SUMMARY The present study was undertaken to answer two questions relating to the exposure of brown trout Salmo trutta to sublethal concentrations of copper and low pH (CLP) for 96h. (1) What is the effect of these pollutants on the rate of oxygen consumption (ṀO2) at different levels of exercise and (2) why does ammonia accumulate within these fish, when the low external pH should favour the diffusion of NH3 across the gills? Mean ṀO2 of fish in CLP and control (normal pH and no added copper) conditions were not significantly different from each other at any level of exercise. This suggests that exposure to CLP was not a `loading' factor at any level of activity. However, both maximum ṀO2 and critical swimming speed (Ucrit) were significantly lower in the CLP trout (5.5±1.6 mmol O2 kg-1 h-1 and 1.12±0.06 BL s-1, respectively) than in control fish (18.5±2.3 mmol O2 kg-1 h-1 and 2.04±0.11 BL s-1, respectively). There was no evidence from cardiovascular variables, such as heart rate and cardiac output, to suggest any changes in the oxygen transport system to compensate for any possible reduction in branchial gas exchange. Thus, it is suggested that oxygen exchange and transport do not limit the swimming performance of CLP trout, but that exposure to CLP reduces the maximum demand for O2, i.e. it is a limiting factor. The accumulation of ammonia in the plasma and white muscles during exposure to CLP has already been implicated in reducing the swimming performance of brown trout. Inhibition of cortisol synthesis abolished a large proportion of the increases in both the accumulation and excretion of ammonia that occurred during the second 48 h of the exposure to CLP, but did not inhibit ammonia accumulation completely. It is suggested that CLP not only causes an increase in the rate of production of ammonia, which is enhanced when the level of cortisol starts to increase after 48 h, but that it also inhibits an excretory mechanism (most probably Na+/NH4+ exchange) that is non-obligatory under `normal' conditions (when passive diffusion is sufficient), but is required in order to respond to unusually high ammonia loads.

Brown trout acclimated to soft water and exposed for 96 h to a sub-lethal concentration of copper at low pH (0.08 μmol l−1 Cu, pH 5) have a lower critical swimming speed than fish from copper-free water at neutral pH. This loss of performance is not due to difficulties in oxygen transfer resulting from gill damage since arterial oxygen and carbon dioxide levels remain unaffected. Both red and white muscle showed some metabolic disruptions consistent with local hypoxia, namely a high lactate concentration at rest and, in the white muscle, depletion of glycogen and phosphocreatine. However, a putative role of increased blood viscosity following haematological changes in reducing the supply of oxygen to the tissues is not supported by the current study. Haematocrit, haemoglobin and plasma protein concentrations were not affected by this treatment and a lack of further change in variables such as lactate at the onset of exercise led one to look for an alternative explanation for the effects of copper and low pH upon tissue metabolites. Ammonia concentration, both in the plasma and muscles, is significantly higher in trout exposed to copper and low pH. Ammonia plays a role in the regulation of a number of metabolic pathways and could contribute to the altered metabolic status of these fish. In addition, ammonium ions are known to cause electrophysiological disruptions, particularly the displacement of K+ in ion exchange mechanisms that could lead to the observed loss of swimming performance. Using the measured distribution of ammonia between intracellular and extracellular compartments to estimate membrane potential of resting muscle, a significant depolarisation is predicted in both red and white muscle of fish exposed to copper and low pH.

Previously, the distribution of ammonia between the intracellular and extracellular compartments has been used to predict a significant depolarisation of the resting membrane potential (E(M)) of white muscle from brown trout (Salmo trutta) exposed to a sub-lethal combination of copper and low pH. However, this prediction is based upon two assumptions (i) a relatively high membrane permeability for the ammonium ion with respect to that for ammonia gas and (ii) that this is unaltered by exposure to copper and low pH. Since there is conflicting evidence in the literature of the validity of these assumptions, in the present study E(M) was directly measured in white muscle fibres of trout exposed to copper and low pH (E(M)=-52.2+/-4.9 mV) and compared with that of unexposed, control animals (E(M)=-86.5+/-2.9 mV) (means +/- s.e.m., N=6). In confirming the predicted depolarisation, these data support the hypothesis of electrophysiological impairment as a factor in the reduction in the swimming performance of trout exposed to these pollutants. In addition, the results of this study support the role of a significant permeability of the muscle membrane to NH(4)(+) in determining the distribution of ammonia in fish.

