Modes of metal toxicity and impaired branchial ionoregulation in rainbow trout exposed to mixtures of Pb and Cd in soft water

Models such as the Biotic Ligand Model (BLM) predict how natural organic matter (NOM) and competing ions (e.g., Ca²⁺, H⁺ and Na⁺) affect metal bioavailability and toxicity in aquatic organisms. However, such models focus upon individual metals, not metal mixtures. This study determined whether Pb and Cd interact at the gill of rainbow trout (Oncorhynchus mykiss) when trout were exposed to environmentally relevant concentrations of these metals (Cd < 100 nmol L^-1; Pb < 500 nmol L^-1) in soft (<100 μmol Ca²⁺ L^-1), moderately acidic (pH 6.0) water. The 96-h LC50 for Pb was 482 nmol L^-1, indicating that Pb was one-order of magnitude more toxic in soft, acidic water than in harder, circumneutral pH waters. The LC50 for Cd alone was also low, 6.7 nmol L^-1. Surprisingly, fish acclimated to soft water had multiple populations of Pb-gill and Cd-gill binding sites. A low capacity, high affinity population of Pb-gill binding sites had a B_max of 18.2 nmol g^-1 wet weight (ww) and apparent log K_Pb-gill = 7.05, but a second low affinity population could not be saturated up to free Pb concentrations approaching 4000 nmol L^-1. Two populations of Cd-gill binding sites were characterized: a high affinity, low capacity population with an apparent log K_Cd-gill = 7.33 and B_max = 1.73 nmol g^-1 ww, and a low affinity, high capacity population with an apparent log K_Cd-gill = 5.86, and B_max = 13.7 nmol g^-1 ww. At low concentrations, Cd plus Pb accumulation was less than additive because Cd out-competed Pb for gill binding sites, which were likely apical Ca²⁺-channels. While disturbances to Ca²⁺ influx were caused by Cd alone, Pb alone had no effect. However, Pb exacerbated Cd-induced disturbances to Ca²⁺ influx demonstrating that, although Pb- plus Cd-gill binding was less than additive due to competition, the effects (ionic disturbances) were more than additive (synergistic). Pb was also likely binding to intracellular targets, such as branchial carbonic anhydrase, which led to inhibited Na⁺ influx. This ionic disturbance was exacerbated by Cd. We conclude that exposure to environmentally relevant concentrations of Pb plus Cd results in less than additive metal-gill binding in soft, moderately acidic waters. However, ionic disturbances caused by Cd plus Pb are greater than additive, and this may ultimately increase the toxicity of Cd-Pb mixtures to fishes. Our findings suggest that it may be necessary to re-evaluate water quality criteria and assumptions of the BLM for fish exposed to mixtures of Pb and Cd in the acidic, soft waters found in the Canadian Shield, Scandinavia and other sensitive regions.