Mechanisms of trace metal and diclofenac toxicity in inanga (Galaxias maculatus): contextualising responses of a non-model native New Zealand species to standard fish models.

Pharmaceuticals and trace metals are increasingly prevalent in the aquatic environment, due to anthropogenic pressures. The essential trace metal zinc (Zn) and the non-essential trace metal cadmium (Cd) are particularly enriched in New Zealand settings owing to factors such as galvanised roof runoff, and superphosphate fertiliser application, respectively. The emerging pharmaceutical contaminant diclofenac is increasing in waters worldwide due to heavy usage and lack of breakdown in waste water treatment. Although present at low concentrations, environmental persistence and high bioactivity of these contaminants results in toxicological impacts on aquatic biota. However, most toxicity studies in fish are conducted on model Northern Hemisphere species, and almost nothing is known regarding the sensitivity of widespread Southern Hemisphere fish such as the inanga (Galaxias maculatus). This species exhibits a number of unusual physiological traits that may alter their responses to environmental contamination. Furthermore, as one of the few amphidromous fish species they move freely through estuaries and near-coastal streams that are compromised by the presence of agricultural, urban and industrial effluents containing key contaminants such as pharmaceuticals and trace metals. In order to adopt regulations that adequately protect New Zealand’s freshwater fish fauna, it is important to determine that mechanisms of toxicity and the biological foundations of regulatory modelling tools established in model species, still apply to fish such as inanga. To investigate mechanisms of trace metal toxicity, inanga were exposed to graded concentrations of Zn or Cd for 96 h. Whole body metal accumulation, ionoregulation (calcium and sodium influx) oxidative stress (catalase and lipid peroxidation), and metabolism (respirometry) endpoints were examined. Zn exposure caused increases in catalase activity and lipid peroxidation, but only at 1000 μg L-1, a concentration at which Zn also significantly inhibited calcium influx, and stimulated sodium influx. Cd induced lipid peroxidation and inhibited catalase in the liver after exposure to concentrations as low as 2.5 μg L-1. Measures of ionoregulatory function were not impaired. In general, inanga was shown to be tolerant to waterborne metals, with mechanisms of toxicity conserved relative to better-studied Northern Hemisphere species. This suggests that mechanistic-based regulatory tools are applicable for the environmental protection of this species. Further, the mechanisms of diclofenac toxicity to inanga were explored, by examining accumulation, and its effects on metabolic rate, ionoregulation, and oxidative stress at environmentally-relevant (0.17 μg L-1) and elevated (763 μg L-1) concentrations. Following an acute 96 h exposure, a bioconcentration factor of 87 was derived for the 0.17 μg L-1 exposure concentration, approaching values where transfer through the food chain may be important. Lipid peroxidation in inanga liver was significantly elevated at both exposure concentrations, but lipid peroxidation in kidney and gill decreased after diclofenac exposure. Catalase activity was also elevated in the liver of inanga, but activity decreased in the gill. There were no effects of diclofenac on metabolic rate or ion (sodium and calcium) influx rates. These data indicate toxicologically-relevant adverse outcomes and bioconcentration of diclofenac at environmentally-relevant levels. The finding that oxidative stress was a major mode of diclofenac impact, led to an examination of whether this mode of action was also prevalent in the more traditional models from the Northern Hemisphere (zebrafish embryos, Danio rerio; larval fathead minnow, Pimephales promelas). Significant effects on lipid peroxidation were noted, but only at concentrations higher than those found in the environment (0.01, 1, 100 mg L-1), and only in the fathead minnow. This research showed distinct species-specific effects, a finding that deserves additional consideration in the development of predictive approaches for the protection of aquatic biota from adverse outcomes elicited by pharmaceuticals. Given the finding that Cd causes pro-oxidant effects, and diclofenac generally behaves as an antioxidant in inanga, the effects of binary mixtures of these two contaminants, which are likely to co-occur in wastewater effluents, were examined. Antioxidant defence (catalase, superoxide dismutase, glutathione S-transferase) and oxidative damage (protein carbonylation, lipid peroxidation) were assessed in exposures of Cd, diclofenac and these contaminants in combination, at concentrations previously shown to induce impacts on oxidative stress. Relative to singular exposures, mixtures of Cd and diclofenac caused significant changes in patterns of oxidative stress, indicating a clear interaction between the two toxicants. In particular, diclofenac exposure reduced Cd-induced impairment of antioxidant defence and the induction of oxidative damage, suggesting that where these two toxicants co-occur traditional models of predicting toxicity based on individual contaminants may be compromised. The results from this thesis contribute significantly to a limited body of research regarding the impacts of environmental contaminants on an important Southern Hemisphere fish species, and are among the first data looking at the effects of simple trace metal/pharmaceutical mixtures in any fish. This research also contributed significant new knowledge regarding the comparative effects of diclofenac in two important model species. As such, the results from this thesis will provide data that can be utilised by regulatory bodies in their adoption and/or development of regulatory tools for protection of freshwater fish fauna