Uptake and Biotransformation of the Tire Rubber-derived Contaminants 6-PPD and 6-PPD Quinone in the Zebrafish Embryo (Danio rerio)

Abstract

N-(1,3-Dimethylbutyl)-N'-phenyl-p-phenylenediamine (6-PPD) is a widely used antioxidant in tire rubber known to enter the aquatic environment via road runoff. The associated transformation product (TP) 6-PPD quinone (6-PPDQ) causes extreme acute toxicity in some fish species (e.g., coho salmon). To interpret the species-specific toxicity, information about biotransformation products of 6-PPDQ would be relevant. This study investigated toxicokinetics of 6-PPD and 6-PPDQ in the zebrafish embryo (ZFE) model. Over 96 h of exposure, 6-PPD and 6-PPDQ accumulated in the ZFE with concentration factors ranging from 140 to 2500 for 6-PPD and 70 to 220 for 6-PPDQ. A total of 22 TPs of 6-PPD and 12 TPs of 6-PPDQ were tentatively identified using liquid chromatography coupled to high-resolution mass spectrometry. After 96 h of exposure to 6-PPD, the TPs of 6-PPD comprised 47% of the total peak area (TPA), with 4-hydroxydiphenylamine being the most prominent in the ZFE. Upon 6-PPDQ exposure, >95% of 6-PPDQ taken up in the ZFE was biotransformed, with 6-PPDQ + O + glucuronide dominating (>80% of the TPA). Among other TPs of 6-PPD, a reactive N-phenyl-p-benzoquinone imine was found. The knowledge of TPs of 6-PPD and 6-PPDQ from this study may support biotransformation studies in other organisms.

Keywords: aquatic organisms; phase II metabolism; suspect and nontarget screening; tire and road wear particles; water quality.

Figure 1

Time course of internal concentrations of 6-PPD and 6-PPDQ in ZFE exposed from 4 to 100 hpf. Data are shown as individual replicates, with means connected by a line. (A) Relative internal concentrations of 6-PPD of all experiments. Analytically determined exposure concentrations of 6-PPD were 6.3 and 1.28 μg/L, respectively. Relative internal concentrations are presented on a logarithmic scale. (B) Relative internal concentrations of 6-PPDQ of all experiments. Analytically determined exposure concentrations of 6-PPDQ were 20.9, 11.3, and 4.8 μg/L, respectively.

Figure 2

TPs detected in ZFE exposed to 6-PPD and hypothetical transformation pathways based on the structural change and putative enzymatic reactions. Proposed intermediates are marked in blue. CYP—cytochrome P450; UGT—UDP-glucuronosyltransferase; SULT—sulfotransferase; MT—methyltransferase; AT—acetyltransferase; NAT—N-acetyltransferase. GSH – glutathione; GST – glutathione-S-transferase; deg. – degradation. The arrows indicate the involved enzymes but not the sequence of the reactions. Detailed mass spectrometric data of TPs are given in the Excel file attached to the Supporting Information.

Figure 3

TPs detected in ZFEs exposed to 6-PPDQ and proposed transformation pathway based on chemical structures and known enzymatic reactions. The arrows indicate the potentially involved enzymes but not the sequence of reactions. Proposed intermediates are marked in blue. CYP—cytochrome P450; NAT—N-acetyltransferase; UGT—UDP-glucuronosyltransferase; SULT—sulfotransferase; QR—quinone reductase. GSH – glutathione; GST – glutathione-S-transferase; deg. – degradation. Detailed mass spectrometric data of TPs are given in the Excel file attached to Supporting Information.

Figure 4

Distribution of TPs of 6-PPD in extracts of ZFE exposed to 6-PPD after 96 h of exposure. (A) Peak area of 6-PPD and its TPs normalized to the TPA. ZFE were only exposed to 6-PPD. TPs based on 6-PPD, 4-HDPA, and 6-PPDQ are shown separately. (B) Distribution of TPs of 6-PPDQ in extracts of ZFE exposed to 6-PPDQ after 96 h of exposure. The exposure was started at 4 ± 1 hpf. Gluc—glucuronide; GSH—glutathione; AcGlu—acylglucuronide; Cys—cysteine; NAcCys—N-acetyl-cysteine. TPA is related to the sum of all of the TPs detected and their corresponding parental compounds. Letters refer to the transformation pathways indicated in Figure 3.

Figure 5

Time course of TPs of 6-PPD and 6-PPDQ. (A) Peak areas for phase II TPs of 6-PPD in ZFE over 96 h of exposure (analytically determined exposure concentration of 6.3 μg/L); (B) peak areas of phase I and phase II metabolites of 6-PPDQ over 96 h of exposure (analytically determined exposure concentration of 11.3 μg/L). Samples are as in Figure 3. Peak areas were normalized to the number of ZFE per sample and its volume at the respective life stage.