Dose-Dependent Toxicity Assessment of Metronidazole in Developing Zebrafish (Danio rerio)

Antibiotics are extensively used in human and veterinary medicine and livestock production. Antibiotics are considered as pseudo-persistent compounds, and a significant portion is excreted  in original form and enters the aquatic environment. Persistence of these chemicals in aquatic environments poses a significant risk to aquatic organisms. Metronidazole is used to treat various protozoal and bacterial infections in humans and animals. In this study, larval zebrafish were exposed to environmentally relevant concentrations of metronidazole to assess its potential toxic impacts. The primary objectives were to assess the effects of metronidazole on the survival rate, oxidative stress and transcriptome of zebrafish embryos. Larval zebrafish were exposed to 0.1, 1.0, 10, 100 and 1000 µg/L metronidazole continuously for five days. There were no significant effects on survival rate, however metronidazole exposure induced antioxidant and redox-regulatory pathways, particularly at higher concentrations, as evidenced by significant upregulation of nrf, cat, sod1, and sod2 due to activation of oxidative stress against high exposure concentration whereas lower exposure concentrations (0.1 μg/L) have caused minimal response showing no transcriptional changes in zebrafish. Locomotor activity was also observed as embryos showed hypoactivity during 1st dark period at 0.1 and 1000 µg/L. In the transcriptome analysis, 1000µg/L metronidazole caused upregulation of pathways related to circadian rhythm, fat digestion and absorption whereas, 0.1µg/L altered fat digestion and absorption and caused downregulation of il1b (Interleukin-1 beta), klf4, chia, ela2 and arg2. Furthermore, Transcriptomics analysis also revealed significant perturbations in processes involved with cholesterol and lipid homeostasis, inflammatory response and cytoskeletal processes which provide plausible mechanistic explanation for the observed induced deformities in embryos including pericardial edema and yolk sac edema. Overall, metronidazole exposure altered locomotor activity, gene expression and biological pathways in larval zebrafish. This study contributes to improved understanding of antibiotic-related toxicity in non-target species.