Structurally Selective Ozonolysis of p-Phenylenediamines and Toxicity in Coho Salmon and Rainbow Trout

The tire rubber-derived ozonation product of N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine (6PPD), N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine-quinone (6PPD-Q), was recently discovered to cause acute mortality in Coho Salmon (Oncorhynchus kisutch). para-Phenylenediamines (PPDs) with variable side chains distinct from 6PPD have been identified as potential replacement commercial antioxidants, but their structure-related ozone reactivities and toxicities remain unexplored. We herein tested the multiphase gas-surface ozone reactivity of four select PPDs and evaluated the toxicity of their reaction mixtures in Coho Salmon and Rainbow Trout (Oncorhynchus mykiss). 6PPD and N-Isopropyl-N'-phenyl-p-phenylenediamine (IPPD) were found to rapidly react with ozone to form 22 and 16 transformation products, respectively, including PPD-Qs. No significant multiphase ozone reactivity was observed for N,N'-Diphenyl-p-phenylenediamine (DPPD) or N-Cyclohexyl-N'-phenyl-p-phenylenediamine (CPPD) despite their structural similarity to 6PPD. The viability of Coho Salmon CSE-119 cells was strongly affected by the ozonolysis products of 6PPD, but not by those of the other three PPDs. The cytotoxicity of the 6PPD reaction mixture increased with ozonolysis time, with the strongest toxicity being observed after 7 days of oxidation by 100 ppb of ozone. As with Coho Salmon cells, acute mortality was only observed in juvenile Rainbow Trout that were exposed to the oxidized 6PPD reaction mixture, suggesting a common mechanism of toxic action in the two salmonid fish species. Compound- and regio-selective formation of hydroxylated metabolites of 6PPD-Q were detected in Rainbow Trout exposed to the 6PPD reaction mixture, which may be related to its selective toxicity. This study reports the structurally selective ozone reactivity of PPDs, and the unique toxicity of 6PPD ozonolysis mixtures, which demonstrates that other PPDs are potential alternative antioxidants.