Stepwise screening with LC-Q/TOF-MS and signal subtraction with GC×GC-TOFMS enables rapid identification of 6PPD-Q and 6PPD metabolites in Raphidocelis subcapitata.

Widespread environmental detection of tire additive N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine (6PPD) and its toxic metabolite N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine-quinone (6PPD-Q) pose a significant threat to aquatic ecosystems. However, a comprehensive understanding of their biotransformation pathways remains elusive. This study investigates the phase I and phase II metabolism of 6PPD and 6PPD-Q in freshwater microalga Raphidocelis subcapitata, revealing their rapid transformation under microalgae aquatic environment. To elucidate the transformation fate of these compounds, we developed a suite of complementary non-targeted strategy to identify potential metabolites, including stepwise screening with liquid chromatography quadrupole-time of flight mass spectrometry (LC-Q/TOF-MS) and signal subtraction with comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry (GC×GC-TOFMS). The stepwise screening approach enabled rapid identification of transformation product (TP) structure especially the previous unknown TPs, while the introduction of GC×GC-TOFMS facilitated the identification of additional TPs. A total of 24 metabolites of 6PPD-Q and 15 TPs of 6PPD were identified, including several novel compounds. Notably, we observed the first evidence of quinone decomposition and phosphorylation of 6PPD-Q, as well as methylation of 6PPD during biotransformation. While the majority of identified TPs suggest detoxification, four TPs of 6PPD-Q (TP270, TP312, TP314 and TP328) showed comparable or even amplified toxicity compared to the parent compound. These findings provide new insights into the environmental fate and ecological risks of 6PPD and 6PPD-Q, particularly their potential impacts on primary producers in freshwater ecosystems.