40 CFR Part 797 — Environmental Effects Testing Guidelines

PART 797—ENVIRONMENTAL EFFECTS TESTING GUIDELINES Authority: 15 U.S.C. 2603. Source: 50 FR 39321, Sept. 27, 1985, unless otherwise noted. Subpart A [Reserved] Subpart B—Aquatic Guidelines § 797.1050 Algal acute toxicity test. (a) Purpose. et seq. (b) Definitions. (1) Algicidal (2) Algistatic (3) ECx (4) Growth (5) Static system (c) Test procedures Summary of the test. (ii) At the end of 96 hours enumerate the algal cells in all containers to determine inhibition or stimulation of growth in test containers compared to controls. Use data to define the concentration-response curve, and calculate the EC 10 50 90 (2) [Reserved] (3) Range-finding test. (A) If definitive testing is necessary. (B) Test chemical concentrations for the definitive test. (ii) Algae are exposed to a widely spaced (e.g., log interval) chemical concentration series. The lowest value in the series, exclusive of controls, should be at the chemical's detection limit. The upper value, for water soluble compounds, should be the saturation concentration. No replicates are required; and nominal concentrations of the chemical are acceptable unless definitive testing is not required. (iii) The test is performed once for each of the recommended algal species or selected alternates. Test chambers should contain equal volumes of test solution and approximately 1 × 10 4 Selenastrum 4 Skeletonema (iv) Definitive testing is not necessary if the highest chemical concentration tested (water saturation concentration or 1000 mg/l) results in less than a 50 percent reduction in growth or if the lowest concentration tested (analytical detection limit) results in greater than a 50 percent reduction in growth. (4) Definitive test. 10 50 90 (ii) Algae should be exposed to five or more concentrations of the test chemical in a geometric series in which the ratio is between 1.5 and 2.0 (e.g., 2, 4, 8, 16, 32, and 64 mg/l). Algae shall be placed in a minimum of three replicate test containers for each concentration of test chemical and control. More than three replicates may be required to provide sufficient quantities of test solution for determination of test substance concentration at the end of the test. Each test chamber should contain equal volumes of test solution and approximately 1 × 10 4 Selenastrum 4 Skeletonema (iii) Every test shall include a control consisting of the same nutrient medium, conditions, procedures, and algae from the same culture, except that none of the test substance is added. If a carrier is present in any of the test chambers, a separate carrier control is required. (iv) The test begins when algae from 5- to 10-day-old stock cultures are placed in the test chambers containing test solutions having the appropriate concentrations of the test substance. Algal growth in controls should reach the logarithmic growth phase by 96 hours. If logarithmic growth cannot be demonstrated, the test shall be repeated. At the end of 24, 48, 72, and 96 hours the algal growth response (number or weight of algal cells/ml) in all test containers and controls shall be determined by an indirect (spectrophotometry, electronic cell counters, dry weight, etc.) or a direct (actual microscopic cell count) method. Indirect methods shall be calibrated by a direct microscopic count. The percentage inhibition or stimulation of growth for each concentration, EC 10 50 90 (v) At the end of the definitive test, the following additional analyses of algal growth response shall be performed: (A) Determine whether the altered growth response between controls and test algae was due to a change in relative cell numbers, cell sizes or both. Also note any unusual cell shapes, color differences, flocculations, adherence of algae to test containers, or aggregation of algal cells. (B) In test concentrations where growth is maximally inhibited, algistatic effects may be differentiated from algicidal effects by the following two methods for Skeletonema Selenastrum. ( 1 ( 2 1 (5) [Reserved] (6) Analytical measurements Chemical. (B) At the end of the test and after aliquots have been removed for algal growth-response determinations, microscopic examination, mortal staining, or subculturing, the replicate test containers for each chemical concentration may be pooled into one sample. An aliquot of the pooled sample may then be taken and the concentration of test chemical determined. In addition, the concentration of test chemical associated with the algae alone should be determined. Separate and concentrate the algal cells from the test solution by centrifuging or filtering the remaining pooled sample and measure the test substance concentration in the algal-cell concentrate. (ii) Numerical. (d) Test conditions Test species. Selenastrum capricornutum, Skeletonema costatum. (2) Facilities General. (B) Disposal facilities should be adequate to accommodate spent glassware, algae and test solutions at the end of the test and any bench covering, lab clothing, or other contaminated materials. (ii) Test containers. (iii) Cleaning and sterilization. (iv) Conditioning. (v) Nutrient medium. Selenastrum Skeletonema (B) Dilution water used for preparation of nutrient medium and test solutions should be filtered, deionized or glass distilled. Saltwater for marine algal nutrient medium and test solutions should be prepared by adding a commercial, synthetic, sea salt formulation or a modified synthetic seawater formulation to distilled/deionized water to a concentration of 30 parts per thousand. (vi) Carriers. (3) Test parameters. Selenastrum Skeletonema. (ii) Test chambers containing Selenastrum Skeletonema 2 (iii) Stock algal cultures should be shaken twice daily by hand. Test containers shall be placed on a rotary shaking apparatus and oscillated at approximately 100 cycles/minute for Selenastrum Skeletonema (iv) The pH of nutrient medium in which algae are subcultured shall be 7.5 (±0.1) for Selenastrum Skeletonema, (v) Light intensity shall be monitored at least daily during the test at the level of the test solution. (e) Reporting. Good Laboratory Practice Standards of this Chapter, (1) Detailed information about the test organisms, including the scientific name, method of verification, and source. (2) A description of the test chambers and containers, the volumes of solution in the containers, the way the test was begun (e.g., conditioning, test substance additions, etc.), the number of replicates, the temperature, the lighting, and method of incubation, oscillation rates, and type of apparatus. (3) The concentration of the test chemical in the control and in each treatment at the end of the test and the pH of the solutions. (4) The number of algal cells per milliliter in each treatment and control and the method used to derive these values at the beginning, 24, 48, and 72 hours, and end of the test; the percentage of inhibition or stimulation of growth relative to controls; and other adverse effect in the control and in each treatment. (5) The 96-hour EC 10 50 90 50 (6) Methods and data records of all chemical analyses of water quality and test substance concentrations, including method validations and reagent blanks. (7) The results of any optional analyses such as: Microscopic appearance of algae, size or color changes, percent mortality of cells and the fate of subcultured cells, the concentration of test substance associated with algae and test solution supernate or filtrate. (8) If the range-finding test showed that the highest concentration of the chemical tested (not less than 1000 mg/l or saturation concentration) had no effect on the algae, report the results and concentration and a statement that the chemical is of minimum phytotoxic concern. (9) If the range-finding test showed greater than a 50 percent inhibition of algal growth at a test concentration below the analytical detection limit, report the results, concentration, and a statement that the chemical is phytotoxic below the analytical detection limit. [50 FR 39321, Sept. 27, 1985, as amended at 52 FR 19058, May 20, 1987] § 797.1300 Daphnid acute toxicity test. (a) Purpose. et seq. Daphnia magna D. pulex (b) Definitions. Good Laboratory Practice Standards (1) Brood stock (2) EC 50 (3) Ephippium (4) Flow-through (5) Immobilization (6) Loading (7) Static system (c) Test procedures Summary of the test. (ii) Dissolved oxygen concentration, pH, temperature, the concentration of test chemical and other water quality parameters are measured at specified intervals in selected test chambers. Data are collected during the test to develop concentration-response curves and determine EC 50 (2) [Reserved] (3) Range-finding test. (ii) The daphnids should be exposed to a series of widely spaced concentrations of the test chemical (e.g., 1, 10, 100 mg/1, etc.), usually under static conditions. (iii) A minimum of five daphnids should be exposed to each concentration of test chemical for a period of 48 hours. The exposure period may be shortened if data suitable for the purpose of the range-finding test can be obtained in less time. No replicates are required and nominal concentrations of the chemical are acceptable. (4) Definitive test. 50 (ii) A minimum of 20 daphnids per concentration shall be exposed to five or more concentrations of the chemical chosen in a geometric series in which the ratio is between 1.5 and 2.0 (e.g., 2, 4, 8, 16, 32, and 64 mg/l). An equal number of daphnids shall be placed in two or more replicates. If solvents, solubilizing agents or emulsifiers have to be used, they shall be commonly used carriers and shall not possess a synergistic or antagonistic effect on the toxicity of the test chemical. The concentration of solvent should not exceed 0.1 mg/l. The concentration ranges shall be selected to determine the concentration-response curves and EC 50 (iii) Every test shall include controls consisting of the same dilution water, conditions, procedures and daphnids from the same population (culture container), except that none of the chemical is added. (iv) The dissolved oxygen concentration, temperature and pH shall be measured at the beginning and end of the test in each chamber. (v) The test duration is 48 hours. The test is unacceptable if more than 10 percent of the control organisms are immobilized during the 48-hour test period. Each test chamber shall be checked for immobilized daphnids at 24 and 48 hours after the beginning of the test. Concentration-response curves and 24-hour and 48-hour EC 50 (vi) In addition to immobility, any abnormal behavior or appearance shall also be reported. (vii) Test organisms shall be impartially distributed among test chambers in such a manner that test results show no significant bias from the distributions. In addition, test chambers within the testing area shall be positioned in a random manner or in a way in which appropriate statistical analyses can be used to determine the variation due to placement. (viii) The concentration of the test chemical in the chambers should be measured as often as is feasible during the test. In the static test the concentration of test chemical shall be measured, at a minimum, at the beginning of the test and at the end of the test in each test chamber. In the flow-through test the concentration of test chemical shall be measured at a minimum: (A) In each chamber at the beginning of the test and at 48 hours after the start of the test; (B) In at least one appropriate chamber whenever a malfunction is detected in any part of the test substance delivery system. Among replicate test chambers of a treatment concentration, the measured concentration of the test chemical shall not vary more than ±20 percent. (5) [Reserved] (6) Analytical measurements. Test chemical. (ii) Numerical. 