First assessment of wastewater effluent and contaminated surface waters using the invertebrate cell line (C6/36)

The increasing complexity of chemical mixtures in aquatic environments, particularly from wastewater treatment plant (WWTP) effluents, drives the need for sensitive, ethical, and efficient toxicity assessment tools. While in vitro methods using fish and mammalian cell lines are well established, invertebrate cell lines remain underutilised despite their ecological relevance. This study aimed to compare the sensitivity of an invertebrate cell line (C6/36, Aedes albopictus) with two commonly used fish cell lines (RTgill-W1 and PLHC-1) across three toxicological endpoints: cytotoxicity, oxidative stress, and genotoxicity. A total of 24 environmentally relevant chemicals and five whole water samples (four WWTP effluents and one contaminated surface water) were tested using a suite of in vitro assays. C6/36 cells exhibited greater sensitivity than the fish-derived cell lines, with lower effect concentrations observed for the majority of chemicals and water samples. C6/36 cells showed the greatest overall sensitivity, with measurable effects observed for 71% of chemicals in the cytotoxicity assay, 25% in the oxidative stress assay, and 63% in the genotoxicity assay. Additionally, C6/36 cells were more sensitive than RTgill-W1 cells across all endpoints and PLHC-1 cells across two of the three assays (cytotoxicity and oxidative stress). When applied to whole samples, C6/36 cells detected effects at lower concentrations, resulting in higher toxic units and earlier detection potential. These findings support the integration of invertebrate-derived cell lines, particularly C6/36, into broader water quality monitoring frameworks and underscore their value in identifying ecological risks posed by complex contaminant mixtures.