Combined effects of copper, nickel, and zinc on growth of a freshwater mussel (Villosa iris) in an environmentally relevant context

Trace metals rarely contaminate freshwaters independently, hence regulatory limits based on single-metal toxicity may be underprotective of aquatic life. This could be especially the case for rare and sensitive fauna like freshwater mussels, such as those suppressed in the Clinch and Powell Rivers in eastern USA where trace metals are long-term contaminants but at concentrations below regulatory limits. We hypothesized metal mixtures may be exerting combined effects on mussels, resulting in greater toxicity than would be predicted based on single-metal exposures. To test that hypothesis, we conducted two experiments exposing juvenile rainbow mussels (Villosa iris) for 42 days to dissolved copper, nickel, and zinc, individually and in three-metal mixtures, in an environmentally-relevant context of water with chemistry (hardness 155 mg/L as CaCO3, dissolved organic carbon 1.7-2.3 mg/L, pH 8.4) similar to that of the Clinch River, which receives alkaline mine drainage. We used a toxic unit approach, selecting test concentrations based on literature values for the lower of 28-day survival or growth (length) effect concentrations for Villosa iris or Lampsilis siliquoidea (fatmucket). Our first experiment confirmed survival and growth effects when acute and chronic water quality criteria, respectively, are approached and/or exceeded. Our second experiment, at lower concentrations, showed no effects on survival but combined effects on growth were evident: a mixture of Cu, Ni, and Zn (7.2 +/- 1.2, 65.3 +/- 6.1, 183 +/- 32 mu g/L, respectively) inhibited growth (dry weight) by 95% versus 73%, 74%, and 83% inhibition for single-metal exposures to Cu, Ni, and Zn of similar concentration (8.0 +/- 1.1, 63.5 +/- 4.8, 193 +/- 31 mu g/L, respectively). Furthermore, a mixture of Cu, Ni, and Zn with individual concentrations 21%, 29%, and 37% of their water quality criteria (3.4 +/- 1.2, 21.8 +/- 1.8, and 62.1 +/- 8.4 mu g/L, respectively) inhibited growth (dry weight) by 61% relative to controls. Our observation of combined effects suggests that regulatory limits based on single-metal toxicity may be underprotective of freshwater mussels when multiple metals are present.