Photometric application of the MTT- and NRR-assays as biomarkers for the evaluation of cytotoxicity ex vivo in Eisenia andrei

In soils, the toxicity of noxious substances such as metals is determined by a number of different factors of chemical, physical, biological, and environmental origin. Addressing these factors as a sum of different parameters, we studied these effects in soil-dwelling indicator species by using ‘sub-cellular’ biomarkers, to see if they can provide an early stage response to environmental disturbances. ‘Sub-cellular’ biomarkers such as the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and the Neutral Red Retention (NRR) assays are well established for the assessment of cytotoxicity of toxicants on cell cultures. Both assays can be carried out in 96-well microplates and evaluated photometrically. To be able to assess the risk potential of contaminated soils using these assays, without changing the soils chemically or physically to accommodate cell cultures, the assays were adapted for ex vivo use. The assays were performed on coelomic liquid extracted from exposed earthworms. Eisenia andrei was exposed in a standardized laboratory soil substrate (OECD soil) spiked with cadmium at a range of concentrations known to be cytotoxic to earthworms. Earthworms were also exposed to ultramafic soils as field-relevant soil substrates, containing high concentrations of a mixture of metals of natural origin such as chromium, cobalt, manganese, and nickel. A significant response was measured with both assays in the coelomocytes of the earthworms exposed to the Cd-spiked OECD soil as well as to the ultramafic soils. It was shown that as biomarkers for the assessment of ‘sub-cellular’ toxicity, the photometrical application of the MTT and the NRR assay promised to be rapid, objective, and easily conductible tools.