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journalArticle Multidisciplinary Digital Publishing Institute Sandré Fidji Huynh Nina Gromaire Marie-Christine Varrault Gilles Morin Christophe Moilleron Régis Le Roux Julien Garrigue-Antar Laure HRMS ecotoxicity light–dark transition micropollutants mitochondria non-target screening road runoff stress zebrafish Road Runoff Characterization: Ecotoxicological Assessment Combined with (Non-)Target Screenings of Micropollutants for the Identification of Relevant Toxicants in the Dissolved Phase 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. 2022/1 511 https://www.mdpi.com/2073-4441/14/4/511 2022-02-09 10:51:27 Road Runoff Characterization en www.mdpi.com http://creativecommons.org/licenses/by/3.0/ Number: 4 Water 14 4 DOI 10.3390/w14040511 ISSN 2073-4441