United States Environmental Protection Iml M m Agency Office of Chemical Safety EPA 712-C-16-010 and Pollution Prevention October 2016 (7101) Ecological Effects Test Guidelines OCSPP 850.1025: Oyster Acute Toxicity Test (Shell Deposition) ------- NOTICE This guideline is one of a series of test guidelines established by the United States Environmental Protection Agency's Office of Chemical Safety and Pollution Prevention (OCSPP) for use in testing pesticides and chemical substances to develop data for submission to the Agency under the Toxic Substances Control Act (TSCA) (15 U.S.C. 2601, et seq.), the Federal Insecticide, Fungicide and Rodenticide Act (FIFRA) (7 U.S.C. 136, et seq.), and section 408 of the Federal Food, Drug and Cosmetic 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. The OCSPP harmonized test guidelines serve as a compendium of accepted scientific methodologies and protocols that are intended to provide data to inform regulatory decisions under TSCA, FIFRA, and/or FFDCA. This document provides guidance for conducting the test, and is also used by EPA, the public, and the companies that are subject to data submission requirements under TSCA, FIFRA, and/or the FFDCA. As a guidance document, these guidelines are not binding on either EPA or any outside parties, and the EPA may depart from the guidelines where circumstances warrant and without prior notice. At places in this guidance, the Agency uses the word "should." In this guidance, the use of "should" with regard to an action means that the action is recommended rather than mandatory. The procedures contained in this guideline are strongly recommended for generating the data that are the subject of the guideline, but EPA recognizes that departures may be appropriate in specific situations. You may propose alternatives to the recommendations described in these guidelines, and the Agency will assess them for appropriateness on a case-by-case basis. For additional information about these test guidelines and to access these guidelines electronically, please go to http://www.epa.gov/ocspp and select "Test Methods & Guidelines" on the 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-QPPT-2009-0576. l ------- OCSPP 850.1025: Oyster acute toxicity test (shell deposition) (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.) and the Toxic Substances Control Act (TSCA) (15 U.S.C. 2601, et seq.). It describes procedures that, if followed, would result in data that would generally be of scientific merit for the purposes described in paragraph (b) of this guideline. (2) Background. The source materials used in developing this harmonized OCSPP test guideline are 40 CFR 797.1800 Oyster Acute Toxicity Test; OPP 72-3 Acute Toxicity Test for Estuarine and Marine Organisms (Pesticide Assessment Guidelines, Subdivision E — Hazard Evaluation; Wildlife and Aquatic Organisms, see paragraph (j)(3) of this guideline); Standard Evaluation Procedure: Acute Toxicity Test for Estuarine and Marine Organisms (Mollusc 96-Hour Flow-Through Shell Deposition Study) (see paragraph (j)(4) of this guideline); EPA Pesticide Reregi strati on Rejection Rate Analysis: Ecological Effects (see paragraph (j)(5) of this guideline); and ASTM E729-96 Standard Guide for Conducting Acute Toxicity Tests on Test Materials with Fishes, Macroinvertebrates, and Amphibians (see paragraph (j)(l) of this guideline). (b) Purpose. This guideline is intended for use in developing data on the acute toxicity of chemical substances and mixtures ("test chemicals" or "test substances") subject to environmental effects test regulations. This guideline describes an acute toxicity test in which Eastern oysters, Crassostrea virginica (Gmelin), are exposed to a test substance in a flow- through system. The Environmental Protection Agency will use data from this test to assess the hazards and risks a test substance may present in the aquatic environment. (c) Definitions. The definitions in OCSPP 850.1000 apply to this test guideline. In addition, the following more specific definitions apply to this guideline: Shell deposition is the measured length of shell growth that occurs between the time the shell is ground at test initiation and test termination 96 hours later. The measurement is made from the peripheral ground edge to the tip of the longest finger of newly deposited shell. In this guideline, a reduction in shell deposition is used as the measure of toxicity. Inhibitory concentration, median (IC50) is the experimentally derived concentration of test substance in dilution water that would be expected to cause a 50 percent (%) decrease or inhibition effect on growth of a group of test organisms under specified exposure conditions. In this guideline, the effect measured is inhibition of shell deposition. Umbo is the narrow end (apex) of the oyster shell. Valve height is the greatest linear dimension of the oyster as measured from the umbo to the ventral edge of the valves (the farthest distance from the umbo). 