Abstract
Environmental concerns have consistently been a focal point for the scientific community. Pollution is a critical ecological issue that poses significant threats to human health and agricultural production. Contamination with heavy metals and pesticides is a considerable concern, a threat to the environment, and warrants special attention. In this study, we investigated the significant issues arising from sub-chronic exposure to imidacloprid (IMI), mercury (Hg), and cadmium (Cd), either alone or in combination, using zebrafish (Danio rerio) as an animal model. Additionally, we assessed the potential protective effects of polyfloral honey enriched with natural ingredients, also called honey formulation (HF), against the combined sub-chronic toxic effects of the three contaminants. The effects of IMI (0.5 mg·L-1), Hg (15 μg·L-1), and Cd (5 μg·L-1), both individually and in combination with HF (500 mg·L-1), on zebrafish were evaluated by quantifying acetylcholinesterase (AChE) activity, lipid peroxidation (MDA), various antioxidant enzyme activities like superoxide dismutase and glutathione peroxidase (SOD and GPx), 2D locomotor activity, social behavior, histological and immunohistochemical factors, and changes in body element concentrations. Our findings revealed that all concentrations of pollutants may disrupt social behavior, diminish swimming performances (measured by total distance traveled, inactivity, and swimming speed), and elevate oxidative stress (OS) biomarkers of SOD, GPx, and MDA in zebrafish over the 21-day administration period. Fish exposed to IMI and Hg + Cd + IMI displayed severe lesions and increased GFAP (Glial fibrillary acidic protein) and S100B (S100 calcium-binding protein B) protein expression in the optic tectum and cerebellum, conclusively indicating astrocyte activation and neurotoxic effects. Furthermore, PCNA (Proliferating cell nuclear antigen) staining revealed reduced cell proliferation in the IMI-exposed group, contrasting with intensified proliferation in the Hg + Cd group. The nervous system exhibited significant damage across all studied concentrations, confirming the observed behavioral changes. Moreover, HF supplementation significantly mitigated the toxicity induced by contaminants and reduced OS. Therefore, the exposure to chemical mixtures offers a more complete picture of adverse impacts on aquatic ecosystems and the supplementation with bioactive compounds can help to reduce the toxicity induced by exposure to environmental pollutants.