Interactive effects of temperature, pH, and antibiotics reshape zebrafish physiological stability

Background: Freshwater organisms are increasingly exposed to multiple stressors, including climate-driven warming, pH shifts, and contamination by antibiotics, which may interact and amplify their effects. Sulfamethoxazole (SMX), trimethoprim (TRIM), and their mixture (MIX) frequently co-occur in aquatic environments, yet their toxicity under projected future environmental scenarios remains poorly understood. This study provides important data to help reduce current knowledge gaps in the combined effects of environmental stressors, particularly in the context of climate change, on Danio rerio. It highlights the need for integrated approaches in environmental research. Methods: A chronic assay was conducted with Danio rerio testing the effects of environmentally relevant concentrations of SMX (150 µg/L), TRIM (30 µg/L), and MIX (150 µg SMX/L + 30 µg TRIM/L) under two environmental scenarios: standard conditions (26 °C + pH 7.5) and a climate-change scenario (28 °C + pH 9.0). A multi-biomarker approach was applied, integrating oxidative stress, lipid peroxidation, neurotransmission, energy metabolism, and DNA damage to evaluate D. rerio health status under different conditions. Results: Antibiotic toxicities were consistently amplified under the climate-change scenario. Under the standard scenario, SMX and TRIM showed limited effects, mainly associated with antioxidant and detoxification responses, whereas MIX caused severe impairment of D. rerio health. Under a climate-change scenario, all antibiotics induced severe alterations in D. rerio, including lipid peroxidation, DNA damage, and disruption of energy metabolism, indicating a clear loss of physiological stability. Conclusions: Overall, these results demonstrate that warming and alkalinization enhanced antibiotic toxicity, both individually and in mixtures, resulting in more pronounced adverse effects on D. rerio. These findings highlight the need for integrative studies that simultaneously address chemical pollution and climate-related stressors to better predict long-term threats to aquatic biodiversity and ecosystem stability.