Exploring the impact of pH on the toxicological response of <i>Lemna minor</i> to pharmaceuticals—insights from sulfamethoxazole exposure

Aquatic acidification driven by increasing atmospheric CO2 is expected to modify the environmental fate and effects of ionizable pharmaceuticals; yet, pH-dependent toxicity is rarely addressed in standard ecotoxicological assessments. Over half of pharmaceuticals are ionizable within environmentally relevant pH ranges, meaning shifts in pH can affect their speciation, bioavailability, and ecotoxicity. Here, we investigated, for the first time, the influence of pH on sulfamethoxazole (SMX) toxicity toward the aquatic macrophyte Lemna minor, following OECD 221 guidelines. Experiments in Steinberg medium (pH 5.0-5.5) revealed increasing SMX toxicity with decreasing pH, consistent with a higher fraction of neutral SMX. Photolysis was a major degradation pathway, while interpretation of pH effects was complicated by pH increases during the test, associated with L. minor growth and medium alkalinization. Our findings demonstrate that the standard Organisation for Economic Co-operation and Development (OECD) Test Guideline 221 is not well-suited for assessing pH-dependent toxicity of ionizable pharmaceuticals without pH control. We highlight the need for buffering strategies and systematic pH monitoring in plant-based ecotoxicity tests, as accounting for pH is crucial for accurate environmental risk assessment of ionizable pharmaceuticals under ongoing aquatic acidification.