Effects of Cadmium on Calcium homeostasis and physiological conditions of the freshwater Mussel Anodonta anatina
Summary As the result of the worldwide decline of freshwater mussels, some significant wetland and riverine habitats are threatened. It has been shown that cadmium (Cd), among other heavy metals, is present at surprisingly high levels in freshwater pearl mussels from mountain brooks in central Europe. This metal is likely to be one of the factors involved in the decline because of its high toxicity, bioaccumulation potential and transfer through food chains. In natural ecosystems, aquatic animals are usually exposed to Cd from both the dissolved- and the particulate-phase, and at lower levels than those used in standard toxicity testing. The aim of this study was to investigate the effects of environment-like Cd levels on its bioaccumulation, tissue distribution and on the redistribution of background Cd (Cd present in animals prior to the experiment) among body compartments of the freshwater mussel Anodonta anatina by exposure to both algae- and water-borne 111Cd (about 0.2 µg/L) for 35 days followed by 120 days of depuration. Furthermore, the effects of Cd on calcium (Ca) homeostasis, condition index, energy reserves, carbonic anhydrase activity and their correlations were examined and evaluated. Before mussels were exposed to Cd, a Cd-exposure experiment with the green algae Parachlorella kessleri was carried out for producing 111Cd-loaded algae suitable for exposed mussels as food of the same quality as non-contaminated algae. P. kessleri were grown at four Cd concentrations (0, 0.5, 2, 8 and 32 µg/L) for 5 days, starting from day 2 after inoculation. At 8 µg Cd/L and higher, Cd showed clear effects on algal growth, cell morphology, size and algal physiological state. The minimum Cd concentration at which the algae were significantly different from the control group was about 3 µg/L. At a Cd concentration of 2 µg/L, algae exhibited a comparable physiological state to the control which was used to grow 111Cd-carrying food for the test mussels. In the mussel exposure experiment, different types of samples were taken. Hemolymph (HML), extrapallial fluid (EPF), gills, mantle, digestive gland, kidney and other tissues were used to determine total Cd, the 114Cd/111Cd isotope ratio for studying Cd accumulation, distribution, elimination and redistribution of background Cd, and its effects on other element profiles (Ca, Mg and Zn). Effects of Cd on energy reserves (glycogen, glucose and proteins) and on carbonic anhydrase were investigated in the body fluids (HML and EPF), gills, mantle and digestive gland. During the exposure phase, newly incorporated Cd increased in all body compartments, the highest levels occurring in the kidney. Elimination of newly incorporated and background Cd was slow and mainly from the digestive gland; the increase of newly incorporated Cd in the mantle and of newly incorporated and background Cd in the kidney during exposure and the first half of depuration indicated the mobilization of the Cd pools from other body compartments and deposition in this organ. Ca concentrations in the body fluids increased during Cd exposure; at the same time, they were lowered in most organs, remaining unchanged only in the gills. Simultaneously, depletion of glycogen in the mantle and digestive gland accompanied by a rise in glucose and a decrease in protein levels in the HML and EPF were observed. This entailed a sharp decrease in condition index and relative dry weight (dw). Significant correlations of Cd with glycogen (mantle, digestive gland) and of Ca with glucose (body fluids) and glycogen (mantle, digestive gland) were found. Concerning enzymatic effects, significantly lower levels of carbonic anhydrase activities were observed in all tested tissues, especially in the gills and digestive gland; strong fluctuations were observed in the HML and EPF with a significant increase right after the rise of glucose in these compartments. The effects of Cd exposure on carbonic anhydrase activities in A. anatina were confirmed by significant negative correlations of both total and cytoplasmic carbonic anhydrase activities with newly incorporated Cd. In conclusion, A. anatina readily accumulates Cd at low exposure levels. This results in unfavorable effects on Ca profiles, energy reserves and carbonic anhydrase activities in tissue-specific and time-dependent manners. The effects are long lasting and entail interactions which finally influence Ca metabolism, e.g. adverse effects on energy reserves and enzyme activities. Among the organs, the digestive gland appears to be actively involved in the uptake of Cd from the outside environment and in distributing it to other organs via the HML. This makes it more sensitive to Cd exposure than other organs, reflected by a strong depression of glycogen, carbonic anhydrase activity and Ca level. HML and EPF are important compartments playing a crucial role in uptake and distribution of Cd, revealed by its effects on all tested parameters, i.e. Ca, glucose, proteins and carbonic anhydrase. The gills seem to be less sensitive due to their high amounts of calcareous concretions. The kidney acts as Cd sink for later excretion. Overall, these findings show that Cd at environment-like levels has distinct biochemical, toxicological and pathological effects which may constitute a critical component in the multitude of environmental factors leading to the observed general decline of freshwater mussel populations. Keywords: Anodonta anatina; Cd bioaccumulation; Cd redistribution; Ca homeostasis; freshwater mussel; stable isotope 111Cd; condition index; glycogen; glucose; protein; energy reserves; carbonic anhydrase; subcellular distribution of carbonic anhydrase; Parachlorella kessleri; Cd-loaded algae.
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