Toxicity of mixture of metal contaminated sediments on the freshwater bivalve Hyridella australis: linking exposure-dose- response Chamani P. M. Marasinghe Wadige, Anne M. Taylor, William A. Maher, Frank Krikowa Ecochemistry Laboratory, Institute for Applied Ecology, University of Canberra, Canberra, ACT 2601, Australia Background Methods The exposure-dose-response (EDR) framework ( Fig. 1) is a prognostic tool in sediment toxicology studies as it integrates the chemistry of abiotic matrices with bioaccumulation and impairment of biological systems. Previous studies showed clear EDR relationship of H. australis to single metal spiked sediments under laboratory condition. In the environment metal are present as metal mixtures, therefore, toxic effects is not a result of a single metal exposure, but is rather a results of exposure to mixtures of metals due to toxicological interactions. In this context, EDR relationship of H. australis to mixture of metal contaminated sediments was investigated under laboratory controlled conditions. Exposure H. australis were exposed to four different concentrations of metal contaminated sediments collected from the bivalve caged sites at the Molonglo River. Seven individuals of H. australis per treatment replicate were exposed for 28 days at 100% air saturation, water temperature 21 ± 1°C with a day / light cycle of 12 / 12 hours (Fig. 2). Response Enzymatic Biomarkers Total antioxidant capacity was determined using Cayman® ABTS assay. Lipid peroxidation was determined as malondialdehyde (MDA) using the Oxitek® TBARS assay. Cellular Biomarkers Integrity of the lysosomal membranes in the hepatopancreas tissues of H. australis were measured using the neutral red retention assay. Fig. 2: Exposure setup Dose Total tissue metal Total metal concentrations in whole soft body tissues of five individuals from each treatment replicate were measured at day 28 (n = 15 per site/treatment). Sub-cellular distribution of metal Sub-cellular fractions (metal rich granules, cellular debris, organelles, heat sensitive proteins and metallothionein like proteins) were separated by differential centrifugation to determine the biologically active lead (BAM) and biologically detoxified metal (BDM) fractions in the hepatopancreas tissues at day 28 (n = 3 per treatment). Characterisation of exposure Exposure Sediment metal concentration Dose Tissue and sub-cellular metal concentration Response Toxic effects: Biochemical, cellular and physiological biomarkers Characterisation of effects Fig. 1 : Exposure-Dose-Response frame work (Adapted from Salazar and Salazar (2000). Results Exposure Dose Response 3A) a b c 3B) a b c a b ab ab b a a 6A) a b ab a b ab a b c a b ab 3C) a b c 3D) a b c a b bc c Fig.4: Metal accumulation (µg/g; µmol/g dry mass) in whole soft tissues of H. australis. 4 A: Zinc; 3 B: Lead; 4 C: Copper; 4 D: Cadmium; 4 E: Total metal. Mean ± SE. n = 15. Different letters denote significant difference between sites/treatments within each metal. Fig.6: Changes in 6A: TAOC, 6B: lipid peroxidation and 6C: percent of cells with unstable lysosomes in hepatopancreas tissues of H. australis. Mean ± SE. n = 15 for TAOC and lipid peroxidation. n = 9 for lysosomal membrane stability . Different letters denote significant differences between sites/treatments. 3E) a b c Sub-cellular distribution of metal 5A) 7A) 7B) Fig.3: Mean metal concentrations (µg/g; µmol/g dry mass) in the exposure medium/sediments of H. australis. 3 A: Zinc; 3 B: Lead; 3 C: Copper; 3 D: Cadmium; 3 E: Total metal. Mean ± SE. n = 6. Different letters denote significant difference between sites/treatments within each metal. Fig.5: Sub-cellular distribution (N + CD–nucleus + cellular debris, n = 3; BAM-Biologically active metals, n = 9; BDM–Biologically detoxified metals, n = 6) of 5 A: zinc and 5 B: total metals in hepatopancreas tissues of H. australis. Mean ± SE. Fig.7: Lysosomal membrane stability assay conducted in the hepatopancreas tissue of H. australis (7A: organisms exposed to highly contaminated sediments; 7B: organisms exposed to the least contaminated sediments). Exposure- Dose Dose-Response 8A) 8B) 9B) 8C) 9A) Fig.8: Regression of H. australis whole organism tissue zinc, cadmium and total metal concentrations with sediment zinc, cadmium and total metal concentration. 8 A: Zinc; 8 B: cadmium; 8 C: Total metal. Mean ± SE. n = 15. Fig.9: Regression of H. australis whole organism tissue cadmium with TAOC (9A; n = 60) and percent of cells with unstable lysosomes (9B; n = 36). Discussion Exposure: Each metal concentration measured at the control sediments was below the ANZECC/ARMCANZ (2000) ISQG low values for freshwater sediment; Cd 1.5 µg/g, Cu 65 µg/g, Pb 50 µg/g and Zn 200 µg/g dry mass (Fig. 3). Except for Cu in medium and low treatments, Zn, Pb and Cd concentrations in the sediments collected from mine affected sites (high, medium and low treatments) exceed the ANZECC/ARMCANZ (2000) ISQG low values for Zn, Pb and Cd (Fig.3). Dose: Even though high concentrations of metals in the sediments H. australis accumulated low concentrations of metals. Zinc and total tissue metal concentrations (when Cd, Cu, Pb and Zn were combined) in body tissues increased with increased zinc and total metal concentrations in the sediments (Figs. 4A and 4E). Accumulated Cu concentrations between treatments were not significantly different between treatments (Figs. 4C). Compared to controls organisms in low, medium and high treatments accumulated significantly high concentrations of Cd and Cd concentrations between medium and high treatments were not significantly different to each other (Fig. 4D). Dose: Sub-cellular distribution of metal Most of the accumulated metals were in the BDM pool and BAM pool between treatments were not different. Exposure-dose: H. australis showed sediment dependent accumulation of zinc , cadmium and total metal (Fig. 8) . Response: Compared to control and low treatments organisms in medium and high treatments had significantly lower levels of TAOC (Fig. 6A). Lipid peroxidation was only significantly higher in the site1/ low treatment, which had the highest percent of unstable lysosomes (Figs. 6B and 6C). Organisms exposed to low , medium and high treatments had significantly higher percentage of lysosomal membrane destabilisation than controls (Fig. 6C). Dose-response: A clear dose-response relationship was only observed for Cd tissue concentrations (Figs. 9A and 9B). Conclusions