Calcium control of zinc uptake in rainbow trout
Introduction
Previous studies have shown that high water hardness reduced the acute toxicity of zinc to fish and lowered the accumulation of zinc in both gills and whole body of fish (Mount, 1966, Solbe, 1974, Zitco and Carson, 1976, Everall et al., 1989). For example, zinc was 27 times more toxic to rainbow trout (Oncorhynchus mykiss) in hard water (390 mg CaCO3/l) than in soft water (31 CaCO3/l) (Bradley and Sprague, 1985a). Greater influx of zinc in soft water compared to hard water has also been observed in vivo in brown trout (Salmo trutta) (Everall et al., 1989) and in the isolated perfused head of rainbow trout (Spry and Wood, 1988). There has been much speculation in the literature on the predominant mechanism of water hardness effects on zinc toxicity and accumulation. Suggestions have included a biological mechanism (e.g. decreased membrane permeability, fewer uptake sites, or lower intrinsic toxicity) or a chemical mechanism (e.g. competition for absorption sites or complexation) (Zitco and Carson, 1976, Bradley and Sprague, 1985b, Everall et al., 1989, Alsop et al., 1999). Recent research indicates that both zinc and calcium competitively inhibit the uptake of each other across the gill and compete for the same uptake site (Hogstrand et al., 1994, Hogstrand et al., 1995, Hogstrand et al., 1998). The apical membrane of the chloride cells appear to be the site of entry of both calcium and zinc across the gill (Spry and Wood, 1988, Hogstrand et al., 1995, Galvez et al., 1998). Current research on zinc–water hardness interactions is focusing on the mechanisms of zinc acclimation in fish (Galvez et al., 1998, Alsop et al., 1999). The dominant mechanism of calcium control of zinc uptake has not been elucidated, nor has the effect of calcium acclimation and exposure on zinc uptake been investigated independently of other cations.
The objective of this study was to determine calcium effects on the uptake of zinc in the gill and whole body of rainbow trout by altering calcium concentrations and maintaining magnesium and bicarbonate concentrations constant. Zinc uptake was measured in four groups of rainbow trout acclimated and/or exposed to calcium concentrations of 6.5 mg/l Ca (160 μM) or 131 mg/l Ca (3300 μM) and sublethal concentrations of radiolabeled zinc (100 μg 65Zn/l; 1.5 μM). These treatments encompassed ranges of calcium and water hardness typical of fresh waters, and were selected to allow discrimination of calcium control of zinc uptake from biological processes (low vs. high Ca acclimation) and chemical processes (low vs. high Ca exposure). An additional group of trout was acclimated and exposed to high bicarbonate and low calcium as a negative control. Changes in alkalinity without concomitant changes in water hardness and calcium were hypothesized to have no effect on zinc uptake. 65Zn was selected for this study because of the high endogenous levels of zinc in fish, and zinc speciation is less affected by changes in water quality at near neutral pHs than many other metals (Stumm and Morgan, 1966, Bradley and Sprague, 1985a, Alsop et al., 1999). Zinc uptake in the gill was studied because the gill is the site of action of acute zinc toxicity to fish (Skidmore, 1970), and understanding the kinetics of metal binding to gills has been identified as a critical research need (Wood et al., 1996).
Section snippets
Fish acclimation
Rainbow trout weighing approximately 0.5 g were obtained from the Spokane Fish Hatchery, Spokane, Washington, and then were maintained in recirculating raceways with laboratory water (deionized tap water; 6.5 mg Ca/l) for at least 2 weeks at 15±1°C (pre-acclimation period). Fish (∼1 g each) were then acclimated for 14–22 days at 15±1°C to either low (6.5 mg Ca/l; 160 μM) or high (131 mg Ca/l; 3300 μM) calcium treatments (Table 1). The high calcium treatment water was prepared by mixing CaSO4
Water chemistry
Concentrations of 65Zn in all exposures were similar to the nominal level of 100 μg/l and were relatively constant over the 24 h exposure period (Table 2). Maximum declines in 65Zn levels were less than 5% between 0 and 24 h. Calcium and total hardness concentrations were significantly greater in the high calcium treatments, whereas pH, magnesium, and bicarbonate levels were similar in low and high calcium treatments (Table 1). The molar ratio of calcium to magnesium was 24 times higher in the
Discussion
Previous studies have demonstrated that high water hardness reduced the acute toxicity of zinc to fish and lowered the accumulation of zinc in both gills and whole body of fish (Mount, 1966, Solbe, 1974, Zitco and Carson, 1976, Bradley and Sprague, 1985a, Bradley and Sprague, 1985b, Everall et al., 1989, Alsop et al., 1999). We observed that elevated calcium significantly lowered 65Zn uptake in both the gills and whole body of rainbow trout independently of other cations and bicarbonate. The
Conclusions and research recommendations
Despite numerous studies, the predominant mechanism of water hardness effects on zinc toxicity and accumulation has remained uncertain. Our study is the first to clearly separate biological acclimation from chemical effects, and to demonstrate that calcium can mediate these effects independently of other cations. The specific biological and chemical mechanisms remain to be elucidated. Future research could be directed at evaluating biological mechanisms of calcium protection, such as
Acknowledgements
We thank Catherine Pagano, Paul Welsh, and Richard Playle for advice and assistance. Sigma Xi provided funding for the experimental phase of this study, and Stratus Consulting/Hagler Bailly supported the data evaluation phase of this work.
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