50 (d) Test conditions Test species Selection. Daphnia magna D. pulex, (B) Daphnids to be used in acute toxicity tests should be cultured at the test facility. Records should be kept regarding the source of the initial stock and culturing techniques. All organisms used for a particular test shall have originated from the same culture population. (C) Daphnids shall not be used for a test ( 1 2 3 4 5 (ii) Acclimation. (B) During culturing and acclimation to the dilution water, daphnids should be maintained in facilities with background colors and light intensities similar to those of the testing area. (iii) Care and handling. (B) Smooth glass tubes (I.D. greater than 5 mm) equipped with rubber bulb should be used for transferring daphnids with minimal culture media carry-over. Care should be exercised to introduce the daphnids below the surface of any solution to avoid trapping air under the carapace. (iv) Feeding. (2) Facilities Apparatus. 1 2 3 4 (B) Facilities should be well ventilated and free of fumes and disturbances that may affect the test organisms. (C) Test chambers shall be loosely covered to reduce the loss of test solution or dilution water due to evaporation and to minimize the entry of dust or other particulates into the solutions. (ii) Construction materials. (B) For static tests, daphnids can be conveniently exposed to the test chemical in 250 ml beakers or other suitable containers. (C) For flow-through tests, daphnids can be exposed in glass or stainless steel containers with stainless steel or nylon screen bottoms. The containers should be suspended in the test chamber in such a manner to insure that the test solution flows regularly into and out of the container and that the daphnids are always submerged in at least 5 centimeters of test solution. Test chambers can be constructed using 250 ml beakers or other suitable containers equipped with screened overflow holes, standpipes or V-shaped notches. (iii) Dilution water. Substance Maximum concentration Particulate matter 20 mg/liter. Total organic carbon or 2 mg/liter. Chemical oxygen demand 5 mg/liter. Un-ionized ammonia 1 µg/liter. Residual chlorine <3 µg/liter. Total organophosphorus pesticides 50 ng/liter. Total organochlorine pesticides plus polychlorinated biphenyls (PCBs) or 50 ng/liter. Organic chlorine 25 ng/liter. (B) The above water quality parameters under paragraph (d)(2)(iii)(A) of this section shall be measured at least twice a year or whenever it is suspected that these characteristics may have changed significantly. If dechlorinated tap water is used, daily chlorine analysis shall be performed. (C) If the diluent water is from a ground or surface water source, conductivity and total organic carbon (TOC) or chemical oxygen demand (COD) shall be measured. Reconstituted water can be made by adding specific amounts of reagent-grade chemicals to deionized or distilled water. Glass distilled or carbon-filtered deionized water with a conductivity less than 1 µohm/cm is acceptable as the diluent for making reconstituted water. (iv) Cleaning. (v) Test substance delivery system. (3) Test parameters. (i) The test temperature shall be 20 °C. Excursions from the test temperature shall be no greater than ±2 °C. (ii) Dissolved oxygen concentration between 60 and 105 percent saturation. Aeration, if needed to achieve this level, shall be done before the addition of the test chemical. All treatment and control chambers shall be given the same aeration treatment. (iii) The number of daphnids placed in a test chamber shall not affect test results. Loading shall not exceed 40 daphnids per liter test solution in the static system. In the flow-through test, loading limits will vary depending on the flow rate of dilution water. Loading shall not cause the dissolved oxygen concentration to fall below the recommended levels. (iv) Photoperiod of 16 hours light and 8 hours darkness. (e) Reporting. Good Laboratory Practice Standards (1) The name of the test, sponsor, testing laboratory, study director, principal investigator, and dates of testing. (2) A detailed description of the test chemical including its source, lot number, composition (identity and concentration or major ingredients and major impurities), known physical and chemical properties and any carriers or other additives used and their concentrations. (3) The source of the dilution water, its chemical characteristics (e.g., conductivity, hardness, pH, etc.) and a description of any pretreatment. (4) Detailed information about the daphnids used as brood stock, including the scientific name and method of verification, age, source, treatments, feeding history, acclimation procedures, and culture method. The age of the daphnids used in the test shall be reported. (5) A description of the test chambers, the volume of solution in the chambers, the way the test was begun (e.g., conditioning, test chemical additions), the number of test organisms per test chamber, the number of replicates per treatment, the lighting, the method of test chemical introduction or the test substance delivery system and the flow rate (in flow-through test) expressed as volume additions per 24 hours. (6) The concentration of the test chemical in each test chamber at times designated for static and flow-through tests. (7) The number and percentage of organisms that were immobilized or showed any adverse effects in each test chamber at each observation period. (8) Utilizing the average measured test chemical concentration, concentration-response curves should be fitted to immobilization data at 24 and 48 hours. A statistical test of goodness-of-fit should be performed and the results reported. (9) The 24- and 48-hour EC 50 50 (10) All chemical analyses of water quality and test chemical concentrations, including methods, method validations and reagent blanks. (11) The data records of the culture, acclimation and test temperatures. (12) Any deviation from this test guideline and anything unusual about the test, e.g., diluter failure, temperature fluctuations, etc. [50 FR 39321, Sept. 27, 1985, as amended at 52 FR 19059, May 20, 1987] § 797.1330 Daphnid chronic toxicity test. (a) Purpose. et seq. (b) Definitions. Good Laboratory Practice Standards (1) Brood stock (2) Chronic toxicity test (3) EC 50 (4) Ephippium (5) Flow-through (6) Immobilization (7) Loading (8) MATC (Maximum Acceptable Toxicant Concentration) (9) Renewal system (c) Test procedures Summary of the test. (ii) The test is started within 30 minutes after the test substance has been added and uniformly distributed in the test chambers in the renewal test or after the concentration of test substance in each test chamber of the flow-through test system reaches the prescribed level and remains stable. At the initiation of the test, daphnids which have been cultured or acclimated in accordance with the test design, are randomly placed into the test chambers. Daphnids in the test chambers are observed periodically during the test, immobile adults and offspring produced are counted and removed, and the findings are recorded. Dissolved oxygen concentration, pH, temperature, the concentration of test substance, and other water quality parameters are measured at specified intervals in selected test chambers. Data are collected during the test to determine any significant differences (p≤0.05) in immobilization and reproduction as compared to the control. (2) [Reserved] (3) Range-finding test. (ii) The daphnids should be exposed to a series of widely spaced concentrations of the test substance (e.g., 1, 10, 100 mg/l), usually under static conditions. (iii) A minimum of five daphnids should be exposed to each concentration of test substance for a period of time which allows estimation of appropriate chronic test concentrations. No replicates are required and nominal concentrations of the chemical are acceptable. (4) Definitive test. 50 (ii) A minimum of 20 daphnids per concentration shall be exposed to five or more concentrations of the chemical chosen in a geometric series in which the ratio is between 1.5 and 2.0 (e.g., 2, 4, 8, 16, 32, 64 mg/l). An equal number of daphnids shall be placed in two or more replicates. The concentration ranges shall be selected to determine the concentration-response curves, EC 50 (iii) Every test shall include controls consisting of the same dilution water, conditions, procedures and daphnids from the same population (culture container), except that none of the chemical is added. (iv) The test duration is 21 days. The test is unacceptable if: (A) More than 20 percent of the control organisms appear to be immobilized, stressed or diseased during the test. (B) Each control daphnid living the full 21 days produces an average of less than 60 young. (C) Any ephippia are produced by control animals. (v) The number of immobilized daphnids in each chamber shall be recorded on day 21 of the test. After offspring are produced, they shall be counted and removed from the test chambers every 2 or 3 days. Concentration-response curves, EC 50 (vi) In addition to immobility, any abnormal behavior or appearance shall also be reported. (vii) Test organisms shall be impartially distributed among test chambers in such a manner that test results show no significant bias from the distributions. In addition, test chambers within the testing area shall be positioned in a random manner as in a way in which appropriate statistical analyses can be used to determine the variation due to placement. (5) [Reserved] (6) Analytical measurements. Test chemical. (ii) Numerical. 