Environmental Toxicology SALINITY ACCLIMATION MODULATES COPPER TOXICITY IN THE SHEEPSHEAD MINNOW, CYPRINODON VARIEGATUS JOSEPH A. ADEYEMI and PAUL L. KLERKS * Department of Biology, University of Louisiana at Lafayette, Lafayette, Louisiana, USA (Submitted 24 November 2011; Returned for Revision 9 January 2012; Accepted 20 February 2012) Abstract —The sheepshead minnow (Cyprinodon variegatus) is able to withstand a wide range of salinities. Salinity acclimation involves physiological and biochemical changes, which may inﬂuence how organisms respond to a stressor. The present study investigated effects of salinity acclimation on subsequent Cu toxicity. In experiment 1, ﬁsh were acclimated to a hyposmotic, isosmotic, or hyperosmotic salinity for 14 d and then exposed at these salinities to 16.6 mM Cu 2þ for 12 h. Survival differed during this Cu challenge; ﬁsh acclimated to 2.5 ppt salinity were much more sensitive to Cu than those acclimated to 10.5 or 18.5 ppt seawater. In experiment 2, ﬁsh were exposed to 14.6 mM Cu 2þ for 6 h after the 14-d salinity acclimation. Whole-body Cu, whole-body Na, liver lipid peroxidation (LPO), liver catalase activity, and liver glucose levels were determined before and after Cu exposure. Prior to Cu exposure, the acclimation groups differed only for liver glucose levels, which were higher in the 2.5 ppt acclimated ﬁsh than in the others. These same 2.5 ppt acclimated ﬁsh were markedly affected by Cu, having increased whole-body Cu and liver LPO and decreased whole-body Na levels. Copper exposure had generally insigniﬁcant effects for the 10.5 ppt and the 18.5 ppt acclimated ﬁsh. This study showed that even in euryhaline ﬁsh, salinity acclimation can have a drastic effect on Cu toxicity. Environ. Toxicol. Chem. # 2012 SETAC Keywords —Salinity acclimation Lipid peroxidation Glucose level Survival Copper accumulation INTRODUCTION Euryhaline teleost ﬁsh possess the ability to adjust quickly and effectively to ﬂuctuations in environmental salinity [1,2]. This rapid adjustment has been linked to various physiological and biochemical changes, such as activation of osmoregulatory mechanisms, augmented antioxidant defenses, and alteration of energy metabolites [3–5]. It is largely unknown, however, whether these changes provide any advantages or disadvantages in terms of tolerance to subsequent contaminant exposure. For example, key enzymes involved in salinity acclimation may be inhibited by contaminants [6–8]. Fish in coastal waters often encounter both salinity changes and elevated contaminant levels, rendering the interaction between salinity acclimation and toxicant response important [9,10]. The present study was designed to determine the effects of acclimation to different salinities on the toxicity of Cu in the sheepshead minnow, Cyprinodon variegatus. One likely target for interaction between salinity acclimation and Cu toxicity is the Na þ /K þ -ATPase; a membrane protein that is highly conserved in animals. In ﬁsh, it is abundant in the basolateral region of the gill [8]. It is possible that the increased activity and elevated expression of Na þ /K þ -ATPase during salinity acclimation is actually beneﬁcial to this ﬁsh during subsequent Cu exposure. For example, because Cu is known to cause impairment of ionoregulation by inhibiting Na þ /K þ -ATPase [6–8], an increased activity and expression of Na þ /K þ -ATPase resulting from the salinity acclimation may buffer the delete- rious effects of Cu. Various studies have reported interactions between salinity and Cu toxicity in ﬁsh [6,11,12]. Salinity has been shown to affect Cu complexation in water [13,14], and it has been shown that Cu toxicity depends on the concentration of free cupric ions [Cu 2þ ] rather than on total Cu levels [15,16]. Although salinity acclimation and ionic- and osmotic regulation in ﬁsh are well understood [17–20], the effect of salinity acclimation on sub- sequent Cu exposure is not. Our prediction is that ﬁsh accli- mated to suboptimal salinities (hyposmotic and hypersomotic) are more likely to exhibit increased tolerance to subsequent Cu exposure compared with ﬁsh that were acclimated to an isosmotic condition. This prediction is based on the fact that salinity acclimation may result in elevated expression of Na þ /K þ -ATPase in ﬁsh acclimated to suboptimal salinities compared with those acclimated to the isosmotic salinity. For the present study, sheepshead minnows were acclimated for two weeks to hypo- smotic (2.5 ppt), isosmotic (10.5 ppt), or hyperosmotic (18.5 ppt) salinity. These ﬁsh were then exposed to Cu at these salinities, at the same free cupric ion concentration. The following toxicity indices were measured before and after the Cu exposure: survival, whole-body Cu, whole-body Na, liver lipid peroxida- tion, liver catalase activity, and liver glucose levels. MATERIALS AND METHODS Fish collection Sheepshead minnows (mixed sexes; mean  standard deviation [SD] for standard length and wet wt were 3.17  0.28 cm and 1.16  0.35 g wet wt, respectively) used in this study were collected near Avery Island, Louisiana (N 298 56.155 0 ; W 918 54.388 0 ). The salinity at the time of collection was 7 ppt and was measured using a salinometer (YSI 30, Yellow Springs Instruments). Fish were netted and transported to the laboratory in coolers containing aerated ambient water. To quantify the salinity that corresponds to an isosmotic con- dition for these ﬁsh, a vapor pressure osmometer (Wescor) was used to determine the plasma osmolality of the ﬁsh. The latter Environmental Toxicology and Chemistry # 2012 SETAC Printed in the USA DOI: 10.1002/etc.1850 * To whom correspondence may be addressed (klerks@louisiana.edu). Published online 17 April 2012 in Wiley Online Library (wileyonlinelibrary.com). 1