronic tests may be used to investigate potential chronic cumulative toxicity and bioconcentration potential of industrial chemicals. For example, microcosm tests (850.1900, 850.1925) and field testing may also be conducted, if necessary. Sediment acute (850.1735, 850.1740) and chronic toxicity testing  may also be conducted.
            (xv) While performing field tests, all necessary measures should be taken to ensure that nontarget organisms, 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 A 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 160 (Good Laboratory Practice Standards); and in TSCA Section 3 regulations 40 CFR 792 (Good Laboratory Practice Standards); and the Agency's "Terms of Environment, Glossary, Abbreviations and Acronyms" (see paragraph (1)(33) of this guideline). The definitions in this section apply to the OCSPP Series 850, Group A test guidelines and 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 (Anabaena flos-aquae) toxicity). Where applicable, the individual test guidelines contain additional or test-specific definitions.
      Acclimation is the physiological or behavioral adaptation by test organisms to one or more new environmental conditions (e.g., temperature, hardness, pH).
      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 CFR 152.3).
      Acute toxicity is the discernible adverse effects (lethal or sublethal) induced in an organism within a short exposure period (usually not constituting a substantial portion of the total life cycle or life span, e.g., days).
      Acute toxicity test is a comparative test in which organisms are subjected to a severe, short-term stimulus (test substance). The organisms, exposed to different concentrations of the test substance (except in a limit test), are observed for a short period usually not constituting a substantial portion of the total life cycle or life span. Acute exposure typically includes a lethal biological response of relatively quick progression. 
      Adjuvant is a subsidiary ingredient or additive in a mixture that 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 include 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 include those plants that are totally aquatic (free-floating or attached, submersed and immersed) and may inhabit still or flowing water bodies.
      Axenic is a culture of one organism free from other organisms. 
      Brood stock refers to the animals that are cultured or maintained to produce, through reproduction, young test organisms (e.g., gametes, larvae, nauplii, juveniles).
      Carrier is any material including, but not limited to, feed, water, soil, and nutrient media, with which the test substance is combined for administration to a test system (40 CFR 160).
      Chemical oxygen demand (COD) is the amount of oxygen required under specified test conditions for the oxidation of water borne organic and inorganic matter.
      Chronic toxicity test is a comparative test in which organisms are exposed to different concentrations of the test substance generally for a relatively long period that constitutes a substantial, nearly complete, or complete portion of the total life cycle or life span. Chronic exposure typically induces a sublethal biological response of relatively slow progression, or which is cumulative in nature. For some chemicals with certain modes-of-action, shorter-term exposure may result in chronic or latent effects, and continued or cumulative exposure is therefore not necessary.
      Concentration-response curve is the graphical and mathematical relationship between the concentration of a test substance and a specific biological response produced from toxicity tests when response (e.g., proportion or percent mortality) 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.
      Conditioning refers to the exposure of construction materials, test vessels, and testing apparatus to dilution water or test solution prior to the start of a test in order to minimize the sorption of the test substance onto the test apparatus or the leaching of substances from the test apparatus into the dilution water or test solution.
      Control refers to test organisms exposed to test conditions and test matrix (water, sediment, growth 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 that 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.
      Ecosystem refers to a community of organisms and its interrelated physical and chemical environment functioning as a unit.
      Effect concentration (ECx) is the experimentally derived concentration of a test substance in a test matrix (e.g., water, sediment, growth medium) that would be expected to cause a specified effect in x percent (%) 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, sediment, growth medium) that would be expected to cause a specified effect (e.g., mortality, immobility) 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.
      Flow-through refers to a continuous or very frequent intermittent passage of test solution or dilution water through a test vessel or holding, acclimation, or culture tank with no recycling of water.
      Generic microcosm refers to a general representation of an aquatic ecosystem in which a microcosm is maintained under constant laboratory conditions, and no attempt is made to simulate the physical/chemical environment of the natural system.
      Holding refers to the period from the time test organisms are received in the laboratory until they are used in testing or begin acclimation to test conditions. Holding conditions may include quarantine, lower temperatures to minimize disease, different dilution water, or other conditions that are different from test conditions. Where holding conditions are different from test conditions, the test organisms should be acclimated to test conditions prior to testing so as not to stress the organisms.
      Inert ingredient is any substance (or group of structurally similar substances if designated by the Agency), other than an active ingredient, that 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, sediment, growth medium) that would be expected to cause an x% inhibition or reduction in a non-quantal response from the smoothed mean control response in a group of test organisms under specified exposure conditions.
      Inhibition concentration (IC50) is the experimentally derived concentration of a test substance in a test matrix (e.g., water, sediment, growth medium) that would be expected to cause a 50% inhibition or reduction in a non-quantal response from the smoothed mean control response in a group of test organisms under specified exposure conditions.
      Immobilization refers to the lack of movement by the test organism and serves as a surrogate measure of death for organisms in which death is difficult to ascertain without prolonged handling of the organisms. Organism-specific operational definitions of immobilization (e.g., lack of response to gentle prodding) are provided in organism-specific guidelines.
      Lethal concentration (LCx) is the experimentally derived concentration of a test substance in a test matrix (e.g., water, sediment, growth medium) that would be expected to cause death in x% of a group of test organisms under specified exposure conditions. 
      Lethal concentration, median (LC50) is the experimentally derived concentration of a test substance in a test matrix (e.g., water, sediment, growth medium) that would be expected to kill 50% of a group of test organisms under specified exposure conditions. 
      Limit of detection (LOD) is the analytic level below which the qualitative presence of the material is uncertain. This is typically defined operationally by the lowest concentration producing a signal two standard deviations above the background noise from a matrix blank sample.
      Limit of quantification (LOQ) is the analytic level below which the quantitative amount of the material is uncertain. This is typically defined operationally by the lowest concentration of fortified matrix successfully analyzed.
      Limit test is a toxicity test performed with a single test substance concentration and control(s) to establish that the value for the measurement endpoint of concern (e.g., LC50) is greater than the test substance concentration (limit concentration).
      Loading refers to the ratio of organism biomass (grams, wet weight) to the volume (liters) of test solution in a test vessel at a point in time or passing through the test vessel during a specific interval. For small organisms, loading may be expressed by basing it on the number of organisms rather than the weight. 
      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.
      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 quantitative measure of a test substance that lies 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, LC50 for mortality; NOEC for number of young produced; NOEC for organism dry weight; etc. Each test-specific guideline identifies the measurement endpoint(s) to be determined by the prescribed test. 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.
      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). 
      Microcosm means a miniaturized model of a natural ecosystem.
      Nominal concentration is, for aquatic tests, the calculated concentration of test substance that would exist if all test substance added to the test solution was completely dissolved and did not dissipate in any way.
      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). Statistically, the NOEC is the test concentration immediately below the LOEC. Throughout these guidelines, the terms NOEC and no observed adverse effect concentration (NOAEC) have the same meaning. 
      Raw data are any laboratory worksheets, records, memoranda, notes, or exact copies thereof, that are the result of original observations and activities of a study and are necessary for the reconstruction and evaluation of the report of that study. In the event that exact transcripts of raw data have been prepared, the exact copy or exact transcript may be substituted for the original source as raw data (40 CFR 160).
      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. For example, in a flow-through test where test solutions can flow from one retention chamber to another within a test vessel but not among test vessels, the test vessel is the experimental unit. Retention chambers are not independent from one another; they are in the same test vessel, and the same portion of test solution flows among them. Test vessels, on the other hand, can be independently randomized and exposed to test solutions.
      Retention chamber is a structure within a flow-through test vessel that confines the test organisms, facilitating observation of test organisms and eliminating loss of organisms in outflow water.
      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 (filtration of test medium or water may also be appropriate). 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. 
      Solvent refers to a substance (e.g., acetone) that is combined with the test substance to facilitate introduction of the test substance into the dilution water. A solvent is a type of vehicle.
      Static-renewal system is a static system in which test organisms are either periodically transferred to fresh solutions of the same composition, or the test solution is renewed at specified intervals during the test.
      Static system is a test system in which no flow of test solution occurs and in which the test solution is not renewed during the test.
      Stock solution is a concentrated solution of the test substance that is dissolved and introduced into the dilution water or test medium.
      Test vessel is the smallest physical container in which the test organisms are maintained during the test period but between which there are no water connections (i.e., test solution cannot flow from one vessel to another).
      Test substance is the specific form of a chemical substance or mixture being evaluated (e.g., pesticide active ingredient or formulation, industrial chemical). 
      Test solution is the test substance and the dilution water or growth medium in which the test substance is dissolved or suspended.
      Total organic carbon (TOC) is the sum of all dissolved, particulate, and suspended organic carbon molecules occurring in a given environmental medium (e.g., water, sediment, soil).
      Total organophosphorus pesticides is the sum of the concentrations of the organophosphorus pesticides chlorpyrifos, demeton, diazinon, disulfoton, fenitrothion, malathion, methyl parathion, and parathion.
      Total organochlorine pesticides is the sum of the concentrations of the organochlorine pesticides aldrin, chlordane, DDD, DDE, DDT, dieldrin, endosulfan, endrin, heptachlor, heptachlor epoxide, lindane, methoxychlor, mirex, and toxaphene. 
      Treatment group is 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.
      Vehicle is any agent that facilitates the mixture, dispersion, or solubilization of a test substance with a test matrix or carrier (e.g., water, sediment, growth medium) used to expose the test organisms (40 CFR 160.3, 40 CFR 792.3). As used within the OCSPP Series 850, Group A guidelines, vehicle does not refer to the inert liquid or solid ingredients in a pesticide product or an adjuvant. 
(c) Apparatuses, 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. Housing and maintenance conditions should be in accordance with acceptable animal husbandry practices (e.g., United States Department of Agriculture Animal Care Regulations (see paragraph (l)(29) of this guideline)). In general, the laboratory toxicity tests require normal laboratory glassware, supplies, and equipment as well as equipment for holding organisms and controlling temperature and lighting and equipment for determination of water quality parameters (e.g., hardness, pH, dissolved oxygen, salinity, etc.). In the laboratory, temperature control is usually accomplished by keeping the test vessels in a constant temperature room or water bath. Equipment that contacts stock solutions, test solutions, or any water into which test organisms will be placed should not contain substances that can be leached or dissolved into aqueous solutions in quantities that can affect the test results. Construction materials and equipment that contact stock and test solutions or sediment should be chosen to minimize sorption of test substances. Glass, number 316 stainless steel, nylon screen, and perfluorocarbon plastic (e.g., Teflon) are acceptable construction materials and should be used whenever possible, except that stainless steel should not be used in tests in saltwater, especially on metals. For substances with high adsorption coefficients, such as the synthetic pyrethroids, silanized glass may be required. In these situations, the equipment will likely have to be discarded after use. Concrete, fiberglass, or rigid plastic (e.g., PVC) may be used for holding tanks, acclimation tanks, and water supply systems, but they should be thoroughly conditioned (soaked, preferably in flowing dilution water, for a week or more) before use. Rubber, copper, brass, galvanized metal, epoxy glues, lead, and flexible tubing should not come in contact with the dilution water, stock solutions, and test solutions or sediment. Test vessels made of stainless steel should be welded, not soldered. Test vessels made of glass should be fused or bonded using clear silicone adhesive. As little adhesive as possible should be left exposed in the interior of the vessel. See specific OCSPP Series 850, Group A guidelines for identification of any atypical facility, equipment, or supplies used in the test. 
     (2) Cleaning and conditioning. All materials that will come in contact with the test organisms, dilution water, test solutions or sediments, and stock solutions should be cleaned before use. Cleaning procedures should be appropriate to remove known or suspected contaminants. For guidance, see paragraph (l)(2) of this guideline. Test containers should be conditioned, if needed, by rinsing with the appropriate test solution prior to the start of the test. After an appropriate conditioning period for the test substance, the test solution used for conditioning should be decanted, and fresh test solution should be added.