50 (d) Test conditions Test species Selection. Daphnia magna D. pulex, (B) First instar daphnids, ≤24 hours old, are to be used to start the test. (ii) Acquisition. (B) Daphnids shall not be used for a test if: ( 1 ( 2 ( 3 ( 4 ( 5 (iii) Feeding. (B) The food concentration depends on the type used. Food concentrations should be sufficient to support normal growth and development and to allow for asexual (parthenogenic) reproduction. For automatic feeding devices, a suggested rate is 5 to 7 mg food (either solids or algal cells, dry weight) per liter dilution water or test solution. For manual once-a-day feeding, a suggested rate is 15 mg food (dry weight) per liter dilution water or test solution. (iv) Loading. (v) Care and handling of test organisms. (B) Organisms should be handled as little as possible. When handling is necessary it should be done as gently, carefully, and quickly as possible. During culturing and acclimation, daphnids should be observed carefully for ephippia and other signs of stress, physical damage, and mortality. Dead and abnormal individuals shall be discarded. Organisms that touch dry surfaces or are dropped or injured during handling shall be discarded. (C) Smooth glass tubes (I.D. greater than 5mm) equipped with a rubber bulb can be used for transferring daphnids with minimal culture media carry-over. (D) Care should be exercised to introduce the daphnids below the surface of any solution so as not to trap air under the carapace. (vi) Acclimation. (B) During culturing and acclimation to the dilution water, daphnids should be maintained in facilities with background colors and light intensities similar to those of the testing area. (2) Facilities General. ( 1 ( 2 ( 3 ( 4 ( 5 ( 6 (B) Facilities should be well ventilated and free of fumes and other disturbances that may affect the test organisms. (ii) Test chambers. (B) For renewal tests, daphnids can be conveniently exposed to the test solution in 250 ml beakers or other suitable containers. (C) For flow-through tests daphnids can be exposed in glass or stainless steel containers with stainless steel or nylon screen bottoms. Such containers shall be suspended in the test chamber in such a manner to ensure that the test solution flows regularly into and out of the container and that the daphnids are always submerged in at least 5 centimeters of test solution. Test chambers can be constructed using 250 ml beakers or other suitable containers equipped with screened overflow holes, standpipes or V-shaped notches. (D) Test chambers shall be loosely covered to reduce the loss of test solution or dilution water due to evaporation and to minimize the entry of dust or other particulates into the solutions. (iii) Test substance delivery system. (B) The test substance delivery system shall be calibrated before each test. Calibration includes determining the flow rate through each chamber and the concentration of the test substance in each chamber. The general operation of the test substance delivery system should be checked twice daily during a test. The 24-hour flow rate through a test chamber shall be equal to at least five times the volume of the test chamber. During a test, the flow rates shall not vary more than 10 percent from any one test chamber to another. For the renewal test, test substance dilution water shall be completely replaced at least once every 3 days. (iv) Dilution water. Substance Maximum concentration Particulate matter 20 mg/l. Total organic carbon or 2 mg/l. Chemical oxygen demand 5 mg/l. Un-ionized ammonia 20 µg/l. Residual chlorine <3 µg/l. Total organophosphorus pesticides 50 ng/l. Total organochlorine pesticides plus polychlorinated biphenyls (PCBs) 50 ng/l. or organic chlorine 25 ng/l. (B) The water quality characteristics listed above shall be measured at least twice a year or when it is suspected that these characteristics may have changed significantly. If dechlorinated tap water is used, daily chlorine analysis shall be performed. (C) If the diluent water is from a ground or surface water source, conductivity and total organic carbon (TOC) or chemical oxygen demand (COD) shall be measured. Reconstituted water can be made by adding specific amounts of reagent-grade chemicals to deionized or distilled water. Glass distilled or carbon filtered deionized water with a conductivity of less than 1 microohm/cm is acceptable as the diluent for making reconstituted water. (D) If the test substance is not soluble in water an appropriate carrier should be used. (v) Cleaning of test system. (3) Test parameters. (A) The test temperature shall be 20 °C. Excursions from the test temperature shall be no greater than ±2 °C. (B) Dissolved oxygen concentration between 60 and 105 percent saturation. Aeration, if needed to achieve this level, shall be done before the addition of the test substance. All treatment and control chambers shall be given the same aeration treatment. (C) Photoperiod of 16-hours light and 8-hours darkness. (ii) Additional measurements include: (A) The concentration of the test substance in the chambers shall be measured during the test. (B) At a minimum, the concentration of test substance should be measured as follows: ( 1 ( 2 ( 3 ( 4 (C) The dissolved oxygen concentration, temperature and pH shall be measured at the beginning of the test and on days 7, 14, and 21 in at least two chambers of the high, middle, low, and control test concentrations. (e) Reporting. Good Laboratory Practice Standards (1) The name of the test, sponsor, testing laboratory, study director, principal investigator, and dates of testing. (2) A detailed description of the test substance including its source, lot number, composition (identity and concentration of major ingredients and major impurities), known physical and chemical properties, and any carriers or other additives used and their concentrations. (3) The source of the dilution water, its chemical characteristics (e.g., conductivity, hardness, pH), and a description of any pretreatment. (4) Detailed information about the daphnids used as brood stock, including the scientific name and method of verification, age, source, treatments, feeding history, acclimation procedures, and culture methods. The age of the daphnids used in the test shall be reported. (5) A description of the test chambers, the volume of solution in the chambers, the way the test was begun (e.g., conditioning, test substance additions), the number of test organisms per test chamber, the number of replicates per treatment, the lighting, the renewal process and schedule for the renewal chronic test, the test substance delivery system and flow rate expressed as volume additions per 24 hours for the flow-through chronic test, and the method of feeding (manual or continuous) and type of food. (6) The concentration of the test substance in test chambers at times designated for renewal and flow-through tests. (7) The number and percentage of organisms that show any adverse effect in each test chamber at each observation period. (8) The cumulative adult and offspring immobilization values and the progeny produced at designated observation times, the time (days) to first brood and the number of offspring per adult in the control replicates and in each treatment replicate. (9) All chemical analyses of water quality and test substance concentrations, including methods, method validations and reagent blanks. (10) The data records of the culture, acclimation, and test temperatures. (11) Any deviation from this test guideline, and anything unusual about the test, (e.g., dilution failure, temperature fluctuations). (12) The MATC to be reported is calculated as the geometric mean between the lowest measured test substance concentration that had a significant (p≤0.05) effect and the highest measured test substance concentration that had no significant (p≤0.05) effect on day 21 of the test. The most sensitive of the test criteria (number of adult animals immobilized, the number of young per female and the number of immobilized young per female) is used to calculate the MATC. The criterion selected for MATC computation is the one which exhibits an effect (a statistically significant difference between treatment and control groups; p≤0.05) at the lowest test substance concentration for the shortest period of exposure. Appropriate statistical tests (analysis of variance, mean separation test) shall be used to test for significant test substance effects. The statistical tests employed and the results of these tests shall be reported. (13) Concentration-response curves utilizing the average measured test substance concentration shall be fitted to cumulative adult immobilization data at 21 days. A statistical test of goodness-of-fit shall be performed and the results reported. (14) An EC 50 [50 FR 39321, Sept. 27, 1985, as amended at 52 FR 19060, May 20, 1987] § 797.1400 Fish acute toxicity test. (a) Purpose. et seq. (b) Definitions. Good Laboratory Practice Standards (1) Acclimation (2) Acute toxicity test (3) Carrier (4) Conditioning (5) Death (6) Flow-through (7) Incipient LC 50 (8) LC 50 (9) Loading (10) Static (11) Test solution (c) Test procedures Summary of the test. (ii) The test substance is introduced into each test chamber. In a flow-through test, the amount of test substance which is added to the dilution water is adjusted to establish and maintain the desired concentration of test substance in each test chamber. (iii) Test fish which have been acclimated in accordance with the test design are introduced into the test and control chambers by stratified random assignment. (iv) Fish in the test and control chambers are observed periodically during the test; dead fish are removed at least twice each day and the findings are recorded. (v) The dissolved oxygen concentration, pH, temperature and the concentration of test substance are measured at intervals in selected test chambers. (vi) Concentration-response curves and LC 50 (2) [Reserved] (3) Range finding test. (4) Definitive test. (ii) For exposure to each concentration of a test substance, an equal number of test fish shall be placed in two or more replicate test chambers. Test fish shall be impartially distributed among test chambers in such a manner that test results show no significant bias from the distributions. (iii) Every test shall include a control consisting of the same dilution water, conditions, procedures, and fish from the same group used in the test, except that none of the test substance is added. (iv) Mortality data collected during the test are used to calculate a 96-hour LC 50 50 50 (v) Test fish shall not be fed while they are being exposed to the test substance under static conditions or during the first 96 hours of flow-through testing. If the test continues past 96 hours, the fish should be fed a suitable food at a maintenance level every other day beginning on test day 5. Any excess food and the fecal material should be removed when observed. (5) Test results. (ii) In addition to death, any abnormal behavior such as, but not limited to, erratic swimming, loss of reflex, increased excitability, lethargy, or any changes in appearance or physiology such as discoloration, excessive mucous production, hyperventilation, opaque eyes, curved spine, or hemorrhaging shall be recorded. (iii) Observations on compound solubility shall be recorded. The investigator shall report the appearance of surface slicks, precipitates, or material adhering to the sides of the test chamber. (iv) Each test and control chamber shall be checked for dead fish and observations recorded at 24, 48, 72, and 96 hours after the beginning of the test or within one hour of the designated times. If the test is continued past 96 