Universität Bayreuth
Lehrstuhl für Umweltchemie und Ökotoxikologie

Diplomarbeit im Studiengang Geoökologie

Effects of Cd on the Ca metabolism of freshwater mussels

David Faubel

Index

1 Introduction ... 1

2 Material and methods ... 5

2.1 Animals ... 5
2.2 Materials ... 5
2.3 Cleaning of equipment for sample storage ... 6
2.4 Exposure experiments ... 7
2.5 Analysis of body fluids ... 11
    2.5.1 Sampling ... 11
    2.5.2 Sample analysis ... 13
2.6 Analysis of soft tissues ... 13
    2.6.1 Sampling ... 13
    2.6.2 Sample preparation ... 13
    2.6.3 Sample analysis ... 14
2.7 Data preparation ... 14

3 Results ... 16

3.1 Element distribution ... 16
3.2 Treatment effects ... 17
    3.2.1 Treatment with Cd ... 22
    3.2.2 Treatment with vitamin D ... 22
    3.2.3 Treatment with SEA0400 ... 22
3.3 Correlation between Ca and Cd ... 22

4 Discussion ... 26

4.1 Quality control ... 26
4.2 Troubleshooting ... 26
    4.2.1 Tank water pH and temperature ... 26
    4.2.2 Feeding ... 27
    4.2.3 Cadmium exposure concentration ... 28
    4.2.4 Injection of test substances ... 28
    4.2.5 Kidney tissue sampling ... 29
    4.2.6 Tissue digestion ... 30
4.3 Ion fluxes in a model freshwater mussel ... 30
    4.3.1 Treatment with Cd ... 32
    4.3.2 Treatment with vitamin D ... 33
    4.3.3 Treatment with SEA0400 ... 34
4.4 Literature review ... 35

5 Conclusion ... 40

6 Appendix ... 42

6.1 Experimental protocols ... 42
    6.1.1 Experiment a ... 42
    6.1.2 Experiment b ... 46
    6.1.3 Experiment c ... 48
    6.1.4 Experiment d ... 50
6.2 Database ... 52
    6.2.1 Experiment a ... 52
    6.2.2 Experiment b ... 59
    6.2.3 Experiment c ... 62
    6.2.4 Experiment d ... 70
6.3 Calculation results ... 77
    6.3.1 Cadmium in fluid samples ... 78
    6.3.2 Cadmium in tissue samples ... 79
    6.3.3 Cadmium in kidney samples ... 80
    6.3.4 Calcium in fluid samples ... 81
    6.3.5 Calcium in tissues samples ... 82
    6.3.6 Calcium in kidney samples ... 83
    6.3.7 Magnesium in fluid samples ... 84
    6.3.8 Magnesium in tissue samples ... 85
    6.3.9 Sodium in fluid samples ... 86
    6.3.10 Sodium in tissue samples ... 87
    6.3.11 Potassium in fluid samples ... 88
    6.3.12 Potassium in tissue samples ... 89
    6.3.13 Chlorine in fluid samples ... 90
    6.3.14 Osmolarity of fluid samples ... 91
6.4 References ... 92
6.5 Index of tables ... 95
6.6 Index of figures ... 97
6.7 Index of abbreviations ... 100
6.8 Index of symbols ... 100

