Integrating morphological, biochemical, behavioural, and molecular approaches to investigate developmental toxicity...

Tebuthiuron (TBU), a phenylurea herbicide, is widely applied in agricultural and non-agricultural soils. Because TBU resists degradation, it can contaminate water and reach the biota once it is released into the environment. However, the potential toxic effects of TBU on aquatic developing organisms have been poorly studied. By taking advantage of the early-life stages of zebrafish (Danio rerio), we have combined morphological, biochemical, behavioural, and molecular approaches to investigate the developmental toxicity triggered by environmentally relevant concentrations (from 0.1 to 1000 μg/L) of TBU. Exposure to TBU did not elicit morphological abnormalities but it significantly delayed hatching. In addition, TBU altered the frequency of tail coils in one-day post-fertilization (dpf) old embryos. Moreover, TBU exposure during four days significantly inhibited the whole body AChE activity of larvae. At the molecular level, TBU did not significantly affect the mRNA levels of four genes (elavl3, gfap, gap43, and shha) that play key roles during the neurodevelopment of zebrafish. By assessing the motor responses to repeated light-dark stimuli, 6 dpf larvae exposed to TBU displayed hyperactivity, showing greater travelling distance during the dark periods. Our categorization of swimming speed revealed an interesting finding - after the light was turned off, the exposed larvae abandoned the freezing mode (<2 mm/s) and travelled mainly at cruising speed (2-20 mm/s), showing that the larval hyperactivity did not translate into higher swimming velocity. Overall, our results offer new insights into the TBU toxicity to developing organisms, namely effects in AChE activity and hyperactivity, providing support data for future studies considering environmental risk assessment of this herbicide.