2761 Environmental Toxicology and Chemistry, Vol. 22, No. 11, pp. 2761–2767, 2003  2003 SETAC Printed in the USA 0730-7268/04 $12.00 + .00 EFFECT OF LOW SALINITY ON CADMIUM ACCUMULATION AND CALCIUM HOMEOSTASIS IN THE SHORE CRAB (CARCINUS MAENAS) AT FIXED FREE Cd 2+ CONCENTRATIONS JONATHAN BURKE,* RICHARD D. HANDY, and STEPHEN. D. ROAST School of Biological Sciences, University of Plymouth, Drake Circus, Plymouth PL4 8AA, United Kingdom ( Received 4 September 2002; Accepted 24 March 2003) Abstract—Increased Cd toxicity at low salinity has been attributed to increased free Cd 2+ ion concentration ([Cd 2+ ] sw ), but transfer to dilute seawater also stimulates physiological ionic regulation in crabs. In this study, Cd accumulation and Ca homeostasis in the shore crab (Carcinus maenas) were explored at fixed [Cd 2+ ] sw to reveal the physiological events during sublethal Cd exposure. Crabs were exposed to 3.4 or 34 g/L [Cd 2+ ] sw in both 100% seawater (SW) and 33% SW for up to 10 d and sampled for hemolymph composition as well as gill and hepatopancreas Ca, Cd, and Ca-ATPase activity. Cadmium exposure ameliorated the expected fall in hemolymph osmotic pressure and NaCl at low salinity and generally protected tissue Ca from decline. Cadmium exposure alone (within salinity) inhibited Ca-ATPase, but this was offset by stimulation of Ca-ATPase at low salinity. The Ca-ATPase activityin the anterior and posterior gills showed different responses to Cd/low salinity stress. Crabs were more sensitive to a 10-fold increase in [Cd 2+ ] sw at low salinity. Overall, we conclude that exposure to a fixed sublethal [Cd 2+ ] sw reveals a compensatory physiological response that is driven primarily by salinity rather than Cd 2+ free ion concentration. Physiological responses are therefore important during low-level Cd exposure in dilute seawater. Keywords—Carcinus Cadmium Salinity Free ion concentration Calcium ATPase INTRODUCTION Cadmium (Cd) toxicity to aquatic biota is well known [1], and estuarine crustacea have received particular attention [2,3], partly because Cd toxicity increases with decreasing salinity [4–8]. This increased Cd toxicity at low salinity is associated with elevated Cd accumulation in crustacea [2], suggesting increased Cd bioavailability at low salinity. Cadmium forms a variety of chloride complexes in seawater (SW), including CdCl + , CdCl 3 - , CdCl 2 · , CdCl 4 - , and these dominate in 100% SW so that free Cd 2+ concentrations ([Cd 2+ ] sw ) are low (around 2.5%; [9–11]). With decreasing salinity, the concentration of Cl - ions declines, and this results in a higher proportion of Cd 2+ ions (around 20% in 1/3 SW), which is assumed to be available for uptake [11]. However, physicochemical events alone do not explain salinity-dependent Cd toxicity because calculation of median lethal concentrations on the basis of [Cd 2+ ] sw does not fit the expected salinity trend [12]. Alter- natively, physiological status may be important in Cd toxicity [13–15]. Many estuarine crustaceans ionoregulate in response to sa- linity changes and are able to maintain relatively stable osmotic pressure in the hemolymph by increasing or decreasing active ion uptake from the medium [16,17]. In the case of the shore crab (Carcinus maenas), ionoregulation includes increased up- take of Ca at low salinity, which has been correlated with Cd accumulation [4,18]. Indeed, the process of Cd uptake on the Ca-transport system has been well characterized, especially in fish gills, where about a third to a half of the total Cd uptake is as free Cd 2+ [19]. This Cd 2+ competes with Ca 2+ for entry into the gill cells via apical Ca channels, and, once accumulated in the cells, may inhibit basolateral Ca-ATPase to reduce Ca * To whom correspondence may be addressed (j.burke-2@plymouth.ac.uk). influx [20]. Similar mechanisms operate in crustacean gills, with additional Cd 2+ entry via stimulation of apical and/or basolateral Na/Ca exchangers [21–23]. Stimulation of these Ca-influx pathways by low salinity therefore might cause in- cidental uptake of Cd 2+ . However, the relative importance of modified ionoregula- tory physiology compared with metal speciation effects in the seawater for Cd accumulation has not been clarified for most crustaceans. In this experiment, we explore Cd accumulation and Ca homeostasis in the shore crab (Carcinus maenas) at normal and low salinity. Additionally, unlike previous studies on the shore crab, we fix the [Cd 2+ ] sw at all salinities used so that the physiological contribution to Cd uptake is revealed. Furthermore, we explore these events using low sublethal Cd concentrations so that Ca homeostasis, as measured by Ca- ATPase activity, remains physiologically responsive. We also explore Cd 2+ concentration effects on these revealed physio- logical components. Finally, for the first time, we demonstrate that Ca homeostasis in the anterior (respiratory) and posterior (ionoregulatory, e.g., [24]) gills differ in response to Cd ex- posure, confirming the notion of an ionoregulatory-driven pro- cess for Cd accumulation at low salinity. MATERIALS AND METHODS Experimental design Shore crabs, Carcinus maenas (mean wt  standard error [SE] was 45.6 g  1.15; n = 68), were collected from clean open seawater sites off Plymouth, United Kingdom. The an- imals were not fed prior to or during exposure. Crabs were kept in recirculating, filtered seawater at 14°C  1°C for at least 48 h before experiments started. The main purpose of the experiment was to determine Cd accumulation while iono- regulatory physiology was stimulated by transfer to dilute sea-