Effects of Cadmium on Calcium homeostasis and physiological conditions of the freshwater Mussel Anodonta anatina

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Ngo, Thi Thuy Huong:
Effects of Cadmium on Calcium homeostasis and physiological conditions of the freshwater Mussel Anodonta anatina.
Bayreuth , 2008
( Dissertation, 2008 , Universität Bayreuth, Fakultät für Biologie, Chemie und Geowissenschaften)

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Abstract

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.

Abstract in weiterer Sprache

Zusammenfassung Flussperlmuscheln sind europaweit von Aussterben bedroht. In Zentraleuropa sind sie überraschend hoch mit Cadmium (Cd) und anderen Schwermetallen belastet sind. Cd ist aufgrund seiner hohen Toxizität und seines Bioakkumulations-Potentials in Nahrungsketten mit hoher Wahrscheinlichkeit am Populations Rückgang beteiligt. Aquatische Organismen sind diesem Metall in der gelösten und in der festen Phase ausgesetzt, normalerweise in Konzentrationen, die weit niedriger sind als die bei üblichen Expositionsexperimenten. Das Ziel der vorliegenden Studie war es, die Wirkung von Cd bei Konzentrationen, wie sie in der Umwelt auftreten, auf die Süßwassermuschel Anodonta anatina zu untersuchen. Dies betraf besonders die Bioakkumulation, Gewebeverteilung und Diposition des im Tier vorhanden Cd und des neu aufgenommenen. Dazu wurden die Muscheln 35 Tage über Algen und Wasser mit 111Cd (ca. 0,2 µg/L) exponiert, gefolgt von 120 Tagen Depuration. Die Auswirkungen der Cd-Exposition auf die Calcium-(Ca)-Homöostase, Gewebe-Trockenmasse in Bezug auf das Schalenlange (Konditions-Index), Energiereserven, Carboanhydrase-(CA)-Aktivität wurden untersucht. Zuerst wurden die Bedingungen zur Kultivierung der Grünalge Parachlorella kessleri etabliert, um 111Cd-beladene Algen zu züchten, die als Futter für die Muscheln ebenso geeignet sind wie unbelastete Algen. Dazu wurde P. kessleri 2 Tage nach Inokulation über 5 Tage mit 4 verschiedenen Cd-Konzentrationen (0; 0,5; 2; 8 und 32 µg/L) kultiviert. Ab einer Konzentration von 8 µg/L zeigten sich starke Wirkungen auf Wachstum, Zellmorphologie, Größe und Physiologie. Die niedrigste Cd-Konzentration, bei der sich exponierte Algen von Kontrollen unterschieden, betrug 3 µg/L. Bei 2 µg/L kultivierte Algen entsprechen bezüglich des Gehaltes photosythentischer Pigmente und Morphologie unbelasteten Algen und sind als Futter für die Muschel Expositionsexperimente geeignet. Im Verlauf dieser Experimente wurden Proben von Hämolymphe (HML), extrapalliale Flüssigkeit (EPF), Kiemen, Mantel, Verdauungsdrüse, Niere und restliche Gewebe genommen, in denen das 114Cd/111Cd-Isotopenverhältnis, Gesamt-Cd, Cd-Akkumulation, Verteilung und Umverteilung des bereits zu Beginn im Körper vorhandenen Cd, sowie seine Effekte auf andere Elementprofile (Ca, Mg, Zn) bestimmt wurden. Die Wirkungen auf Energiereserven (Glykogen, Glukose, Proteine) und die Aktivitäten von CA in HML, EPF, Kiemen, Mantel und Verdauungsdrüse wurden ebenfalls untersucht. Während der Exposition stiegen die Konzentrationen an neu akkumuliertem Cd in allen Körper-Kompartimenten, mit höchsten Werten in der Niere. Die Elimination von neu aufgenommenem und vorhandenem Cd war langsam und verlief zum Teil über die Verdauungsdrüse, zum Teil über die Niere. Die starke Zunahme von Cd im Mantel und in der Niere während der Depuration zeigten die Mobilisierung von Cd-Reservoirs aus anderen Körper-Kompartimenten. Die Ca-Konzentrationen nahmen während der Cd-Exposition in den Körperflüssigkeiten zu, ab im Gewebe; in den Kiemen blieb sie fast unverändert. Damit ging eine Glykogen-Verarmung des Mantels und der Verdauungsdrüse einher, simultan stiegen die Glukose-Gehalte in HML und EPF, während Protein-Gehalte sowie relatives Trockengewicht fielen. Korrelationen von Cd zu Glykogen (Mantel, Verdauungsdrüse) und von Ca zu Glukose (Körperflüssigkeiten) und Glykogen (Mantel, Verdauungsdrüse) waren hoch signifikant. Besonders in den Kiemen und der Verdauungsdrüse wurden signifikant niedrige CA-Aktivitäten beobachtet. In den Körperflüssigkeiten HML und EPF traten starke Fluktuationen auf; besonders kam es direkt nach der Glukose-Zunahme zu einem signifikanten CA-Anstieg. Die Auswirkungen der Cd-Exposition spiegelten sich in seiner negativen Korrelation zur CA-Gesamt-Aktivität im Gewebe und im Cytoplasma wieder. A. anatina akkumuliert bei niedrigen Expositions-Konzentrationen Cd stark. Dies hat biochemisch-physiologische Effekte zur Folge, besonders in Bezug auf Ca-Profile, Energiereserven und CA-Aktivitäten. Die Auswirkungen sind langanhaltend und ziehen Interaktionen nach sich, die letztendlich den Ca Metabolismus beeinflussen, z.B. gegensätzliche Effekte auf Energiereserven und Enzymaktivitäten. Die Verdauungsdrüse ist das wichtigste Organ für Cd-Aufnahme aus der Umgebung; von dort wird Cd über die HML in andere Organe verteilt. Dies macht die Verdauungsdrüse empfindlicher als andere Organe, wie starke Rückgange von Glykogen, CA-Aktivität und Ca-Konzentration zeigen. HML und EPF sind Kompartimente, die bei Cd-Aufnahme und Verteilung eine wichtige Rolle spielen. Dies zeigt sich in den Auswirkungen auf Ca, Glucose und Protein sowie auf die CA-Aktivität. Die Kiemen sind am unempfindlichsten, wahrscheinlich wegen ihres hohen Anteils an Calciumcarbonat-Ablagerungen. Die Niere ist eine Cd-Senke. Insgesamt zeigen die Ergebnisse, dass Cd in Konzentrationen, wie sie in der Umwelt auftreten, toxische Wirkungen hat. Es ist wahrscheinlich, dass die Cd-Belastung von Süßwassermuscheln einen wesentlichen Anteil am beobachteten Rückgang der Populationen beteiligt sind. Schlüsselwörter: Anodonta anatina, Cd-Bioakkumulation, Cd-Umverteilung, Ca-Homöostase, Süßwassermuschel, stabiles Isotop 111Cd, Konditions-Index, Glykogen, Glukose, Protein, Energiereserve, Carboanhydrase, subzelluläre Distribution, Parachlorella kessleri, Cd-belastete Algen 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.