Biological test method for determining acute lethality of sediment to amphipods: chapter 5

Section 5: Procedure for Testing a Reference Toxicant

The routine use of a reference toxicant is necessary to assess the relative sensitivity of the populations of amphipods used, and the precision and reliability of data produced by the laboratory personnel for that reference toxicant, under standardized test conditions. When determining the toxicity of samples of marine or estuarine sediment to marine or estuarine amphipods according to this reference method, a static, water-only reference toxicity test must be performed on each batch of field-collected organisms used for testing. A guidance document on controlling the precision of toxicity tests using water-only reference toxicity tests has been published by Environment Canada (1990), and provides useful background information and instructions in this respect.

The reference toxicity test to be conducted with each batch of field-collected amphipods must be a static, 96-h LC50 using reagent-grade cadmium chloride. This test must be initiated within one day of the start of the 10-day test for sediment toxicity, and is normally started on the same day (EC, 1992).

The reference toxicity test requires a minimum of six treatments (i.e., a control and five concentrations of cadmium in water), and one or more replicates per treatment (USEPA, 1994a). The test is performed in 1-L glass beakers or jars, using ≥800 mL of test solution and a minimum of 10 amphipods per test chamber. Unless otherwise described, all applicable conditions and procedures for preparing for and undertaking the test must be identical to those defined in Sections 2, 3, and 4 of this report, except that sediment is not added to the test chambers and replicates are not required for each test concentration. One distinction is that, unlike the sediment toxicity test which requires continuous overhead illumination of test chambers, the reference toxicity test is to be performed in the dark (USEPA, 1994a). This can be achieved by covering test chambers with opaque material (e.g., aluminum foil), or by undertaking the test in a separate, enclosed testing facility where the lights are left off. A second distinction is that, unlike the sediment toxicity test, which requires gentle aeration of the overlying water throughout the test, the solutions of cadmium or water (control) in the test chambers are not aerated since the concentrations of dissolved oxygen that are present in each test solution (including the controls) are adequate to satisfy the oxygen requirements of the test organisms. Each test chamber is covered to minimize contamination and losses due to evaporation.

When undertaking a reference toxicity test, a series of concentrations should be chosen which, based on preliminary and/or previous tests performed using the same conditions and procedures, will provide partial mortalities in two or more concentrations and enable calculation of a 96-h LC50 with acceptably narrow 95% confidence limits. The selected test concentrations should bracket the predicted LC50 for the test species (see Appendices D, E, F, and G for species-specific guidance). An appropriate dilution series in which each successive concentration of cadmium is at least 50% of the previous concentration may be used. Test concentrations may also be selected from other appropriate logarithmic dilution series (see Environment Canada, 1992; Appendix L).

The same type (i.e., natural or reconstituted seawater), source, and pretreatment of the control/dilution water should be used for each reference toxicity test performed by the laboratory using this procedure and a single species of test organisms. Salinity of this water must be 28 ± 2‰, and should be the same for each reference toxicity test performed with a particular species at each test facility. The control/dilution water must be temperature adjusted (i.e., 10 ± 2°C if A. virginiana; 15 ± 2°C if R. abronius, E. washingtonianus, or E. estuarius) and aerated as required to achieve a dissolved oxygen content of 90 to 100% saturation, before test solutions are made up and before each group of animals is introduced. The temperature of the solution in each test chamber should be measured daily, and must be measured at the beginning and end of the test. Mean daily temperature during the test must be 15 ± 2°C for R. abronius, E. washingtonianus, or E. estuarius; and 10 ± 2°C for A. virginiana. Dissolved oxygen, salinity, and pH in each test chamber must be measured at the beginning and end of the test.

At the end of the 96-h exposure period, the number of amphipods alive and the number dead are determined (see Section 4.9) and recorded for each treatment including the control group. Biological endpoints for this test are percent survival for each treatment, and the 96-h LC50. Environment Canada (1998a) provides definitive direction and advice for calculating LC50s, which should be followed. Results must be calculated and reported as mg Cd/L.

For tests using R. abronius, E. estuarius, or A. virginiana, the results of the reference toxicity test are only valid and acceptable if control survival at 96 h is ≥90% (EC, 1992; USEPA, 1994a). For tests using E. washingtonianus, the results of the reference toxicity test are only valid and acceptable if control survival at 96 h is ≥85% (see Appendix E).

It is the responsibility of laboratory personnel to demonstrate their ability to obtain consistent, precise results with the reference toxicant before definitive sediment assays are conducted using this reference method. To meet this responsibility, the laboratory personnel should initially determine their intralaboratory precision, expressed as percent coefficient of variation (% CV), by performing five or more reference toxicity tests with different batches of test organisms of the same species, using cadmium chloride and the procedures and conditions defined herein. This should be conducted to gain experience with the test procedure, and as a point of reference for future tests (USEPA, 1994a).

While routinely performing this reference toxicity test with each batch of field-collected amphipods of the same species, laboratory personnel should continue to follow this same procedure. Once sufficient data are available (EC, 1990), LC50s derived from these tests must be plotted successively on a species-specific warning chart, and examined to determine whether the results are within ± 2 SD of values obtained in previous tests using the same species, reference toxicant (i.e., cadmium chloride), and test procedure. A separate warning chart must be prepared and updated for each species of marine or estuarine amphipod used with this reference method.

The warning chart should plot logarithm of concentration on the vertical axis against date of the test or test number on the horizontal axis. Each new LC50 for the reference toxicant must be compared with established limits of the chart; the LC50 is acceptable if it falls within the warning limits. All calculations of mean and standard deviation should be made on the basis of log(LC50).

The logarithm of concentration (including LC50) should be used in all calculations of mean and standard deviation, and in all plotting procedures. This simply represents continued adherence to the assumption by which each LC50 was estimated based on logarithms of concentrations. The warning chart may be constructed by plotting the logarithmic values of the mean and ±2 SD on arithmetic paper, or by converting them to arithmetic values and plotting those on the logarithmic scale of semi-log paper. If it were demonstrated that the LC50s failed to fit a log-normal distribution, an arithmetic mean and SD might prove more suitable. The mean of the available values of log(LC50), together with the upper and lower warning limits (±2 SD), should be recalculated with each successive LC50 until the statistics stabilize (EC, 1990; 1998a; USEPA, 1994a).

If a particular LC50 fell outside the warning limits, the sensitivity of the test organisms and the performance and precision of the test would be suspect. Since this might occur 5% of the time due to chance alone, an outlying LC50 would not necessarily mean abnormal sensitivity of the batch of test organisms or unsatisfactory precision of toxicity data. Rather, it would provide a warning that there might be a problem. A thorough check of all acclimation and test conditions and procedures should be carried out. Depending on the findings, it might be necessary to repeat the reference toxicity test, or to obtain a new batch of field-collected organisms for evaluating the toxicity of the samples of test material (together with a new reference toxicity test using the new batch of test organisms).

Results that remained within the warning limits might not necessarily indicate that a laboratory was generating consistent results. Extremely variable data for a reference toxicant would produce wide warning limits; a new data point could be within the warning limits but still represent undesirable variation in test results. A coefficient of variation of no more than 30%, and preferably 20% or less, is suggested as a reasonable limit by Environment Canada (1990).