§ 797.1600 Fish early life stage toxicity test.
(a) Purpose. This guideline is intended to be used for assessing the propensity of chemical substances to produce adverse effects to fish during the early stages of their growth and development. This guideline describes the conditions and procedures for the continuous exposure of several representative species to a chemical substance during egg, fry and early juvenile life stages. The Environmental Protection Agency (EPA) will use data from this test in assessing the potential hazard of the test substance to the aquatic 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, apply to this section. In addition, the following definitions are applicable to this specific test guideline:
(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 CaCO3/liter).
(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—(1) Summary of test. (i) The early life stage toxicity test with fish involves exposure of newly fertilized embryos to various concentrations of a test substance. Exposure continues for 28 days post hatch for the minnows and 60 days post hatch for the trout species. During this time various observations and measurements are made in a specific manner and schedule in order to determine the lowest effect and highest no-effect concentrations of the test substance.
(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. (A) For fathead minnow and sheepshead minnow a test begins when the newly fertilized minnow embryos (less than 48-hours old) are placed in the embryo cups and are exposed to the test solution concentrations. The test terminates following 28 days of post-hatch exposure, i.e., 28 days after the newly hatched fry are transferred from the embryo cups into the test chambers.
(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. (i) A range finding test is normally performed with the test substance to determine the test concentrations to be used in the early life stage toxicity test, especially when the toxicity is unknown. It is recommended that the test substance concentrations be selected based on information gained from a 4- to 10-day flow-through toxicity test with juveniles of the selected test species.
(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—(i) General. (A) A test shall not be initiated until after the test conditions have been met and the test substance delivery system has been observed functioning properly for 48-hours. This includes temperature stability, flow requirements of dilution water, lighting requirements, and the function of strainers and air traps included in the water-supply system, and other conditions as specified previously.
(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. (A) Embryos can be distributed to the embryo cups or screen trays using a pipette with a large bore or a similar apparatus. Newly-hatched silverside fry are very sensitive to handling; the egg incubation cups should not be handled at all the first 5 days after hatching begins. Just before hatching is expected to begin, the embryos should be transferred to clean incubation cups. Trout embryos can be distributed by using a small container which has been precalibrated to determine the approximate number of embryos it can hold; embryos are measured volumetrically in this manner, and are then poured onto the screen tray (or embryo cup). Trout embryos should be separated on the screen tray so that they are not in contact with each other. A final count will ensure the actual number on the screen tray. After random assignment, the screen trays or embryo cups are placed in the test chambers.