Yan Xiaopeng, Zhang Yuanyuan, Kiki Claude, Zhao Shengwen, Zhao Guangpu, Xiao Jin, Sun Qian
State Key Laboratory of Advanced Environmental Technology, Institute of Urban Environment, Chinese Academy of Sciences, Xiamen 361000, China; University of Chinese Academy of Sciences, Beijing 100043, China.
SCNU Environmental Research Institute, Guangdong Provincial Key Laboratory of Chemical Pollution and Environmental Safety & MOE Key Laboratory of Theoretical Chemistry of Environment, South China Normal University, Guangzhou 510006, China.
J Hazard Mater. 2025 Sep 5;495:138805. doi: 10.1016/j.jhazmat.2025.138805. Epub 2025 Jun 4.
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.
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）在环境中广泛存在，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欠缺。本研究调查了淡水微藻头状针杆藻中6PPD和6PPD-Q的I相和II相代谢，揭示了它们在微藻水生环境中的快速转化。为阐明这些化合物的转化归宿，我们开发了一套互补的非靶向策略来鉴定潜在代谢物，包括采用液相色谱四极杆飞行时间质谱（LC-Q/TOF-MS）进行逐步筛选以及采用全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，尤其是先前未知的TP，而引入GC×GC-TOFMS则有助于鉴定更多的TP。共鉴定出24种6PPD-Q的代谢物和15种6PPD的TP，包括几种新化合物。值得注意的是，我们首次观察到6PPD-Q的醌分解和磷酸化以及6PPD在生物转化过程中的甲基化证据。虽然大多数鉴定出的TP表明具有解毒作用，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更强的毒性。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。