     (3) Dilution water. In toxicity tests with aquatic organisms, dilution water refers to the water that is used to prepare the various test solution concentrations. Dilution water also serves as a control in water only tests and as overlying water in spiked-sediment tests. Although it has the same purpose, "dilution water" in toxicity tests with aquatic plants and in aquatic microcosms is more properly referred to as growth medium, since it typically has various nutrients added to support plant growth. However, in other sections of OCSPP 850.1000, references to dilution water are inclusive of references to growth medium. Specifications for preparation of aquatic microcosm and aquatic plant growth media are test-specific and are not discussed in OCSPP 850.1000.
            (i) Source. For freshwater organisms, clean surface water, ground water, or synthetic/reconstituted water should be used as dilution water. Synthetic/reconstituted dilution water is prepared by adding reagent-grade chemicals to reagent water (deionized, distilled, or reverse osmosis water). For saltwater organisms, clean natural or artificial seawater should be used as dilution water. Natural seawater should be from uncontaminated surface-water upstream of known discharges. Artificial seawater is prepared by adding commercially available formulations or specific amounts of reagent-grade chemicals to reagent water, surface water, or ground water. If necessary, artificial salts may be added to natural seawater prior to use in test vessels during periods of low salinity. Refer to the individual test-specific OCSPP Series 850, Group A (also applies to some OCSPP Group D) guidelines for any recommended pretreatment (filtration, sterilization, etc.) and desirable water quality characteristics (hardness, pH, salinity, etc.).
            Dilution water is acceptable if the test species will survive in it for the duration of the holding, acclimation, and testing periods without showing any abnormal appearance or behavior. Ideally, it should be demonstrated that the test species can survive, grow, and reproduce in the dilution water (e.g., in laboratory culture or a life-cycle toxicity test). However, for some test species, it is recognized that demonstrating reproduction in the test facility may not be possible due to necessary culturing practices for that species (e.g., bluegill, salmonids). Dilution water should be aerated prior to use to bring the pH and concentrations of dissolved oxygen and other gases into equilibrium with air and to ensure sufficient dissolved oxygen during the test. 
                  (A) Reagent water. The reagent water used to prepare dilution water should meet the specifications in Table 1 of this guideline, in accordance with the specifications given for Type II reagent water in ASTM D-1193 (see paragraph (l)(5) of this guideline). Conductivity of the reagent water should be determined for each batch of reconstituted water prepared, whereas the other constituents should be checked several times each year or more often if there is a documented or suspected change in the water source (see paragraph (c)(3)(ii) of this guideline).
	Table 1.--Specifications for Reagent Water Used to Prepare Dilution Water
Conductivity at 25 degrees Celsius (°C)
Less than (<) 1 microsiemens per centimeter (uS/cm)
Resistivity at 25 °C
Greater than (>) 1 megaohm-centimeter (MΩ-cm)
Total organic carbon (TOC)
<50 micrograms per liter (ug/L)
Sodium
<5 ug/L
Chlorides
<5 ug/L
Total silica
<3 ug/L
                  (B) Synthetic or reconstituted water. For the preparation of synthetic/reconstituted water (both freshwater and saltwater), see the references in paragraphs (l)(1), (l)(2), (l)(35), (l)(36), and (l)(37) of this guideline. Historically, a type of reconstituted water that has been used successfully in 10-day round-robin testing with H. azteca and C. dilutus is described in paragraph (l)(25) of this guideline. It is noted, however, that modifications to dilution water with respect to Cl and Br concentrations have been recommended to improve performance for 10-d and chronic (life cycle) testing with H. azteca (see guideline OCSPP 850.1735).
                  (C) Chlorinated water. Chlorinated water should not be used as, or in the preparation of, dilution water because residual chlorine and chlorine by-products are toxic, and this can bias results. 
                  (D) Dechlorinated water. Dechlorinated water is not recommended for preparation of dilution water and should only be used as a last resort because some forms of chlorine are difficult to remove adequately. If dechlorinated tap water is used, it should be demonstrated that test organism survival, growth, and reproduction are not affected and that test organisms do not show other signs of stress. Additionally, chlorine analysis should be performed on each batch of dilution water. 
                  (E) Facultative pathogens. If the source water is contaminated with facultative pathogens, it should be UV-irradiated using intensity meters and flow controls or filtered through 0.45 micron (um) pore size filters.
            (ii) Characterization of dilution water. Dilution water should meet the specifications listed in Table 2 of this guideline. Other parameters that may be suspected to influence the health of test organisms (e.g., metals) should not exceed National Ambient Water Quality chronic criteria values where available. The dilution water should be periodically analyzed to ensure that pesticides, PCBs, and toxic metals are not present at concentrations that are considered toxic. Analyses for parameters listed in Table 2 of this guideline or other parameters that may be suspected to influence the health of the organisms should be conducted at a frequency dependent upon the constancy of the water source. A minimum frequency of once or twice a year is recommended for sources with well-established low variability. The analysis frequency should increase if it is suspected that the characteristics of the water source may have changed significantly, or when substantial changes are expected (e.g., seasonal). For new sources or most surface waters, analyses will typically be needed more frequently to characterize magnitude and variability than for established sources or well waters. For dilution water prepared from surface water, conductivity and total organic carbon (TOC) or chemical oxygen demand (COD) should be measured on each batch.
                  (A) Constancy specifications for freshwater. For hardness, alkalinity, conductivity, TOC, COD, and pH, freshwater used as dilution water is considered of constant quality if the monthly ranges of hardness, alkalinity, conductivity, and TOC or COD are less than 10% of the average values, and the monthly range of pH is less than 0.4 units (see paragraph (l)(1) of this guideline). 
                  (B) Total suspended solids (TSS) and TOC maximum concentrations. TSS as well as TOC of the dilution water should be as low as possible to avoid adsorption of the test substance to organic matter which may reduce its bioavailability. The maximum recommended values are 5 milligrams per liter (mg/L) for TSS (i.e., particulate matter that is retained on a glass fiber filter and dried to a constant weight; see paragraph (l)(6) of this guideline) and 2 mg/L for TOC. 
                  (C) Constancy specifications for seawater. In terms of salinity, natural seawater used as dilution water is considered to be of constant quality if the weekly range of the salinity is less than 10 parts per thousand (ppt), and on any given day, the range of salinity is not more than plus or minus (+-) 2 ppt, Natural seawater should have a monthly range in pH of less than 0.8 pH units, and artificial seawater pH should not vary more than 0.5 pH units over a month.
             Table 2.--Specifications for Dilution Water Quality
Parameter
Maximum Value or Range
Freshwater hardness as calcium carbonate (CaCO3) for testing organic chemicals
250 milligrams per liter (mg/L), 
preferably <180 mg/L
Freshwater hardness as calcium carbonate (CaCO3) for testing metals and metal compounds
40  -  50 mg/L
TSS
20 mg/L
COD
5 mg/L
TOC
2 mg/L
Freshwater pH
6.0  -  8.5 
Natural or artificial seawater pH
7.5  -  8.5 
Total residual chlorine
0.01 mg/L
Un-ionized ammonia
0.02 mg/L
Total organophosphorus pesticides
50 nanograms per liter (ng/L)
Total organochlorine pesticides plus PCBs or organic chlorine
50 ng/L
      (4) Sediment. Clean natural sediment or formulated sediment should be used in spiked sediment testing. For industrial chemicals, the use of natural sediment or formulated sediment depends on the objective of the test and is determined on a case-by-case basis. In general, formulated sediment is recommended for routine testing with pesticides. Except as modified in a specific guideline, a formulated sediment that is recommended for use in sediment guideline tests in the OCSPP Series 850, Group A is described in paragraph (c)(4)(i) in this guideline. If natural sediment is used, it should be fully characterized and consistent with characteristics of the formulated sediment.
      The characteristics of sediment used in a test should be determined. At a minimum, the following factors should be measured for a batch of sediment: pH and ammonia concentration of pore water (natural sediment), TOC, particle size distribution (percent sand, silt, clay), and percent water content. In addition, for natural sediment, background metals, synthetic organic compounds, oil and grease, and petroleum hydrocarbons should be determined in a sediment batch prior to testing to ensure that effects noted in the test sediments are a result of the test substance and not from extraneous sources. For formulated sediment, the lack of contaminants in the constituents composing the sediment should be documented by reporting the analytic grade of the constituent used or by providing analytical results for potential metal and synthetic organic compounds. Additional suggested analyses include biological oxygen demand (BOD), COD, cation exchange capacity, redox potential (Eh), total inorganic carbon, total volatile solids, and acid volatile sulfides. Sediment characterization should also include qualitative parameters such as color, texture, and the presence and types of macrophytes or animals. Ammonia and pH should be determined for interstitial water. 
      Interferences in sediment are defined as those characteristics of sediment or the sediment test system that are unrelated to the test substance but have the potential to affect the survival and growth of test organisms. These factors include, but are not limited to, altered bioavailability (e.g., due to sediment manipulation, storage, preparation, etc.), competition with or injury from indigenous species present in natural sediment, sediment avoidance, lighting, and geomorphological and physico-chemical characteristics of the sediment (e.g., texture, grain size, and organic carbon content). The spiked sediment test procedures and selected test species were designed to minimize impacts due to interferences and are suitable for providing direct measures of test substance effects on benthic organisms. Natural geomorphological and physico-chemical characteristics of the sediment should be within the tolerance limits of the test species. The handling, storage, and preparation of test sediment should be as consistent as possible to avoid disruption of the equilibrium of organic carbon and the pore water-particle system resulting in the increased availability of organic compounds. While food addition is necessary in most cases, the quantity and composition of food added should be carefully considered because adjuncts such as food, water, or solvents may alter bioavailability and promote the growth of microorganisms (e.g., algae, fungi, and bacteria). Laboratory sediment testing with test substances that are photoinduced by ultraviolent (UV) light may necessitate the use of specialized lighting because typical laboratory lighting (i.e., fluorescent) does not emit UV radiation.
            (i) Formulated sediment. Formulated sediment consists of a mixture of materials designed to mimic natural sediment. The use of formulated sediment eliminates or controls the variation in sediment physico-chemical characteristics and provides a consistent method for evaluating the fate and effects of chemicals in sediments. Detailed information on formulating sediment can be found in paragraphs (j)(2) and (j)(12) of this guideline. The formulated sediment should meet the specifications in Table 3 of this guideline. 
               Table 3.--Specifications for Formulated Sediment
Content
Material
5% (dry weight)
Dried (air) sphagnum moss peat (powder form, finely ground to 1 millimeter (mm)); alpha cellulose can be used as an alternative
20% (dry weight)
Kaolin clay (kaolinite content preferably >30%)
75% (dry weight)
Industrial sand (fine sand should predominate with >50% of the particles between 50 and 200 micrometers (um))
30  -  50% (wet weight)
Deionized water
As needed
Calcium carbonate (CaCO3) of chemically pure quality should be added to adjust the pH of the final mixture of the sediment to 7.0 +- 0.5
2(+- 0.5)%
Organic carbon content of the final mixture, adjusted by the use of appropriate amounts of peat and sand

             (ii) Natural sediment. Procedures for handling natural sediment should be established prior to collection. Pertinent data such as location, time, core depth, water depth, and collection equipment should be recorded. While some disruption of the sediment is inevitable regardless of the sampling equipment used, disruption of sediment should be kept to a minimum. Several devices are available for collecting sediment, but benthic grab or core samplers are recommended. The depth of sediment collected should reflect the expected exposure of the test organism (e.g., burial depth). During sediment collection, exposure to direct sunlight should be kept to a minimum. Cooling of sediment to 4 °C is recommended. Sediments collected from multiple locations or sites should be pooled and mixed using a suitable apparatus (e.g., stirring, rolling mill, feed mixer).
            Storage of sediment may affect bioavailability and toxicity. Long storage may result in changes of sediment properties. For spiked sediment tests, holding clean sediments at 4 °C in the dark, and testing within 8 weeks after collection is recommended. Water that may have settled above sediment during shipment should be mixed back into the sediment before storage (or whenever observed during storage). 