hours, additional observations shall be made every 24 hours until termination. (v) The mortality data is used to calculate LC 50 50 (vi) A test is unacceptable if more than 10 percent of the control fish die or exhibit abnormal behavior during a 96-hour test. If a flow-through test is continued past 96 hours, the maximum allowable additional mortality is 10 percent. (6) Analytical measurements Water quality analysis. (B) During static tests, the dissolved oxygen concentration, temperature, and pH shall be measured in each test chamber at the beginning and end of the test. The test solution volume shall not be reduced by more than 10 percent as a result of these measurements. (C) During flow-through tests, dissolved oxygen, temperature and pH measurements shall be made in each chamber at the beginning and end of the test. (ii) Collection of samples for measurement of test substance. (iii) Measurement of test substance. ( 1 ( 2 ( 3 ( 4 (B) Filters and their holders used for determining the dissolved test substance concentrations should be prewashed with several volumes of distilled water and undergo a final rinse with test solution. Glass or stainless steel filter holders are best for organic test substances, while plastic holders are best for metals. The sample should be filtered within 30 minutes after it is taken from the test chamber. (C) The analytical methods used to measure the amount of test substance in a sample shall be validated before beginning the test. The accuracy of a method should be verified by a method such as using known additions. This involves adding a known amount of the test substance to three water samples taken from a chamber containing dilution water and the same number and species of fish as are used in the test. The nominal concentration of the test substance in those samples should span the concentration range to be used in the test. (D) An analytical method is not acceptable if likely degradation products of the test substance give positive or negative interferences, unless it is shown that such degradation products are not present in the test chambers during the test. (E) In addition to analyzing samples of test solution, at least one reagent blank, containing all reagents used, should also be analyzed. (F) If the measured concentrations of dissolved test substance are considerably lower (e.g., <50 percent) than the nominal concentrations, the total test substance concentration should be measured in the highest test concentration. (G) Among replicate test chambers, the measured concentrations shall not vary more than 20 percent. The measured concentration of the test substance in any chamber during the test should not vary more than 30 percent from the measured concentration at time 0. (H) The mean measured concentration of test substance shall be used to calculate all LC 60 (d) Test conditions Test species Selection. Salmo gairdneri Lepomis macrochirus Pimephales promelas (ii) Age and condition of fish. (B) All newly acquired fish should be quarantined and observed for at least 14 days prior to use in a test. (C) Fish shall not be used for a test if they appear stressed or if more than five percent die during the 48 hours immediately prior to the test. (iii) Acclimation of test fish. (B) During the final 48-hours of acclimation, fish should be maintained in facilities with background colors and light intensities similar to those of the testing area and should not be fed. (2) Facilities General. (A) Flow-through tanks for holding and acclimating fish. (B) A mechanism for controlling and maintaining the water temperature during the holding, acclimation and test periods. (C) Apparatus for straining particulate matter, removing gas bubbles, or insufficient dissolved oxygen, respectively. (D) Apparatus for providing a 16-hour light and 8-hour dark photoperiod with a 15- to 30-minute transition period. (E) Chambers for exposing test fish to the test substance. (F) A test substance delivery system for flow-through tests. (ii) Construction materials. (iii) Test substance delivery system. (iv) Test chambers. (v) Cleaning of test system. (vi) Dilution water. Substance Maximum Particulate matter 20 mg/liter. Total organic carbon or 2 mg/liter. chemical oxygen demand 5 mg/liter. Un-ionized ammonia 1 µg/liter. Residual chlorine 1 µg/liter. Total organochloring pesticides 50 µg/liter. Total organocholorine pesticides plus polychlorinated biphenyls (PCBs) 50 µg/liter. or organic chlorine 25 µg/liter. (B) The concentration of dissolved oxygen in the dilution water should be between 90 and 100 percent saturation; 9.8 to 10.9 mg/l for tests with trout, and 8.0 to 8.9 mg/l for tests with bluegill or fathead minnow at sea level. If necessary, the dilution water can be aerated before the addition of the test substance. All reconstituted water should be aerated before use. Buffered soft water should be aerated before but not after the addition of buffers. (C) If disease organisms are present in the dilution water in sufficient numbers to cause infection, they should be killed or removed by suitable equipment. (D) Glass distilled or carbon filtered deionized water with a conductivity less than 1 micromho/cm is acceptable for use in making reconstituted water. If the reconstituted water is prepared from a ground or surface water source, conductivity, and total organic carbon (TOC) or chemical oxygen demand (COD) should be measured on each batch. (vii) Carriers. (B) Triethylene glycol and dimethyl formamide are the preferred carriers, but acetone may also be used. The concentration of triethylene glycol in the test solution should not exceed 80 mg/1. The concentration of dimethyl formamide or acetone in the test solution should not exceed 5.0 mg/1. (3) Test parameters Loading. (ii) Dissolved oxygen concentration. (B) During flow-through tests the dissolved oxygen concentration shall be maintained above 8.2 mg/1 in tests with trout and above 6.6 mg/l in tests with bluegills or fathead minnows. (iii) Temperature. (iv) Light. (e) Reporting. Good Laboratory Practice Standards (1) The source of the dilution water, a description of any pretreatment, and the measured hardness, acidity, alkalinity, pH, conductivity, TOC or COD and particulate matter. (2) A description of the test chambers, the depth and volume of solution in the chamber, the specific way the test was begun (e.g., conditioning, test substance additions), and for flow-through tests, a description of the test substance delivery system. (3) Detailed information about the test fish, including the scientific name and method of verification, average weight (grams, wet weight), standard length, age, source, history, observed diseases, treatments, and mortalities, acclimation procedures, and food used. (4) The number of replicates used, the number of organisms per replicate, the loading rate, and the flow rate for flow-through tests. (5) The measured DO, pH and temperature and the lighting regime. (6) The solvent used, the test substance concentration in the stock solution, the highest solvent concentration in the test solution and a description of the solubility determinations in water and solvents if used. (7) The concentrations of the test substance at each test concentration just before the start of the test and at all subsequent sampling periods. (8) The number of dead and live tests organisms, the percentage of organisms that died, and the number that showed any abnormal effects in the control and in each test chamber at each observation period. (9) The 96-hour LC 50 50 50 (10) When observed, the observed no effect concentration (the highest concentration tested at which there were no mortalities or abnormal behavioral or physiological effects). (11) The concentration-response curve at each observation period for which a LC 50 (12) Methods and data records of all chemical analyses of water quality parameters and test substance concentrations, including method validations and reagent blanks. [50 FR 39321, Sept. 27, 1985, as amended at 52 FR 19062, May 20, 1987; 54 FR 29715, July 14, 1989; 54 FR 33148, Aug. 11, 1989] § 797.1600 Fish early life stage toxicity test. (a) Purpose. (b) Definitions. Good Laboratory Practice Standards, (1) “Acclimation” physiological or behavioral adaptation of organisms to one or more environmental conditions associated with the test method (e.g., temperature, hardness, pH). (2) “Carrier” solvent or other agent used to dissolve or improve the solubility of the test substance in dilution water. (3) “Conditioning” exposure of construction materials, test chambers, and testing apparatus to dilution water or to the test solution prior to the start of the test in order to minimize the sorption of test substance onto the test facilities or the leachig of substances from test facilities into the dilution water or the test solution. (4) “Control” an exposure of test organisms to dilution water only or dilution water containing the test solvent or carrier (no toxic agent is intentionally or inadvertently added). (5) “Dilution water” the water used to produce the flow-through conditions of the test to which the test substance is added and to which the test species is exposed. (6) “Early life stage toxicity test” a test to determine the minimum concentration of a substance which produces a statistically significant observable effect on hatching, survival, development and/or growth of a fish species continuously exposed during the period of their early development. (7) “Embryo cup” a small glass jar or similar container with a screened bottom in which the embryos of some species (i.e., minnow) are placed during the incubation period and which is normally oscillated to ensure a flow of water through the cup. (8) “Flow through” refers to the continuous or very frequent passage of fresh test solution through a test chamber with no recycling. (9) “Hardness” the total concentration of the calcium and magnesium ions in water expressed as calcium carbonate (mg CaCO 3 (10) “Loading” the ratio of biomass (grams of fish, wet weight) to the volume (liters) of test solution passing through the test chamber during a specific interval (normally a 24-hr. period). (11) “No observed effect concentration (NOEC)” the highest tested concentration in an acceptable early life stage test: (i) which did not cause the occurrence of any specified adverse effect (statistically different from the control at the 95 percent level); and (ii) below which no tested concentration caused such an occurrence. (12) “Observed effect concentration (OEC)” the lowest tested concentration in an acceptable early life stage test: (i) Which caused the occurrence of any specified adverse effect (statistically different from the control at the 95 percent level); and (ii) above which all tested concentrations caused such an occurrence. (13) “Replicate” two or more duplicate tests, samples, organisms, concentrations, or exposure chambers. (14) “Stock solution” the source of the test solution prepared by dissolving the test substance in