Biotransformation of Tire-Derived 6PPD and 6PPD-Q in Soil Nematode Caenorhabditis elegans: Unraveling Novel Phosphorylation Products and Distinct Kinetic Profiles

The extensive use of tire antioxidant N-(1,3-dimethylbutyl)-N′-phenyl-p-phenylenediamine (6PPD) has raised significant environmental concerns, given the ubiquity and severe toxicity of 6PPD and its quinone derivative (6PPD-Q). While their hazards could be mediated through biotransformation pathways of detoxification and/or bioactivation and associated metabolites, the biotransformation of these tire-derived contaminants in soil organisms, key environmental compartments directly exposed to tire wear, remains unexplored. In this work, we investigated the biotransformation of 6PPD and 6PPD-Q in soil nematode Caenorhabditis elegans (C. elegans). We identified 9 transformation products (TPs) of 6PPD and 26 of 6PPD-Q using suspect and nontargeted screening methods, providing the first comprehensive metabolic profile of these contaminants in soil nematodes. Novel in vivo metabolites including phosphorylated and monohydroxy phosphorylated 6PPD-Q were first discovered, revealing unique metabolic pathways of these contaminants in C. elegans compared to other eukaryotes. Kinetic profiling delineates heterogeneous temporal patterns among TPs, with specific derivatives showing progressive accumulation over the exposure duration. Notably, isomers such as dihydroxy 6PPD-Q exhibit distinct substitution-site-dependent dynamics. Furthermore, in silico toxicity prediction indicated that certain TPs exhibited earthworm toxicity comparable to 6PPD-Q, implying potential contributions to the discernible adverse effects observed in C. elegans. Our findings elucidate the biotransformation of tire-derived contaminants in soil nematodes, suggest potential ecological concerns posed by 6PPD and 6PPD-Q to soil organisms, and underscore the necessity for further evaluation of their bioactivities and environmental impacts.