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.

中文翻译：

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使用 LC-Q/TOF-MS 进行逐步筛选并使用 GC×GC-TOFMS 进行信号扣除，能够快速鉴定 Raphidocelis subcapitata 中的 6PPD-Q 和 6PPD 代谢物

轮胎添加剂 N-（1,3-二甲基丁基）-N′-苯基-对苯二胺 （6PPD） 及其有毒代谢物 N-（1,3-二甲基丁基）-N′-苯基-对苯二胺-醌 （6PPD-Q） 的广泛环境检测对水生生态系统构成重大威胁。然而，对其生物转化途径的全面了解仍然难以捉摸。本研究调查了淡水微藻 Raphidocelis subcapitata 中 6PPD 和 6PPD-Q 的 I. 期和 II. 期代谢，揭示了它们在微藻水生环境中的快速转化。为了阐明这些化合物的转化命运，我们开发了一套互补的非靶向策略来识别潜在的代谢物，包括使用液相色谱四极杆飞行时间质谱 （LC-Q/TOF-MS） 进行逐步筛选和使用综合二维气相色谱飞行时间质谱 （GC×GC-TOFMS） 进行信号扣除。逐步筛选方法能够快速鉴定转化产物 （TP） 结构，尤其是以前的未知 TPs，而 GC×GC-TOFMS 的引入有助于鉴定其他 TPs。共鉴定出 24 种 6PPD-Q 代谢物和 15 种 6PPD TPs，包括几种新型化合物。值得注意的是，我们观察到 6PPD-Q 的醌分解和磷酸化，以及生物转化过程中 6PPD 甲基化的第一个证据。虽然大多数已鉴定的 TP 表明解毒，但 6PPD-Q 的四种 TP（TP270、TP312、TP314 和 TP328）与母体化合物相比显示出相当甚至放大的毒性。 这些发现为 6PPD 和 6PPD-Q 的环境归宿和生态风险提供了新的见解，特别是它们对淡水生态系统中初级生产者的潜在影响。