dilution water or a carrier which is then added to dilution water at a specified, selected concentration by means of the test substance delivery system. (15) “Test chamber” the individual containers in which test organisms are maintained during exposure to test solution. (16) “Test solution” dilution water with a test substance dissolved or suspended in it. (17) “Test substance” the specific form of a chemical substance or mixture that is used to develop data. (c) Test Procedures Summary of test. (ii) A minimum of five exposure (treatment) concentrations of a test substance and one control are required to conduct an early life stage toxicity test. The concentration of the test substance in each treatment is usually 50 percent of that in the next higher treatment level. (iii) For each exposure concentration of the test substance and for each control (i.e., regular control and carrier control is required) there shall be: (A) At least two replicate test chambers, each containing one or more embryo incubation trays or cups; and there shall be no water connections between the replicate test chambers; (B) At least 60 embryos divided equally in such a manner that test results show no significant bias from the distributions, between the embryo incubation trays or cups for each test concentration and control (i.e., 30 per embryo cup with 2 replicates); (C) All surviving larvae divided equally between the test chambers for each test concentration and control (e.g., 30 larvae per test chamber with 2 replicates). (iv) Duration. (B) For brook trout and rainbow trout a test begins when newly fertilized trout embryos (less than 96-hours old) are placed in the embryo trays or cups and are exposed to the test solution concentrations. The test terminates following 60 days of post-hatch exposure (for an approximate total exposure period of 90 days). (C) For silverside a test begins with newly fertilized embryos (less than or equal to 48 hours old) and is terminated 28 days after hatching. The chorionic fibrils should be cut before randomly placing the embryos in the egg incubation cups. (2) [Reserved] (3) Range-finding test. (ii) The highest concentration selected for the early life stage toxicity test should approximate the lowest concentration indicated in any previous testing to cause a significant reduction in survival. The range of concentrations selected is expected to include both observed effect and no-observed effect levels. The dilution factor between concentrations is normally 0.50, however, other dilution factors may be used as necessary. (4) Definitive test General. (B) New holding and test facilities should be tested with sensitive organisms (i.e., juvenile test species or daphnids) before use to assure that the facilities or substances possibly leaching from the equipment will not adversely affect the test organisms during an actual test. (C) Embryos should be acclimated for as long as practical to the test temperature and dilution water prior to the initiation of the test. (D) When embryos are received from an outside culture source (i.e., rainbow and brook trout) at a temperature at variance with the recommended test temperature they shall be acclimated to the test temperature. When eggs are received, they should be immediately unpacked and the temperature of the surrounding water determined. Sudden temperature changes should be avoided. Acclimation to the appropriate test temperature should be accomplished within a period of 6 hours, and should incorporate the use of dilution water. (E) Embryos should be visually inspected prior to placement in the embryo cups or screen trays. All dead embryos shall be discarded. Dead embryos can be discerned by a change in coloration from that of living embryos (e.g., trout embryos turn white when dead). During visual inspection, empty shells, opaque embryos, and embryos with fungus or partial shells attached shall be removed and discarded. If less than 50 percent of the eggs to be used appear to be healthy, all embryos in such a lot shall be discarded. (ii) Embryo incubation procedures. (B) Each day until hatch the embryos are visually examined. Minnow embryos may be examined with the aid of a magnifying viewer. Trout embryos should not be touched. Trout embryos should be maintained in low intensity light or in darkness until 1-week post hatch, and are usually examined with the aid of a flashlight or under low intensity light. Dead embryos should be removed and discarded. Any embryos which are heavily infected with fungus shall be discarded and shall be subtracted from the initial number of embryos used as a basis for the calculations of percentage hatch. (C) When embryos begin to hatch they should not be handled. (iii) Initiation of fry exposure. (B) If necessary, fry can be transferred from one replicate embryo cup to the other replicate within a test concentration to achieve equal numbers in each replicate chamber. (C) The number of live fry, live normal fry, live embryos, dead embryos and unaccounted for embryos for each cup shall be recorded when hatching is deemed complete. Those fry which are visibly (without the use of a dissecting scope or magnifying viewer) lethargic or grossly abnormal (either in swimming behavior or physical appearance) shall be counted. Late hatching embryos shall be left in the embryo cups to determine if they will eventually hatch or not. The range of time-to-hatch (to the nearest day) for each cup shall be recorded. (iv) Time to first feeding. ad libitum, Artemia salina (B) For the first seven days, feeding shall be done at minimum intervals of four hours (i.e., 8 am, 12 noon, and 4 pm); thereafter the fry shall be fed as indicated below. (v) Feeding. Brachionus plicatilis, Artemia (B) An identical amount of food should be provided to each chamber. Fish should be fed ad libitum (C) Fish should not be fed for the last 24 hours prior to termination of the test. (vi) Carriers. (vii) Controls. (viii) Randomization. (ix) Observations. (x) Biological data. (B) When hatching commences, daily records of the number of embryos remaining in each embryo cup are required. This information is necessary to quantify the hatching success. A record of all deformed larvae shall be kept throughout the entire post-hatch exposure. Time to swim-up shall be recorded for the trout. Upon transfer of fry from the embryo cups to the test chambers, daily counts of the number of live fish should be made. At a minimum, live fish shall be counted on days 4, 11, 18, 25 and (weekly thereafter for the trout species) finally on termination of the test. (C) The criteria for death of young fish is usually immobility, especially absence of respiratory movement, and lack of reaction to gentle prodding. Deaths should be recorded daily and dead fish removed when discovered. (D) Daily and at termination of the test, the number of fish that appear (without the use of a magnifying viewer) to be abnormal in behavior (e.g., swimming erratic or uncoordinated, obviously lethargic, hyperventilating, or over excited, etc.) or in physical appearance (e.g., hemorrhaging, producing excessive mucous, or are discolored, deformed, etc.) shall be recorded and reported in detail. (E) All physical abnormalities (e.g., stunted bodies, scoliosis, etc.) shall be photographed and the deformed fish which die, or are sacrificed at the termination of the test, shall be preserved for possible future pathological examination. (F) At termination, all surviving fish shall be measured for growth. Standard length measurements should be made directly with a caliper, but may be measured photographically. Measurements shall be made to the nearest millimeter (0.1 mm is desirable). Weight measurements shall also be made for each fish alive at termination (wet, blotted dry, and to the nearest 0.01 g for the minnows and 0.1 g for the trout). If the fish exposed to the toxicant appear to be edematous compared to control fish, determination of dry, rather than wet, weight is recommended. (G) Special physiological, biochemical and histological investigations on embryos, fry, and juveniles may be deemed appropriate and shall be performed on a case by case basis. (5) Test results. (ii) The significance level for all statistical testing shall be a minimum of P = 0.05 (95 percent confidence level). (A) Example of statistical analysis. 1 ( 2 ( 3 ( 4 (B) Test data to be analyzed. ( 1 ( 2 ( 3 ( 4 ( 5 ( 6 ( 7 (C) It is important that fish length and weight measurements be associated with individual test chambers since the density of the fish and available food should be considered in the growth of the organism. (iii) Acceptability criteria. (B) In addition to obtaining significant effects on the exposed test species, a measure of acceptability in the response of control fish is also required. (C) A test is not acceptable if the average survival of the control fish at the end of the test is less than 80 percent or if survival in any one control chamber is less than 70 percent. For silversides, a test is not acceptable if the average overall survival of the control embryos and fish at the end of the test is less than 60 percent. (D) If a carrier is used, the criteria for effect (mortality of embryos and fry, growth, etc.) used in the comparison of control and exposed test organisms shall also be applied to the control and control with carrier chambers. For the test to be considered acceptable, no significant difference shall exist between these criteria. (E) A test is not acceptable if the relative standard deviation (RSD = 100 times the standard deviation divided by the mean) of the weights of the fish that were alive at the end of the test in any control test chamber is greater than 40 percent. (6) Analytical measurements Analysis of water quality. (ii) Dissolved oxygen measurement. (iii) Temperature measurement. (iv) Test substance measurement. (B) The measured concentration of test substance in any chamber should be no more than 30 percent higher or lower than the concentration calculated from the composition of the stock solution and the calibration of the test substance delivery system. If the difference is more than 30 percent, the concentration of test substance in the solution flowing into the exposure chamber (influent) should be analyzed. These results will indicate whether the problem is in the stock solution, the test substance delivery system or in the test chamber. Measurement of degradation products of the test substance is recommended if a reduction of the test substance concentration occurs in the test chamber. (v) Sampling and analysis methodology. (B) For measurement of the test substance, water samples shall be taken midway between the top, bottom, and sides of the test chamber and should not include any surface scum or material stirred up from the bottom or sides. Samples of test solutions shall be handled and stored appropriately to minimize loss of test substance by microbial degradation, photodegradation, chemical reaction, volatilization, or sorption. (C) Chemical