1 ------- (d) General considerations. (1) Summary of the test. Oysters (Crassostrea virginica), which previously had a portion of the shell periphery ground away, are exposed to the test substance and to appropriate controls {i.e., dilution water control and vehicle (solvent) control, if a vehicle is used) for 96 hours, during which observations of any toxic effects are made on the organisms. At test termination, the increment of new shell growth is measured on all live oysters. The results of the test are expressed as the shell deposition 96-hour median inhibition concentration (96-h IC50), i.e., the concentration resulting in a 50% decrease or inhibition effect on growth, and the slope of the concentration-response relationship. Although the 96-h IC50 for shell deposition is the primary toxicity endpoint, information on other signs of toxicity such as abnormal appearance and behavior and concentration- response curves is useful in understanding the toxic response. (2) General test guidance. The general guidance in OCSPP 850.1000 applies to this guideline except as specifically noted herein. (3) Range-finding test. A range-finding test is usually conducted to establish the appropriate test solution concentrations to be used for the definitive test. In the range- finding test, the test organisms are generally exposed to a series of widely-spaced concentrations of the test substance {e.g., 1, 10, 100 milligrams per liter (mg/L)). The details of the range-finding test do not have to be the same as those of definitive testing in that the number of replicates, the number of test organisms, and duration of exposure may be less than that used in definitive testing. In addition, the types of observations made on test organisms may not be as detailed or as frequently observed as that of a definitive test. (4) Definitive test. The primary goal of the definitive test is to determine the 96-hour concentration-response curve for shell deposition; the 96-h IC50, its standard error and 95% confidence interval; and the slope of the concentration-response curve, its standard error and 95% confidence interval. A minimum of 5 concentrations of the test substance, plus appropriate controls, should be tested. Concentrations should be selected so that: very little shell growth relative to control(s) occurs in oysters at the highest test concentration; shell growth is slightly less than control(s) at the lowest test concentration; and there are at least two additional test concentrations that bracket the 96-h IC50. Clinical signs of toxicity such as abnormal appearance and behavior, if any, should be reported. Analytical confirmation of dissolved test concentrations should be performed as described in OCSPP 850.1000. Summaries of the test conditions are presented in Table 1 of this guideline. Test validity elements are listed in Table 2. (5) Limit test. In some situations, it is only necessary to ascertain that the shell deposition 96-h IC50 is above a certain limit {i.e., 96-h IC50 greater than (>) limit concentration). In a limit test, at least 32 oysters, divided into 4 replicates of 8 organisms each, are exposed to a single "limit concentration," with the same number of organisms in appropriate controls. For most industrial chemicals, the lower of 100 mg/L or the limits of water solubility or dispersion is considered appropriate as the limit concentration. For pesticides, the lower of 100 milligrams active ingredient per liter (mg a.i./L), when estimated environmental concentrations are not expected to exceed 100 mg/L, or the limit 2 ------- of water solubility may be used as the limit concentration. Except for the number of test concentrations and the number of replicates, limit tests should follow the same test procedures, have the same duration as the multiple-concentration definitive test (see Table 1 of this guideline), and have both a dilution water control and a vehicle (solvent) control, if a vehicle is used. Limit tests, like definitive tests, should include analytical confirmation of the dissolved concentration of the test substance. Clinical signs of toxicity such as abnormal appearance and behavior, if any, should be reported. If the effect level for the inhibition of shell growth at the limit concentration compared to the control(s) is 50% or greater, then a multiple-concentration 96-hour test should be conducted. For pesticides, if there is a statistically significant reduction (inhibition) in shell growth at the limit concentration as compared to the control(s) (i.e., no observed effect concentration (NOEC) less than (<) limit concentration), a multiple-concentration 96-hour test should be conducted. (e) Test standards. (1) Test substance. The substance to be tested should be technical or reagent grade unless the test is designed to evaluate a specific formulation, mixture, or end-use product. For pesticides, if more than one active ingredient constitutes a technical product, the technical grade of each active ingredient should be tested separately, in addition to the combination, if applicable. OCSPP 850.1000 lists the type of information that should be known about the test substance before testing and discusses methods for preparation of test solutions. (2) Test duration. The test duration is a minimum of 96 hours. (3) Test organism. (i) Species. The test species is the Eastern oyster, Crassostrea virginica. Oysters used in the same test should be 30 to 50 millimeters (mm) in valve height and should be as similar in age and size as possible to reduce variability. The standard deviation of the valve height should be <20% of the mean. Small individuals are preferred because they are less affected by temperature and thus continue to grow in spite of temperature fluctuations as opposed to older individuals that tend to become less active at temperature extremes. Oysters should be in a pre-spawn condition of gonadal development prior to and during the test. This may be determined by direct or histological observation of the gonadal tissue for the presence of gametes or may be inferred based upon size and condition. If evidence of spawning is observed during the test, the test should be repeated. Oysters may be cultured in the laboratory, purchased from culture facilities or commercial harvesters, or collected from a natural population in an unpolluted area free from epizootic diseases. Records should be kept regarding their source and/or culturing techniques. Oysters used in the test should be from the same source and from the same holding and acclimation tanks. 