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(a) Purpose. This guideline may be used to develop data on the acute toxicity of chemical substances and mixtures ("chemicals") subject to environmental effects test regulations under the Toxic Substances Control Act (TSCA) (Pub. L. 94-469, 90 Stat. 2003, 15 U.S.C. 2601 et seq.). This guideline prescribes tests to be used to develop data on the acute toxicity of chemicals to fish. The United States Environmental Protection Agency (EPA) will use data from these tests in assessing the hazard of a chemical to the environment.
(b) Definitions. The definitions in section 3 of the Toxic Substances Control Act (TSCA), and the definitions in Part 792—Good Laboratory Practice Standards of this chapter apply to this test guideline. The following definitions also apply to this guideline:
(b)(1) Acclimation means the physiological compensation by test organisms to new environmental conditions (e.g., temperature, hardness, pH).
(b)(2) Acute toxicity test means a method used to determine the concentration of a substance that produces a toxic effect on a specified percentage of test organisms in a short period of time (e.g., 96 hours). In this guideline, death is used as the measure of toxicity.
(b)(3) Carrier means a solvent used to dissolve a test substance prior to delivery to the test chamber.
(b)(4) Conditioning means the exposure of construction materials, test chambers, and testing apparatus to dilution water or to test solutions prior to the start of a test in order to minimize the sorption of the test substance onto the test facilities or the leaching of substances from the test facilities into the dilution water or test solution.
(b)(5) Death means the lack of opercular movement by a test fish.
(b)(6) 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.
(b)(7) Incipient LC50 means that test substance concentration, calculated from experimentally-derived mortality data, that is lethal to 50 percent of a test population when exposure to the test substance is continued until the mean increase in mortality does not exceed 10 percent in any concentration over a 24-hour period.
(b)(8) LC50 means that test substance concentration, calculated from experimentally-derived mortality data, that is lethal to 50 percent of a test population during continuous exposure over a specified period of time.
(b)(9) Loading means the ratio of fish biomass (grams, wet weight) to the volume (liters) of test solution in a test chamber or passing through it in a 24 hour period.
(b)(10) Static means the test solution is not renewed during the period of the test.
(b)(11) Test solution means the test substance and the dilution water in which the test substance is dissolved or suspended.
(c) Test procedures—(1) Summary of the test. (i) Test chambers are filled with appropriate volumes of dilution water. If a flow-through test is performed, the flow of dilution water through each chamber is adjusted to the rate desired.
(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 LC50 values for the test substance are developed from the mortality data collected during the test.
(c)(2) [Reserved]
(c)(3) Range finding test. If the toxicity of the test substance is not already known, a range finding test should be performed to determine the range of concentrations to be used in the definitive test. The highest concentration of test substance for use in the range finding test should not exceed its solubility in water or the permissible amount of the carrier used.
(c)(4) Definitive test. (i) A minimum of 20 fish should be exposed to each of five or more test substance concentrations. The range of concentrations to which the fish are exposed should be such that in 96 hours there are at least two partial mortality exposures bracketing 50 percent survival.
(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 LC50. The 24-, 48-, and 72-hour values should be calculated whenever there is sufficient mortality data to determine such values. If the 96-hour LC50 is less than 50 percent of the estimated 48-hour LC50 in a flow-through test, the test shall be continued until the mean increase in mortality at any test concentration does not exceed 10 percent over a 24-hour period or until 14 days.
(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.
(c)(5) Test results. (i) Death is the primary criterion used in this test guideline to evaluate the toxicity of the test substance.
(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 LC50's and their 95 percent confidence limits, and to plot concentration-response curves for each time interval whenever sufficient data exists. The methods recommended for use in calculating LC50's include probit, logit, binomial, and moving average angle.
(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.
(c)(6) Analytical measurements—(i) Water quality analysis. (A) The hardness, acidity, alkalinity, pH, conductivity, TOC or COD, and particulate matter of the dilution water should be measured at the beginning of each static test and at the beginning and end of each flow-through test. The month to month variation of the above values should be less than 10 percent and the pH should vary less than 0.4 units.
(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. Test solution samples to be analyzed for the test substance should be taken midway between the top, bottom, and sides of the test chamber. These samples should not include any surface scum or material dislodged from the bottom or sides. Samples should be analyzed immediately or handled and stored in a manner which minimizes loss of test substance through microbial degradation, photodegradation, chemical reaction, volatilization, or sorption.
(c)(6)(iii) Measurement of test substance. (A) For static tests, the concentration of the test substance shall be measured at a minimum in each test chamber at each test concentration at the beginning (0-hour, before fish are added) and at the end of the test. During flow-through tests, the concentration of test substance shall be measured as follows:
(1) In at least the chamber of each test concentration at 0-hour.
(2) In at least the chamber of each test concentration at 96-hours and every 4 days thereafter, as long as the test is continued.
(3) In at least one appropriate chamber whenever a malfunction is detected in any part of the test substance delivery system.
(4) Equal aliquots of test solution may be removed from each replicate chamber and pooled for analysis.
(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 LC60's and to plot all concentration-response curves.
