Toxicity of 6PPD alternatives to salmonid cell lines

Abstract

Stormwater runoff in urban areas introduces numerous anthropogenic chemicals into surrounding aquatic environments. One such chemical is 6PPD ( N -(1,3-Dimethylbutyl)- N ’-phenyl- p- phenylenediamine), the primary antidegradant responsible for protecting tire rubber from ozone-induced degradation and cracking. When exposed to ozone on the road surface 6PPD is transformed into the toxic transformation product 6PPD-quinone (6PPDQ). This compound is highly lethal to coho salmon ( O ncorhynchus kisutch ), and 24 hour 50% lethal concentrations (LC 50 ; 41-95 ng/L) are frequently detected in urban streams during stormwater runoff events 1-5 . The highest stream concentrations of 6PPDQ and most extensive coho salmon mortality occur in high-traffic urban areas, where tire wear particles accumulate due to the frequent braking and acceleration of cars 6 . In the Seattle area, some coho salmon-bearing streams can lose over 90% of returning females prior to spawning due to 6PPDQ-containing runoff 7 . Prespawn loss of this magnitude could result in the extinction of some urban spawning populations 8 . Importantly, the lethal effects of 6PPDQ have been observed in several other salmonid species, indicating that its impact may threaten fish health for urban aquatic environments globally 9-11 . The ubiquity of 6PPD in automobile tires, high toxicity of the transformation product 6PPDQ, and prevalence of 6PPD and 6PPDQ in environmental matrices has spurred investigations into alternative rubber antiozonants to replace 6PPD that retain tire performance while reducing ecological harm. A technical memorandum provided to the legislature by the Washington State Department of Ecology summarized a list of potential 6PPD alternatives requiring further study, including several structurally similar p-phenylenediamines (PPDs) 12 . However, the structural similarity of other PPDs to 6PPD and their known production of quinone transformation products 13 raises concerns that they may elicit similar toxic effects as 6PPDQ. Currently, investigations into the toxicity of proposed PPD alternatives or their ozonated transformation products in coho salmon are limited, representing a significant data gap in evaluating whether they offer improved environmental safety over 6PPD. This project investigated the toxicity of proposed alternative rubber antiozonants in vitro using immortalized cell lines derived from three salmonid species with known differences in sensitivity to 6PPDQ (coho salmon, Chinook salmon ( O. tshawytscha ), rainbow trout ( O. mykiss )) 1 . These immortalized cell lines replicate toxic responses observed in in vivo studies, while enabling higher-throughput testing and reducing the need for animal use and other resource-intensive procedures. The antiozonants and their transformation products selected for testing were chosen based on multiple criteria, including their inclusion in the Ecology technical memorandum, structural substitutions (e.g., branched/cyclic alkyl vs aryl substitutions), commercial availability, and input from the Washington State Department of Ecology and other industry experts.