3 ------- (ii) Holding and acclimation. Oysters should be attended to immediately upon arrival at the test facility. Oyster shells should be brushed clean of fouling organisms. Oysters should be held and acclimated in dilution water from the same origin as that used for testing. If the organisms arrive in water, the transfer of the oysters to the holding water should be gradual to reduce stress caused by differences in water quality characteristics and temperature. Oysters should be held for a period of time long enough to demonstrate that they are not diseased or stressed and are capable of obtaining minimum control shell growth after 96 hours. Holding times of 10 to 12 days before testing have been used. During holding and acclimation, the oysters should not be crowded, and the dissolved oxygen concentration should be above 60% saturation. Holding tanks should be kept clean and free of debris. All oysters should be maintained in dilution water at the test temperature for at least 48 hours before they are used. Mortalities should be recorded, and the following recommendations should be applied: (A) Mortalities of greater than 10% of the population in the 7 days directly preceding the test: rejection of entire batch; (B) Mortalities of between 5 and 10% of the population during the 7 days directly preceding the test: acclimation/holding continued for additional 7 days; (C) Mortalities of less than 5% of the population during the 7 days directly preceding the test: acceptance of batch. (iii) Health status and condition. Oysters should not receive treatment for disease during a test. Oysters should not be used in a test: (A) If more than 5% of the culture or stock dies or shows signs of stress (e.g., exhibits abnormal behavior; excessive mucus) during the 48 hours preceding the test; (B) If the shell is cracked, chipped, bored, or gaping; (C) If the adductor muscle is damaged during grinding; (D) If so much of the shell rim has been removed after grinding that an opening into the shell cavity is visible; (E) If infested with mudworms (Polydora sp.) or boring sponges (Cliona celata); (F) If they have been used in a previous test, either in a treatment or in a control group. 4 ------- (iv) Care and handling. Oysters should be handled as little as possible, but when necessary, it should be done as carefully and as quickly as possible. To prepare each oyster for testing, within 24 hours of test initiation (preferably within 1-2 hours of test initiation), approximately 3 to 5 mm of the shell periphery should be ground away at the rounded (ventral) end using a small electric disc grinder or other appropriate device, taking care to remove the shell rim uniformly to produce a smooth, rounded, blunt profile. The oyster's valves should be held together tightly during grinding to avoid vibrating the shell and injuring the adductor muscle. (v) Diet and feeding. Oysters should be provided enough food to support survival and growth during holding, acclimation, and testing. Holding and acclimating oysters in natural seawater that is not expected to contain disease-causing organisms at adverse concentrations and contains as much natural phytoplankton as possible is advantageous. Cultured algae may be added to the water as necessary to support oyster survival and growth. If natural seawater that is not supplemented with an additional food source is used, it should not be passed through an ultraviolet sterilizer or a filter of <20 micrometers (|im). If unsterilized and unfiltered natural seawater is used without adding algae, at least 1 liter per hour per individual (L/h/individual) is usually the minimum flow rate for mollusks of the size 40-50 mm (umbo to distal valve edge) to provide an adequate food supply that supports the desired growth rate. If the presence of disease- causing organisms is suspected in natural seawater, then filtration through a smaller sized filter and addition of a supplemental algal source to the test system may be necessary. (4) Administration of test substance. (i) Preparation of test solutions. Preparation of test solutions depends on the solubility and stability of the test substance. Guidance for preparation of test solutions, especially for difficult or low solubility test substances, is provided in OCSPP 850.1000. Dilution water source and quality used in the test are described in OCSPP 850.1000 and paragraph (e)(7)(vi) of this guideline. The concentration of vehicle solvent should not exceed 0.1 milliliters per liter (mL/L). A previous review recommends that solvent concentrations as low as 0.02 mL/L of dilution water be used (see paragraph (j)(2) of this guideline). 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 (HC1) and sodium hydroxide (NaOH) may be used for this adjustment if warranted. See additional information about pH during testing in (e)(8)(ii). 5 ------- (ii) Exposure technique. The test should be conducted using the flow-through exposure technique. Additional guidance on flow-through exposures is provided in OCSPP 850.1000. (iii) Treatment concentrations. At least 5 test solution concentrations should be for definitive testing, plus the appropriate control(s). A range-finding test can be used to establish the appropriate test solution concentrations for the definitive test (see paragraph (d)(3) of this guideline). For scientifically sound estimates of a given point estimate (e.g., IC50), test substance concentrations should immediately bracket the point estimate(s) of concern. OCSPP 850.1000 provides guidance on selection of test concentrations. For a limit test, there is a single treatment concentration, plus the appropriate control(s). Guidance on the limit concentration is provided in paragraph (d)(5) of this guideline. (5) Controls. Every test includes a dilution water control and a vehicle (solvent) control, if a vehicle is used. Controls consist of the same dilution water, conditions, procedures, and