1 Introduction

Freshwater mussels live as filter-feeders (Watters, 1998a). They filter plankton, suspended and dissolved organic matter out the aqueous medium to use it as a food source (Russell-Pinto, 1998). For this they take up water from the exterior into their mantle cavity through their inhalant aperture. The inhalant water current is produced by cilia on the gills. Due to the ciliary movement the water streams by the ctenidia, which secrete mucus to bind the food particles in the water. The mucus is then transported with the water current to the mouth at the end of the mantle cavity (Moore, 2001). From there the water returns through the suprabranchial chamber, where it takes up the faeces and excretion products, just before leaving the mussel again through the exhalant aperture (Morton, 1983). As the water circulates through the whole mantle cavity, all body parts of the mussel are exposed to pollutants possibly carried by the water. These pollutants can be taken up by the mussel, and if not metabolised and excreted, they accumulate. In fact, the elimination rates of these organisms are very low (Fent, 1998). Pollutant concentrations in mussels reflect the pollution accumulated over a certain period, rather than momentary environmental concentrations (Gundacker, 1999). This property of mussels makes them useful for the biomonitoring of chronic pollution (Franco, 2002).
One pollutant freshwater mussels are exposed to is cadmium. This heavy metal is used in dry batteries, alloys and anticorrosive coatings. Cadmium compounds are also used as heat resistant colour pigments and stabilizers for PVC. It is released into the environment mainly from smelteries and waste incinerating plants. World-wide emissions are around 8000 t/a, 90% of which are of anthropogenic origin (Bliefert, 1995). In the atmosphere cadmium is transported bound to small aerosol particles. The atmospheric deposition of these particles onto rivers and lakes is only one way cadmium can enter freshwater systems. Run-off from fields fertilized with sewage sludge or other fertilizers containing cadmium, precipitation water from galvanised gutters as well as leachate from landfills are other possible sources (Bliefert, 1995).
Cadmium is a non-essential metal that is accumulated by animals and plants. Its biological half-life in humans can be of up to 30 years (Timbrell, 1993), where it accumulates especially in the kidney, leading to renal failure. Cadmium is also known to disturb the calcium metabolism of humans, causing osteoporosis and making the bones brittle, symptoms of the Itai-Itai disease. This disease appeared during the 1940s in Japan, where people were eating rice contaminated with cadmium (Bliefert, 1995).
In a similar way, cadmium could interfere with the calcium metabolism of freshwater mussels. These animals need calcium primarily to form their shells, which are made of calcium carbonate. Besides calcium is also stored as extracellular concretion spherules (Dube, 1997), 75% of which are found in the gills (Pynnönen, 1987). These concretions are made of calcium phosphate, which is less soluble than calcium carbonate (Dube, 1997). Hence, the shell material dissolves first when body fluid pH goes down, releasing carbonate ions, which act as a pH-buffer (Greenaway, 1971). In fact the carbonate buffer represents the most important pH-buffer in mussels, since the haemolymph contains very low amounts of proteins, which have a buffering capacity as well. To conserve the calcium ions released, they are immobilized in the phosphate concretions (Dube, 1997). Under normal pH conditions the shell is then again regenerated using the calcium ions from the concretion spherules. These have the enzyme carbonic anhydrase on their surface (Istin, 1970), which catalyses the formation of carbonic acid out of water and carbon dioxide (Wilbur, 1983). The protons resulting out of the dissociation of the carbonic acid dissolve material from the phosphate concretions thereby releasing calcium, which becomes available again for shell regeneration (Istin, 1970). Tracer experiments with radio-calcium revealed a half-life of calcium in the phosphate concretions of Velesunio angasi of 106 days (Brown, 1996). The calcium from the concretion spherules is also used to form the glochidial shells (Watters, 1998b). To maintain all these functions, freshwater mussels have to take up calcium from their environment. Even though they live in a medium with low calcium concentrations, it has been estimated that freshwater molluscs take up 80% of the calcium they need from the water (van der Borght, 1966); freshwater Ca concentrations are below 3 mmol/L (Bliefert, 1995). The remaining 20% are taken up with the food. There are three sites in their body, where freshwater mussels take up calcium, namely the gills, the mantle and the intestine, the mantle, however, not being the main uptake route (Coimbra, 1993). The mechanism, however, by which calcium is taken up is still unclear. Due to the different Ca concentrations in the external medium and the body fluids, the latter being around 6 mmol/L in Anodonta cygnea (Coimbra, 1988), it must be an energy-driven mechanism. Little is also known about the transport of calcium within the body. The transfer of calcium from one compartment to another is linked to the transport across the epithelia that border the different organs. All these uptake and transport mechanisms can potentially be disturbed by cadmium, that has been reported to compete with calcium for the transport sites of Ca-ATPases (Long, 1997) and to block calcium channels (Kochegarov, 2003). Likewise, the calcium channel blockers verapamil and lanthanum have been shown to inhibit the Cd uptake in mussels (Sidoumou, 1997; Qiu, 2005), showing the similarity of both ions’ uptake pathways. The Antagonism between calcium and cadmium was also observed by Mungkung (2001), who found that the cadmium toxicity and accumulation in freshwater fish decreased with increasing water hardness, which is primarily controlled by calcium. He traced this finding back to the competition between calcium and cadmium for the binding sites of the same uptake systems. It was found green mussels collected from low salinity sites accumulated six time more cadmium than individuals from high salinity sites. This observation was also attributed to the competition of cadmium with major ions like Ca2+ for the uptake through calcium channels (Blackmore, 2002). These findings have been confirmed recently as well by Qiu (2005), who found the cadmium uptake of Asiatic clams to be inhibited by high calcium concentrations. He describes this competition between calcium and cadmium using a Michaelis-Menten uptake inhibition model. Brown (1996) suggested that 109Cd, among other radio-nuclides, is absorbed by freshwater bivalves in a similar manner to Ca with subsequent deposition into the concretion spherules.
Another group of membrane proteins that transport calcium are the sodium-calcium exchangers (NCX). Under normal physiological conditions the sodium concentration outside the cell is higher than inside of it. Driven by this concentration gradient across the cell membrane the NCX in the plasma membrane pump calcium to the outside of the cell in exchange for sodium, which is transferred to the inside of the cell (Ca efflux mode). This is, for instance, the case at the basolateral membrane of intestinal cells, where NCX extrude calcium to the extracellular medium (Larsson, 2002). Under special conditions in which sodium accumulates inside the cell, the NCX work inversely (Ca influx mode), pumping calcium from the outside to the inside of the cell (Iwamoto, 2003). NCX are inhibited selectively by the novel compound 2-[4-[(2,5-difluorophenyl)methoxy]phenoxy]-5-ethoxyaniline (Fig. 1.1) also known as SEA0400 (Matsuda, 2001). The inhibitory potency of SEA0400 is strongly dependent on the intracellular sodium concentration. Only when high sodium concentrations are present within the cell and the NCX work in the Ca influx mode, SEA0400 develops its inhibitory potential (Lee, 2004). SEA0400 scarcely inhibits other receptors, channels and transporters at the concentrations it inhibits NCX (Iwamoto, 2003). The IC50 values of SEA0400 for the sodium-dependent radio-calcium uptake in cultured rat neurons, astrocytes, and microglia are between 5 and 33 nmol/L (Matsuda, 2001); the value obtained for ventricular cardiomyocytes of guinea-pigs is between 30 and 40 nmol/L (Tanaka, 2002).