            Indigenous species existing in field-collected sediments could negatively affect the growth rate or survival of test organisms. Because oligochaetes may inhibit the growth of test organisms, removing them as well as other macroorganisms, rocks, wood, and the like by sieving may be advantageous. If sieving is used (before and/or during storage), samples of the sediment before and after sieving should be chemically characterized (see paragraphs (j)(1), (j)(2) and (j)(12) of this guideline) to document the influence of sieving on sediment characteristics. Any manipulation or treatment of the sediment prior to testing and its impact on sediment characteristics should be reported.
(d) Experimental design and data analysis.
      (1) Design elements. Elements of experimental design such as the number of treatment concentrations, progression factor between treatment concentrations, number of replicates, and number of organisms per replicate and 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 laboratory ecotoxicity tests can be found in paragraphs (l)(1), (l)(3), (l)(19), (l)(21), (l)(23), (l)(35), (l)(36), and (l)(37) of this guideline. Test design and statistical analysis of aquatic microcosm and field tests are generally developed on a case-by-case basis. 
      (2) Calculation of endpoints.
            (i) Background. 
                  (A) Data generated in toxicity tests with aquatic and sediment-dwelling fauna may be of three types: 
                        (1) 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;
                        (2) 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 young produced); or 
                        (3) Continuous, where the variable can assume a continuum of possible outcomes (e.g., height and weight). 
                  (B) These data may be analyzed using regression-based techniques or hypothesis-testing procedures depending on the objectives and endpoints of a specific test guideline. Traditionally, the results of acute toxicity tests have been expressed as point estimates (e.g., LC50 for lethality, or EC50 or IC50 for other effects), whereas the results of chronic toxicity tests have been expressed as the results of hypothesis-testing procedures to determine the NOEC and LOEC. Regarding terminology, the term ICx is more appropriately used for continuous endpoints, rather than ECx. For information on the advantages and disadvantages of these approaches, see paragraphs (l)(8), (l)(11), (l)(23), (l)(24), and (l)(28) of this guideline. Specific test guideline objectives, either point estimates or hypothesis-based endpoints or both, are identified in each specific test guideline.
            (ii) Point estimate and concentration-response tests. This type of toxicity test is designed to allow calculation of a concentration-response curve (mathematical model) and to estimate one or more specific points (point estimates) on the curve, such as an LC50. 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 use (see paragraphs (l)(1), (l)(7), (l)(9), (l)(10), (l)(12), (l)(13), (l)(15), (l)(18), (l)(28), and (l)(39) of this guideline). Not all methods suitable for continuous data are appropriate for quantal data (see paragraphs (l)(7), and (l)(18) of this guideline). For point estimate tests, regression-based methods (e.g., probit) that model the full concentration-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., LC50), 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., binomial, moving average, trimmed Spearman-Karber, linear interpolation (e.g., Bootstrap ICp)) are available (see paragraphs (l)(34), (l)(36) and (l)(37) of this guideline). Which of these other methods is selected is dependent upon the shape of the concentration-response curve, the number of treatments with partial mortalities (i.e., where mortality is greater than 0% but less than 100%), the magnitude of these mortalities, and the number of replicates. The method used to estimate the endpoint and, if applicable, the 95% confidence interval for the point estimate should be recorded. 
                  (B) Concentration-response 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 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 are calculated by determining statistically non-significant and 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(s) in an adverse biological effect relative to the control. Parametric and nonparametric analysis of variance (ANOVA) tests and multiple-comparison tests are often appropriate for continuous data and for count 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)(iv)(A) of this guideline), or non-parametric 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 that assume a concentration-response relationship in the data. When the assumption of a monotonic concentration-response holds, Williams' and Jonckheere's tests are examples of parametric and nonparametric tests, respectively, that can be used. When the assumption of a monotonic concentration-response fails, Dunnett's t-test and either Steel's many-one rank test or the Wilcoxon rank sum test with Bonferroni adjustment are examples of parametric and nonparametric multiple-comparison tests, respectively, requiring no assumption about the concentration-response but which restrict comparisons of treatments to the 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 4 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 4. For multiple-comparison tests, the Type I error rate is controlled to account for multiple test comparisons.
Table 4.--Types of Errors and the Probabilities of Making Correct and Incorrect Decisions Based on the Results of Testing
Test Decision Outcome:
Actual (or True) Condition:

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. Information regarding power of the test versus percent reduction in treatment response relative to the control mean at various coefficients of variation is provided in paragraph (l)(34) 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 paragraph (l)(34) of this guideline for various low α and beta (β) values (i.e., α + β = 0.25). See also paragraphs (l)(12) and (l)(28) of this guideline.
                  (D) Limit test. In a limit test, it is only necessary to ascertain that a fixed point standard (e.g., LC50) is greater than a given "limit" concentration and/or 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 standard. For a fixed point limit test, the null hypothesis is that the mean "limit" concentration response as compared to the control response is greater than or equal to the fixed point response of concern (e.g., 50% mortality). The alternative hypothesis is that the mean "limit" concentration response as compared to the control response is less than the fixed point response of concern (where response is inhibition relative to the control, switch hypotheses around). Examples of statistical approaches for analyzing fixed point limit tests are the one sample binomial test or one sample t-test.
                        (2) Difference between two means (or medians). For testing if the treatment concentration affects the test organism, the null hypothesis is that the mean (or median) treatment response is equal to the mean (or median) control response, and the alternative hypothesis is that the mean (or median) treatment response differs from the mean (or median) control response. The direction of the alternative hypothesis depends on what is considered an adverse direction for the specific response being evaluated (e.g., decreased survival and weight or increased number deformed) as compared to the control response. Examples of parametric and nonparametric two-group comparison tests are Student's t-test and Wilcoxon signed ranks test, respectively.
            (iv) Transformations, outliers, and non-detects.
                  (A) Transformations. Transformation 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 that are not satisfied by the original data 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 paragraph (l)(28) 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) (e.g., 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 paragraph (l)(28) of this guideline). However, the analyst may conduct all statistical analyses of the data with both a full and truncated (i.e., presumed outliers are discarded) data set so that the effect of the presumed outlier(s) on the conclusion may be assessed.
                  (C) Non-detects. Data generated from chemical analysis that fall below the LOD of the analytical procedure are generally described as not detected or non-detects 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 paragraph (l)(28) of this guideline). However, generally test substance concentrations should not be below the LOD, except in the control(s).
      (3) Selection of test concentrations. The highest test concentration should not exceed the test substance's aqueous solubility if the test substance is not a surfactant, or the test substance's self-dispersibility limit if the test substance is a surfactant or charged polymer. Exceptions may be required in testing certain pesticide active ingredients as products. Product formulations may increase the solubility of the active ingredient beyond its aqueous solubility limit. Also, preparing a test solution at a concentration above the test substance's reported aqueous solubility limit may be necessary to obtain a measured concentration at the aqueous solubility limit.
            (i) Point estimate and concentration-response test. Toxicity tests where the objective is a concentration-response curve (mathematical model) and specific point(s) on the curve (e.g., LC50, EC50, IC50, IC05) usually consist of a control and at least 5 test concentrations which should bracket the specific point(s) of concern for the test. For example, to obtain a reasonably precise estimate of the LC50 or EC50 using probit analysis, one or more test concentrations should be between, but not include, 0 and 50% of the test organisms, and one or more test concentrations should be between, but not include, 50 and 100% of the test organisms. Where the objective of the test is to derive a concentration-response curve and determine more than one specific point response on the curve (e.g., IC50 and IC05 or LC50 and LC05), the use of additional test concentrations is recommended to ensure that both point response values are bracketed. The spacing between test concentrations depends on the expected slope of the concentration-response curve, information about which can be gained during a range-finding test. The test concentrations are usually selected in a geometric series in which the ratio is between 1.5 and 3.2. When the objective of the test is to determine a regression-based estimate and sample size constraints apply, the use of more test concentrations is preferable to the use of more replicates. The inclusion of additional test concentrations rather than additional replicates results in better characterization of the overall concentration-response relationship.
            (ii) Hypothesis-based test.
                  (A) Multiple-concentration definitive test. Each test usually consists of one or more control groups and at least 5 test concentrations that span the expected environmental concentrations and where at least the lowest test concentration is the NOEC. The test concentrations are usually selected in a geometric series in which the ratio is between 1.5 and 3.2. For sediment tests, while a two-fold progression is preferred, alternate progressions may be acceptable if accompanied by appropriate rationale, and the effect on NOAEC and LOAEC (as well as point estimates(s), if applicable) should be reported. If the failure to achieve statistical difference from the control 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 test concentration and the appropriate control(s). Individual OCSPP Series 850, Group A guidelines identify the limit concentration that satisfies the limit test for that guideline.
      (4) Randomization. For test results to be satisfactory, test treatments should be randomly or indiscriminately assigned to individual test vessels and the test vessels randomly or indiscriminately assigned to locations. Randomized block designs may be used. For test results to be satisfactory, test organisms should ideally be randomly assigned to the test vessels; where this is not practical, indiscriminate assignment can be used (with the exception of assignment intentionally according to sex). It should be noted that random assignment as used here implies a mathematically-based unbiased assignment method, and indiscriminate assignment implies a non-mathematically-based unbiased assignment procedure. All test vessels should be treated as similarly as possible to eliminate potential bias in test results. The methods used to randomize treatments among test vessels and test vessels among locations as well as methods of indiscriminate organism assignment to test vessels should be recorded. 
      (5) Number of replicates. The number of replicate test vessels for a given treatment is dependent upon the objective of the specific guideline test. Except for aquatic microcosm and 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 A guideline.
            (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 test concentrations rather than additional replicates results in better characterization of the overall concentration-response relationship. In most acute definitive tests, a minimum of 2 replicates per treatment is preferred to increase the confidence in the concentration-response relationship resulting from the test by providing a check that responses at a given test concentration are consistent and allowing for some estimate of experimental variation. The objective of some OCSPP Series 850, Group A guideline tests includes determination of both a regression-based point estimate (e.g., LD50) 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 for a given response, the size of effect to be detected, the minimum detectable difference from the control sought, and the acceptable error rate. It should be noted that several of the recommended non-parametric multiple-comparison tests cannot be performed without at least a minimum of 4 replicates. Sufficient replication should be used to provide the statistical power to detect adverse effects on the test organisms. For parametric tests, the minimum detectable difference achieved in an actual test should be reported. For non-parametric tests, at a minimum, the actual percent differences should be defined. Where the minimum replication number identified in a test-specific guideline is not sufficient to provide the statistical power to detect adverse effects on the test organisms, the number of replicates should be adjusted upward, or if appropriate, any environmental, handling, and culturing conditions, etc. that are contributing to the high variability should be identified and corrected.
      (6) Controls. Control groups are used to ensure that the observed effects are associated with or attributed only to the test substance. The appropriate control group should be similar in every respect to the test group except for exposure to the test substance. Within a given test, all test organisms including the control organisms should be from the same source. 
      If a vehicle (solvent) is used to facilitate the mixing of a test substance with a carrier (e.g., water, sediment, growth medium), a vehicle (solvent) control should be tested in addition to a dilution water control. If using a vehicle in a spiked sediment test, the appropriate control(s) should be included in the test design (see OCSPP 850.1735 and 1740 for additional details).  As prescribed by Good Laboratory Practice (GLP) regulations (40 CFR 160.113; 40 CFR 792.113), assurance should be provided that the vehicle does not interfere with the integrity of the test; OECD Principles of GLP (see paragraph (l)(22) of this guideline) also states that materials used in the test should not interfere adversely with the test systems. The highest concentration of vehicle added to any of the test vessels should be used in the vehicle control to demonstrate that the vehicle has no unacceptable affect. Use of the same vehicle concentration at each treatment level is recommended.