test population as the test solutions, except that no test substance is added. A test is not acceptable if: (i) More than 10% of the organisms in any control showed signs of disease, stress (e.g., abnormal behavior, excessive mucus), and/or death. (ii) An overall mean of at least 2 mm of new shell growth (i.e., overall mean of all replicates) was not observed in each control group (dilution water control and vehicle (solvent) control, if a vehicle was used). (6) Number of test organisms and replicates. For definitive tests, the minimum number of test organisms per test concentration is 20 divided into a minimum of 2 replicates, each with 10 oysters. For limit tests, the minimum number of test organisms is 32 divided into 4 replicates, each with 8 oysters. Each test vessel should contain an equal volume of test solution and an equal number of oysters. Replicate test vessels should be physically separated, since the test vessel is the experimental unit. (i) Loading. The loading rate should not crowd oysters and should permit adequate circulation of water around each shell while avoiding physical agitation of the oysters by water currents. Flow rates should be sufficient to promote adequate oyster shell growth and maintain environmental conditions. A flow rate of 1 L/h/individual has been demonstrated to allow for adequate shell growth and is recommended as the minimum flow rate if using unfiltered natural seawater that is not supplemented with additional algae. (ii) Introduction of test organisms. The test should be started by introducing oysters that have been acclimated to test conditions and have had approximately 3 to 5 mm of the shell periphery ground away at the rounded (ventral) end into the test vessels after the test system has equilibrated. Within a test vessel, the oysters should be spread out equidistant from one another so that the entire test vessel is used. The oysters should all be placed with the left (cupped) valve down and the 6 ------- open, unhinged ends all oriented in the same direction facing the incoming flow of test solution. Test vessels for treatment levels should be randomly or indiscriminately located within the test area, and test organisms should be randomly or indiscriminately distributed among test vessels. Further guidance is provided in OCSPP 850.1000. (7) Facilities, apparatuses, and supplies. Normal laboratory equipment should be used, especially the following: (i) Facilities. Facilities for culturing, holding, acclimating, and testing oysters that are well ventilated and free of fumes and disturbances which may affect the test organisms. There should be flow-through tanks for holding and acclimating oysters and a system for culturing algae, if prepared on-site. (ii) Environmental control equipment. Mechanisms for controlling and maintaining the water temperature, lighting, salinity, and flow during the culturing, holding, acclimation and test periods. Apparatus for aerating dilution water and removing gas bubbles as necessary. Apparatus for aerating the dilution water in the head box before mixing with the test substance or delivery to test vessels. An apparatus providing a 30-minute lighting transition period may be needed. (iii) Water quality testing instruments. Equipment for determination of water quality characteristics (pH, salinity, temperature, etc). (iv) Cleaning of test system. Test substance delivery systems and test vessels should be cleaned before each test. See OCSPP 850.1000 for further information. (v) Test containers and delivery system. Construction materials and equipment that may contact the stock solution, test solution, or dilution water 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 or test solutions should be chosen to minimize sorption of test substances. Refer to OCSPP 850.1000 for additional information on appropriate construction materials. Test vessels, which should be constructed of chemically inert material, should be of a capacity to maintain the loading rate and environmental conditions. Test vessels should be loosely covered to reduce the loss of test solution or dilution water due to evaporation and to minimize entry of dust and other particles into the solutions. The flow-through system should contain an appropriate test substance delivery system. (vi) Dilution water. A dilution water is acceptable if oysters will survive in it for the duration of the culturing, holding, acclimation, and testing periods without showing signs of stress. Clean natural unfiltered seawater may be used; such water should come from a thoroughly mixed common source to ensure each oyster is provided equal amounts of food. Clean artificial seawater or filtered natural seawater with food (algae) added may be used especially if the presence of 7 ------- disease organisms is suspected in natural seawater. Natural seawater should be filtered through a filter with a pore size of <20 |im prior to use in a test. Artificial seawater can be prepared by adding commercially available formulations or specific amounts of reagent-grade chemicals to reagent water (deionized, distilled, or reverse osmosis water), surface water, or well water. Dechlorinated tap water is not recommended for preparation of artificial seawater (or dilution of natural seawater) because some forms of chlorination are difficult to remove adequately. If dechlorinated tap water is used, recommended maximum chlorine levels as well as other ways to demonstrate suitability as a dilution water source are in OCSPP 850.1000. The recommended salinity is 20 parts per thousand (ppt). However, if unfiltered natural seawater is used, greater variability may be unavoidable but still allow for adequate control performance due to the euryhaline life history of oysters. For unfiltered natural seawater that is not diluted