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R9 Laboratory SOP 1001
SEA URCHIN (Strongylocentrotus purpuratus) AND SAND DOLLAR (Dendraster excentricus}) FERTILIZATION TOXICITY TEST
Summary
This method estimates the chronic toxicity of environmental samples (e.g. effluents, receiving waters) to the gametes of sea urchins, (Strongylocentrotus purpuratus), or sand dollars (Dendraster excentricus). The test measures fertilizing capacity of the sperm with a 20 minute sperm exposure to the sample and a subsequent 20 minute exposure period following the addition of eggs.
The purpose of the test is to determine the concentrations of a test substance that reduce egg fertilization by exposed sperm relative to that attained by sperm in control solutions. Concentrations of materials adversely affecting egg fertilization under the conditions of this test are usually acutely and chronically toxic to one or more of several common marine test species and, by extension, are presumably acutely and chronically toxic to other of the many untested marine species.
The procedure follows methods specified in Short-Term Methods for Estimating the Chronic Toxicity of Effluents and Receiving Waters to West Coast Marine and Estuarine Organisms, EPA/600/R-95/136 (USEPA, 1995). U.S. EPA Region 9 Laboratory staff perform toxicity tests on samples from NPDES facilities or Superfund sites.
This method measures the toxicity of a sample to sea urchin or sand dollar sperm by exposing known sperm suspensions to various dilutions of the sample for 20 minutes. Known densities of eggs are then added to the exposed sperm to allow the eggs to fertilize for 20 minutes. The sperm to egg dilution must be 3000:1 or can be determined by a trial fertilization test with different sperm densities. The test is terminated by the addition of a preservative and the percent fertilization is determined by microscopic examination of 100 eggs in an aliquot of eggs from each treatment. The test endpoint is normal egg fertilization shown by the presence of a fertilization membrane around the egg. Point estimates and hypothesis-derived endpoints are calculated using the TOXIS computer program to determine the samples in which fertilization is reduced or statistically significantly different from the control. A reference toxicant test is conducted with copper chloride.