      If either the results of the dilution water control or vehicle control (or the negative sediment control or vehicle sediment control for spiked sediment testing) are not satisfactory for the test (e.g., control survival or reproduction are below test standards), the test results should be considered scientifically unsound and unacceptable. Control standards for a given test are described in each individual OCSPP Series 850, Group A guideline. If results of the dilution water control and the vehicle control (or the negative sediment control and vehicle sediment control for spiked sediment testing) meet the control standards for a given test, the results of dilution water control and vehicle control (or negative sediment control and vehicle sediment control for spiked sediment testing) should be compared using an appropriate statistical method to determine if there is an effect of the vehicle on the test organisms (i.e., the vehicle potentially confounded the test results). If there is a statistically significant positive or negative difference (typically at an α-level of 0.05) between the dilution water control and the vehicle control (or the negative sediment control and vehicle sediment control for spiked sediment testing) for any of the measured response variables, the test may be considered unacceptable. Such occurrences will be considered on a case-by-case basis to determine if the vehicle interfered with the integrity of the test and if the results meet the purposes for which the data are intended.
(e) Test substance characterization.
      (1) Background information on the test substance. The following information 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 active ingredient (a.i.); identities and concentrations of major ingredients and major impurities; radiolabeling, if any, including location of label(s) and radiopurity; and 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 levels, pKa; etc. Of particular relevance are rates for processes such as hydrolysis, photolysis, and volatilization.
            (iv) Solubility and stability of the test substance under actual test conditions. Determining the solubility and stability of the test substance in the test system is an important part of these tests (see paragraph (e)(2) of this 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 exposure matrix (e.g., test solutions, growth medium, bulk sediment, pore-water, etc.). Analyses are conducted with the specific media that will be used during the test, i.e., under test conditions.
      (2) Preliminary analyses. 
            (i) The Agency recommends preliminary testing of the test substance. Information about the stability and solubility of the test substance should be developed under actual test conditions. This information can be gained while doing the range-finding tests. 
            (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., saltwater, freshwater, growth medium, sediment).
                  (B) Temperature, pH, conductivity, lighting.
                  (C) With test organisms in place (when practical).
                  (D) Use of the same test vessels.
                  (E) Exposure technique of test (static, static-renewal, flow-through).
            (iii) The following tests should be performed:
                  (A) Stability trials under actual test conditions. 
                  (B) Solubility trials under actual test conditions. Surfactants and charged polymers are self-dispersing in water and should be tested at or below their dispersibility limits (critical micelle concentration). If the aqueous solubility of the test substance is less than 100 or 10 mg/L for pesticides or industrial chemical (acute or chronic testing, respectively), trials should be conducted under test conditions using various vehicles (solvents) that are most likely to be effective at enhancing solubility in the dilution water and that are widely recognized as being nontoxic. These trials may include other means to enhance solubility during the laboratory tests. A condition or mechanism increases solubility if the increase in the dissolved concentration is 2 times or greater. Once a vehicle (solvent) is chosen based on comparative evaluations, the decision should be confirmed in the preliminary analyses with only that vehicle.
                  (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. This includes determining whether and how samples can be stored for future analysis.
      (3) Sample storage. If samples of the test 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, including storage of the test substance before testing or storage of samples awaiting analysis, is required by GLP regulation and should be documented.
      (4) Aqueous exposure techniques. Laboratory or aquatic microcosm or field tests should be designed taking into account preliminary test information. Therefore, the preliminary tests should ideally be conducted before the definitive tests are initiated. At the discretion of the investigator, some or all of the recommended preliminary tests may be conducted concurrently with the definitive test; however, the risk of having to repeat the definitive test increases if the resulting information indicates different procedures should have been used. There are three commonly-used exposure techniques for aquatic toxicity tests. In the static exposure technique, test solutions and organisms are placed in the test vessels and kept there for the duration of the test, and the test solutions are not changed during the test. In the static-renewal (sometimes called `renewal') exposure technique, test organisms are periodically exposed to fresh test solutions of the same composition either by transferring the organisms from one test vessel to another or by replacing nearly all of the test solution, usually once every 24 to 72 hours depending on duration of test. In the flow-through exposure technique, test solution flows through the test vessel on a once-through basis throughout the test, and the displaced test solutions are discarded. In the flow-through exposure technique, test solutions are added to the test vessels on either an intermittent or continuous basis.
            (i) Selection of technique. Characteristics of the test organisms and properties and stability of the test substance under test conditions (see paragraph (e)(5) of this guideline) will dictate the exposure technique (static, static-renewal, or flow-through) to be used and the frequency of sampling needed to confirm exposure concentrations. All algae and cyanobacteria tests may be conducted as static. Flow-through and static-renewal systems are not recommended for these tests since they are conducted with microscopic organisms that cannot be protected from loss when renewing or draining water from test vessels. 
                  (A) Static tests. Static tests may be conducted only if, among other things, the test substance has been shown to be stable under test conditions, as defined in paragraph (e)(5) of this guideline. Even if the test substance is stable under test conditions, other factors not addressed in this guideline may preclude conducting a static test. These include, but are not limited to, problems in maintaining dissolved oxygen levels, feeding requirements, and concern for bacterial/microbial contaminants.
                  (B) Static-renewal tests. Static renewal is one method to ensure relatively continuous concentrations when the test substance is not or will not be stable under static test conditions. At a minimum, the renewal cycle should be based on the stability of the test substance under test conditions. The time to renewal (renewal cycle) should be shorter than the time it takes for the concentration of the test substance to decline to less than 80% of the initial- measured concentration. The renewal cycle may be shorter than required by stability characteristics of the test substance because of other factors (e.g., dissolved oxygen, feeding). However, a balance should be struck so that renewal is not frequent enough to stress the test organisms from the procedure. 
                  (C) Flow-through tests. The flow-through exposure technique is used to maintain constant test solution concentrations. Flow-through tests are particularly applicable when the chemical has a high oxygen demand, is highly volatile, can be quickly transformed, or can be sorbed to components of the test system (e.g., glass). If this exposure technique is used, guideline-specific flow parameters for flow-through tests should be followed. These parameters are described in each specific OCSPP Series 850, Group A test guideline and consist of discussions on monitoring of the flow-through system, measurement of the flow rate, variation in flow rates during the test, and test vessel volume replacements that are recommended during flow-through testing. 
                        (1) In flow-through tests, diluters, metering pump systems, or other suitable devices should be used to deliver the test substance to the test chambers. The choice of a specific delivery system depends on the specific properties of the test substance.
                        (2) The system should be calibrated before each test. Calibration includes determining the flow rate through each test vessel and the concentration of test substance in each test vessel. The measured flow rate should be within 10% of nominal.
                        (3) A closed flow-through system may be used to test volatile compounds when more than 20% of the test substance would be lost through volatility.
            (ii) Introduction of test organisms. Acclimated test organisms should be added to the test vessels subsequent to the addition of the test substance and/or test solutions to the test vessels. For static tests and initiation of static-renewal tests, the test organisms should be added as soon as possible after the test solutions are prepared. For flow-through tests, the test organisms should be added after the test system has been equilibrated (see paragraph (e)(6)(iv) of this guideline). 
      (5) Stability of test substance during test. A test substance is considered to be stable under actual test conditions if, under those conditions, it does not degrade, volatilize, dissipate, precipitate, sorb to test vessel walls, or otherwise decline to concentrations generally less than 80% of the initial-measured (day 0) concentration during the test period. (Refer to paragraph (j) of this guideline for additional information on measuring test concentrations). If the concentration of the test substance is expected to decline to less than generally 80% of the initial-measured (day 0) concentration during the test period, either a static-renewal or flow-through design should be used to ensure that the test concentration is maintained at a level greater than or equal to generally 80%. The only exception is testing with algae and cyanobacteria which cannot be tested in static-renewal or flow-through systems based on the experimental design described in OCSPP 850.4500 and OCSPP 850.4550 (see discussion in paragraph (j)(2)(ii) of this guideline on testing with algae and cyanobacteria). It should be noted that the most important consideration is maintenance of a constant exposure, regardless of the percentage of the nominal concentration that is attained. Whenever possible, the exposure scenario should be selected so that the measured concentration of test substance at each treatment level remains within plus or minus (+-) 20% of the time-weighted average concentration for the duration of the test.
            (i) To the extent possible, the exposure design should minimize variability within a given replicate over time and between replicates of the same treatment level. The goal is to maintain the ratio of the highest measured concentration to the lowest measured concentration at 1.5:1 or less within a replicate and between replicates of the same treatment level. Variability above this amount demonstrates that a constant exposure was not maintained.
            (ii) An important factor in considering the limits of variability is the avoidance of overlapping measured test concentrations between test levels. High variability calls into question the reliability of the environmental chemistry method, the stability of the test substance, and the concentrations on which to base test conclusions. Variability beyond the 1.5:1 ratio should be justified. This justification should clearly state the problem, explain why it occurred, provide a scientific explanation, and identify all measures taken to mitigate the problem. The justification should also include the fully developed chemistry method, including documentation necessary for a bench chemist to review and evaluate it.
            (iii) For cases in which variability problems are expected to occur, preliminary analyses are strongly recommended. If it becomes clear that high variability cannot be avoided, an exception should be justified. Any justification should be provided to the Agency in advance of conducting the definitive test. Agency scientists will decide on the validity of the rationale for the exception and may recommend other methods to reduce potential variability.
	(6) Analytical test substance determinations.
            (i) Nominal versus measured concentrations. Pesticides and other chemicals that are used at very low levels tend to have high biological activity. For this reason, it is imperative that the toxicity data developed for these test substances be accurate and scientifically defensible. Toxicity data should also be precise (repeatable and reproducible).
            Measured concentrations should be used over nominal concentrations when they are available because measured concentrations indicate what the actual exposure was for the test organisms in the test vessels. Concentrations of the test substance in aqueous media are expressed as the dissolved form. This form 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. The results of the toxicity test may be expressed based on the time-weighted average concentration, which may be calculated following the methods described in paragraph (l)(21) or other similar methods. An explanation of the rationale and method used should be provided in the study report. When measured concentrations are indicated (see paragraph (e)(6)(ii) of this guideline), they are considered necessary because:
                  (A) There are concerns that the actual dissolved concentrations to which the test organisms are exposed may differ from nominal concentrations. This variation may be due to chemical characteristics, test conditions, or mechanical apparatus.
                  (B) Measured concentrations confirm that the test system was designed appropriately and is operating acceptably. 
                  (C) Exposure estimates using measured concentrations account for characteristics of the test substance that make testing difficult (e.g., low solubility, short half-life, high binding potential). 
                  (D) Measurement of test concentrations is not performed solely to determine if the technician knows how to mix the test solution once. Among other reasons, this measurement also ensures that the test solution was mixed correctly each time. Measurement of test concentrations corroborates the precision of the technician or mechanics of the test system. 
            (ii) Sampling frequency. Selection of the appropriate frequency of sampling to confirm exposure concentrations throughout the duration of the test should be dictated by the exposure technique and the stability of the test substance. 
                  (A) Acute static test. In an acute static test with a test substance that is demonstrated to be stable, not sorptive, and readily soluble at the test concentrations and under the test conditions, measurements of each test concentration are not absolutely required (but is preferred). With these considerations in mind, sampling should be as follows for acute static test designs:
                        (1) The concentration of test substance in each test vessel should be measured at the beginning and end of the test (except as provided in (e)(6)(iii) for sampling at test initiation). Further, measurement of the test substance's degradation products is desirable, but not necessary, for a satisfactory test.
                        (2) If variability is expected to be a problem, measuring test concentrations midway through 96-hour tests at 48 hours is recommended. Replicate test vessels should be measured separately.
                  (B) Acute static-renewal test. The concentration of test substance in each test vessel should be measured at the beginning of the test (except as provided under (e)(6)(iii)), at the end of the first (or longest) renewal cycle, and at the end of the test. Measuring test concentrations at the beginning and end of each renewal cycle is also recommended. For sampling replicate test vessels at test initiation and the beginning of a renewal cycle, refer to the general discussion of replicates under paragraph (e)(6)(iii) of this guideline.
