Road runoff (RR) is an important vector of micropollutants towards groundwater and soils, threatening the environment and ecosystems. Through combined chemical and biological approaches, the purpose of this study was to get insights on specific toxicants present in RR from two sites differing by their traffic intensity and their toxicological risk assessment. Non-target screening was performed by HRMS on RR dissolved phase. Ecotoxicological risk was evaluated in a zebrafish embryos model and on rat liver mitochondrial respiratory chain. Specific HRMS fingerprints were obtained for each site, reflecting their respective traffic intensities. Several micropollutants, including 1,3-diphenylguanidine (DPG) and benzotriazole (BZT) were identified in greater concentrations at the high-traffic site. The origin of DPG was confirmed by analyzing HRMS fingerprints from shredded tires. RR samples from each site, DPG and BZT were of relatively low toxicity (no mortality) to zebrafish embryos, but all generated distinct and marked stress responses in the light–dark transition test, while DPG/BZT mixes abolished this effect. The moderate-traffic RR and DPG inhibited mitochondrial complex I. Our study highlights (i) the unpredictability of pollutants cocktail effect and (ii) the importance of a multi-approaches strategy to characterize environmental matrices, essential for their management at the source and optimization of depollution devices.
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