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, [Ca(2+)]=0.15, [Mg(2+)]=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 (J(Na)net) by increasing unidirectional Na(+) efflux rates (J(Na)out) and inhibiting unidirectional Na(+) uptake rates (J(Na)in). The effect on J(Na)out was virtually immediate, whereas the effect on J(Na)in developed progressively over 24h and was associated with an inhibition of branchial Na(+), K(+) ATPase activity. The J(Na)in inhibition was eventually significant at a lower Cu threshold concentration (15μgCuL(-1)) than the J(Na)out 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.