                  (C) Acute flow-through test. The frequency of sampling depends on the stability of the test substance in stock solution(s) feeding the diluter system and the frequency of renewal of the stock solution(s) and should be sufficient to document the stability of test substance exposure throughout the duration of the test. With these considerations in mind, sampling should be as follows for acute flow-through exposure designs:
                        (1) The concentration of test substance in each test vessel should be measured at the beginning and end of the test (except as provided in (e)(6)(iii) for sampling at test initiation). 
                        (2) If variability is expected to be a problem, measuring test concentrations midway through 96-hour tests at 48 hours is recommended. Replicate test vessels should be measured separately.
                        (3) If the stock solutions feeding the diluter system are renewed during the test, they should be measured at the beginning and end of the longest renewal period. 
                        (4) If a metering fluctuation or malfunction is detected or observed, test concentrations should be measured. 
                  (D) Chronic static-renewal test. The concentration of test substance in each test vessel should be measured at the beginning of the test, at the beginning and end of a renewal cycle at least once per week, and at the end of the test. The longest renewal cycle in a sequence should be used if variable-cycle periods are employed. However, for some chronic static-renewal tests (e.g., Daphnia magna chronic toxicity, 850.1300), it is recognized that this may result in a large of samples at each sampling period. In such cases, at least two alternating replicates per treatment level should be analyzed at each sampling period (e.g., if there are 10 replicates (A through J) per treatment level, replicates A and B should be analyzed in the first week and replicates C and D in the second week, and replicates E and F in the third week, and so on) to demonstrate acceptable variability of measured concentrations between replicates during the test (see paragraph (e)(5) of this guideline). Refer to the general discussion of replicates under paragraph (e)(6)(iii) of this guideline. 
                  (E) Chronic flow-through test. The frequency of sampling depends on the stability of the test substance in stock solution(s) feeding the diluter system and the frequency of renewal of the stock solution(s) and should be of sufficient design to document the stability of the test substance exposure throughout the duration of the test. With these considerations in mind, sampling should be as follows for chronic flow-through exposure designs:
                        (1) The concentration of test substance in each test vessel should be measured at a minimum at the beginning of the test, once every 7 days, and at the end of the test (except as provided under (e)(6)(iii)). 
                        (2) The stock solution(s) feeding the diluter system should be measured at a minimum at the beginning and end of the longest renewal period. If the test substance is stable, the low, medium, and high stocks should be measured at the start of a renewal cycle at least once a week. 
                        (3) If a metering fluctuation or malfunction is detected or observed, test concentrations should be measured. 
                  (F) Microcosm and field studies. For microcosm and field studies, the frequency of sampling depends on the objective of the study and is determined on a case-by-case basis.
            (iii) Exceptions to sampling and analyzing each replicate test vessel separately. When measurement of replicate test vessels of a treatment level is indicated (see paragraph (e)(6)(ii) of this guideline), each replicate should be analyzed separately because the responses in each replicate are viewed as independent, and knowing the concentration in each replicate allows for determination of variation. Exceptions to this occur when:
                  (A) Static test. Replicate test vessels in static tests are filled from a bulk preparation. In this case, only samples from the bulk preparation for each treatment level should be analyzed at the beginning of the test.
                  (B) Static-renewal test. Replicate test vessels in static-renewal tests are filled from a bulk preparation at the beginning of the test and at renewal. In this case, only samples from the bulk preparation for each treatment level should be analyzed at the beginning of the test and at renewal. Also see discussion on static-renewal chronic test in paragraph (e)(6)(ii)(D).
                  (C) Flow-through test. A "splitter" is used in a flow-through test to feed more than one replicate test vessel. In this case, only samples from one replicate per treatment level should be analyzed. Collecting and storing samples from all replicates is recommended in case anomalous concentrations are measured in the one that is analyzed. Analyzing the other replicates may shed light on the cause and extent of the anomalous measurements. Replicates receiving flow from a splitter should be sampled and analyzed alternately (e.g., if there are 2 replicates (A and B), replicate A should be analyzed in the first week and replicate B in the second week). 
                  (D) Insufficient volume. Insufficient volume is available from individual replicates to conduct the required analysis. This may occur when samples need to be concentrated and extracted to attain the detection limit objective, or where the test solution volume is relatively small (as in algal and daphnid studies). In this case, samples for each treatment level may be pooled.
            (iv) Pretest initiation analysis in flow-through and sediment tests. In some instances, a flow-through test system may need to operate for a set period of time prior to the addition of test organisms to obtain constant and representative concentrations (as defined in paragraph (e)(5) of this guideline). Also, spiked sediments may require a significant period of equilibration before the concentrations can be confirmed and the test can be initiated. 
(f) Preparation of test substances.
      (1) Aqueous exposure medium. In some cases, the test substance is added directly to the dilution water or growth medium on a weight to volume or volume to volume basis. Alternatively, a stock solution of the test substance is prepared, and aliquots of the stock solution or secondary stock solutions are added to the dilution water or growth medium. The preferred practice may be to make a bulk preparation of each test solution and distribute portions to each replicate test vessel to minimize variation between replicates and reduce the number of analytical samples. 
            (i) Direct addition. If it is not possible to prepare a homogeneous stock solution of the test substance, the test substance may be added directly to intermediate mixing containers or test vessels either by weight or volume.
            (ii) Stock solutions.
                  (A) Reagent water. Provided that the test substance can be dissolved in water and does not readily hydrolyze and that the amount of stock solution added to the dilution water or growth medium will be less than 10% of the total volume (to avoid changes in the dilution water or growth medium), the preferred choice for preparation of a stock solution is to use reagent water (deionized, distilled, or reverse osmosis water). To avoid alterations in the dilution water or growth medium (e.g., unacceptable change in the salinity of seawater or concentration of nutrients in growth medium), the stock solution may also be prepared in dilution water or growth medium. 
                  The pH of stock solutions may be adjusted to match the pH of dilution water or to a neutral pH if pH change does not affect the stability of the test substance in water. The pH of test solutions may be adjusted after the addition of the test substance or stock solution into the dilution water. However, all pH adjustments need to be made prior to the addition of test organisms. Hydrochloric acid (HCl) and sodium hydroxide (NaOH) may be used for this adjustment if warranted. 
                  (B) Vehicle. The use of a vehicle should be avoided if possible and only be considered as a last resort when all other chemical delivery options have been considered. However, if the test substance cannot be dissolved in dilution water or growth medium, a water-miscible solvent is often used. Because there is the potential for interaction between the vehicle and test substance resulting in an altered test response, the use of a vehicle should be restricted to situations where no other acceptable method of media preparation is available. If a vehicle, i.e., a solvent (or potentially a dispersant for industrial chemical testing), is absolutely necessary to dissolve the test substance, the amount used should not exceed the minimum volume necessary to dissolve or suspend the test substance in dilution water. If the test substance is a mixture, formulation, or commercial product, none of the ingredients is considered to be a vehicle unless an additional amount is used to prepare the stock solution. 
                        (1) Preferred vehicle solvents are dimethylformamide, triethylene glycol, methanol, acetone, and ethanol. The use of solvents with high purity (e.g., analytical grade) are preferred. It should be noted that the use of solvents such as acetone, ethanol, and methanol in aquatic test systems can be problematic due to the growth of bacteria and resulting depletion of oxygen and/or unequal food resources among and between treatments. See paragraph (f)(1)(C) for other solubility enhancement methods.
                        (2) If a vehicle is used to prepare the test substance, a vehicle control  should also be included in the test design in addition to the dilution water control. If using a vehicle in a spiked sediment test, the appropriate control(s) should be included in the test design (see OCSPP 850.1735 and 1740 for additional details). The same batch of vehicle used to prepare the treatment levels should be used in to prepare the vehicle control. The selected vehicle should not confound the test results or affect the test organisms at the concentration used. As prescribed by Good Laboratory Practice regulations (40 CFR 160.113; 40 CFR 792.113), assurance should be provided that the vehicle does not interfere with the integrity of the test; OECD Principles of GLP (see paragraph (l)(22) of this guideline) also states that materials used in the test should not interfere adversely with the test systems. 
                        (3) Ideally, the vehicle concentration should be kept constant in the vehicle control (or vehicle sediment control for spiked sediment testing) and all treatment levels. If the concentration of vehicle is not constant, the vehicle control (or vehicle sediment control for spiked sediment testing) should contain the highest concentration of vehicle used in any treatment level. The concentration of vehicle should not exceed 0.1 mL/L. A recent review recommends that solvent concentrations as low as 0.02 mL per liter of dilution water be used (see paragraph (l)(14) in this guideline). 
                        (4) Solvents are not appropriate for mixtures where the use of a solvent can give preferential dissolution of one or more components and thereby affect the toxicity, or where the solvent is suspected of chemically reacting with the test substance.
                  (C) Alternative testing methods for poorly soluble materials. 
                        (1) Methods for solubility enhancement in dilution water. The technique used to maximize test substance dissolution in the test media under standard conditions should be demonstrated. Consideration of the optimum technique should include use of nontoxic solvents, saturation (solubility) columns, sonication, and minor adjustments to environmental conditions (i.e., temperature, pH, etc.), as appropriate. Minor adjustments to environmental conditions should not extend outside the recommended range of conditions for the specific test organism. Additional guidance can be found in paragraph (l)(20) of this guideline.
                              (i) Saturator columns. The use of saturation columns as an aid in the dissolution of test substance and in maximizing the solubility of nonvolatile test substances with test media solubilities of 10 mg/L or less is recommended. Methods for using these columns in aquatic toxicity tests can be adapted from methods established for their use in determining water solubility under OECD's Column Elution Method (see OCSPP 830.7840). Saturator columns may be considered to generate test solutions for static or for flow-through tests.
                              (ii) Effect of environmental conditions. Solubility is a function of temperature and is especially sensitive to temperature at the limit of solubility. Generally, below saturation, increases of as much as 10 °C may affect the solubility up to a factor of two. However, if test solutions are close to saturation, small changes in temperature may result in supersaturated solutions. In addition, control of temperature is important because of its well-known effects on the actual toxicity of the compound. Water hardness can also affect the solubility of certain test substances. Studies that involve radical changes in environmental test conditions outside the recommended range of values for temperature, salinity, pH, etc., will be considered by the Agency on a case-by-case basis.
                        (2) Emulsifiers and formulation testing. The use of emulsifiers and dispersants when testing industrial chemicals or pesticide technical grade active ingredients is strongly discouraged. When specifically testing pesticide formulations, it is understood that emulsifiers, dispersants, solubilizing agents, etc., may be part of the test substance. In such cases, the test concentrations should be expressed in terms of the concentration of pesticide active ingredient, and it should be clearly indicated that the test was performed on the formulated product.  
                        (3) Separation of soluble fraction prior to testing. There is the potential for undissolved test substance to cause physical effects to test organisms (e.g., blocking of fish or aquatic invertebrate gill membranes, encapsulation/entrapment of daphnids, or the reduction of light intensity in algal tests). Therefore, when undissolved test substance, precipitate, flocculant, or colloidal suspension (except for surfactants or charged polymers) is observed in test solutions prior to introduction to test vessels despite efforts to obtain homogeneity, separating the soluble fraction of test substance from the particulates prior to use in testing may be advisable. Separation may be accomplished by conventional centrifugation or filtration. If one test solution is centrifuged or filtered, all test solutions should be treated similarly. The effect of centrifugation or filtration on the accuracy and precision of the analytical method used to verify dissolved concentrations of the test substance should be known. The use of centrifugation or filtration in the preparation of test solutions does not necessarily obviate the need for centrifugation or filtration of samples prior to analytical confirmation of dissolved test concentrations.
       (2) Sediment exposure medium. 
            (i) Preparation of spiked sediment.
                  (A) Spiking sediment. The method used for preparing spiked sediment depends on the properties of the test substance. Stock solutions of the test substance in organic solvents should not be added to the sediment mixture because such solvents affect the concentration of dissolved organic carbon in pore water.
