THE MICRONUCLEUS ASSAY IN CRASSOSTREA-GIGAS FOR THE DETECTION OF SEAWATER GENOTOXICITY

The micronucleus (MN) test was performed in vivo and in vitro on the oyster Crassostrea gigas to evaluate the genotoxic effect of the marine environment. In vitro tests were carried out on adult and young (spat) specimens exposed to benzo[a]pyrene (BaP:0.5, 5, 500 and 1000 mu g.l(-1)) and an effluent (5, 50, 75 and 100%) of Seine Bay, one of the most highly contaminated sites in France. MN frequency observed after 48 h exposure to the two pollutants was much greater in adults than spats. A preliminary test of the genotoxic effect of BaP (0.05, 0.5, 1 and 500 mu g.l(-1)), cupric sulfate (10, 25, 50 and 100 mu g.l(-1)) and a paper mill effluent (1, 3, 10 and 30 mg.l(-1)) was performed in C. gigas heart cells cultured for 6 days. Comparison of the MN assay with the C. gigas larva test showed the clastogenic action of BaP and the toxic effect of cupric sulfate on culture cells as well as the slighter toxic effect of paper mill effluent on spats. An in vivo study was conducted in an oyster-farming area contaminated by cadmium and copper. MN frequency was not very sensitive to a pollution gradient but showed high interindividual variability. The absence of precise criteria for MN identification in mollusks and the identification of highly basophilic spherical inclusions in the cytoplasm of gill tissue hemocytes in oysters during viral infection are handicap for application of the micronuclei assay in the marine environment. Another limitation of the assay is the particularly onerous requirement for manual observation. Optimization of the assay by automated analysis is necessary but can only be achieved if cytologic preparations are of good quality.