Cytotoxicity of single and bimetallic mixtures of heavy metals and antioxidant de-fenses in the soil ciliated protist Rigidohymena tetracirrata

Ciliated protists are ubiquitous eukaryotic microorganisms which are well adapted for life in soil ecosystems. Ciliates exert a key role in soil microbial food webs by mediating the fluxes of nutrients and energy between different trophic levels. Further, they promptly sense and react to various soil perturbations and toxicants. All together, this made them valuable bioindicators of soil health. In this study, we analysed the toxicity (LC20 and LC50 at 24hrs) of single metal (Cd, Cu, and Zn) and bimetallic mixtures (Cd + Zn, Cd + Cu, and Cu + Zn) and, antioxidant responses in the soil ciliate Rigidohymena tetracir-rata. The LC20 values for Cd, Cu, and Zn were 0.53, 0.22 and 23.0 mg l-1, respectively; LC50 values were 1.16, 0.37 and 32.7 mg l-1, respectively. The order of decreasing toxicity was Zn >> Cd > Cu. Analysis of the bimetallic mixture treatments using the Concentration Ad-dition (CA) model, unveiled that the Cd + Zn mixtures were able to prevalently generate antagonistic effects as compared to the other mixtures (Cd + Cu, and Cu + Zn). Antioxi-dant activities were measured in (intra- and extra-) cellular extracts using different in vitro tests. The total phenolic contents (TPC) were significantly higher in extra- Cu LC20 (p ≤ 0.01) and intra-cellular Cd LC20 extracts (p ≤ 0.001). The intra- Zn LC50 and extra- cellular Cd LC50 extracts showed significantly higher α,α-diphenyl-β-picrylhydrazyl (DPPH) scav-enging activities (p ≤ 0.05). The extra- Cd LC50 and intra-cellular Cu LC50 extracts showed significantly higher hydroxyl radical scavenging (HRSA) activities (Cd LC50 p ≤ 0.001; Cu LC50 p ≤ 0.001). Overall, R. tetracirrata seem to have a good potential to be used as test organism in ecotoxicological analysis of soil contaminated with heavy metals.