                        (1) Water soluble test substance. For a water soluble test substance, spiked sediment of a given concentration is prepared by adding the test substance directly to the sediment as an aqueous solution. Aqueous stock solutions of metals and metal compounds and sufficiently water soluble organic chemicals can be prepared using the overlying water source or deionized water. Small volumes of aqueous stock solutions are added or spiked directly into sediment and then mixed.
                        (2) Water insoluble test substance.
                              (i) Solids. For a water insoluble test substance existing as a solid, spiked sediment of a given concentration may be prepared by adding the test substance to the sediment in a dry form. Water insoluble solids may be prepared for spiking of sediment by grinding or other methods to reduce the particles to smaller than 200 micrometers (um) in diameter. Appropriate weighed amounts are then mixed with the sediment.
                              (ii) Solids and Liquids. For a water insoluble test substance, spiked sediment of a given concentration may be prepared by sorbing the test substance to sand and then mixing the treated sand into the sediment. Stock solutions of water insoluble test substances may be prepared by dissolving the test substance in a volatile solvent. Small volumes of the stock solution are then mixed with fine quartz sand, (e.g., approximately 10 grams (g) of quartz sand per test vessel). The solvent is then slowly evaporated off the sand, and the sand (e.g., 10 g per test vessel) is mixed with a suitable amount of sediment per test vessel. All of the vehicle should be totally evaporated from the coated sand before mixing with the sediment. Only those vehicles that volatize readily (e.g., acetone) should be used to prepare stock solutions. When using this method, the amount of sand added to formulate artificial sediment should be adjusted downward to accommodate the amount of coated sand that will be added. In some cases, analyzing the test substance concentration on the sand may be appropriate.
                  (B) Mixing. Mixing the test substance with sediment following the methods described in paragraph (f)(2)(i)(A) of this guideline can be accomplished using various methods such as a rolling mill, feed mixer, or hand mixing. Care should be taken to ensure complete and homogenous mixing of the test substance with sediment, and analyses of the test substance in subsamples of bulk sediment should be conducted to ensure that spiking is uniform in the mixed material. To minimize potential changes in the physico-chemical and microbial characteristics of the sediment, the mixing time should be limited from minutes to a few hours, and the temperature should be kept low.
                  (C) Equilibration. Aging of sediment after spiking is dependent on the objective of the test. In general, once a sediment has been spiked with the test substance, the mixture should be allowed to reach equilibrium to the extent practical prior to beginning the test. The time to reach equilibrium (i.e., between the concentration sorbed to the sediment particles and the concentration in the pore water) will be depend to a large extent on the properties of the test substance and sediment. For metals, 1 to 2 weeks of aging may be sufficient. For persistent, highly hydrophobic organic chemicals (e.g., Log KOW > 6), longer equilibration times may be needed.  It is noted, however, that the persistence of the chemical in sediment and pore water will also need to be considered in determining the most appropriate equilibration times. Shorter equilibration times may be needed for chemicals that degrade more rapidly in sediment in order to ensure that sufficient test material remains for testing. Periodic monitoring of chemical concentrations in pore water during sediment aging is highly recommended as a means to assess the equilibration of the spiked sediments. Prepared sediments should be aged at 4 °C.
                  (D) Placement of sediment in test vessels. Equilibrated sediment (control sediment and sediment spiked at various test concentrations and aged as described in paragraph (f)(2)(i)(C) of this guideline) should be thoroughly mixed and added to test vessels the day before (day -1) the start of the test. The same amount of sediment should be added to each test vessel on the basis of volume or dry weight. The degree of homogeneity should be inspected visually. Homogeneity may be quantified by taking replicate subsamples and analyzing for TOC, test substance sediment concentration, and particle size.
            (ii) Overlying water. Spiked sediment tests should be conducted under static-renewal or flow-through conditions of the overlying water. Various types of apparatuses have been used in sediment toxicity tests as described in paragraph (l)(34) of this guideline. The water delivery system should be designed to minimize sediment resuspension. To minimize disturbance of sediment, overlying water should be poured gently along the sides of the test vessels or poured over a turbulence reducer (e.g., a disk cut from polyethylene, nylon, or Teflon, or a glass Petri dish attached to a glass pipet) positioned above the sediment. Turbulence reducers should be rinsed with overlying water between replicates, and a different turbulence reducer should be used for each treatment group. 
                  (A) Static-renewal conditions. Renewal of overlying water should be started on generally day -1 before the addition of test organisms or food on day 0. The rate of overlying water renewal may vary depending on test conditions (i.e., need to maintain environmental conditions), but generally, each test vessel should receive no more than 2 volume additions per day of overlying water.
                  (B) Flow-through conditions. Renewal of overlying water should be started on generally day -1 before the addition of test organisms or food on day 0. Each test vessel should receive 2 volume exchanges per day of overlying water. Flow rates through any 2 test vessels should not differ by more than 10% at any time during the test. Each water delivery system should be calibrated prior to test initiation to verify that the system is functioning properly. The flow rates of stock solutions and dilution water should be checked, visually or mechanically, at least daily during the test.
(g) Analytical methods and sampling for verification of exposure.
      (1) Method validation. 
            (i) The analytical method used to measure the amount of test substance in the exposure matrix (e.g., test solutions, growth medium, bulk sediment, pore water) should be validated by appropriate laboratory practices before beginning the definitive test. An analytical method is not acceptable if likely degradation products of the test substance, such as hydrolysis and oxidation products, give positive or negative interferences that cannot be systematically identified and mathematically corrected, unless it is shown that such degradation products are not present in the test vessels during the test. 
            (ii) Method validation should be 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 (dilution water, test solutions, growth medium, etc.) as will be used in the definitive test. 
            (iii) The method validation should include a determination of linearity between detector response and test substance concentration, the limit of quantification (LOQ), the method detection limit (MDL), method accuracy (average percent recovery), and precision (relative standard deviation). The method validation should establish the acceptance criteria for the quality control samples that will be prepared and analyzed during the test. 
      (2) Collection of samples. Samples should be collected in such a manner so as to provide an accurate representation of the matrix being sampled. 
            (i) Water samples for analysis should be obtained by siphoning through inert tubing from a central point in the test vessel, not from inflow or outflow points. These samples should not contain any surface particulates or material dislodged from the bottom or sides of the test vessels. 
            (ii) Samples should be processed and analyzed immediately, or handled and stored in a manner that minimizes loss of test substance through microbial degradation, photodegradation, chemical reaction, volatilization, sorption, or other processes. 
            (iii) The test solution volume should not be reduced during the test by more than 10% as a result of sampling. 
            (iv) Samples from each replicate of a treatment level should not be pooled for analysis, except as described under paragraph (e)(6)(iii) of this guideline. 
            (v) Sample collection for sediment toxicity tests is described in the applicable guidelines. 
      (3) Analysis of samples.
            (i) Aqueous media. Concentrations of the test substance in aqueous test media should be determined from centrifuged supernatant or other appropriate separated fraction (e.g., filtrate) except when testing self-dispersing industrial chemicals (e.g., surfactants, detergents, or charged polymers), which should be measured directly. Conventional centrifugation or filtration should be performed for all test media where undissolved test substance, precipitate, flocculant, and/or colloidal suspension are observed in any stock solution, mixing chamber(s), or test vessel(s) or where solubility, and hence bioavailability, is in question. For example, where a solvent or alternative method is used to enhance solubility, a clear test medium should not be assumed to indicate a true solution since crystals, aggregates, micelles, etc., cannot easily be detected by visual observation. 
                  (A) Measurement of dissolved form. In aqueous media (e.g., dilution water, test solutions, growth medium, pore water), determination is made on the concentration of the dissolved form of the test substance. This form 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. If test concentrations are not near the functional water solubility limit (in the test media), and TOC is less than or equal to (<=) 2 mg/L, it reasonable to assume that the results are equivalent to the dissolved form such that no further sample preparation (i.e., centrifugation, filtration) is necessary. In a bioconcentration study, the concentrations used should be below the solubility limit under test conditions, and samples may not be subjected to centrifugation or filtration. Instead, during a bioconcentration study, measures should be taken to keep the tanks as clean as possible and the TOC low.
                  (B) Separation techniques. Possible separation techniques include centrifugation and filtration. Separation techniques should not be applied to solutions containing self-dispersing industrial chemicals, including when such chemicals are added for end-use product testing either as an adjuvant or contained within the formulation. 
                        (1) Centrifugation. The preferred separation method is centrifugation, but there may be practical difficulties in applying the technique to large volumes of test medium. As a guide, centrifugation at 100,000 to 400,000 meters per second square (m·s[-2]) for 30 minutes may achieve adequate separation. It should be noted that most centrifuge containers are made of various sorts of plastics that may adsorb the test substance and that glass containers are more likely to break. 
                        (2) Filtration. Although less widely advocated because of the potential for losses due to adsorption onto the filter matrix, filtration through a membrane filter is another possible separation method. Filtration may represent the only practical option where large volumes of test medium are required. Filter sizes of 0.22 to 0.45 μm may be suitable for achieving adequate separation. Filter matrix should be made of inert materials (i.e., materials that are chemically and physically non-reactive with the test substance). Filters should be rinsed with high purity water prior to use to reduce the risk of contamination of test solutions with toxic residues. Adsorption of the test substance to the filter matrix may be reduced to insignificant levels by preconditioning filters with solutions of the test substance prepared at appropriate test concentrations. Filtration under pressure is preferable to vacuum filtration due to potential losses by evaporation.
            (ii) Sediment. In bulk sediment, determination is made on the total mass of the test substance. 
            (iii) Quality control. Concurrent with each analysis of test samples, quality control (fortified) samples should be analyzed. Quality control samples should be 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. To determine compliance with the provisions of paragraph (e)(5) of this guideline, correcting test sample recoveries for inherent method bias as determined from the concurrent analysis of freshly fortified quality control samples may be appropriate. For example, if the average recovery from the test samples is 73%, and the average recovery from the freshly fortified controls is 80%, then the true recovery from the test samples is 91%. However, before a correction factor is applied, it should be adequately demonstrated that the bias is indeed from the method and not another source (e.g., instrument or technician error). 
(h) Reference toxicants. Historically, reference toxicity testing has been thought to provide 3 types of information relevant to the interpretation of toxicity test data: an indication of the relative "health" of the organisms used in the test; a demonstration that the laboratory can perform the test procedure in a reproducible manner over a period of time; and information to indicate whether the sensitivity of a particular strain or population in use at a laboratory is comparable to those used in other facilities and how intra- and inter-laboratory 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, as discussed in paragraph (l)(34) of this guideline. 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 tests in this guideline series, a quarterly or semiannual positive control (on a guideline-specific basis) can serve as a means of detecting possible inter-laboratory or temporal variation. A reference toxicant might also be desirable when there is any significant change in source or maintenance of test organisms or other test conditions. Despite these potential uses of reference toxicants, alternative means of assessing organism health, test reproducibility, and intra- and inter-laboratory variation should be considered in order to minimize of the use of test animals.
(i) Monitoring environmental test conditions. Test conditions are specified in each test-specific guideline in the OCSPP Series 850, Group A. These conditions include environmental factors such as temperature, dissolved oxygen, pH, and lighting among others. Methods used for monitoring test conditions should be in accordance with established methods (e.g., those published by U.S. EPA, ASTM, APHA; see paragraphs (l)(1), (l)(2), (l)(3) of this guideline) and should be referenced in the study report. The test solution volume should not be reduced by more than 10% as a result of these measurements. While the frequency and where to monitor environmental conditions are described in subsequent paragraphs, if during a test it appears that environmental conditions may have changed (e.g., unexpected abnormal behavior or mortality observed), it is recommended that environmental conditions be checked. 
      (1) Temperature. Temperature should preferably be monitored continuously (recorded at least hourly). Alternatively, temperature should be measured daily, which is defined as a minimum of two times during each 24-hour period of the test. If measurements are not made in each test vessel, they should be made in a sufficient number of representative test vessels, and they should be made near the beginning and end of the test in all test vessels or in various parts of the water bath, incubator, environmental chamber, or constant temperature room. To meet test-specific environmental conditions, the more temperature measurements that are made, the less likely it is that any single deviation from the specified temperature will lessen confidence in the results of the test. Conversely, if the temperature is measured only a minimal number of times, any deviation might infer that more deviations would have been detected if measurements had been taken more frequently. 
