Mixture Matters: Exploring the Overlooked Toxicity of Sulfamethoxazole and Trimethoprim in Aquatic Environments

Sulfamethoxazole (SMX) and trimethoprim (TRIM) are two antibiotics included in the third Watch list to be monitored in inland waters by the Water Framework Directive. However, their mixture (MIX) is overlooked and is especially concerning because it mirrors the real conditions in natural aquatic ecosystems. This study aimed to evaluate the toxic effects of environmentally relevant concentrations of SMX (150 μg/L), TRIM (30 μg/L), and MIX (150 μg/L SMX + 30 μg/L TRIM) in different standard species: Aliivibrio fischeri (bioluminescence inhibition), Escherichia coli ATCC 25922 (growth inhibition), Lemna minor (growth inhibition and biochemical biomarkers), Daphnia magna (immobilization/mortality, reproductive effects, and biochemical biomarkers), and Danio rerio (survival, hatchability, abnormalities, and biochemical biomarkers). Considering all the parameters evaluated, and using the Independent Action model and the antibiotics ecotoxicological assessment approach, the results reveal that: (i) all antibiotics affect the bacteria A. fischeri and E. coli differently, causing a pronounced E. coli inhibition growth; (ii) SMX and MIX were slightly toxic to D. magna (after 48 h) and D. rerio (after 96 h), and moderately toxic to L. minor (after 7 days) and D. magna (after 10 days); (iii) TRIM was slightly toxic to all organisms tested, except for L. minor , which exhibited moderate toxicity; (iv) the interaction of the antibiotics in mixture varied significantly depending on the parameter; however, it revealed similar toxicity to SMX. Significant ecotoxicological data were obtained, clarifying the ecological risks of these antibiotics in aquatic ecosystems. Remaining vigilant regarding environmental contamination linked to levels of SMX and TRIM in surface waters is crucial. Assessing the combined impacts of multiple stressors is essential for prioritizing environmental safety and ecotoxicity assessments for antibiotics amidst climate change, preserving the health and integrity of aquatic ecosystems.