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A spatial and temporal survey of six sites in the Saguenay Fjord and of one adjacent site in the St. Lawrence River estuary (Quebec, Canada) was undertaken to study the possible effects of anthropogenic contaminant input on soft-shell clam (Mya arenaria) populations. Bivalve sampling sites were selected because they reflected a range of areas representative of either no known (or apparent) pollution sources or of areas potentially influenced by different gradients and types of contamination sources. The most upstream site selected in the Saguenay Fjord, nearest to a highly populated and industrialized sector, and the most downstream site, near its mouth with the St. Lawrence River estuary, spanned a distance of some 70 km and encompassed the entire intertidal area suitable for Mya arenaria habitat. To measure effects in collected animals, we used a comprehensive battery of biomarkers composed of metallothionein-like proteins (MT), 7-ethoxyresorufin O-deethylase activity (EROD), DNA damage (DD), lipid peroxidation (LPO), vitellinlike proteins (Vn), phagocytosis (PHAG), nonspecific esterase (NspE) activity, and condition factor (weight-to-length ratio of clams). Vn, PHAG, DD, and NspE biomarkers were assayed in hemolymph (or hemocytes), whereas others (MT, EROD, LPO) were determined in the digestive gland. Whole-tissue metal content was also quantified in clams collected in the spatial survey. The spatial survey conducted in June 1997 showed significant effects at all sites, and principal component analysis indicated in addition that the more important responses were linked to the MT, LPO, and NspE biomarkers. Clams collected from sites closest to the upstream reaches of the fjord generally displayed higher levels of tissue metals (cadmium, manganese), as well as greater responses of NspE activity, MT, LPO, and PHAG. Animals collected from sites influenced by municipal wastewaters had higher levels of Vn, suggesting the presence of environmental estrogens. The results of the temporal survey (six monthly samplings of clams at three sites from May through October, 1997) showed that the bivalve reproductive cycle (vitellogenesis and spawning) can modulate the expression of several biomarkers. Vn levels, for example, were positively correlated with DD and EROD and negatively correlated with MT, suggesting that reproduction can influence the susceptibility of clams to some contaminants. Discrimination analysis over the 6 months of sampling revealed that the mean value of the discriminant function changed significantly over time, suggesting important changes in the relative contribution of each biomarker. In short, this study has provided evidence that clam populations in the Saguenay Fjord are impacted by multiple sources of contamination whose effects can be modulated by reproduction.

Immune function in bivalves can be adversely affected by long-term exposure to environmental contaminants. Investigating alterations in immunity can therefore yield relevant information about the relationship between exposure to environmental contaminants and susceptibility to infectious diseases. We have developed a rapid, cost-effective, and miniaturized immunocompetence assay to evaluate the phagocytic activity, viability, and concentration of hemocytes in freshwater and marine bivalves. Preliminary experiments were performed to optimize various aspects of the assay including 1) the time required for adherence of hemocytes to polystyrene microplate wells, 2) the time required for internalization of fluorescent bacteria, 3) the ratio of hemocytes to fluorescent bacteria in relation to phagocytosis, 4) hemolymph plasma requirements, and 5) the elimination of fluorescence from (noninternalized) bacteria adhering to the external surface of hemocytes. The results of these experiments showed the optimal adherence time for hemocytes in microplate wells to be 1 h, that phagocytosis required at least 2 h of contact with fluorescently labeled E. coli cells, that the number of fluorescent E. coli cells had a positive effect on phagocytic activity, that at least 2.5 million cells/mL were required to measure a significant intake, and that a linear increase in uptake of bacteria (R = 0.91; p < 0.01) could be obtained with concentrations of up to 1.3 x 10(6) hemocytes/mL. Afterward, the assay was used in two field studies to identify sites having the potential to affect the immunocompetence of bivalves. The first study was conducted on Mya arenaria clams collected at selected contaminated sites in the Saguenay River (Quebec, Canada), and the second examined Elliptio complanata freshwater bivalves that had been exposed to a municipal effluent plume in the St. Lawrence River (Quebec, Canada). In the Saguenay River field study a significant increase in phagocytosis was observed at sites closest to polluted areas. Phagocytotic activity varied over time and was highest during the warmest months (June, July, and August), closely paralleling the spawning period of Mya arenaria clams. In contrast, a drop in phagocytic activity was observed in Elliptio complanata mussels exposed to surface water 4 km downstream of a major municipal effluent plume, with a concomitant increase in the number of hemocytes in the hemolymph. It appears that both immunosuppressive and immunostimulative effects are likely to occur in the field and that responses will be influenced by the type and intensity of contaminants at play.

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