with freshwater to reduce salinity, salinity of >12 ppt is recommended with a weekly range in salinity of <5 ppt. For artificial seawater or natural seawater that is diluted with freshwater, salinity should be maintainable within a weekly range of 2 ppt. Dissolved oxygen in the dilution water (prior to use in a test) should be between 90 and 100% saturation. If necessary, the dilution water can be aerated before the addition of the test substance. Measurement of total organic carbon (TOC) or chemical oxygen demand (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. Specifications for dilution water quality and constancy are described in OCSPP 850.1000. (8) Environmental conditions. Environmental parameters during the test should be maintained as specified below. The number and frequency of measurements recommended for documenting and confirming the magnitude and variability of water quality parameters (e.g., temperature, dissolved oxygen, pH, and salinity) in test solutions during the test are described in detail in OCSPP 850.1000. (i) Temperature. The recommended water temperature is 20 °C. During a given test, the temperature should be constant within plus or minus (±) 2 °C. However, if unfiltered natural seawater that has not been previously held is used, temporary fluctuations (less than 8 hours) of ±5 °C may occur and be tolerated by oysters (i.e., not affect control performance) due to their adaptations to fluctuating tidal habitats. 8 ------- (ii) pH and salinity. The pH should be between 7.5 and 8.5 and vary less than 1 pH unit during the test within a test vessel and between test concentrations (including control(s)). For unfiltered natural seawater that is not diluted with freshwater to reduce salinity, a salinity of >12 ppt, with a range of <5 ppt, is recommended. For artificial seawater or natural seawater that is diluted with freshwater, salinity should be 20 ppt and constant within ±2 ppt during the test. (iii) Lighting and photoperiod. A photoperiod should be selected from regimes of 12 hours light: 12 hours dark to 16 hours light: 8 hours dark. For any given test, the light regime should be constant. Light intensity should range from 540 to 1080 lux (approximately 50-100 foot-candles (ft-c)). A 15- to 30-minute transition period between light and dark is suggested. (iv) Dissolved oxygen. The dissolved oxygen concentration should be between 60 and 100% saturation during the test. If aeration is needed to achieve an appropriate dissolved oxygen level, it should be done before addition of the test substance. The dilution water may be aerated vigorously prior to delivery to the test vessels (e.g., in the diluter head box) such that the dissolved oxygen concentration is at or near 90 to 100% saturation. If the water is heated, precautions should be taken to ensure that supersaturation of dissolved gases is avoided. Aeration of the test solutions during the test is not recommended. Gentle aeration of test vessels during the exposure period is permitted only in cases where the dissolved oxygen levels are in danger of dropping below 60% saturation. In such cases, assurances should be made that the use of aeration does not stress the test organisms; test substance concentrations should be measured during the test to ensure that they are not affected by the use of aeration; and all treatment and control vessels should be given the same aeration treatment. (v) Flow in a flow-through system. During a test, the flow rates should not vary more than 10% between any one replicate and another. The flow rate should be sufficient to promote adequate oyster shell growth and maintain environmental conditions. A flow rate of 1 L/h/individual has been shown to provide adequate environmental conditions. If unfiltered natural seawater that is not supplemented with additional algae is used, then the recommended flow rate is 1 L/hour/individual. It is recommended that diluter systems be monitored for proper adjustment and operation at least twice daily throughout the test period to better ensure that the target test concentrations are achieved and maintained. The flow rate to each test vessel should be measured at the beginning and end of the test. (9) Observations. (i) Measurement of test substance. OCSPP 850.1000 describes the recommended sampling methods, frequency of sampling, and sample processing (especially of low solubility test substances) for analytical confirmation of dissolved test concentrations and characterization of test substance stability throughout the test. The analytical methods used to measure the amount of dissolved test substance in a sample should be validated before beginning the test, 9 ------- as described in OCSPP 850.1000, and the relevant method detection limit(s) and limit(s) of quantification should be reported. (ii) Test solution appearance. Observations on test solution appearance and test substance solubility should be made daily and at the beginning and end of the test. The appearance of surface slicks, precipitates, or material adhering to the sides of the test vessels or in any part of the mixing and delivery system should be recorded at a minimum at the beginning and end of the test and during the test when the test solution appearance changes. (iii) Measures of effect. (A) Mortality. The oysters should be inspected at least after 24, 48, 72, and 96 hours. An observation period at <12 hours is desirable. Oysters should be considered dead if touching of the gaping shell produces no reaction. Dead oysters should be removed from the test vessels at the time of observation, and mortalities should be recorded. (B) Appearance and behavior. Visible abnormalities such as loss of feeding activity (failure to deposit feces), excessive mucus production (stringy material floating suspended from oysters), spawning, or