ABSTRACT Adult brown trout (300–600 g) were acclimated for 2 weeks to an artificial soft water (Ca2+, 50 μmol l−1) and maintained at either 5 °C (October to March) or 15 °C (May to August). Following insertion of a cannula into the dorsal aorta under MS-222 anaesthesia and a recovery period of 2 days, the fish were exposed to a 4 day episode of sub-lethal copper levels at pH 5 or kept at control conditions of pH 7 without copper. The copper concentrations had been predetermined by toxicity testing and were approximately 0.47 μmol l−1 at 5 °C and 0.08 μmol l−1 at 15 °C. At 5 °C, a group of fish was also exposed to approximately 0.08 μmol l−1 copper at pH 5. Plasma total ammonia (Tamm) concentration was significantly elevated by exposure to copper and pH 5. In resting trout exposed to the appropriate sub-lethal copper concentration at pH 5, Tamm was six and 7.5 times greater at 5 and 15 °C, respectively, than those of control trout at the respective temperatures. Although unconfirmed, an elevation of ammonia production alone seems unlikely to account for such substantial increases. From previous studies, there is little evidence of impairment of respiratory gas exchange in trout exposed to these copper concentrations and yet, in the acidic test waters, the gradient of NH3 partial pressure between fish and water was 5.5–6 times greater than that under control conditions. Swimming performance determined by the critical swimming speed (Ucrit) was reduced by copper and acid exposure, and a significant relationship existed between Ucrit and the plasma ammonia concentration of exercised trout. Ammonium ions influence several key enzymes involved in energy metabolism, and elevated ammonia levels might, therefore, reduce the capacity of muscle to exercise. Alternatively, ammonia may have affected the nervous coordination of exercise either centrally or by disrupting peripheral motor innervation.

Toxicity tests were performed to determine the 96 h sub-lethal copper concentration at pH 5 (SLCC) for adult brown trout acclimated to 5 °C (Oct.–Mar.) or 15 °C (May–Aug.) in an artificial softwater (Ca2+ 50 μmol l−1). These were found to be 0.47 and 0.08 μmol l−1 Cu2+ at 5 and 15 °C respectively. Routine oxygen consumption (Mo2) and critical swimming speed (Ucrit) of groups of trout exposed to the SLCC for each temperature were measured and compared to trout maintained in the absence of copper and at pH 7. At 5 °C, an additional group was exposed to the summer copper level of 0.08 μmol l−1 Cu2+ at pH 5. At the end of each experiment, samples of arterial blood were taken from the dorsal aorta via chronically indwelling cannulae. Copper exposure in acidic water significantly reduced swimming performance. Trout exposed to 0.08 μmol l−1 Cu2+ at pH 5 achieved a critical swimming speed some 25–50% slower than control trout with no significant temperature effect. Of the six winter trout exposed to 0.47 μmol l−1 Cu2+ at pH 5, only one swam steadily at the lowest test speed of 0.3 m s−1, the remainder achieving at most a brief burst of activity. Earlier workers suggested that observed increases in aerobic maintenance costs and decreased maximum metabolism arose from copper-induced inefficiencies in ionoregulation and gas exchange and reduced Ucrit to be a consequence of this lower scope for activity. In the present study, plasma sodium and chloride concentrations were reduced some 25–35%, regardless of acclimation temperature or copper concentration. The routine Mo2 of trout exposed to the SLCC at either temperature was elevated (by 72% and 38% at 5 and 15 °C respectively). However, there was no change in that of winter trout exposed to only 0.08 μmol l−1 Cu2+ at pH 5 although their swimming performance was also reduced. More significantly, there were no changes in the blood oxygen content or lactate concentration of exercised trout at any copper concentration as might be expected if Mo2max was limited by an impaired oxygen uptake. There were, however, some effects of copper and acid exposure, such as increases in haemoglobin and plasma protein concentrations, which may have elevated blood viscosity and which could have led to a disruption of oxygen transport to the tissues. Although the occurrence and magnitude of these effects were not consistent between treatment groups, there is some evidence for a tissue hypoxia from a parallel study.