Methods for assessing fertilization and embryonic/larval development in toxicity tests using the california mussel (Mytilus californianus)

Abstract

AbstractThe fertilized eggs, embryos and larvae of the California mussel (Mytilus californianus) were used to develop methods for assessing fertilization and larval development rates, and for conducting toxicity tests in standard exposure chambers and small volume (3 ml) chambers. Because the sperm/egg ratio resulting in high monospermic fertilization rates varied among females, the “optimal” ratio for each female was predetermined in an aliquot of eggs prior to fertilizing the entire batch for bioassay use. The fluorescent DNA probe Hoechst 33342 was used to rapidly differentiate unfertilized, monospermic and polyspermic eggs. Following fertilization of entire batches of eggs based on the predetermined sperm/egg ratio, monospermic fertilization rates of >90% were readily achieved. Fertilized embryos were then used in bioassays conducted in beakers or tissue‐culture chamber slides (mini chambers), which could be viewed directly on the microscope. The response of the embryos and larvae to the metabolic inhibitor sodium azide was assessed in both types of test chambers. At 96 h postinsemination at 12°C, veliger or nonveliger larvae were assessed using polarization microscopy to determine the presence or absence of a complete shell. No significant difference in the response to sodium azide was detected between the beakers and mini chambers. This study demonstrates that M. californianus is suitable for static embryo/larval toxicity tests, and that small volume chambers allow successful larval development and eliminate the need for subsampling of larger test chambers. We also demonstrated the value of assessing fertilization rates prior to conducting the bioassay, and present an improved method for examining larval shell development using polarization microscopy.