Assessment of the sub-lethal effects induced by three tire rubber-derived contaminants on zebrafish larvae

This study focuses on the sub-lethal effects of three key tire rubber-derived contaminants (TRDCs) used as vulcanizing agents and antioxidants in the tire production: 1,3-diphenylguanidine (DPG), N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine (6PPD), and its byproduct 6PPD-quinone (6PPD-q). Effects were assessed at the concentration of 500 ng/L on Danio rerio (zebrafish) larvae, from 0 to 120 h post fertilization, through a wide battery of biomarkers, proteomics, as well as imaging techniques for the inflammation detection. No significant effects were observed on larvae concerning apical endpoints, such as viability and morphological alterations, in all experimental groups. However, DPG exposure led to the most widespread biomarker responses among the tested compounds, with a significant induction of superoxide dismutase, ethoxyresorufin-O-deethylase and glutathione-S-transferase. To obtain a toxicity scale of tested TRDCs, based on used biomarkers, each endpoint was integrated in the Biomarker Response Index. This approach indicated that 6PPD-q and DPG were more toxic than 6PPD. Coherently, proteomic analysis revealed the modulation of 8 proteins with the DPG treatment, 7 with 6PPD-q and only 4 impacted proteins in the treatment with 6PPD. Other evidence, which considers also the effects of TRDC mixture, is necessary to better investigate the main toxic chemicals related to tire in aquatic ecosystems.