An Integrative Toxicological Assessment of the Herbicide Tebuthiuron: Elucidating Biochemical and Behavioral Responses in Developing Zebrafish

Abstract

Pesticides represent a significant threat to aquatic ecosystems due to their persistence and widespread use in agricultural areas, altering environments and exerting adverse effects on non-target organisms. Tebuthiuron is a phenylurea herbicide extensively used as an agrochemical to control pests and weeds in various crops, which often leads to contamination of aquatic environments. Despite its high water solubility and relatively long half-life in soil, studies on Tebuthiuron toxicity in fishes at environmental concentrations are limited. This study aimed to unravel the toxicity mechanisms of Tebuthiuron using the zebrafish model. Zebrafish embryos were exposed to Tebuthiuron (one concentration of either 0.1, 10, 1000 and 5000 µg/L) for 5 days and assessed for hatchability, heart rate, locomotor activity, oxygen reactive species, apoptosis and gene expression. There was no change in frequency of hatch, heart rate, or apoptosis. However, behavioral changes were noted, with hyperactivity in zebrafish larvae during the first light (at 10 µg/L), second light (at 10 and 1000 µg/L) and third dark (10 µg/L) periods of the Visual Motor Response assay. Biochemically, a significant depletion of basal ROS levels was observed at 1000 µg/L. At the molecular level, downregulation of oxidative stress-related genes (cat, sod1, and sod2) in larval fish was noted with exposure to 10 and 5000 µg/L Tebuthiuron, suggesting a depletion of antioxidant enzymes. In addition, some neurotoxicity-related genes were downregulated, such as acetylcholinesterase (ache), while some genes were upregulated, like microtubule-associated protein tau b (maptb) and synapsin 2 alpha (syn2a), with 5000 µg/L Tebuthiuron exposure. In conclusion, Tebuthiuron induces sublethal toxicity characterized by irregular, concentration-specific disruptions to photomotor behavior, basal ROS levels, and transcriptional markers of oxidative stress and synaptic function, even in the absence of acute morphological defects. Future research should prioritize functional assays targeting mitochondrial bioenergetics and antioxidant enzyme activities, alongside evaluations of later developmental stages, to fully elucidate the long-term ecological risks of phenylurea herbicides to non-target aquatic species.