INVESTIGATIONS OF WATERBORNE METAL INTERACTIONS IN THE GILLS OF RAINBOW TROUT USING SYNCHROTRON-BASED TECHNIQUES

Most of our current understanding of metal toxicity in aquatic organisms has been based on single metal toxicity. However, metals do not exist singly in the aquatic environment, but rather as mixtures. As mixtures, they are subject to complex interactions for uptake and/or intracellular handling. An enhanced understanding of intracellular metal interactions could help in developing predictive models (e.g., biotic ligand model) for assessing the toxicity of metals in mixture. To this end, I adopted a suite of synchrotron-based techniques to investigate the acute interactions of waterborne Zn, Cd and Cu in binary mixtures in situ at the gills of a model teleost species, rainbow trout (Oncorhynchus mykiss). Gills (the biotic ligand) of freshwater fish are primary site of toxic action for metals during acute waterborne exposures, and thus were chosen as the target tissue for this investigation. Micro X-ray fluorescence imaging (µ-XRF) and micro X-ray absorption near-edge spectroscopy (µ-XANES) were employed to investigate: (i) the spatial distribution and chemical speciation of Zn and its co-localization pattern with other essential elements (Ca, S and Fe), (ii) the effect of competing metals (Cd and Cu) on Zn distribution and speciation in the gills of rainbow trout. In addition, Fourier transform infrared micro-spectroscopy (FTIRM) was employed to examine the effects of Zn, Cd and Cu, singly and in binary mixtures, on biochemical constituents (e.g., proteins, lipids and nucleic acids) of the gills of rainbow trout. Fish (~200g) were exposed to acutely toxic concentrations (1x 96h LC50) of each metal, alone and in binary combination, in dechlorinated municipal water for 24h. Following exposure, gills were dissected and 10 um thick sections were prepared and analyzed at the VESPERS and Mid-IR beamlines of the Canadian Light Source. My findings indicated that Zn accumulated in high proportions in the primary lamellae (the primary site of mitochondria-rich (chloride) cell localization) of the fish gill. Zinc was also found to predominantly co-localize with Ca and S, but not with Fe, indicating that Ca and S binding intracellular ligands play a crucial role in Zn handling in the gill. The distribution of Zn in the gill was markedly reduced during co-exposure to Cd, but not to Cu, suggesting a competitive interaction between Zn and Cd for uptake. Exposure to Zn, alone and in combination with Cd or Cu, was found to decrease Ca from the gill tissue. The speciation of Zn in the gill was dominated by Zn-phosphate, Zn-histidine and Zn-cysteine; however, the interactions of Zn with Cd or Cu resulted in the loss of Zn-cysteine. The composition of the biochemical constituents of the fish gill was also found to be altered by the exposure to metals, both individually and in binary mixture, and this was apparent both on the primary and secondary lamellae. These alterations were mainly characterized as degradations of proteins and lipids. Generally, exposure to Cu, alone and in mixture with Cd, was found to induce maximum adverse effects in the fish gill, which was followed by exposure to Cd and Zn alone. Interestingly though, the adverse effects of Cu and/or Cd in the gill were ameliorated by the presence of Zn in the co-exposure. Collectively, my findings indicated that the interactions of Zn and Cd or Cu in the fish gill could occur via both common and disparate mechanisms, and their interactions elicit antagonistic toxicity in the fish gill