Introduction: 4-chloroaniline (4-CA) is an aromatic amine widely used in the production of dyes, pharmaceuticals, agrochemicals, and polymers. It is considered a potential freshwater contaminant due to its ecological risk and uncertainty surrounding the predicted no-effect concentration [1–3]. In aquatic systems, 4-CA can disrupt physiological pathways in non-target species, causing sublethal stress and biodiversity loss [4]. Given that temperature can reshape toxicodynamic and toxicokinetic processes, 4-CA toxicity may be underestimated in a warming climate. This study assessed whether elevated temperature modulates the chronic sub-individual toxicity of 4-CA in zebrafish [5].

Methods: Juvenile D. rerio were chronically exposed (28 days) to environmentally relevant concentrations of 4-CA (≤ 100 µg/L), at two temperatures: standard and projected warming scenario (25 and 30 ºC, respectively). Several biological responses were evaluated: antioxidant defence and detoxification, cellular energy allocation, neurotoxicity, and genotoxicity.

Results: Temperature significantly modulated 4-CA toxicity, with the combined factors disrupting antioxidant and detoxification capacity, enhancing oxidative stress, altering energy metabolism, and increasing neuro- and genotoxic damage. Temperature alone affected all biological responses, except lipid peroxidation. Significant effects were observed at 25 ºC included oxidative stress (≥ 19.75 µg/L), energy imbalance (29.63 and 66.67 µg/L), neurotoxicity (44.44 µg/L), and genotoxicity (≥ 29.63 µg/L); and at 30 ºC, oxidative stress (≥ 19.75 µg/L), detoxification disruption (100 µg/L), and neurotoxicity (29.63 µg/L).

Conclusions: Chronic exposure to 4-CA disrupted redox balance, antioxidant and detoxification capacity, and energy metabolism in zebrafish, with warming exacerbating oxidative stress, neuro-, and genotoxic damage. Although some compensatory antioxidant and metabolic responses were triggered, they were insufficient to prevent cellular damage, suggesting increased energetic costs under combined stressors. At the ecosystem level, enhanced contaminant toxicity under warming scenarios may alter species interactions and weaken the resilience of freshwater communities, suggesting that current ecological risk assessment may underestimate the future impacts of 4-CA.