closure or gaping of shell should be recorded. (C) Shell growth. Shell growth is the primary criterion used in this test to evaluate the toxicity of the test substance. Shell growth increments in all oysters should be measured after 96 hours of exposure. The length of the longest "finger" of new shell growth, from the ground edge to the tip, should be recorded to the nearest 0.1 mm. (f) Treatment of results. (1) Summary statistics. (i) Mortality and shell growth. The number of oysters exposed at test initiation in each treatment and replicate and the cumulative number of dead oysters should be summarized in tabular form by time of observation, treatment, and replicate. Shell growth (mean, standard deviation) after 96 hours of exposure should be summarized in tabular form by treatment and replicate. (ii) Appearance and behavior. The number of oysters exhibiting abnormal appearance or behavioral symptoms should be summarized in tabular form by time of observation, treatment, and replicate. (2) Percent mortality and shell growth inhibition. (i) The percent mortality at each treatment level and in the controls at 24, 48, 72, and at test termination (96 hours) should be calculated. 10 ------- (ii) The mean percent inhibition of shell growth at each treatment level compared to the control(s) at test termination (96 hours) should be calculated. (3) Evaluation of limit test results. If the effect level for the inhibition of shell growth at the limit concentration compared to the control(s) is 50% or greater, then a multiple- concentration 96-hour test should be conducted. For pesticides, if there is a statistically significant reduction (inhibition) in shell growth at the limit concentration compared to the control(s) (i.e., NOEC < limit concentration), then a multiple-concentration 96-hour test should be conducted. (4) Evaluation of multiple-concentration definitive test. (i) Concentration-response curve, slope, and ICso. Statistical procedures should be employed to calculate the 96-h IC50 (standard error and 95% confidence interval) based on reduction in shell deposition. If a concentration-response curve model was fit to the data to determine the IC50, the model parameters (e.g., slope) and their uncertainty estimates (e.g., standard error, 95% confidence interval) should be recorded. (ii) NOEC. While calculation of the NOEC and lowest observed effect concentration (LOEC) is usually not part of the experimental design for regression-based definitive tests, reporting these values when possible is useful when testing industrial and pesticide chemicals for understanding the toxic response. The limit test for shell deposition is designed for hypothesis testing. (iii) Statistical methods. Statistical procedures for modeling continuous data should be used. Additional discussion about endpoints and statistical procedures can be found in OCSPP 850.1000. (g) Tabular summary of test conditions. Table 1 lists the important conditions that should prevail during the multiple-concentration definitive test. The same conditions are recommended for a limit test, except for differences in the number of test concentrations and replicates. Meeting these test conditions will help ensure the satisfactory performance of the test. Table 1.—Summary of Test Conditions for Oyster Acute Toxicity Test (Shell Deposition) Test type Flow-through Test species Crassostrea virginica Test duration 96 hours Temperature 20 °C (constant during test within ±2 °C) However, if unfiltered natural seawaterthat has not been previously held is used, temporary fluctuations (less than 8 hours) of ±5 °C may occur and be tolerated by oysters (i.e., not affect control performance) due to their adaptations to fluctuating tidal habitats. Light quality Ambient laboratory illumination Light intensity 540-1080 lux (approximately 50-100 ft-c) 11 ------- Photoperiod Selected from among 12 hours light: 12 hours dark to 16 hours light:8 hours dark schemes Salinity Artificial or natural seawaterthat is diluted with freshwater: 20 ppt (range of ±2 ppt during test); Unfiltered natural seawaterthat is not diluted with freshwater to reduce salinity: >12 ppt (range of <5 ppt during test) PH Between 7.5 and 8.5 (constant during test within ±1 pH unit) TOC <2 mg/L Size of test organisms 30-50 mm in valve height; similar in age and size; standard deviation of valve height <20% of the mean Number of organisms per concentration Multiple-concentration definitive test: 20 (minimum) Limit test: 32 (minimum) Number of replicate test vessels per concentration Multiple-concentration definitive test: 2 (minimum) Limit test: 4 (minimum) Loading Flow rate is adequate to promote adequate shell growth and maintain environmental conditions. Flow rate of 1 L/h/individual has been shown to be adequate when using unfiltered natural seawaterthat is not supplemented with additional algae Feeding regime Phytoplankton naturally occurring in the dilution water (if using unfiltered, unsterilized natural seawater) or supplemented (needed if using artificial seawater) Test vessel aeration Not recommended; gentle aeration of test vessels may only be used in cases where the dissolved oxygen levels are in danger of dropping below 60% saturation. In such cases, assurances should be made that the use of aeration does not stress the test organisms; test substance concentrations should be measured during the test; and all treatment and control vessels should be given the same aeration treatment. Test concentrations Definitive test: minimum of 5 test concentrations chosen in a geometric series plus a dilution water control and a vehicle (solvent) control, if a vehicle is used Vehicle concentration, if used <0.1 mL/L for recommended solvents (see OCSPP 850.1000) Measure of effect or measurement endpoint 96-h IC50 based on reduction in shell