and physical analyses shall be performed using standardized methods whenever possible. The analytical method used to measure the concentration of the test substance in the test solution shall be validated before the beginning of the test. At a minimum, a measure of the accuracy of the method should be obtained on each of two separate days by using the method of known additions, and using dilution water from a tank containing test organisms. Three samples should be analyzed at the next-to-lowest test substance concentration. It is also desirable to study the accuracy and precision of the analytical method for test guideline determination by use of reference (split) samples, or interlaboratory studies, and by comparison with alternative, reference, or corroborative methods of analysis. (D) 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, unless it is shown that such degradation products are not present in the test chambers during the test. In general, atomic absorption spectrophotometric methods for metals and gas chromatographic methods for organic compounds are preferable to colorimetric methods. (E) In addition to analyzing samples of test solution, at least one reagent blank also should be analyzed when a reagent is used in the analysis. Also, at least one sample for the method of known additions should be prepared by adding test substance at the concentration used in the toxicity test. (d) Test conditions Test species. (A) Fathead minnow ( Pimephales promelas (B) Sheepshead minnow ( Cyprinodon variegatus (C) Brook trout ( Salvelinus fontinalis (D) Rainbow trout ( Salmo gairdneri (E) Atlantic silverside ( Menidia menidia (F) Tidewater silverside ( Menidia peninsulae (ii) Embryos used to initiate the early life stage test shall be less than 48 hours old for the fathead and sheepshead minnows, silversides, and less than 96 hours old for the brook trout and rainbow trout. In addition, the following requirements shall be met: (A) All embryos used in the test shall be from the same source. Embryos shall be obtained from a stock cultured in-house when possible, and maintained under the same parameters as specified for the test conditions. When it is necessary to obtain embryos from an external source, caution should be exercised to ensure embryo viability and to minimize the possibility of fungal growth. A description of the brood stock history or embryo source shall be made available to EPA upon request. (B) Test species shall be cared for and handled properly in order to avoid unnecessary stress. To maintain test species in good condition and to maximize growth, crowding shall be prevented, and the dissolved oxygen level shall be maintained near saturation. (C) Embryos and fish shall be handled as little as possible. Embryos shall be counted and periodically inspected until hatching begins. When larvae begin to hatch, they shall not be handled. Transfer of minnow larvae from embryo cups to test chambers shall not involve the use of nets. No handling is necessary following introduction into the test chambers until termination of the test. (D) If fathead minnow embryos are obtained from in-house culture units, the embryos should be gently removed from the spawning substrate. The method for separating the fertilized eggs from the substrate is important and can affect the viability of the embryos; therefore the finger-rolling procedure is recommended. (E) Disease treatment. Chemical treatments to cure or prevent diseases should not be used before, and should not be used during a test. All prior treatments of brood stock should be reported in detail. Severely diseased organisms should be destroyed. (2) Test facilities Construction materials. (ii) Test chambers (B) Many different sizes of test chambers have been used successfully. The size, shape and depth of the test chamber is acceptable if the specified flow rate and loading requirements can be achieved. (C) The actual arrangement of the test chambers can be important to the statistical analysis of the test data. Test chambers can be arranged totally on one level (tier) side by side, or on two levels with each level having one of the replicate test substance concentrations or controls. Regardless of the arrangement, it shall be reported in detail and considered in the data analysis. (iii) Embryo incubation apparatus. (B) The trout embryo incubation trays can be made from stainless steel screen (or other acceptable material such as plastic) of about 3-4 mm mesh. The screen tray should be supported above the bottom of the test chamber by two folds of screen or other devices which function as legs or supports. The edges of the screen tray should be turned up to prevent bump spills and to prevent the embryos from rolling off in the event of excessive turbulence. Suspending or supporting the screen tray off the bottom ensures adequate water circulation around the embryos and avoids contact of embryos with possible bottom debris. (iv) Test substance delivery system. (B) The proportional diluter and modified proportional diluter systems and metering pump systems have proven suitable and have received extensive use. (C) Mixing chambers shall be used between the diluter and the test chamber(s). This may be a small container or flow-splitting chamber to promote mixing of test substance stock solution and dilution water, and is positioned between the diluter and the test chambers for each concentration. If a proportional diluter is used, separate delivery tubes shall run from the flow-splitting chamber to each replicate test chamber. Daily checks on this latter system shall be made. (D) Silverside fry are injured easily and are susceptible to impingement on the mesh of the incubation cups. Consequently, water flow into and out of the cups when counting fry must be at a slow rate. This can be accomplished by using small diameter (e.g., 2 mm I.D.) capillary tubes to drain the test solution from spitter boxes into the replicate test chambers. The use of a self-starting siphon to gradually lower (i.e., less than or equal to 1 min.) the water level approximately 2 cm in the test chamber is recommended. A minimum water depth of 5 cm should be maintained in the cups. Although it may be satisfactory, a rocker-arm type apparatus has not yet been used with silversides. (v) Other equipment required. (B) An apparatus for aerating water. (C) A suitable magnifying viewer for examination of minnow embryos. (D) A suitable apparatus for the precise measurement of growth of the fish, including both length (e.g., with metric or ruler caliper or photographic equipment) and weight. (E) Facilities for providing a continuous supply of live brine shrimp nauplii ( Artemia salina (F) For silversides, facilities for providing a supply of rotifers ( Brachionus plicatilis (G) Facilities (or access to facilities) for performing the required water chemistry analyses. (vi) Cleaning of equipment. (B) Debris can be removed with a rubber bulb and large pipette or by siphoning with a glass tube attached to a flexible hose. Debris should be run into a bucket light enough to observe that no live fish are accidentally discarded. (vii) Dilution water General. 1 ( 2 ( 3 (B) Freshwater. 1 ( 2 Substance Concentration maximum Particulate matter <20 mg/liter. Total organic carbon (TOC) <2 mg/liter. Chemical oxygen demand (COD) <5 mg/liter. Un-ionized ammonia <1 µg/liter. Residual chlorine <1 µg/liter. Total organoposphorus pesticides <50 ng/liter. Total organochlorine pesticides plus polychlorinated biphenyls (PCBs) <50 ng/liter. Total organic chlorine <25 ng/liter. ( 3 (C) Saltwater. 1 0 00 0 00 ( 2 0 00 (3) Test parameters Dissolved oxygen concentration. (ii) Loading and flow rate. (B) A lower loading or higher flow rate or both shall be used if necessary to meet the following three criteria at all times during the test in each chamber containing live test organisms: ( 1 ( 2 ( 3 (iii) Temperature. ( 1 ( 2 ( 3 ( 4 (B) Excursions from the test temperature shall be no greater than ±2.0 °C. It is recommended that the test system be equipped with an automatic alarm system to alert staff of instantaneous temperature changes in excess of 2 °C. If the water is heated (i.e., for minnow species), precautions should be taken to ensure that supersaturation of dissolved gases is avoided. Temperatures shall be recorded in all test chambers at the beginning of the test and weekly thereafter. The temperature shall be recorded at least hourly in one test chamber throughout the test. (iv) Light. (B) For fathead and sheepshead minnows, a 16-hour light and 8-hour dark (or 12:12) photoperiod shall be used throughout the test period. (C) For silversides, a 14-hour light and 10-hour dark photoperiod shall be used throughout the test period. (D) A 15-minute to 30-minute transition period between light and dark is optional. (E) Light intensities ranging from 30 to 100 lumens at the water surface shall be provided; the intensity selected should be duplicated as closely as possible for all test chambers. (e) Reporting. (1) Name of test, sponsor, investigator, laboratory, and dates of test duration. (2) Detailed description of the test substance including its source, lot number, composition (identity and concentration of major ingredients and major impurities), known physical and chemical properties, and any carriers (solvents) or other additives used. (3) The source of the dilution water, its chemical characteristics, and a description of any pretreatment. (4) Detailed information about the test organisms including scientific name and how verified and source history, observed diseases, treatments, acclimation procedure, and concentration of any contaminants and the method of measurement. (5) A description of the experimental design and the test chambers, the depth and volume of the solution in the chambers, the way the test was begun, the number of organisms per treatment, the number of replicates, the loading, the lighting, a description of the test substance delivery system, and the flow rate as volume additions per 24 hours. (6) Detailed information on feeding of fish during the toxicity test, including type of food used, its source, feeding frequency and results of analysis (i.e., concentrations) for contaminants. (7) Number of embryos hatched, number of healthy embryos, time to hatch, mortality of embryos and fry, measurements of growth (weight and length), incidence of pathological or histological effects and observations of other effects or clinical signs, number of healthy fish at end of test. (8) Number of organisms that died or showed an effect in the control and the results of analysis for concentration(s) of any contaminant in the control(s) should mortality occur. (9) Methods used for, and the results of (with standard deviation), all chemical analyses of water quality and test substance concentration, including validation studies and reagent blanks; the average and range of the test temperature(s). (10) Anything unusual about the test, any deviation from these