Combined toxicity of therapeutic pharmaceuticals to duckweed, Lemna minor

Pharmaceuticals, which are designed to be biologically active at low concentrations, are found in surface waters, meaning aquatic organisms can be exposed to complex mixtures of pharmaceuticals. In this study, the adverse effects of four pharmaceuticals, 17α-ethynylestradiol (synthetic estrogen), methotrexate (anticancer drug), diclofenac (nonsteroidal anti-inflammatory drug) and fluoxetine (antidepressant), and their binary mixtures at mg/L concentrations were assessed using the 7-day Lemna minor test, with both apical and biochemical markers evaluated. The studied biochemical markers included chlorophyll a, chlorophyll b, carotenoids and oxidative stress enzymes catalase, glutathione-S-transferase and glutathione reductase, with effects compared to solvent controls. The adverse effects on Lemna minor were dose-dependent for frond number, surface area, relative chlorophyll content and activity of glutathione S-transferase for both individual pharmaceuticals and binary mixtures. According to the individual toxicity values, all tested pharmaceuticals can be considered as toxic or harmful to aquatic organisms, with methotrexate considered highly toxic. The most sensitive endpoints for the binary mixtures were photosynthetic pigments and frond surface area, with effects observed in the low mg/L concentration range. The concentration addition model and toxic unit approach gave similar mixture toxicity predictions, with binary mixtures of methotrexate and fluoxetine or methotrexate and 17α-ethynylestradiol exhibiting synergistic effects. In contrast, mixtures of diclofenac with fluoxetine, 17α-ethynylestradiol or methotrexate mostly showed additive effects. While low concentrations of methotrexate are expected in surface water, chronic ecotoxicological data for invertebrates and fish are lacking, but this is required to better assess the environmental risk of methotrexate.