Joint toxicity of permethrin and cypermethrin at sublethal concentrations to the embryo-larval zebrafish

Pyrethroids, the widely used pesticides, are highly toxic to aquatic organisms. However, little information is so far available regarding the joint toxicity of type I and type II pyrethroids to fish. Zebrafish is a well-accepted aquatic vertebrate model for toxicity assessment due to small size, easy husbandry, high fecundity and transparent embryos. In this study, we utilized embryo-larval zebrafish to elucidate the combined effects of sublethal concentrations of permethrin (PM) and cypermethrin (CP), which are the most frequently used type I and type II pyrethroids, respectively. Fish were exposed from 3 h postfertilization (hpf) to 144 hpf to binary mixtures of nominal concentrations of 100, 200, 300 μg L^1 PM (PM100, PM200, PM300) and 10, 20, 30 μg L^1 CP (CP10, CP20, CP30). Analytical data of the real concentrations of the chemicals showed a significant degradation of the pyrethroids but an obvious recovery after the renewal of the exposure solution. Defect rates of embryos exposed to these low concentrations of single PM or CP exhibited no statistically significant difference from the control,while the application of combination of PM and CP resulted in deleterious effects on zebrafish embryonic development. In all PM200 and PM300 exposure groups, increasing CP concentrations acted additively to the action of PM in terms of all sublethal endpoints. Co-treatment of embryos with the specific sodium channel blocker MS-222 and pyrethroids (individuals or the mixture) caused a decline in the incidences of body axis curvature and spasms compared to treatment of animals with pyrethroids alone, suggesting that the developmental toxicity of PM and CP to zebrafish was related to disruption of ion channels. We further revealed that mixture of the two pyrethroids caused greater down-regulation in the mRNA levels of proneural genes. The individual pesticides had no effect on the activity of superoxide dismutase (SOD), while the mixture exposure caused significant induction. Treatment with CP or the mixture increased the activity of catalase (CAT). Taken together, our data indicated that the mixture of PM and CP caused higher incidence of morphological defects, greater inhibition in proneural gene expression and more oxidative stress, compared to the single chemical at the corresponding doses. Our findings suggest that the combination of type I and type II pyrethroids poses a greater risk to fish in the water column.