| Popis: |
The toxicology of a widely used fungicide, chlorothalonil (TCIN), was investigated in three species of native Australian galaxiid fish and rainbow trout, Salmo gairdneri Rich., after measuring environmental levels in two aerially sprayed north-eastern Tasmanian catchment streams. TCIN residues (0 - 5 ug/l) were detected intermittently in stream waters after spraying events. LC50 values determined in flow through tests were from 12 - 21 ug/l. Investigation of sublethal effects showed short term anaemia, haemolysis and long-term gill damage quantified by morphometric analysis. No anaerobic glycolysis was measurable on short-term lethal exposure, despite a synergistic effect of low oxygen levels measured in an LC50 test at 50% saturation. A device counting opercular rhythms by Fast Fourier Transform analysis was designed and tested in conjunction with experiments to determine respiratory rate and responses of S. gairdneri to low TCIN levels. A distinct respiratory response was measured, characterized by elevated ventilation rates, with a threshold of 20 ug/1 during 2 h exposure. Environmental degradation of TCIN was studied in stream waters with a variety of substrates, temperatures and water types. TCIN was found to be a non-persistent pollutant whose disappearance rates were markedly enhanced by the presence of algal substrate and the presence of fish. Sediment partition coefficients were established. In a stream-dosing study, TCIN was found to be highly associated with suspended sediment. In order to study metabolism of chlorothalonil (TCIN) in more detail, a synthetic route was designed and used successfully to produce (C\\(^{14}\\)-CN) TCIN. Tracing experiments were carried out in 10 ug/1 exposed rainbow trout. Significant levels built up in all organs. Protein binding occurred in the liver. The primary concentration of label occurred in the gall bladder (up to 4000 ug/g in 96h). Sephadex filtration and TLC showed that glutathione conjugates were the primary metabolites. Structural studies were carried out using synthesized model compounds, and the structures of TCIN glutathione conjugates were fully elucidated by spectral methods. Catalysis of the conjugation reaction by hepatic glutathione S-transferase (GST) enzymes was studied in detail in the three Galaxias species, S. gairdneri and S. trutta. Molecular weights, comparative activities to another substrate (CDNB), pH optima, induction by TCIN exposure, binding and inhibition, reaction order, and organ specificity were all studied. All species were found to have two GST enzymes of different molecular weight with peak activity toward the two substrates, C\\(^{14}\\)-TCIN TCIN and CDNB. Galaxias GST's were consistently lower in molecular weight than those of the salmonids, indicating a major phylogenetic difference in this enzyme group. Induction and binding of metallo-thioneins by TCIN and co-induction with Zn exposure were also studied. Hepatic GST activity was promoted in S. gairdneri, G. maculatus and G. truttaceus exposed to low levels of TCIN over four days, and the response was dose dependent. Confirmation of detoxication by glutathione (GSH) depletion was obtained by measuring GSH levels in livers of exposed fish. Despite in vitro inhibition at low GAPDH levels, GAPDH inhibition was prevented by the presence of glutathione and was low in hepatic cytosol preparations of similar low GAPDH levels. Inhibition of GAPDH in livers of TCIN exposed fish was also studied, and found to be only transitorily affected by exposure to lethal levels. No similar work on either toxicology, degradation or metabolism of pesticides in Australia has been reported in the literature. Apart from same heavy metal and hydrocarbon toxicology studies, almost no such work has been performed in Australia, most information being derivative from overseas. This study shows that the toxicological response of the galaxiids appears to be as sensitive as that of rainbow trout. A case is made for aquatic toxicological information applied within Australia to be directly related to information on native and not overseas species. A proposal is made to screen native fish, both marine and freshwater, before time-consuming, expensive toxicity tests, possibly detrimental to native species populations, are carried out. The screening process involves selecting species \at risk\" to classes of pollutants by studying levels and activities of detoxication enzyme systems. This proposed procedure is illustrated with the example of a correlation between GST and GSH levels in Salmo gairdneri Galaxias auratus and Galaxias maculatus and the LC50 values for TCIN for these species determined in this work. A paper on the synthesis of C\\(^{14}\\)-TCIN has been accepted for publication. The manuscript is contained in Appendix 5. Six other papers have been submitted for publication on the toxicology and metabolism of chlorothalonil in fish." |
