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

Abstract

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+]=0.05mmolL−1) and then exposed to Cu with or without elevated [Ca] but at constant titratable alkalinity (0.27mmolL−1). The 96-h LC50 was 7-fold higher (63.8 versus 9.2μgCuL−1; 1.00 versus 0.14μmolCuL−1) at [Ca]=3.0 versus 0.15mmolL−1. Gill Cu burden increased with exposure concentration, and higher [Ca] attenuated this accumulation. At 24h, the gill Cu load (LA50≈0.58μgCug−1; 9.13nmolCug−1) predictive of 50% mortality by 96h was independent of [Ca], in accord with Biotic Ligand Model (BLM) theory. Cu exposure induced net Na+ losses (JNanet) by increasing unidirectional Na+ efflux rates (JNaout) and inhibiting unidirectional Na+ uptake rates (JNain). The effect on JNaout was virtually immediate, whereas the effect on JNain developed progressively over 24h and was associated with an inhibition of branchial Na+, K+ ATPase activity. The JNain inhibition was eventually significant at a lower Cu threshold concentration (15μgCuL−1) than the JNaout stimulation (100μg Cu L−1). Elevated Ca protected against both effects, as well as against the inhibition of Na+, K+ ATPase activity. Branchial V-type H+ ATPase activity was also inhibited by Cu exposure (100μgCuL−1), but only after 24h at high [Ca] (3.0mmolL−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.