Revisiting the mechanisms of copper toxicity to rainbow trout: Time course, influence of calcium, unidirectional Na+ fluxes, and branchial Na+, K+ ATPase and V-type H+ ATPase activities

In order to resolve uncertainties as to the mechanisms of toxic action of Cu and the protective effects of water [Ca], juvenile rainbow trout were acclimated to baseline soft water (SW, [Na+] = 0.07, [Ca2+] = 0.15, [Mg2+] = 0.05 mmol L-1) and then exposed to Cu with or without elevated [Ca] but at constant titratable alkalinity (0.27 mmol L-1). The 96-h LC50 was 7-fold higher (63.8 versus 9.2 mu g Cu L-1; 1.00 versus 0.14 mu mol Cu L-1) at [Ca] = 3.0 versus 0.15 mmol L-1. Gill Cu burden increased with exposure concentration, and higher [Ca] attenuated this accumulation. At 24 h, the gill Cu load (LA(50) approximate to 0.58 mu g Cu g(-1); 9.13 nmol Cu g(-1)) predictive of 50% mortality by 96 h was independent of [Ca], in accord with Biotic Ligand Model (BLM) theory. Cu exposure induced net Na+ losses (J(net)(Na)) by increasing unidirectional Na+ efflux rates (J(out)(Na)) and inhibiting unidirectional Na+ uptake rates (J(in)(Na)). The effect on J(out)(Na) out was virtually immediate, whereas the effect on J(in)(Na) developed progressively over 24 h and was associated with an inhibition of branchial Na+, K+ ATPase activity. The J(in)(Na) inhibition was eventually significant at a lower Cu threshold concentration (15 mu gCu L-1) than the J(out)(Na) stimulation (100 mu g Cu L-1). Elevated Ca protected against both effects, as well as against the inhibition of Na+, ATPase activity. Branchial V-type H+ ATPase activity was also inhibited by Cu exposure (100 mu g Cu L-1), but only after 24 h at high [Ca] (3.0 mmol L-1). These novel results therefore reinforce the applicability of BLM theory to Cu, clarify that whether Na+ influx or efflux is more sensitive depends on the duration of Cu exposure, show that elevated water [Ca], independent of alkalinity, is protective against both mechanisms of Cu toxicity, and identify V-type H(+)ATPase as a new Cu target for future investigation. (C) 2016 Elsevier B.V. All rights reserved.