Copper exposure of freshwater mussels (Anodonta anatina): Some physiological effects
Copper (Cu), a transition metal, has the tendency to increase in its concentration in freshwater ecosystems over natural levels, due to industrial and other anthropogenic sources. In water, copper can exist in dissolved form or associated with suspended food particles. Freshwater mussels living at the interface of the free-flowing water and the sediment phase can take up copper directly from the water or by consumption of lower trophic level organisms laden with copper. For mussels, copper is essential at low concentration as cofactor of metalloenzymes involved in growth regulation and development, but it may be toxic at higher levels by disturbing calcium (Ca) homeostasis. The duck mussel Anodonta anatina is a freshwater species found in abundance in limnic and lotic European ecosystems and is used as test organism in ecotoxicological studies. The potential involvement of Cu in the general decline of many European freshwater mussel species is the major motivation for this work. This research aims to study the relevance of Cu exposure pathways on its uptake, distribution, bioaccumulation, and elimination in the freshwater mussel A. anatina and its various potential physiological impacts. The work is started with raising Cu-loaded algae using the stable isotope 63Cu as marker for feeding of mussels without affecting the nutritional value of the algal food. In these latter experiments, mussels are exposed to 63Cu via water or via food to investigate the relative importance of Cu uptake to its distribution and accumulation among the mussel’s organs. Its consequences on calcium homeostasis, soluble carbohydrate and protein levels in various tissues, metallothionein induction, glutathione levels, activities of antioxidative enzymes and glutathione reductase, and on lipid peroxidation are examined. In the algal experiment, Parachlorella kessleri is grown at six 63Cu concentrations (0, 5.9, 11.7, 23.5, 47, and 94 µmol L-1) for 4 days, starting from day 3. When exposed to Cu at a level of up to 6 µmol L-1, P. kessleri is largely unchanged in its nutritional values; so this concentration is used to grow 63Cu-carrying food for mussel experiment. Concentrations above 6 µmol L-1 decrease significantly in the algal growth and alter the other physiological parameters. Three groups of 21 mussels each are used, one as control and two for exposure, receiving copper as the stable isotope 63Cu via the water at 0.3 µmol L-1 or via the food (1.5 mg L-1 freeze-dried Cu-loaded algae, equivalent to 0.06 µmol L-1 Cu) for 24 days, followed by 12 days of depuration. For analysis, three mussels each are taken randomly from every group at days 0, 6, 12, 18, 24, 30, and 36. The mussels are anaesthetized and hemolymph and extrapallial fluid are sampled before the mussels are dissected into gills, mantle, kidney, digestive gland, foot, adductors, intestines, and the remainder (gonads, heart, and labial palps). During copper exposure, the levels of exogenous copper (63Cu) and total Cu increase in all body compartments. Uptake via the water leads to higher Cu levels than via the food, but in relative terms food uptake is more efficient taking the five-fold lower nominal concentration of copper into consideration. Upon exposure via the water, the metal is compartmentalized mainly in the mantle, the gills, and the digestive gland, upon exposure via the food the major recipients are the digestive gland and the intestines. Upon depuration for two weeks, copper is quickly but not completely eliminated. Simultaneously with increasing Cu levels, Ca levels are increased in all body compartments, accompanied by decreases in soluble carbohydrates and proteins in the gills, mantle, digestive gland, and kidney. At the same time, Cu exposure results in increases in malondialdehyde levels, decreases in glutathione levels, strong increases in metallothionein levels, and changes in the activities of the antioxidative enzymes superoxide dismutase, catalase, and glutathione peroxidise, and of glutathione reductase in the gills, mantle, digestive gland, and kidney. During depuration, most parameters tend to normalize but do not return to control values. In conclusion, the overall pictures suggest that the considerable physiological stress elicited by low-level copper exposure may contribute to the factors involved in the decline of many European freshwater mussels.
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