Abstract
Evaluation of the effects of linuron and S-metolachlor on apical, biochemical and transcriptional endpoints in zebrafish (Danio rerio) early life stages was the main purpose of this work. Embryos were exposed for 96h to a range of concentrations of each herbicide to determine lethal and sub-lethal effects on apical (e.g. malformations, hatching) and biochemical parameters (cholinesterase, ChE; catalase, CAT; glutathione S-transferase, GST; lipid peroxidation, LPO and lactate dehydrogenase, LDH). To evaluate endocrine disruption effects, embryos were exposed during 96h to 0.88mg/L linuron and 9.66mg/L S-metolachlor, isolated or in binary mixture. Expression of a suite of genes involved in HPT, HPG and HPA-axis was then assessed. Highest concentration of linuron (5.0mg/L) decreased hatching rate to 5% and 70.0mg/L S-metolachlor completely inhibited hatching, about 100%. Both herbicides impaired development by inducing several malformations (100% in 5.0mg/L linuron and 70.0mg/L S-metolachlor). Linuron only affected GST and CAT at concentrations of 0.25 and 0.0025mg/L, respectively. S-metolachlor induced GST (to 256%), inhibited ChE (to 61%) and LDH (to 60%) and reduced LPO levels (to 63%). Linuron isolated treatment seems to have an estrogenic mode of action due to the observed induction of vtg1. Exposure to S-metolachlor seems to interfere with steroidogenesis and with HPT and HPA-axis, since it has inhibited cyp19a2, TSHβ and CRH gene expression. In addition to vtg1 induction and CRH inhibition, herbicide combination also induced sox9b that has a role in regulation of sexual development in zebrafish. This study pointed out adverse effects of linuron and S-metolachlor, namely impairment of neurotransmission and energy production, induction of steroidogenesis, and interference with HPT and HPA-axis. These results contributed to elucidate modes of action of linuron and S-metolachlor in zebrafish embryo model. Furthermore, gene expression patterns obtained are indicative of endocrine disruption action of these herbicides.
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