            (A) Acute test. Temperature should preferably be measured daily in each replicate. However, at a minimum, temperature should be measured in each replicate at the beginning and end of the test and every 24 hours in at least one replicate each of the control(s) and highest, lowest, and middle treatment levels.
            (B) Chronic test. At a minimum, temperature should be measured in each replicate at the beginning and end of the test and at weekly intervals.	
            (C) Sediment test. Temperature should be measured daily in at least one replicate per control and treatment level. 
            (D) The difference between the time-weighted average measured temperature for any two test vessels, from the beginning to the end of the test, should not be greater than 1 °C. 
            (E) Any individual measured temperature in any test vessel should not be more than 3 °C different from the mean of the time-weighted average measured temperatures for the individual test vessels.
            (F) At any moment in time, the difference between the measured temperatures in any two test vessels should not be more than 2 °C.
      (2) Lighting. Guidance for lighting in laboratory toxicity tests can be found in paragraph (l)(4) of this guideline. The recommended photoperiod and light intensity are reported in each test-specific guideline in the OCSPP Series 850, Group A. 
            (A) Acute and chronic tests. Light intensity should be measured from at least one representative location at the beginning of the test. For longer duration chronic tests (i.e., OCSPP 850.1500, Fish Life-cycle), weekly measurements are preferred as well.
      (3) Dissolved oxygen. Dissolved oxygen is necessary to support the respiration of aquatic animals. Specifications for the acceptable dissolved oxygen concentration are provided in each guideline. Dissolved oxygen is maintained through the use of acceptable biomass loading and use of renewal or flow-through exposure scenarios. Additionally, in a flow-through design, dilution water can be aerated prior to use. For some guidelines, dissolved oxygen may be maintained by aeration of the test vessels as a last resort. However, in such cases, maintenance of test concentrations, control performance, loading, and other environmental conditions within acceptable limits should be demonstrated. It should be noted that aeration of test vessels is strongly discouraged in some guidelines as it may greatly stress the test organisms and potentially bias test results. 
            (A) Acute test. Dissolved oxygen should preferably be measured daily in each replicate. However, at a minimum, dissolved oxygen should be measured in each replicate at the beginning and end of the test and every 24 hours in at least one replicate each of the control(s) and highest, lowest, and middle treatment levels.
            (B) Chronic test. At a minimum, dissolved oxygen should be measured in each replicate at the beginning and end of the test and at weekly intervals. However, for some chronic static-renewal tests (e.g., Daphnia magna chronic toxicity, 850.1300), it is recognized that this may result in a large of samples at each sampling period. Additionally, for chronic flow-through tests that use "splitters" to deliver the test solution to more than one replicate test vessel in a consistent manner, dissolved oxygen may not need to be measured in each replicate at weekly intervals.  Refer to paragraph (e)(6)(iii) for examples regarding exceptions to measuring each test vessel.
             (C) Sediment test. Dissolved oxygen should be measured daily in at least one replicate per control and treatment level. For static-renewal tests, the measurement should be taken just before water renewal. 
      (4) pH. The pH in tests should not vary by more than 1.0 units in any one test.
            (A) Acute test. pH should preferably be measured daily in each replicate. However, at a minimum, pH should be measured in each replicate at the beginning and end of the test and every 24 hours in at least one replicate each of the control(s) and highest, lowest, and middle treatment levels.
            (B) Chronic test. At a minimum, pH should be measured in each replicate at the beginning and end of the test and at weekly intervals. However, for some chronic static-renewal tests (e.g., Daphnia magna chronic toxicity, 850.1300), it is recognized that this may result in a large of samples at each sampling period. Additionally, for chronic flow-through tests that use "splitters" to deliver the test solution to more than one replicate test vessel in a consistent manner, dissolved oxygen may not need to be measured in each replicate at weekly intervals.  Refer to paragraph (e)(6)(iii) for examples regarding exceptions to measuring each test vessel.
            (C) Sediment test. pH should be measured daily in at least one replicate per control and treatment level. For static-renewal tests, the measurement should be taken just before water renewal.
      (5) Hardness (freshwater tests).
            (A) Acute test. Total water hardness should be measured in the dilution water at the beginning of the test.
            (B) Chronic test. At a minimum, total water hardness should be measured in at least one replicate in the dilution water control and highest treatment level at the beginning and end of the test and at weekly intervals.
            (C) Sediment test. Total water hardness should be measured in the overlying water in at least one replicate of each control and treatment level at the beginning and end of the test. 
      (6) Salinity (saltwater tests).
            (A) Acute test. Salinity should be measured at the beginning and end of the test in at least one replicate of each control and treatment level. For a static-renewal test, salinity should be measured at the beginning of the test and at the end of the longest renewal period in at least one replicate of each control and treatment level. For flow-through tests, salinity should be measured at the beginning and end of the test in at least one replicate of each control and treatment level. In a flow-through test using natural seawater unadjusted for salinity which can fluctuate during the test, salinity should be measured at the beginning and end of the test in at least one replicate of each control and treatment level, and measuring the salinity in one replicate of each control for the other days is recommended. The specific test guideline should be consulted for further guidance.
            (B) Chronic test. For flow-through tests, salinity should be measured at a minimum at the beginning and end of the test in at least one replicate of each control and treatment level.
            (C) Sediment test. Salinity should be measured in the overlying water in at least one replicate of each control and treatment level at the beginning and end of the test. 
      (7) Ammonia. Exposure to ammonia can have an adverse effect on the test organisms (see paragraphs (l)(30) and (l)(33) of this guideline). Ammonia levels are generally of a greater concern in sediment tests. Therefore, in sediment tests, the ammonia concentration should be measured in the overlying water in at least one replicate of each control and treatment level at the beginning and end of the test. In addition, ammonia should be measured in pore water at the beginning, middle, and end of the test. Total ammonia measurements are recommended. 
       (8) Alkalinity and specific conductance. Alkalinity and specific conductance should be measured in the dilution water at the beginning of the test. For longer duration chronic tests (i.e., OCSPP 850.1400, Fish Early Life-stage), weekly measurements are recommended as well (see paragraph (i)(5) on dilution water hardness for details regarding where to sample).
      (9) TOC and COD. Measurement of TOC or COD in the dilution water at the beginning of the test is recommended, but at a minimum, TOC and COD should be analyzed periodically in the dilution water source to document and characterize their magnitude and variability. For tests with cationic substances, TOC or COD should be measured at the beginning of the test. 
(j) Test validity elements. Test-specific validity elements can be found in each OCSPP Series 850, Group A guideline. Additional test validity elements that apply to all OCSPP Series 850, Group A guidelines are as follows: 
      (1) Test substance exposure concentrations. Refer to the general discussion of replicates under paragraph (e)(6)(iii) of this guideline. 
            (i) A test may be considered to be unacceptable or invalid if:
                  (A) The test substance was not stable as defined in paragraph (e)(5) of this guideline.
                  (B) Solubility was likely to have been a problem at the treatment levels tested (e.g., precipitates formed), and no analytical measurements were performed on the soluble fraction to verify dissolved exposure concentrations. See paragraph (g)(3) of this guideline for methods used to segregate out the soluble fraction.	
      (2) Measured concentrations versus nominal concentrations. This section describes acceptable limits of deviation of measured concentrations from nominal concentrations. Additional information can be found in paragraph (l)(20) of this guideline.
            (i) When a laboratory test design has been specifically modified to accommodate the instability of the test substance or for other factors likely to cause variability in test concentrations, and the design is judged adequate based on sufficient preliminary information, the test will not be considered to be unacceptable or invalid solely on the grounds that measured concentrations deviated by more than 20% from nominal concentrations, provided that the following conditions are met:
                  (A) Preliminary stability information is provided with complete documentation and description of the methods used, and preliminary stability tests are conducted under test conditions essentially identical to the actual test conditions.
                  (B) A reasonable and scientific explanation is provided, and the variability of results produced by the chemical analysis method is adequately characterized.
                  (C) All test vessels exhibit a similar (but not necessarily identical) shift in concentrations. If concentrations in some test vessels increase substantially (greater than 20%), and concentrations in other test vessels decrease substantially (greater than 20%), they will not be considered to have exhibited a similar shift. The most important consideration is that concentrations do not experience a shift in "order," i.e., the highest treatment level should retain its position as the highest, and the next should remain second, and so on. If the order is shifted, the test may be considered to be unacceptable or invalid since regression analysis would not yield statistically sound median lethal concentrations and confidence intervals.
                  (D) The variability of the measured concentrations is acceptable as discussed in paragraph (e)(5) of this guideline.
                  (E) A statistically valid endpoint (e.g., LC50, EC50, NOEC, etc., or LC50, EC50, or NOEC is greater than the limit concentration) can be derived from the measured concentrations.
            (ii) Conducting flow-through or static-renewal tests with algae and cyanobacteria is not feasible with the current state of available methods. Therefore, the following are recommended for a test substance that is expected to degrade or volatilize to less than 80% of the initial-measured concentration based on preliminary stability testing and physico-chemical properties: 
                  (A) The test should be conducted normally, with concentrations measured at the beginning and end of the test, as it is important to understand the behavior of the test substance during the test. If stability is expected to be a problem, measuring test concentrations midway through 96-hour tests at 48 hours may be considered to better understand the rate of dissipation.
                  (B) Based on preliminary analysis, the analytical method should have a detection limit below any anticipated decrease in test concentrations, and test concentrations should not decrease below the LOD. 
                  (C) Procedural modifications may be necessary when testing algae with volatile materials or fast degrading materials. These modifications should still allow for carbon dioxide uptake sufficient for acceptable growth. Possible modifications include shortening the test period, using test flasks with ground glass stoppers, increasing the size of the test flasks to reduce the surface to volume ratio, shaking flasks twice daily instead of continuously when using a rotary shaker, and conducting testing at lower temperatures. 
	(2) Test substance concentration variability. 
            (i) In some cases, high variability in test substance concentrations cannot be avoided because the test concentrations are approaching the LOD or because of unavoidable binding of the test substance to the chemical analysis apparatus. When the ratio of the highest measured concentration to the lowest measured concentration for a treatment level is expected to vary by more than 1.5 or, for sediment testing, if equilibrium or steady state cannot be achieved, providing a justification for an exception to this to the Agency in advance of conducting aquatic laboratory testing is strongly advised. This exception justification should consist of:
                  (A) Documentation of preliminary analyses indicating this problem.
                  (B) The specific steps that will be taken to mitigate the problem.
                  (C) The fully developed chemical analysis method.
                  (D) The raw data, standards, quality control samples, and chromatograms from a representative analysis using the method. For each chemistry method, the MDL and LOQ should be identified.
            (ii) The Agency will decide on each exception justification on a case-by-case basis. However, if a series of aquatic tests are to be conducted with one chemical, and exceeding this limit in all of the tests is anticipated, one exception justification may cover more than one test. 
(k) Reporting.
      (1) Background information. In addition to the reporting requirements prescribed in the Good Laboratory Practices Standards (40 CFR 792 and 40 CFR 160), the study report should include the following information:
            (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. Raw data includes all measurements recorded during the study including, but not limited to, effects (mortality, growth, behavior, etc.), environmental conditions (temperature, dissolved oxygen, pH, etc.), and test substance concentrations 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 study being considered unacceptable or invalid as scientifically unsound.
            (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 792 or 40 CFR 160). Alternatively, the statement can indicate it was conducted under OECD Principles of GLP (see paragraph (l)(22) of this guideline), 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 following:
            (i) Objectives and procedures stated in the approved protocol, including any changes or deviations or occurrences that 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, 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) Source of the dilution water, its chemical characteristics (e.g., conductivity, hardness, TOC, salinity, pH), and a description of any pretreatment; description of growth medium or sediment, if applicable.
