Browsing by Author "Fourie, Frana"
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- ItemComparing the sensitivity of five earthworm species to cadmium exposure using the comet assay(Stellenbosch : Stellenbosch University, 2006-04) Fourie, Frana; Reinecke, S. A.; Reinecke, A. J.; Stellenbosch University. Faculty of Science. Dept. of Botany and Zoology.ENGLISH ABSTRACT: It is known that species differ in their sensitivities to toxicants. This has been exploited to aid in environmental toxicity testing and environmental management. "Sensitivity" in this sense is usually seen as a function of lethality; assessed by determining the toxicant concentration where 50% of the test cohort die. The processes at the sub-organismal or sublethal level are however ignored. Little has been done to combine such sub lethal sensitivities with the concept of species sensitivity differences. The present study therefore focused on the potential use of a cellular biomarker to compare the sensitivities of species. The heavy metal cadmium, which bio-accumulates, is teratogenic, mutagenic and carcinogenic was chosen as toxicant. Earthworms were chosen as experimental animals and species were selected to represent various ecological types that may occur in soils. The species studied were representative of three ecological types: epigeic (Amynthas diffringens, Dendrodrilus rubidus and Eisenia fetida), endogeic (Aporrectodea caliginosa) and anecic (Microchaetus benhami). The alkaline single cell gel electrophoresis assay (SCGE or comet assay), which measures DNA integrity in individual cells, was used as biomarker. Earthworms were exposed to a range of Cd concentrations (2.5, 5, 10 and 20 mg/I Cd) in the form of CdS04, in artificial soil water. A negative control (uncontaminated soil water) and a positive control, nickel (20 mg/I Ni) in the form of NiS04 were used. Exposure to cadmium induced significantly higher levels of DNA damage in the exposed worms than in those exposed to negative controls. Species differed from each other in their sensitivity to Cd. The most sensitive species was E. fetida followed by D. rubidus, A. caliginosa, A. diffringens and M benhami. Ecological type did not predict sensitivity, and it is concluded that physiology and possibly relatedness may provide a possible explanation. All species exhibited a pattern where DNA damage was inhibited at low Cd exposure concentrations, and was increased again at high Cd concentrations. This corresponds with the hormetic dose-response, where a compensatory response is stimulated by low levels of a toxicant, but inhibited at high levels. Two possible compensatory mechanisms are proposed. Firstly, DNA repair could have been upregulated at low Cd concentrations, and inhibited by high Cd concentrations. Secondly, the production of metal-binding metallothioneins, which sequestrate Cd and renders it unavailable to cause toxic responses, could have been increased with low Cd concentrations. At high Cd concentrations, the rate of metallothionein production would not have been high enough to sequestrate Cd before it could cause damage. The exposed earthworms accumulated Cd, but there was no definite relationship between Cd body loads and DNA damage. It is possible that a fraction of the measured Cd in the body was sequestrated, therefore not being available to cause genotoxic effects. It is concluded that the comet assay is a useful biomarker to demonstrate DNA damage and species sensitivity differences in earthworms exposed to cadmium.
- ItemEvaluation of the suitability of responses on various organisational levels in terrestrial Oligochaeta to determine species sensitivity relationships(Stellenbosch : Stellenbosch University, 2011-03) Fourie, Frana; Reinecke, S. A.; Reinecke, A. J.; Stellenbosch University. Faculty of Science. Dept. of Botany and Zoology.ENGLISH ABSTRACT: Species differ in their sensitivities to toxicants and these differences are exploited in ecological risk assessment methods such as species sensitivity distributions (SSDs). The most commonly used endpoints for ecotoxicity testing and thus to generate data for use in SSDs are on the whole-organismal level, and usually include the evaluation of survival and reproduction. However, suborganismal biomarker responses are in many instances more sensitive than these whole-organismal responses. Therefore, this study investigated and compared responses on various biological organisational levels to determine their suitability for use in SSDs. Five terrestrial oligochaete species (earthworms) were selected as model test organisms, and were exposed to a range of concentrations of a well-studied pesticide, copper oxychloride. The investigated responses included survival, biomass change and reproduction on the whole-organismal level. In order to investigate responses on the suborganismal level, cells (coelomocytes) were extracted non-invasively. The spectrophotometric neutral red retention (NRR) assay was used to determine cell survival and the MTT assay to determine mitochondrial metabolic activity of the coelomocytes. The alkaline single cell gel electrophoresis (comet) assay was used to assess DNA integrity in these cells. The amount of Cu taken up by earthworms was also determined and compared to their responses. Species differences were observed in all responses, and EC50 and EC10 values were calculated for the whole-organismal endpoints and used to generate SSDs. From these SSDs, the hazardous concentrations where 5% of all species would be detrimentally affected (HC5) were calculated, which indicated that the most sensitive whole-organismal endpoint was mass change, followed by reproduction and survival. It was found that earthworms avoided feeding on the contaminated substrate in high copper oxychloride concentration exposures. The concentration where this behaviour occurred could be estimated for each species, and an SSD was constructed with these data. The HC5 value indicated that this response is more sensitive than earthworm survival, but less sensitive than the other responses. It was shown that the earthworms regulated their body Cu concentrations in a species-specific manner. This regulation of Cu was reflected in the suborganismal responses, and the species that had taken up the highest amount of Cu was the most sensitive species for all three suborganismal assays. Due to this regulation of Cu, the resulting dose-responses for the suborganismal endpoints did not allow for the calculation of EC50 values in most of the species and such data could thus not be used to generate SSDs. Sufficient EC10 values were however generated to construct SSDs from the results of the NRR and comet assays. The HC5 values obtained from SSDs constructed with EC10 values for both suborganismal and whole-organismal endpoints indicated that the NRR assay was the most sensitive endpoint, followed by both the comet assay and earthworm mass change, and subsequently the other whole-organismal endpoints. In conclusion, the majority of the responses on the various levels of biological organisation investigated during the present study were shown to be suitable to determine species sensitivity relationships in the terrestrial oligochaete species studied.