procedures, and any other relevant information. (11) A description of any abnormal effects and the number of fish which were affected during each period between observations in each chamber, and the average concentration of test substance in each test chamber. (12) Reference to the raw data location. [50 FR 39321, Sept. 27, 1985, as amended at 52 FR 19064, May 20, 1987] § 797.1930 Mysid shrimp acute toxicity test. (a) Purpose. et seq. (b) Definitions. Good Laboratory Practice Standards (1) “Death” means the lack of reaction of a test organism to gentle prodding. (2) “Flow-through” means a continuous or an intermittent passage of test solution or dilution water through a test chamber or a holding or acclimation tank, with no recycling. (3) “LC 50 (4) “Loading” means the ratio of test organisms biomass (grams, wet weight) to the volume (liters) of test solution in a test chamber. (5) “Retention chamber” means a structure within a flow-through test chamber which confines the test organisms, facilitating observation of test organisms and eliminating loss of organisms in outflow water. (6) “Static system” means a test chamber in which the test solution is not renewed during the period of the test. (c) Test procedures Summary of the test. 50 (2) [Reserved] (3) Range-finding test. (A) Which life stage (juvenile or young adult) is to be utilized in the definitive test. (B) The test solution concentrations for the definitive test. (ii) The mysids should be exposed to a series of widely spaced concentrations of test substance (e.g., 1, 10, 100 mg/l, etc.), usually under static conditions. (iii) This test should be conducted with both newly hatched juvenile (<24 hours old) and young adult (5 to 6 days old) mysids. For each age class (juvenile or young adult), a minimum of 10 mysids should be exposed to each concentration of test substance for up to 96 hours. The exposure period may be shortened if data suitable for the purpose of the range-finding test can be obtained in less time. The age class which is most sensitive to the test substance in the range-finding test shall be utilized in the definitive test. When no apparent difference in sensitivity of the two life stages is found, juveniles shall be utilized in the definitive test. No replicates are required, and nominal concentrations of the chemical are acceptable. (4) Definitive test. 50 (ii) The definitive test shall be conducted on the mysid life stage (juveniles or young adults) which is most sensitive to the test substance being evaluated. (iii) A minimum of 20 mysids per concentration shall be exposed to five or more concentrations of the chemical chosen in a geometric series in which the ratio is between 1.5 and 2.0 (e.g., 2, 4, 8, 16, 32, and 64 mg/l). An equal number of mysids shall be placed in two or more replicates. If solvents, solubilizing agents or emulsifiers have to be used, they shall be commonly used carriers and shall not possess a synergistic or antagonistic effect on the toxicity of the test substance. The concentration of solvent shall not exceed 0.1 ml/1. The concentration ranges shall be selected to determine the concentration-response curves and LC 50 (iv) Every test shall include controls consisting of the same dilution water, conditions, procedures, and mysids from the same population or culture container, except that none of the chemical is added. (v) The dissolved oxygen concentration temperature, salinity, and pH shall be measured at the beginning and end of the test in each chamber. (vi) The test duration is 96 hours. The test is unacceptable if more than 10 percent of the control organisms die or exhibit abnormal behavior during the 96 hour test period. Each test chamber should be checked for dead mysids at 24, 48, 72, and 96 hours after the beginning of the test. Concentration-response curves and 24-, 48-, 72- and 96-hour LC 50 (vii) In addition to death, any abnormal behavior or appearance shall also be reported. (viii) Test organisms shall be impartially distributed among test chambers in such a manner that test results show no significant bias from the distributions. In addition, test chambers within the testing area shall be positioned in a random manner or in a way in which appropriated statistical analyses can be used to determine the variation due to placement. (ix) The concentration of the test substance in the chambers should be measured as often as is feasible during the test. At a minimum, during static tests the concentration of test substance shall be measured at each concentration at the beginning and at the end of the test. During the flow-through test, the concentration of test substance should be measured at the beginning and end of the test and in at least one appropriate chamber whenever a malfunction is detected in any part of the test substance delivery system. Equal aliquots of test solution may be removed from each replicate chamber and pooled for analysis. Among replicate test chambers of a treatment concentration, the measured concentration of the test substance should not vary more than 20 percent. (5) [Reserved] (6) Analytical measurements Test chemical. (ii) Numerical. 50 (d) Test conditions Test species Selection. Mysidopsis bahia, (B) Mysids to be used in chronic toxicity tests should originate from laboratory cultures in order to ensure the individuals are of similar age and experimental history. Mysids used for establishing laboratory cultures may be purchased commercially or collected from appropriate natural areas. Because of similarities with other mysids species, taxonomic verification should be obtained from the commercial supplier by experienced laboratory personnel or by an outside expert. (C) Mysids used in a particular test shall be of similar age and be of normal size and appearance for their age. Mysids shall not be used for a test if they exhibit abnormal behavior or if they have been used in a previous test, either in a treatment or in a control group. (ii) Acclimation. (B) During acclimation mysids should be maintained in facilities with background colors and light intensities similar to those of the testing areas. (iii) Care and handling. (iv) Feeding. Artemia (2) Facilities Apparatus. 1 2 3 4 (B) Test chambers shall be loosely covered to reduce the loss of test solution or dilution water due to evaporation and to minimize the entry of dust or other particulates into the solutions. (ii) Cleaning. (iii) Construction materials. (B) For use in the flow-through test, retention chambers utilized for confinement of test organisms can be constructed with netting material of appropriate mesh size. (iv) Dilution water. (B) Natural seawater shall be filtered through a filter with a pore size of <20 microns prior to use in a test. (C) Artificial seawater can be prepared by adding commercially available formulations or by adding specific amounts of reagent-grade chemicals to deionized water. Deionized water with a conductivity less than 1 µohm/cm at 12 °C is acceptable for making artificial seawater. When deionized water is prepared from a ground or surface water source, conductivity and total organic carbon (or chemical oxygen demand) shall be measured on each batch. (v) Test substance delivery system. (3) Test parameters. (i) The test temperature shall be 25 °C. Excursions from the test temperature shall be not greater than ±2 °C. (ii) Dissolved oxygen concentration between 60 and 105 percent saturation. Aeration, if needed to achieve this level, shall be done before the addition of the test substance. All treatment and control chambers shall be given the same aeration treatment. (iii) The number of mysids placed in a test solution shall not be so great as to affect results of the test. Loading shall not exceed 30 mysids per liter for a static test. Loading requirements for the flow-through test will vary depending on the flow rate of dilution water. The loading shall not cause the dissolved oxygen concentration to fall below the recommended levels. (iv) Photoperiod of 14 hours light and 10 hours darkness, with a 15 to 30 minute transition period. (v) Salinity of 20 parts per thousand ±3 percent. (e) Reporting. Good Laboratory Practice Standards (1) The source of the dilution water, its chemical characteristics (e.g., salinity, pH, etc.) and a description of any pretreatment. (2) Detailed information about the test organisms, including the scientific name and method of verification, age, source, history, abnormal behavior, acclimation procedures and food used. (3) A description of the test chambers, the depth and volume of solution in the chamber, the way the test was begun (e.g., conditioning, test substance additions, etc.), the number of organisms per treatment, the number of replicates, the loading, the lighting, the test substance delivery system and the flow rate expressed as volume additions per 24 hours. (4) The measured concentration of test substance in test chambers at the times designated. (5) The number and percentage of organisms that died or showed any other adverse effects in the control and in each treatment at each observation period. (6) Concentration-response curves shall be fitted to mortality data collected at 24, 48, 72, and 96 hours. A statistical test of goodness-of-fit shall be performed and the results reported. (7) The 96-hour LC 50 50 (8) Methods and data records of all chemical analyses of water quality and test substance concentrations, including method validations and reagent blanks. (9) The data records of the holding, acclimation and test temperature and salinity. (f) References. (1) U.S. Environmental Protection Agency, “Bioassay Procedures for the Ocean Disposal Permit Program,” EPA Report No. 600-9-78-010 (Gulf Breeze, Florida, 1978). (2) [Reserved] [50 FR 39321, Sept. 27, 1985, as amended at 52 FR 19068, May 20, 1987; 52 FR 26150, July 13, 1987] § 797.1950 Mysid shrimp chronic toxicity test. (a) Purpose. et seq. (b) Definitions. Good Laboratory Practice Standards (1) “Chronic toxicity test” means a method used to determine the concentration of a substance that produces an adverse effect from prolonged exposure of an organism to that substance. In this test, mortality, number of young per female and growth are used as measures of chronic toxicity. (2) “Death” means the lack of reaction of a test organism to gentle prodding. (3) “Flow-through” means a continuous or an intermittent passage of test solution or dilution water through a test chamber or a holding or acclimation tank, with no recycling. (4) “G1 (Generation 1)” means those mysids which are used to begin the test, also referred to as adults; G2 (Generation 2) are the young produced by G1. (5) “LC 50 (6) “Loading” means the ratio of test organism biomass (gram, wet weight) to the volume (liters) of test solution in a test chamber. (7) “MATC” (Maximum Acceptable Toxicant Concentration) means the maximum concentration at which a chemical can be present and not be toxic to the test organism. (8) “Retention chamber” means a structure within a flow-through