growth (h) Test validity elements. This test would be considered to be unacceptable or invalid if one or more of the conditions in Table 2 occurred. These parameters are not the only elements considered when evaluating the acceptability of a test, and it is possible that a test could be found unacceptable or invalid based on other considerations. However, except for the conditions listed in Table 2 and in OCSPP 850.1000, it is unlikely that a test will be rejected when there are only slight variations from guideline environmental conditions and test design unless the control organisms are significantly affected and/or significant biases are introduced in defining the magnitude of effect on measurement endpoints as compared to guideline conditions. Before departing significantly from this guideline (such as deviating from the organism size), the investigator should contact the Agency to discuss the reason for the departure and the effect the change(s) may have on test acceptability. In the test report, all departures from the guideline should be identified, reasons for the changes given, and any resulting effects on test endpoints noted and discussed. 12 ------- Table 2. —Test Validity Elements for the Oyster Acute Toxicity Test (Shell Deposition) 1. All test vessels were not identical. 2. Treatments were not randomly or indiscriminately assigned to individual test vessel locations, or individual test organisms were not randomly or indiscriminately assigned to test vessels. 3. A dilution water control (and vehicle (solvent) control, if a vehicle was used) was not included in the test. 4. More than 10% of the organisms in either the dilution water or vehicle (solvent) controls showed signs of disease, stress (e.g., shell gaping, excessive mucus), and/or death. 5. An overall mean of at least 2 mm of new shell growth (i.e., overall mean of all replicates) was not observed in each control group (vehicle (solvent) control and dilution water control). 6. A surfactant or dispersant was used in the preparation of a stock or test solution. (However, adjuvants may be used when testing pesticide typical end-use products.) 7. Evidence of spawning was observed. (i) Reporting. (1) Background information. Paragraph (k)(l) of OCSPP 850.1000 describes the minimum background information to be supplied in the report. (2) Guideline deviations. Provide a statement of the guideline or protocol followed. Include a description of any deviations from the test guideline or any occurrences which may have influenced the results of the test, the reasons for these changes, and any resulting effects on test endpoints noted and discussed. (3) Test substance. (i) Identification of the test substance: common name, IUPAC and CAS names, CAS number, structural formula, source, lot or batch number, chemical state or form of the test substance, purity (i.e., for pesticides, the identity and concentration of active ingredient(s)), and radiolabeling, if any, including the location of label(s) and radiopurity. (ii) Storage conditions of the test chemical or test substance and stability of the test chemical or test substance under storage conditions if stored prior to use. (iii) Methods of preparation of the test substance and the treatment concentrations used in the range-finding and definitive tests, or limit test. Identify whether the nominal concentrations are corrected or uncorrected for purity of the test substance. (iv) Physicochemical properties of the test substance such as water solubility, vapor pressure, UV absorption, pKa, and Kow. (v) If a vehicle (solvent) is used to prepare stock or test substance provide: the name and source of the vehicle, the nominal concentration(s) of the test substance in the vehicle in stock solutions or mixtures, and the vehicle concentration(s) used 13 ------- in the treatments and vehicle control. If different vehicle concentrations are used at different treatment levels, the report should, at a minimum, identify the maximum vehicle concentration used. It is helpful to support the vehicle choice by including a description of any measures that were taken to identify an appropriate vehicle for use in the test, such as the types and concentrations of vehicles used and their corresponding effects on solubility during any preliminary work. (vi) If a positive control is used, provide the name and source of positive control and the nominal concentration(s) of the positive control material in stock solutions or mixtures. (4) Test organism. (i) Scientific name and common name. (ii) Method for verifying the species. (iii) Information about the oysters used in the test: source, culture practices, and holding and acclimation procedures and conditions, including acclimation period, water used, feeding history, and health status (mortality before test initiation and any preventative or disease treatments). (iv) Duration of quarantine for test organisms collected from a natural population. (v) For oysters receiving disease treatment, hours prior to testing with no sign of disease. (vi) Age of test organisms and method of confirmation of prespawn condition. (vii) Size of test organisms at test initiation (i.e., mean valve height). (5) Test system and conditions. Provide a description of the test system and conditions used in the definitive or limit test and any preliminary range-finding tests. (i) Description of the test vessels: size, type, material, and fill volume. (ii) Description of the exposure technique: flow-through, open or closed system. A description of the flow-through system, including flow rate and test vessel turnover rate. For closed systems, a description of the closed system design. For all systems, a description of the calibration and validation methods. (iii) Description of the dilution water and any water pretreatment: source/type; temperature; salinity; pH; dissolved oxygen; total organic carbon or chemical oxygen demand; particulate matter; conductivity; metals, pesticides, and residual chlorine concentrations (mean, standard deviation, range). Describe the frequency and sample date(s) for documenting dilution water quality and consistency. 