Fig. 1.1: Chemical structure of SEA0400 (Matsuda, 2001). (only available in download-version)

A third compound known to have an effect on the uptake of calcium is vitamin D3. In the human liver, vitamin D3 is converted to 25-hydroxyvitamin D3, which is transported to the kidney, where it is again converted to 1,25-dihydroxy-vitamin D3 (1,25(OH)2D3). 1,25(OH)2D3 can then be transported to different tissues. In the intestinal epithelial cells, for instance, 1,25(OH)2D3 promotes the expression of genes encoding calcium binding proteins (calbindin). Calbindin binds calcium in the microvillae region and facilitates its transport to the basolateral membrane, where calcium is finally extruded from the cell. The decrease of the calcium concentration near the apical membrane, however, favors the movement of calcium into the cell and thereby the transport across the epithelium (McCarthy, 1999). It is conceivable that vitamin D3 regulates the intestinal absorption of calcium in mussels in a similar way. This mechanism of transcellular calcium transport by calbindin is relatively slow and therefore relevant only for long-term calcium regulation. For the short-term regulation the vesicular transport and the tunnelling through the endoplasmic reticulum are more important (Larsson, 2002).
To achieve a better understanding of how cadmium interferes with the calcium metabolism of freshwater mussels, four cadmium exposure experiments were performed with the freshwater clam A. cygnea, which belongs to the family of the Unionidae (Kaestner, 1993). Separate experimental groups were treated with vitamin D3 and SEA0400 to compare the effects cadmium has with those of other substances influencing the calcium metabolism. The tissue and body fluid samples taken from the animals were not only analysed for cadmium and calcium, but also for other ions of physiological importance, that might elucidate the calcium transport processes further. These additional ions were magnesium, sodium, potassium and chlorine. The osmolarity of the body fluid samples was also measured.

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