Zebrafish (n=420; 70/treatment) were weighed and placed in 8L aerated tanks,<br></br>served with flow-through soft-water at 25ml/min. A combination of Mariotte bottles<br></br>were used to dose tanks with Cu (concentrated Cu solution made from CuSO4 dissolved<br></br>in 0.05% HNO3), Na (concentrated NaCl solution), and Ca (concentrated CaCl2 solution)<br></br>to 6 treatment regimes of either control (Ctrl), Cu only (Cu), high Ca only (Ca), Ca + Cu<br></br>(CaCu), high Na only (Na), and Na + Cu (NaCu; See Table 1 for ion concentrations).<br></br>Nitric acid had no measurable impact on water pH. Fish were fed 2% body weight of<br></br>commercial tropical fish food, once per day. Tanks were monitored daily for mortality<br></br>and cleaned of any food or waste that had accumulated. Water samples (10 ml) were<br></br>taken from each tank, filtered though a 0.45?m filtration disc (Pall Corporation, East<br></br>Hills, NY), added to a plastic tube containing 100?l HNO3 and kept at 4oC for analysis of<br></br>ion content and Cu concentration. Throughout the experiment, there were no mortalities<br></br>in any of the tanks. At the end of the exposure period, fish were quickly euthanized by<br></br>overdose of buffered aesthetic (MS-222, Sigma) and sampled for gill, liver, and gut,<br></br>which were immediately frozen in liquid N2 for further analysis of Cu burden, gene<br></br>expression, and enzyme activity.