Critical Membrane Concentration and Baseline Toxicity of Organic Pollutants in a High-Throughput Rainbow Trout Cell Assay

Abstract A high-throughput RTgill-W1 cytotoxicity assay (RTgillHT) was developed using 384-well plates and live/dead staining combined with high-content imaging to quantify cytotoxicity. RTgillHT facilitated a significant increase in sample throughput compared to the OECD Test Guideline 249 (Fish Cell Line Acute Toxicity: The RTgill-W1 cell line assay) and showed comparable cytotoxic concentrations (IC10) if freely dissolved concentrations (Cfree) were used as dose-metric for more hydrophobic chemicals. A critical membrane concentration for baseline toxicity of 183 mmol/Llip was derived from experimentally measured IC10,free and membrane-water distribution ratios for 11 chemicals. The measurement of Cfree in the assay system is not necessary on a routine basis, because a mass-balance model (MBM) is shown to predict Cfree reliably if the system parameters are well characterized and the physicochemical properties of the test substances are carefully compiled. For most chemicals, this MBM was sufficient to predict Cfree, but a more complex kinetic model helped to sharpen the applicability domain of RTgillHT with respect to loss to the air and binding to well plate plastic. RTgillHT showed a good agreement with in vivo fish acute toxicity if in vivo lethal concentrations (LC50) were compared to freely dissolved concentrations that cause 50% cytotoxicity (IC50,free). Using in vitro data, it is possible to predict acute toxicity concentrations for fish in vivo, which can significantly reduce the number of in vivo tests required.