Metformin in a warming world: The hidden danger to freshwater ecosystems

Background: Metformin (MET), a widely prescribed drug for type-II diabetes mellitus, is increasingly detected in aquatic environments due to its high consumption and inefficient removal during wastewater treatment [1]. Concomitantly, freshwater ecosystems are experiencing intensifying thermal stress driven by climate change, which may modulate contaminant toxicity and challenge ecological risk predictions [2]. Objective: This study evaluated how thermal stress modulates the long-term ecotoxicological effects of MET in Daphnia magna, combining life-history traits with sub-individual physiological responses. Methods: Standard D. magna reproduction tests were conducted following OECD 211 guidelines [3], exposing organisms to 0-100 µg MET/L for 21 days at two temperatures: 20 °C (standard conditions) and 24 °C (warming scenario reflecting the + 4 °C increase projected by the IPCC until 2100). To evaluate potential impairments in feeding behavior after chronic exposure, post-exposure feeding inhibition assays [4] were performed after a 24h depuration period in clean medium, followed by a 4h feeding phase. Biomarkers related to oxidative stress, detoxification and neurotoxicity were quantified. Results: Temperature strongly influenced the reproductive responses of D. magna. At 20 °C, MET exposure induced concentration-dependent responses in key reproductive parameters, including fertility, reproductive output, and first brood fecundity. Under the warming scenario (24 ºC), D. magna exhibited an overall enhancement of reproductive performance, with earlier maturation, increased offspring production and fecundity, higher reproductive output, and greater population growth rates. While MET exposure did not affect D. manga feeding behavior at 20 ºC, a significant decline in feeding rate was observed at all MET concentrations at 24 ºC. Preliminary biomarker analysis further suggests that MET-induced physiological responses are temperature-dependent. Conclusions: Thermal stress appears to reshape the ecotoxicological responses to MET in D. magna, influencing both organismal performance and physiological status. By integrating life-history traits with mechanistic biomarkers, this study provides a multilevel understanding of MET effects under climate-driven warming. These findings highlight the importance of incorporating multi-stressor and multi-level approaches into future ecological risk assessment frameworks.