Introduction Metal bioaccumulation in bivalves may occur as a consequence of the ingestion of inert or living particles which have bound trace elements. The best experiments, such as those carried out by Borchardt (1983, 1985) about Cd in mussels, have shown that suspended matter is only a small part of the source of contamination in molluscs. But whatever the reliability of the experimental methodologies, laboratory conditions never perfectly mimic natural phenomena. Thus, we have planned to restart these works using a large-scale experiment, the procedure of which was characterized by a small number of controlled parameters with a high degree of representativeness of reality. During experimental nursing of young oysters, we have assessed the influence of different controlled quantities of phytoplankton and of various experimental population densities on metal transfer from their environment to molluscs. Materials and methods Oysters (Crassostrea gigas) were distributed in several cylindrical containers the bottoms of which consisted of a sieve. Food and seawater were renewed continuously by means of an ascending current (Baud and Bacher, 1990). Nursing assays were carried out during summer over a 74 day period or during winter over a 91 day period. Twelve groups of oysters were set up according to food supply (0, 1x, 2x and 4x of Skeletonema costatum grown on underground salt-water in addition to natural phytoplankton) and population density (25,000 or 50,000 individuals per experimental container) (Table 1). Young molluscs were fed according to an alternating cycle of 3 h-feeding periods and 2 h-periods without food. The mean concentrations of pigment cells in the rearing seawater of the molluscs were 0, 10, 20 and 40 mug/l. This seawater was renewed at a flow rate of 3 m3/h during the summer experiment and 1 or 3 m3/h during winter. At the end of the nursing period, molluscs exposed to different experimental conditions were separated using sieves with different mesh-sizes (6, 8, 10, 14 and 18 mm; Table 2). Young oysters were purged for 36 h with a view to limiting the overvaluation of bioaccumulated metal levels due to ingested material (Amiard-Triquet et al., 1984; Kennedy, 1986). In each experimental and age-related category, 90 individuals were sampled and divided into 3 groups of 30 specimens. In these groups, soft tissues were separated from the shells and oven-dried at 80-degrees-C for 48 h. The dry samples were powdered and three aliquots of about 1 00 mg each were digested with 1 ml of concentrated nitric acid (HNO3, Suprapur) at 95-degrees-C for 1 h. Then trace element analyses were performed in this solution diluted with deionized water by flame (Zn) or flameless (Cd, Cu, Pb) atomic absorption spectrophotometry using the Zeeman effect (Amiard et al., 1987). The influence of food supplies, flow rate and experimental population density on the dry weight of the soft tissues of young oysters, their metal concentrations and body burdens were examined using multi-linear regression analysis (Tomassone et al., 1983; Dagnelie, 1973). Results and discussion In Table 3 the metal concentrations in the seawater [Table 3(a)], phytoplankton and seston [Table 3(b)] are shown. In summer, the average weight and metal (Cd, Cu, Pb, Zn) burden in the soft tissues of the oysters increased with the quantity of food available and the size of the individuals, whereas it decreased concomitantly with increasing density of the experimental population of oysters (Table 4). The individual weights increased with increasing food supply (Fig. 2). The increase of food quantity and of the size of the oysters was concomitant with enhancement of the weight of their soft tissues and a decrease of metal concentrations (Table 5). In winter, the population density was correlated negatively with the weight of soft tissues but not with metal concentrations or burdens (Table 6). In summer, the experimental population density was correlated with Cu concentration [Table 7 and Fig. 5(B)] but not with Cd, Pb or Zn concentrations [Fig. 5(A) and (B)]. The oyster size was correlated negatively with Cd and Pb concentrations during summer [Figs 3 and 5(B)] and with Cu and Zn concentrations during winter (Fig. 2 and Table 8). The influence of season was clearly established (Fig. 4): in summer, dry weights were two times higher than in winter and Cd, Cu and Zn concentrations and body burdens were more elevated in winter whereas the opposite pattern was shown for Pb. The influences of food supply, population density and individual size on the animal's weight, metal concentrations and corresponding body burdens are summed up in Table 9 for both the studied seasons. Conclusion Increased food supplies induce a biological dilution of Cd and Pb in young oysters. These results are in agreement with previous data about Cd, Cu, Pb and Zn in different species (Mackay et al., 1975; Boyden, 1974; Phelps and Hetzel, 1987; Berthet, 1986). Thus from the sanitary point of view, the use of underground seawater for algal culture is not at risk since metal concentrations in molluscs are not enhanced.