14 ------- (iv) Use of aeration, if any, and location of aeration within exposure system (e.g. test solution or dilution water prior to test substance addition). (v) Number of test organisms added to each test vessel at test initiation. (vi) Number of test vessels (replicates) per treatment level and control(s). (vii) Methods used for treatment randomization and assignment of test organisms to test vessels. (viii) Date of introduction of test organisms to test solutions and test duration. (ix) Loading rate. (x) Photoperiod and light source. (xi) Detailed information on feeding (e.g., type of feed, source, amount given, and frequency). Feed should be analyzed periodically to identify background contaminants such as heavy metals (e.g., arsenic, cadmium, lead, mercury, and selenium) and persistent pesticides, especially chlorinated insecticides. (xii) Methods and frequency of environmental monitoring performed during the definitive or limit test for test solution temperature, dissolved oxygen, pH, salinity, and light intensity. (xiii) Methods and frequency of measuring the dissolved test substance to confirm exposure concentrations. (xiv) Methods and frequency of measuring shell growth and observing mortality or any other toxic symptoms. (xv) For definitive and limit tests, description of all analytical procedures, accuracy of the method, method detection limit, and limit of quantification. (6) Results. (i) Nominal exposure concentrations and a tabulation of test substance analytical results by treatment group and test vessel (provide raw data) and descriptive statistics (mean, standard deviation, minimum, maximum, coefficient of variation). (ii) Environmental monitoring data results (test solution temperature, dissolved oxygen, pH, salinity, and light intensity) in tabular form (provide raw data for measurements not made on a continuous basis) and descriptive statistics (mean, standard deviation, minimum, maximum). (iii) For preliminary range-finding test, if conducted, a tabulation of shell growth (mm) of each oyster and the number and percentage of dead oysters in each test 15 ------- vessel, for all treatment levels and control(s), at each observation period. A description and count of any other appearance or behavioral effects, if recorded, at each treatment level and in the control(s). (iv) For limit test, a tabulation of shell growth (mm) of each oyster and the number and percentage of dead oysters in each test vessel, for the limit concentration and control(s), at each observation period (provide the raw data) and descriptive statistics (mean, standard deviation, minimum, maximum). (v) For definitive test, a tabulation of shell growth (mm) of each oyster and the number and percentage of dead oysters in each test vessel, for all treatment levels and control(s), at each observation period (provide the raw data) and descriptive statistics (mean, standard deviation, minimum, maximum). (vi) For limit and definitive tests, a description and tabulation of abnormal appearance and behavioral signs of toxicity by test vessel, treatment, and observation time (provide raw data). (vii) The percent inhibition of shell growth for organisms in each replicate test vessel for test concentrations compared to the control(s) at each observation period. (viii) Graphs of the concentration-response data for percent inhibition of shell growth. (ix) For limit test, conclusion about the 96-h IC50 for shell growth being above the limit concentration. (x) For definitive test, where sufficient data exist to fit a model, a tabulation of the 96-hour slope of the concentration-response curve, its standard error and 95% confidence interval, and any goodness-of-fit results. (xi) For definitive test, the 96-h IC50 value for shell growth, and its standard error and 95% confidence interval. (xii) For definitive test, the 96-hour NOEC for shell growth, if determined. (xiii) Description of statistical method(s) used for point estimates, including the software package for determining IC50 values and fitting the concentration- response model, and the basis for the choice of method. Provide results of any goodness-of-fit tests. (xiv) Description of statistical method(s) used for NOEC and LOEC determination, including the software package, and the basis for the choice of the method. (j) References. The following references should be consulted for additional background material on this test guideline. 16 ------- (1) American Society for Testing and Materials. ASTM E729-96, Standard Guide for Conducting Acute Toxicity Tests on Test Materials with Fishes, Macroinvertebrates, and Amphibians. In Annual Book of ASTM Standards, Vol. 11.06, ASTM, West Conshohocken, PA. Current edition approved October 1, 2014. (2) 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. (3) U.S. Environmental Protection Agency, 1982. Pesticide Assessment Guidelines Subdivision E, Hazard Evaluation: Wildlife and Aquatic Organisms, Office of Pesticide and Toxics Substances, Washington, DC, EPA 540/9-82-024. (4) U.S. Environmental Protection Agency, 1985. Hazard Evaluation Division Standard Evaluation Procedure, Acute Toxicity Test for Estuarine and Marine Organisms (Mollusc 96-Hour Flow-Through Shell Deposition Study). Office of Pesticides Programs, Washington, DC, EPA-540/9-85-011. (5) U.S. Environmental Protection Agency, 1994. Pesticides Reregi strati on Rejection Rate Analysis: Ecological Effects, Prevention, Pesticides, and Toxic Substances, Washington, DC, EPA 738-R-94-035 17 ------- |