An in vitro approach for modelling branchial copper binding in rainbow trout

The main objective of this study was to characterize the individual effects of water chemistry (Ca(2+), Na(+), dissolved organic matter (DOM), pH, alkalinity) on the rapid binding of copper to the gill surface of rainbow trout using an in vitro gill binding assay. In this assay, individual gill arches were exposed for 5 min to (64)Cu labelled copper solutions ranging from 0.02 to 0.16 microM in water chemistries reflecting the full range of fresh water values for the Great Lakes. The gills displayed saturable Cu binding within this Cu range but gill-Cu binding was completely unaffected over the full range of calcium, sodium and alkalinity concentrations used. Only low pH (pH 4.0) and commercial DOM (Aldrich humic acid at > or =3 mgC/l) altered copper binding to rainbow trout gills in vitro. These findings were consistent with the results of geochemical modelling of our water chemistry (using MINEQL+, Version 4.5) which showed that H(+) and DOM affected the free cupric ion concentration. However, DOM (up to 80 mgC/l) was only able to reduce Cu on the gills by 50%. We hypothesize that in the range of 0.02-0.16 microM Cu there are two high affinity Cu binding sites on the gills, one having a substantially higher affinity for copper than DOM. The absence of a calcium effect on gill copper binding was in accord with in vivo evidence that calcium primarily acts to alter the physiology of the gill binding sites through acclimatory processes, rather than through competitive interactions. It was a surprise that water chemistry parameters influence rapid gill-metal binding in a manner different to their influence on acute toxicity and different from the effects on long-term binding reported in other studies. Currently, the biotic ligand model uses the rapid increase of gill copper (believed to reflect binding to the physiologically active receptor sites) to model gill binding characteristics. The distinction between rapid surface binding and metal uptake obviously plays an important role in determining the toxic effects of copper, especially when regulators need to predict the modifying effects of water chemistry.