The Hidden Threat of Combined Stressors: The Influence of Temperature and pH on Antibiotic Toxicity in Danio rerio

Background: Aquatic ecosystems are increasingly subjected to overlapping stressors, including rising temperatures, pH variations, and antibiotic contamination (e.g., sulfamethoxazole—SMX, trimethoprim—TRIM, and their mixture—MIX). These abiotic factors may interact in complex ways, altering antibiotic toxicity and impairing key biological processes in aquatic organisms. This study aims to contribute significant data to bridge the gap regarding the combined impacts of environmental stressors, particularly in a climate change context, underscoring the need for integrative studies. Methods: We evaluated the chronic effects of environmentally relevant concentrations of SMX (150 µg/L), TRIM (30 µg/L), and their mixture (150 µg SMX/L + 30 µg TRIM/L) on Danio rerio after exposure to various i) temperatures (26, 28, and 32 °C) and ii) pH values (6.5, 7.5, and 9.0). A multi-biomarker approach was applied to assess antibiotic ecotoxicity and the biological health status of D. rerio. Results: Temperature and pH variations affected the antibiotic’s toxicity to D. rerio. At 28 °C, SMX and MIX exhibited moderate toxicity, inducing significant biological alterations (neurotoxicity and DNA damage), while TRIM showed only slight toxicity, mainly altering antioxidant/detoxification defenses. At 32 °C, MIX emerged as the most toxic compound, causing genotoxic and histopathological damage. In terms of the influence of pH, SMX had a greater impact under low-pH conditions (pH 6.5 and 7.5), leading to oxidative stress, lipid peroxidation, and DNA damage. In contrast, TRIM and MIX showed increased toxicity at neutral-to-alkaline pH (7.5 and 9.0), with notable impairment of antioxidant defenses, as well as DNA and histopathological injuries. Conclusions: The results highlight the urgent need for integrated studies that address chemical pollutants and climate change-related stressors. Ignoring these combined pressures could lead to long-term impacts on aquatic biodiversity and ecosystem stability. These findings reinforce the importance of comprehensive risk assessments that consider the potential amplification of pharmaceutical toxicity under changing environmental conditions.