(d) Test conditions—(1) Test species—(i) Selection. The test species for this test are the rainbow trout (Salmo gairdneri), bluegill (Lepomis macrochirus) and fathead minnow (Pimephales promelas). The particular species of fish to be used will be prescribed in the test rule.
(d)(1)(ii) Age and condition of fish. (A) Juvenile fish shall be used. Fish used in a particular test shall be the same age and be of normal size and appearance for their age. The longest fish shall not be more than twice the length of the shortest.
(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.
(d)(1)(iii) Acclimation of test fish. (A) If the holding water is not from the same source as the test dilution water, acclimation to the dilution water should be done gradually over a 48-hour period. The fish should then be held an additional 14 days in the dilution water prior to testing. Any changes in water temperature should not exceed 3°C per day. Fish should be held for a minimum of 7 days at the test temperature prior to testing.
(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.
(d)(2) Facilities—(i) General. Facilities needed to perform this test include:
(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.
(d)(2)(ii) Construction materials. Construction materials and commercially purchased 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 alter the test results. Materials and equipment that contact stock or test solutions should be chosen to minimize sorption of test chemicals. Glass, stainless steel, and perfluorocarbon plastic should be used whenever possible. Concrete, fiberglass, or plastic (e.g., PVC) may be used for holding tanks, acclimation tanks, and water supply systems, but they should be used to remove rust particles. Rubber, copper, brass, galvanized metal, epoxy glues, and lead should not come in contact with the dilution water, stock solution, or test solution.
(d)(2)(iii) Test substance delivery system. In flow-through tests, diluters, metering pump systems, or other suitable devices should be used to deliver the test substance to the test chambers. The system used should be calibrated before each test. Calibration includes determining the flow rate through each chamber and the concentration of the test substance delivered to 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 should be a minimum of 6 tank volumes. During a test, the flow rates should not vary more than 10 percent from one test chamber to another.
(d)(2)(iv) Test chambers. Test chambers made of stainless steel should be welded, not soldered. Test chambers made of glass should be fused or bonded using clear silicone adhesive. As little adhesive as possible should be left exposed in the interior of the chamber.
(d)(2)(v) Cleaning of test system. Test substance delivery systems and test chambers should be cleaned before each test. They should be washed with detergent and then rinsed in sequence with clean water, pesticide-free acetone, clean water, and 5 percent nitric acid, followed by two or more changes of dilution water.
(d)(2)(vi) Dilution water. (A) Clean surface or ground water reconstituted water, or dechlorinated tap water is acceptable as dilution water if the test fish will survive in it for the duration of the holding, acclimating, and testing periods without showing signs of stress, such as discoloration, hemorrhaging, disorientation or other unusual behavior. The quality of the dilution water should be constant and should meet the following specifications measured at least twice a year:
| 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 | 50 µg/liter. |
| polychlorinated biphenyls (PCBs)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.
(d)(2)(vii) Carriers. (A) Distilled water should be used in making stock solutions of the test substance. If the stock volume however is more than 10 percent of the test solution volume, dilution water should be used. If a carrier is absolutely necessary to dissolve the test substance, the volume used should not exceed the minimum volume necessary to dissolve or suspend the test substance in the test solution. If the test substance is a mixture, formulation, or commercial product, none of the ingredients is considered a carrier unless an extra amount is used to prepare the stock solution.
(B) Triethylene glycol and dimethyl formamide are the prefered 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.
(d)(3) Test parameters—(i) Loading. The number of fish placed in a test chamber should not be so great as to affect the results of the test. The loading should not be so great that the test substance concentrations are decreased by more than 20 percent due to uptake by the fish. In static tests, loading should not exceed 0.5 grams of fish per liter of solution in the test chamber at any one time. In flow-through tests loading should not exceed 0.5 grams of fish per liter of test solution passing through the chamber in 24 hours. These loading rates should be sufficient to maintain the dissolved oxygen concentration above the recommended levels and the ammonia concentration below 20 µg/l.
(d)(3)(ii) Dissolved oxygen concentration. (A) During static tests with rainbow trout the dissolved oxygen in each test chamber shall be greater than 5.5 mg/1. In tests with bluegill and fathead minnows, the DO shall be maintained above 4.5 mg/l.
(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.
(d)(3)(iii) Temperature. The test temperature shall be 22°C for bluegill and fathead minnow and 12°C for rainbow trout. Excursions from the test temperature shall be no greater than ±2°C. The temperature shall be measured at least hourly in one test chamber.
(d)(3)(iv) Light. A 16-hour light and 8-hour dark photoperiod should be maintained.
(e) Reporting. The sponsor shall submit to the EPA all data developed by the test that are suggestive or predictive of toxicity. In addition to the reporting requirements prescribed in Part 792—Good Laboratory Practice Standards of this chapter, the reported test data shall include the following:
(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 LC50, and when sufficient data have been generated, the 24- , 48-, 72-, and incipient LC50 values, their 95 percent confidence limits, and the methods used to calculate the LC50 values and their confidence limits.
(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 LC50 was calculated.
(12) Methods and data records of all chemical analyses of water quality parameters and test substance concentrations, including method validations and reagent blanks.