Using the comet assay to assess the combined and separate genotoxic effects of Cd and Zn in Eisenia andrei (Oligochaeta) at different temperatures.

Using the comet assay, the genotoxicity of Cd, Zn and Cd/Zn mixtures in Eisenia andrei was assessed after 4 weeks of exposure at 15, 20 and 25 °C. Relative to the controls, significant increases in TDNA% were observed in exposures to Cd alone at 500 and 1,000 mg/kg soil at both 20 and 25 °C, while a general decrease occurred at 15 °C. For Zn alone, a decreasing trend in TDNA% occurred at all three temperatures with increasing Zn concentration. For the Cd/Zn mixtures at 15 °C, genotoxicity was reduced at all mixture concentrations relative to the control. At 20 °C, the genotoxic response was similar to the control at all exposures. At 25 °C, the response was elevated at the 50 + 50 and 250 + 250 mg/kg mixture concentrations. In the remaining treatments at 25 °C, TDNA% was similar to the values in the respective control. The lack of consistently significant mixture genotoxicity may indicate antagonistic interactions between Cd and Zn in the mixtures. However, this was not conclusively determined because temperature alone had an inconsistent effect upon TDNA% readings in the control exposures.

Using estimates of metal bioavailability in the soil and genetic variation of allozymes to investigate heavy metal tolerance in the earthworm Eisenia fetida (Oligochaeta).

In a recent study, we showed that the earthworm species Eisenia fetida, inhabiting an extremely high metal polluted compost heap on a wine farm, did not have elevated body loads of the metals but exhibited genotoxic tolerance when exposed to Cd in the laboratory (Voua Otomo and Reinecke, 2010). To unravel the mechanism behind the surprisingly low metal body burdens on one hand and genotoxic tolerance on the other hand, we investigated the estimated bioavailability of these metals (Cu, Zn, Pb and Cd) using sequential extraction methods with CaCl(2) and di-ethylene-triamine-pentaacetic acid (DTPA) and allozyme polymorphism in this field population, a laboratory control as well as a long-term Cd exposed population. The amounts of mobile (extracted with CaCl(2)) and mobilizable (extracted with DTPA) metals in relation to the total (extracted with nitric acid) metals were all below 0.05% for all four metals, suggesting low availability for uptake. The low availability of these metals could not be explained by physico-chemical properties of soil but by the phenomenon of aging of the metals. There was no difference in allozyme frequency between metal tolerant and non-metal tolerant populations of E. fetida. This suggested that the tolerance found in earlier studies could be a mere physiological adaptation.

Increased cytotoxic and genotoxic tolerance of Eisenia fetida (Oligochaeta) to cadmium after long-term exposure.

Since life-cycle studies showed that the earthworm species Eisenia fetida can develop increased tolerance after long-term exposure to a sub-lethal concentration of Cd in the laboratory, we assessed both the cytotoxicity and genotoxicity of Cd in a long-term Cd-exposed population. We exposed E. fetida specimens from this population, from a laboratory control population and from a field population to various concentrations of CdSO(4) in artificial soil water. Toxic effects were measured using the MTT test and the comet assay. The group that had been exposed to Cd for more than a decade was found to be more tolerant to the deleterious effects of Cd at both cellular and molecular levels than the laboratory control population. The field population, which came from a severely metal polluted environment, displayed high tolerance at molecular level as well. The results provide novel biomarker evidence of increased Cd tolerance in E. fetida, but the mechanisms supporting the apparent tolerance, still need to be clarified.