Dose-Dependent Neurobehavioral and Molecular Responses to Diclofenac in Zebrafish Larvae

The transport of pharmaceutical compounds into aquatic ecosystems poses a significant environmental threat, particularly due to the presence of drugs that cannot be completely removed during wastewater treatment processes. Diclofenac (DCF), one of the most widely used nonsteroidal anti-inflammatory drugs worldwide, is among the pharmaceuticals frequently detected in aquatic environments due to its high consumption levels and persistence in the environment. It is known that this compound causes neurotoxicity, behavioral disorders, and physiological stress responses in aquatic organisms even at low concentrations. This study aimed to determine the effects of diclofenac exposure on oxidative stress, circadian rhythm, and behavioral parameters in zebrafish larvae. For this purpose, zebrafish embryos and early-stage larvae were exposed to DCF at concentrations of 0.5, 2.5, and 12.5 μg/L for 120 h. Subsequently, to investigate the effect of DCF on oxidative stress, SOD, CAT, GPX, and AChE enzyme activities and gene expression levels were analyzed. To examine its effects on behavior and circadian rhythm, thigmotaxis and locomotor activity analyses were performed. Additionally, to determine the molecular-level effects of behavioral changes, the expression levels of the bdnf, 5ht4, crhr, bmal1, per, and gnat2 genes were analyzed. Overall, our findings indicate that DCF affects behavioral activity, neurotransmitter metabolism, oxidative stress response, circadian rhythm, and retina-related molecular regulators in zebrafish larvae in a multilevel manner. These results highlight the potential risks of pharmaceutical contaminants on neurodevelopmental processes in aquatic ecosystems and demonstrate that even environmental doses can produce complex responses in biological systems.