H9c2(2-1)-based sulforhodamine B assay as a possible alternative in vitro platform to investigate effluent and metals toxicity on fish

Abstract

To overcome restrictions on the use of fish in toxicity testing, the present study proposes to compare the 50% growth inhibition potential (EC50) of four types of effluents on the rat cardiomyoblast H9c2(2-1) cell line by using the sulforhodamine B (SRB) cell mass colorimetric assay, with the corresponding fish lethal test results. Our objective was to evaluate if H9c2(2-1) cells shows comparable sensitivities, in both relative and absolute terms, to those provided by fish. In parallel, this study also compared the results of the chemical characterization with the legislation in force for environmental protection against effluent release into the receiving environment. Moreover, we tested the H9c2(2-1)-based SRB assays with the metals of concern found in the effluent samples. Both fish and cell assays showed the same toxicity rank for effluents: Metal > Oil > Municipal > Paper, and it should be stressed that the complementarity of using chemical and biological data represents a step forward to guarantee both environmental and human safety, since the chemical characterization showed a different toxicity rank: Metal > Municipal > Oil > Paper. Regarding metal elements, the short-term fish results showed a toxicity rank non-comparable with the rank obtained for cells. Nevertheless, the gathered results reveal the potentiality of the in vitro H9c2(2-1) platform as an alternative for fish lethal testing to assess, in absolute terms, the toxicity of effluents, particularly municipal effluents, and metals.