test chamber which confines the test organisms, facilitating observation of test organisms and eliminating washout from test chambers. (c) Test procedures Summary of the test. (ii) Dissolved oxygen concentration, pH, temperature, salinity, the concentration of test substance and other water quality characteristics are measured at specified intervals in selected test chambers. (iii) Data collected during the test are used to develop a MATC (Maximum Acceptable Toxicant Concentration) and quantify effects on specific chronic parameters. (2) [Reserved] (3) Range-finding test. (ii) The mysids should be exposed to a series of widely spaced concentrations of the test substance (e.g., 1, 10, 100 mg/l), usually under static conditions. (iii) A minimum of 10 mysids should be exposed to each concentration of test substance for a period of time which allows estimation of appropriate chronic test concentrations. No replicates are required and nominal concentrations of the chemical are acceptable. (4) Definitive test. 50 (ii) A minimum of 40 mysids per concentration shall be exposed to four or more concentrations of the chemical chosen in a geometric series in which the ratio is between 1.5 and 2.0 (e.g., 2, 4, 8, 16, 32, and 64 mg/1). An equal number of mysids shall be placed in two or more replicates. If solvents, solubilizing agents or emulsifiers have to be used, they shall be commonly used carriers and shall not possess a synergistic or antagonistic effect on the toxicity of the test substance. The concentration of solvent should not exceed 0.1 ml/1. The concentration ranges should be selected to determine the concentration response curves, LC 50 (iii) Every test should include controls consisting of the same dilution water, conditions, procedures and mysids from the same population or culture container, except that none of the chemical is added. (iv) The dissolved oxygen concentration, temperature, salinity, and pH shall be measured weekly in each chamber. (v) The test duration is 28 days. The test is unacceptable if more than 20 percent of the control organisms die, appear stressed or are diseased during the test. The number of dead mysids in each chamber shall be recorded on days 7, 14, 21, and 28 of the test. At the time when sexual characteristics are discernible in the mysids (approximately 10 to 12 days in controls; possible delays may occur in mysids exposed to test substances), the number of males and females (identified by ventral brood pouch) in each chamber shall be recorded. Body length (as measured by total midline body length, from the anterior tip of the carapace to the posterior margin of the uropod) shall be recorded for males and females at the time when sex can be determined simultaneously for all mysids in control and treatment groups. This time cannot be specified because of possible delays in sexual maturation of mysids exposed to test substances. A second observation of male and female body lengths shall be conducted on day 28 of the test. To reduce stress on the mysids, body lengths can be recorded by photography through a stereomicroscope with appropriate scaling information. As offspring are produced by the G1 mysids (approximately 13 to 16 days in controls), the young shall be counted and separated into retention chambers at the same test substance concentration as the chambers where they originated. If available prior to termination of the test, observations on the mortality, number of males and females and male and female body length shall be recorded for the G2 mysids. Concentration-response curves, LC 50 (vi) In addition to death, any abnormal behavior or appearance shall also be reported. (vii) Test organisms shall be impartially distributed among test chambers in such a manner that test results show no significant bias from the distributions. In addition, test chambers within the testing area shall be positioned in a random manner or in a way in which appropriate statistical analyses can be used to determined the variation due to placement. (viii) The concentration of the test substance in the chambers should be measured as often as is feasible during the test. The concentration of test substance shall be measured: (A) At each test concentration at the beginning of the test and on days 7, 14, 21, and 28; and (B) In at least one appropriate chamber whenever a malfunction is detected in any part of the test substance delivery system. Equal aliquots of test solutions may be removed from each test chamber and pooled for analysis. Among replicate test chambers of a treatment concentration, the measured concentration of the test substance should not vary more than 20 percent. (5) [Reserved] (6) Analytical measurements Test chemical. (ii) Numerical. (B) A 7-, 14-, 21- and 28-day LC 50 (d) Test conditions Test species Selection. Mysidopsis bahia, (B) Mysids to be used in chronic toxicity tests should originate from laboratory cultures in order to ensure the individuals are of similar age and experimental history. Mysids used for establishing laboratory cultures may be purchased commercially or collected from appropriate natural areas. Because of similarities with other mysid species, taxonomic verification should be obtained from the commercial supplier, by experienced laboratory personnel, or by an outside expert. (C) Mysids used in a particular test shall be of similar age and be of normal size and appearance for their age. (D) Mysids shall not be used for a test if they exhibit abnormal behavior, or if they have been used in a previous test, either in a treatment or in a control group. (ii) Acclimation. (B) During acclimation mysids should be maintained in facilities with background colors and light intensities similar to those of the testing areas. (iii) Care and handling. (iv) Feeding. Artemia (2) Facilities Apparatus. 1 2 3 4 (B) Facilities should be well ventilated and free of fumes and disturbances that may affect test organisms. (C) Test chambers shall be loosely covered to reduce the loss of test solution or dilution water due to evaporation and to minimize the entry of dust or other particulates into the solutions. (ii) Cleaning. (iii) Construction materials. (B) Retention chambers utilized for confinement of test organisms can be constructed with netting material of appropriate mesh size. (iv) Dilution water. (B) Natural seawater shall be filtered through a filter with a pore size of >20 microns prior to use in a test. (C) Artificial seawater can be prepared by adding commercially available formulations or by adding specific amounts of reagent-grade chemicals to deionized or glass-distilled water. Deionized water with a conductivity less than 1 µohm/cm at 12 °C is acceptable as the diluent for making artificial seawater. When deionized water is prepared from a ground or surface water source, conductivity and total organic carbon (or chemical oxygen demand) shall be measured on each batch. (v) Test substance delivery system. (3) Test parameters. (i) The test temperature shall be 25 °C. Excursions from the test temperature shall be no greater than ±2 °C. (ii) Dissolved oxygen concentration between 60 and 105 percent saturation. Aeration, if needed to achieve this level, shall be done before the addition of the test substance. All treatment and control chambers shall be given the same aeration treatment. (iii) The number of mysids placed in a test solution shall not be so great as to affect results of the test. Loading requirements for the test will vary depending on the flow rate of dilution water. The loading shall not cause the dissolved oxygen concentration to fall below the recommended levels. (iv) Photoperiod of 14 hours light and 10 hours darkness, with a 15-30 minute transition period. (v) Salinity of 20 parts per thousand ±3 percent. (e) Reporting. Good Laboratory Practice Standards (1) The source of the dilution water, its chemical characteristics (e.g., salinity, pH, etc.) and a description of any pretreatment. (2) Detailed information about the test organisms, including the scientific name and method of verification, average length, age, source, history, observed diseases, treatments, acclimation procedures and food used. (3) A description of the test chambers, the depth and volume of solution in the chamber, the way the test was begun (e.g., conditioning, test substance additions, etc.), the number of organisms per treatment, the number of replicates, the loading, the lighting, the test substance delivery system, and the flow rate expressed as volume additions per 24 hours. (4) The measured concentration of test substance in test chambers at the times designated. (5) The first time (day) that sexual characteristics can be observed in controls and in each test substance concentration. (6) The length of time for the appearance of the first brood for each concentration. (7) The means (average of replicates) and respective 95 percent confidence intervals for: (i) Body length of males and females at the first observation day (depending on time of sexual maturation) and on day 28. (ii) Cumulative number of young produced per female on day 28. (iii) Cumulative number of dead adults on day 7, 14, 21 and 28. (iv) If available prior to test termination (day 28), effects on G2 mysids (number of males and females, body length of males and females and cumulative mortality). (8) The MATC is calculated as the geometric mean between the lowest measured test substance concentration that had a significant (P<0.05) effect and the highest measured test substance concentration that had no significant (P<0.05) effect in the chronic test. The most sensitive of the test criteria for adult (Gl) mysids (cumulative number of dead mysids, body lengths of males and females or the number of young per female) is used to calculate the MATC. The criterion selected for MATC computation is the one which exhibits an effect (a statistically significant difference between treatment and control groups; P<0.05) at the lowest test substance concentration for the shortest period of exposure. Appropriate statistical tests (analysis of variance, mean separation test) should be used to test for significant chemical effects. The statistical tests employed and the results of these tests shall be reported. (9) Concentration-response curves shall be fitted to the cumulative number of adult dead for days 7, 14, 21, and 28. A statistical test of goodness-of-fit shall be performed and the results reported. (10) An LC 50 (11) Methods and data records of all chemical analyses of water quality and test substance concentrations, including method validations and reagent blanks. (12) The data records of the holding, acclimation and test temperature and salinity. (f) References. (1) U.S. Environmental Protection Agency, “Bioassay Procedures for the Ocean Disposal Permit Program,” EPA Report No. 600/9-78-010 (Gulf Breeze, Florida, 1978). (2) [Reserved] [50 FR 39321, Sept. 27, 1985, as amended at 52 FR 19069, May 20, 1987]

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