The relationships between metal exposure, dose and response were investigated in two sediment dwelling marine bivalves: a deposit feeder Tellina deltoidalis and a filter feeder Anadara trapezia. The bivalves were exposed in the laboratory to individual metal spiked sediments: Cadmium 10 and 50 Ag/g; lead 100 and 300 Ag/g; selenium 5 and 20 Ag/g dry mass, T. deltoidalis for 28 days A. trapezia for 56 days. A. trapezia was also exposed in the laboratory for 56 days to sediments from three sites along a metal contamination gradient of cadmium, lead, selenium, zinc and copper from Lake Macquarie, NSW. Metal total tissue dose was measured in whole tissue of T. deltoidalis over 28 days and in gill, hepatopancreas and haemolymph tissues in A. trapezia over 56 days. Subcellular metal distribution, biologically active metal (BAM) versus biologically detoxified metal (BDM) was measured in whole tissues of T. deltoidalis at day 28 and in gill and hepatopancreas tissues of A. trapezia at day 56. Biomarkers of response measured in spiked sediment exposed, at day 28 T. deltoidalis and day 56 A. trapezia were: total antioxidant capacity (TAOC); glutathione peroxidase enzyme activity (GPx); total glutathione concentration (GSH+2GSSG); reduced to oxidised glutathione ratio (GSH:GSSG); lipid peroxidation (TBARS); lysosomal membrane stability and micronuclei frequency. Response indices measured in A. trapezia exposed to Lake Macquarie sediments were: TAOC, TBARS, lysosomal membrane stability, micronucleus frequency and condition index. Native A. trapezia and sediments were also collected from Lake Macquarie and measured for sediment and tissue metal concentrations, TAOC, TBARS, lysosomal membrane stability and condition index to allow comparison between chronically exposed and previously unexposed organisms. T. deltoidalis and A. trapezia accumulated metal over time in all sediment metal exposures with most reaching equilibrium tissue metal concentrations by the end of the exposure period. T. deltoidalis generally reached equilibrium with the exposure concentration for cadmium and lead but had significantly higher selenium tissue concentrations than the sediment metal at the 5 Ag/g exposure. A. trapezia tissue lead was below the sediment concentration for all exposures including in the native organisms. A high proportion of accumulated lead and copper in A. trapezia was in the haemolymph, probably associated with haemoglobin which has a high affinity for these metals`. A. trapezia cadmium tissue concentrations were higher than the sediment metal in the 10 Ag/g spiked sediment exposure and between half and one eighth the sediment concentrations in other treatments, including in native organisms. A. trapezia including the native organisms exposed to selenium sediment concentrations at or below 5 Ag/g in the Lake Macquarie mixed metal sediments accumulated significantly higher than ambient selenium tissue concentrations while those exposed to 5 and 20 Ag/g selenium spiked sediments had lower than ambient selenium tissue concentrations. The majority of accumulated cadmium ,selenium and zinc was associated with the gill/mantle tissues. A. trapezia hepatopancreas contributed significant selenium concentrations in the later part of the exposure period indicating and increased contribution from dietary derived selenium. Native A. trapezia had significantly lower tissue concentrations of selenium, copper and zinc, higher cadmium and approximately equal lead compared to organisms exposed to similar sediment metal concentrations in the laboratory. T. deltoidalis detoxified around 50 % of accumulated cadmium and 70 % of lead while A. trapezia detoxified around 70 % of accumulated cadmium and 60 % of lead. Much of T. deltoidalis BDM cadmium was converted to metal rich granules (MRG),while A. trapezia had most in the metallothionein like proteins (MTLP) fraction. The conversion of lead to MRG was 75 % of the total BDM in T. deltoidalis while A. trapezia had an even distribution between MRG and MTLP. The majority of recovered selenium in both species was associated with the nuclei+cellular debris fraction, probably as protein bound selenium associated with plasma and selenium bound directly to cell walls. Selenium exposed organisms had increased BDM selenium burdens which were associated with both MRG and MTLP fractions, indicating selenium detoxification. The majority of BAM cadmium, lead and selenium was associated with the mitochondrial fraction in both species with increases in cadmium burden in this organelle of T. deltoidalis up to 7200 fold; lead 154 fold; and selenium 7 fold and in A. trapezia up to 84 fold cadmium,50 fold lead and selenium 7 fold in exposed organisms compared to controls. The subcellular distribution of all three metals in T. deltoidalis and A. trapezia indicates active metal detoxification processes which at these exposure concentrations were unable to prevent significant metal burdens from accumulating in sensitive organelles. A contamination gradient of zinc, lead, copper, cadmium and selenium was established in Lake Macquarie sediments which emanated from the same source. A. trapezia accumulated all metals in each sediment exposure. Accumulation and tissue distribution patterns of cadmium, lead and selenium were similar to those of the single metal spiked sediment exposures. Cadmium and lead BAM burdens increased at all exposures while selenium, zinc and copper did not. T. deltoidalis and A. trapezia in the spiked sediment metal exposures generally had reduced GPx activity. This resulted in an increase in total glutathione concentrations which the reduced GSH:GSSG ratios indicated was due to a build up of oxidised glutathione. T. deltoidalis and A. trapezia had reduced TAOC in all laboratory sediment metal exposures which corresponded with increased TBARS concentrations, lysosomal destabilisation and micronucleus frequency. A. trapezia exposed to Lake Macquarie metal contaminated sediments also had a reduction in physiological condition, indicated by the reduced condition index, after 56 days at the higher metal exposures. Clear exposure - dose - response relationships have been demonstrated for T. deltoidalis and A. trapezia exposed to single cadmium, lead and selenium spiked sediments and for A. trapezia exposed to Lake Macquarie mixed metal contaminated sediments. Detoxification of all metals was evident in both T. deltoidalis and A. trapezia but detoxification capacity was exceeded for cadmium, lead and selenium leading to significant accumulation of these metals in sensitive organelles. The significant relationships, in the laboratory exposed T. deltoidalis and A. trapezia, between TAOC reduction with increased TBARS, lysosomal destabilisation and micronuclei frequency and between increased TBARS with lysosomal destabilisation and micronuclei frequency indicates that increased tissue metal dose and BAM burdens caused significant impairment of the antioxidant reduction capacity which resulted in a cascade of effects from lipid peroxidation to cellular perturbation and genotoxic damage. The reduction in physiological condition in the organisms with the highest tissue metal doses suggests the response goes beyond subcellular perturbations to whole organism and potentially population effects. Chronically metal exposed native Lake Macquarie A. trapezia did not show a clear metal exposure - dose - response relationship. Accumulation of the essential elements zinc, copper and selenium appeared to be regulated while cadmium and lead were not. TAOC was significantly reduced and TBARS significantly increased compared to reference organisms but lysosomal stability and condition were not significantly affected. The suite of interrelated biomarkers used offers a weight of evidence approach for demonstrating adverse effects of metal tissue accumulation in T. deltoidalis and A. trapezia.
|Date of Award
|Bill Maher (Supervisor) & Anthony Roach (Supervisor)