            (iv) Methods of preparation of the test substance (e.g., stock solution preparation) and the concentrations of test substance used in limit and definitive testing. If vehicles are used, provide the name, composition, and source of the vehicle, the nominal concentration of the test substance in the vehicle, and the vehicle concentration(s) used in the test.
            (v) Information about the test organisms: common name and scientific name, method of species verification, source, life stage, sex (if appropriate), preventative or disease treatments, feeding history, acclimation procedures, and culture method.
            (vi) Description of the test system used in definitive, limit, or any preliminary testing. This includes a description of the test vessels, volume of solution in the test vessels, method of test substance introduction or test substance delivery system, renewal schedule (static-renewal tests), renewal schedule for stock feeding the diluter system (flow-through tests), system flow rate expressed as volume additions per 24 hours (flow-through tests), number of test organisms per test vessel, number of replicates per control and treatment level, all environmental parameters, and description of any feeding during the test (if applicable) including type of food, source, amount given, and frequency.
            (vii) Description of all techniques used in stock solution preparation (e.g., shaking, stirring, sonication, heating, solvent, etc.) and the appearance of the stock solution.
            (vii) Methods and frequency of sampling test media for the test substance and degradates and media sampled. Report the frequency of primary stock and secondary stock solution preparation, and timing of sampling from aquaria in relationship to replacement of stock solutions feeding diluter system (flow-through tests).
            (viii) Description of all analytical chemistry methods used in preliminary trials in establishing percent purity of batches of test substance or in measuring concentrations in range-finding, limit, and definitive tests. Include a complete description of the method so that a bench chemist can independently determine the necessary equipment and perform the analysis. Also include the raw data, standards, quality control samples, and chromatograms from samples taken during either range-finding, definitive, or limit tests, not of standards or samples from recovery tests. Provide the accuracy of the method, LOD, MDL, and LOQ.
            (viii) Results of measurements of test substance. Provide a justification for, or validation of, the separation technique should be provided in the study report. 
            (x) Methods, frequency, and results of environmental monitoring performed during the test (e.g., temperature, dissolved oxygen, pH, water hardness (freshwater), salinity (saltwater), lighting, photoperiod) and other records of test conditions. 
            (xi) Biological observations reported in sufficient detail to allow complete independent evaluation of the results (see specific test guidelines in this group for specific measures of effect to be reported). All data developed during the study that are suggestive or predictive of toxic effects and all concomitant gross toxicological manifestations should be submitted.
            (xii) 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, a measure of the sensitivity of hypothesis tests (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 vessel and treatment randomization as well as methods for random or indiscriminate assignment of test organisms to test vessels.
 (l) References.
      (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. ASTM E729-96, Standard Guide for Conducting Acute Toxicity Tests with Fishes, Macroinvertebrates, and Amphibians. In Annual Book of ASTM Standards, Vol. 11.06, American Society for Testing and Materials, West Conshohocken, PA. Current edition reapproved October 1, 2014.
      (3) American Society for Testing and Materials. ASTM E1847-96, Standard Practice for Statistical Analysis of Toxicity Tests Conducted Under ASTM Guidelines. In Annual Book of ASTM Standards, Vol. 11.06, American Society for Testing and Materials, West Conshohocken, PA. Current edition reapproved March 1, 2013.
      (4) American Society for Testing and Materials. ASTM E1733-95, Standard Guide for the Use of Lighting in Laboratory Testing. In: Annual Book of ASTM Standards, Vol. 11.06, West Conshohocken, PA. Current edition reapproved October 1, 2014.
      (5) American Society for Testing and Materials. ASTM D1193-06. Standard Specification for Reagent Water. In: Annual Book of ASTM Standards, Vol. 11.01, West Conshohocken, PA. Current edition reapproved May 1, 2011. 
      (6) American Society for Testing and Materials. ASTM D5907-13. Standard Test Methods for Filterable Matter (Total Dissolved Solids) and Nonfilterable Matter (Total Suspended Solids) in Water. In: Annual Book of ASTM Standards, Vol. 11.01, West Conshohocken, PA. Current edition reapproved June 1, 2013. 
      (7) Bruce, R.D. and D.J. Versteeg, 1992. A statistical procedure for modeling continuous toxicity data. Environmental Toxicology and Chemistry 11: 1485-1491.
      (8) Chapman, G.A., B.S. Anderson, A.J. Bailer, R.B. Baird, R. Berger, D.T. Burton, D.L. Denton, W.L. Goodfellow, M.A. Heber, L.L. McDonald, T.J. Norberg-King and P.J. Ruffier, 1996. Methods and appropriate endpoints. In Whole Effluent Toxicity Testing, D.R. Grothe, K.L. Dickson and D.K. Reed-Judkins, eds., SETAC Press, Pensacola, FL. 
      (9) Daum, R.J., 1970. Revision of two computer programs for probit analysis. Bulletin of the Entomological Society of America 16:10-15.
      (10) Daum, R.J. and W. Killcreas, 1966. Two computer programs for probit analysis. Bulletin of the Entomological Society of America 12:365-369.
      (11) deBruijn, J.H.M. and M. Hof, 1997. How to measure no effect. Part IV: How acceptable is the ECx from an environmental policy point of view? Environmetrics 8:263-267.
      (12) Fairweather, P.G., 1991. Statistical power and design requirements for environmental monitoring. Aust. J. Mar. Freshwater Res. 42:555-567. 
      (13) Finney, D.J., 1971. Probit Analysis 3rd ed., Cambridge: London and New York.
      (14) Hutchinson, T.H., N. Shillabeer, M.J. Winter and D.B. Pickford, 2006. Acute and chronic effects of carrier solvents in aquatic organisms: A critical review. Aquatic Toxicology 76, 69-92.
      (15) Litchfield, J.T., Jr. and F. Wilcoxon, 1949. A simplified method of evaluating dose-effect experiments. Journal of Pharmacological Experimental Therapy 96:99-133.
      (16) Nabholz, J.V., 1991. Environmental hazard and risk assessment under the Toxic Substances Control Act. Science of the Total Environment 109/110: 649-665.
      (17) Nabholz, J.V., P. Miller and M. Zeeman, 1993. Environmental risk assessment of new chemicals under the Toxic Substances Control Act (TSCA) Section 5, In Environmental Toxicology and Risk Assessment, Landis, W.G., Hughes, J.S., and Lewis, M.A., eds., ASTM STP 1179, American Society for Testing and Materials, Philadelphia, PA, pp. 40 -55.
      (18) Nyholm, N., P.S. Sorenson, K.O. Kusk and E.R. Christensen, 1992. Statistical treatment of data from microbial toxicity tests, Environmental Toxicology and Chemistry 11:157-167.
      (19) Organization for Economic Co-operation and Development, 1998. Report of the OECD Workshop on Statistical Analysis of Aquatic Toxicity Data. OECD Series on Testing and Assessment, No. 10. ENV/MC/CHEM(98)18. 
      (20) Organization for Economic Co-operation and Development, 2000. Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures. OECD Series on Testing and Assessment, No. 23. ENV/JM/MONO (2000)6. 
      (21) Organization for Economic Co-Operation and Development, 2006. Current Approaches in the Statistical Analysis of Ecotoxicity Data: A Guidance to Application. OECD Series on Testing and Assessment, No. 54. ENV/JM/MONO(2006)18. 
      (22) Organization for Economic Co-Operation and Development, 1998. OECD Series on Principles of Good Laboratory Practice and Compliance Monitoring. No. 1. OECD Principles on Good Laboratory Practice (as revised in 1997). ENV/MC/CHEM(98)17.
      (23) Pack, S., 1993. A review of statistical data analysis and experimental design in OECD aquatic toxicology test guidelines. Report to OECD. Paris.
      (24) Rand, G.M., ed., 1995. Fundamentals of Aquatic Toxicology: Effects, Environmental Fate, Risk Assessment, 2[nd] Edition. Taylor and Francis Publishers, Washington, DC, 1125 pp. 
      (25) Smith, M.E., J.M. Lazorchak, L.E. Herrin, S. Brewer-Swartz and W.T. Thoney, 1997. A reformulated, reconstituted water for testing the freshwater amphipod, Hyalella azteca, Environ. Toxicol. Chem. 16:1229-1233. 
      (26) Smrchek, J.C., R. Clements, R. Morcock, and W. Rabert, 1993. Assessing ecological hazard under TSCA: methods and evaluation of data, In Environmental Toxicology and Risk Assessment, Landis, W.G., Hughes, J.S., and Lewis, M.A., eds., ASTM STP 1179, American Society for Testing and Materials, Philadelphia, PA, pp. 22-39.
      (27) Smrchek, J.C. and M.G. Zeeman, 1998. Assessing risks to ecological systems from chemicals. In Handbook of Environmental Risk Assessment and Management, P. Calow, ed., Blackwell Science, Ltd., Oxford, UK, pp. 24-90, Chapter 3. 
      (28) Stephan, C.E., 1997. Methods for calculating an LC50. In Aquatic Toxicology and Hazard Evaluation, ASTM STP 634, F.L. Mayer and J.L. Hamelink, eds., American Society for Testing and Materials, Philadelphia, PA. 
      (29) U.S. Department of Agriculture (USDA), 2001. Information Resources on Amphibians, Fish & Reptiles Used in Biomedical Research. 
      (30) U.S. Environmental Protection Agency, 1989. Ambient Water Quality Criteria for Ammonia: Saltwater, EPA 440/5-88-004.
      (31) U.S. Environmental Protection Agency, 1994. Pesticides Reregistration Rejection Rate Analysis: Ecological Effects, EPA 738-R-94-035, Office of Prevention, Pesticides and Toxic Substances, December 1994. 
      (32) U.S. Environmental Protection Agency, 1997. Terms of Environment, Glossary, Abbreviations, and Acronyms, Communications, Education, and Public Affairs, EPA 175-B-97-001, December 1997.
      (33) U.S. Environmental Protection Agency, 1999. 1999 Update of Ambient Water Quality Criteria for Ammonia, EPA-822-R-99-014.
      (34) U.S. Environmental Protection Agency, 2000. Methods for Measuring the Toxicity and Bioaccumulation of Sediment-Associated Contaminants with Freshwater Invertebrates, Second Edition, EPA 600/R-99/064, March 2000. 
      (35) U.S. Environmental Protection Agency, 2002. Methods for measuring the acute toxicity of effluents and receiving waters to freshwater and marine organisms, Fifth edition, October 2002. EPA-821-R-02-012.
      (36) U.S. Environmental Protection Agency, 2002. Short-term methods for estimating the chronic toxicity of effluents and receiving waters to freshwater organisms, Fourth edition. EPA-821-R-02-013.
      (37) U.S. Environmental Protection Agency, 2002. Short-term methods for estimating the chronic toxicity of effluents and receiving waters to marine and estuarine organisms, Third edition. EPA-821-R-02-014.
      (38) U.S. Environmental Protection Agency, Code of Federal Regulations (CFR) Title 40 - Pesticide Programs Subchapter E - Pesticide Programs. Part 158 - Data Requirements for Pesticides. 
      (39) VanEwijk, P.H. and J.A. Hoekstra, 1993. Calculation of the EC50 and its confidence interval when a subtoxic stimulus is present. Ecotoxicology and Environmental Safety 25:25-32.
      (40) Zeeman, M. and J. Gilford, 1993. Ecological hazard evaluation and risk assessment under EPA's Toxic Substances Control Act (TSCA): an introduction. In Environmental Toxicology and Risk Assessment, Landis, W.G., Hughes, J.S., and Lewis, M.A., eds., ASTM STP 1179, American Society for Testing and Materials, Philadelphia, PA, pp. 7-21. 
      (41) Zeeman, M.G., 1995. Ecotoxicity testing and estimation methods developed under Section 5 of the Toxic Substances Control Act (TSCA), In Fundamentals of Aquatic Toxicology, 2[nd] Edition, G.M. Rand, ed., Taylor and Francis, Washington, DC, pp. 703-715. 
      
