The Cocktail Effects on the Acute Cytotoxicity of Pesticides and Pharmaceuticals Frequently Detected in the Environment

Xenobiotics never appear as single, isolated substances in the environment but instead as multi-component mixtures. However, our understanding of the ecotoxicology of mixtures is far from sufficient. In this study, three active pharmaceutical ingredients (carbamazepine, diclofenac, and ibuprofen) and three pesticides (S-metolachlor, terbuthylazine, and tebuconazole) from the most frequently detected emerging micropollutants were examined for their acute cytotoxicity, both individually and in combination, by bioluminescence inhibition in Aliivibrio fischeri (NRRL B-11177). Synergy, additive effects, and antagonism on cytotoxicity were determined using the combination index (CI) method. Additionally, PERMANOVA was performed to reveal the roles of these chemicals in binary, ternary, quaternary, quinary, and senary mixtures influencing the joint effects. Statistical analysis revealed a synergistic effect of diclofenac and carbamazepine, both individually and in combination within the mixtures. Diclofenac also exhibited synergy with S-metolachlor and when mixed with ibuprofen and S-metolachlor. S-metolachlor, whether alone or paired with ibuprofen or diclofenac, increased the toxicity at lower effective concentrations in the mixtures. Non-toxic terbuthylazine showed great toxicity-enhancing ability, especially at low concentrations. Several combinations displayed synergistic effects at environmentally relevant concentrations. The application of PERMANOVA was proven to be unique and successful in determining the roles of compounds in synergistic, additive, and antagonistic effects in mixtures at different effective concentrations.