Assessing the ecotoxicity of wastewater effluents contaminated with illicit drugs and benzodiazepines: Insights from ZFL cell exposure

Ecotoxicity of wastewater effluent contaminated with illicit drugs and benzodiazepines was evaluated by utilizing Zebrafish Liver cells (ZFL) as a streamlined and economical model for efficiently assessing aquatic toxicity, compared to other tiers such as Effect Directed Analysis (EDA). Rather than applying EDA, which aims to identify causative toxicants, this study provides a complementary line of evidence by directly assessing the cumulative biological effects of the complex mixture using in vitro bioassays. Detection and quantification of illicit drugs and benzodiazepines, even at trace levels, were conducted using HPLC-MS/MS. Cellular toxicity was determined by using MTT and flow cytometry assays. Bioanalytical equivalent concentrations were expressed in terms of Relative Enrichment Factor (REF) to standardize the biological responses and facilitate interpretation. The results indicate that the mixture of illicit drugs and benzodiazepines tested accounts for approximately 10 % of the total toxicity observed in the wastewater samples, studied mainly in terms of mitochondrial metabolic damage. This suggests that other compounds or synergistic effects between contaminants may also contribute to cytotoxicity. Additionally, the effect of filtration prior to cell exposure was studied and a decrease in sample toxicity was observed because of this process. In conclusion, these findings contribute to a better understanding of the potential risks raised by the presence of benzodiazepines and illicit drugs in wastewater. This approach provides complementary information to chemical analysis and mode of action-based profiling, supporting the use of integrative strategies for environmental risk assessment. These insights can inform policy decisions and environmental management strategies aimed at protecting aquatic ecosystems.