Zinc accumulation as a primary contributor to co-exposure toxicity with 6PPD-quinone in earthworms: Multi-level...

Tire and road wear particles introduce complex mixtures of metals and organic additives into soils, yet the primary drivers of their combined toxicity remain poorly resolved. Here, we investigated the individual and combined effects of zinc oxide (ZnO), a major inorganic tire additive, and 6PPD-quinone (6PPD-Q), a toxic transformation product of the tire antioxidant 6PPD, using a soil invertebrate exposure model. A gradient of ZnO concentrations was tested alone and in combination with an environmentally relevant level of 6PPD-Q to simulate co-contaminated soils. While single exposures induced oxidative stress, metabolic perturbation, and gut microbiome alteration, co-exposure under high Zn conditions resulted in increased mortality and growth inhibition relative to single treatments. Chemical analysis revealed enhanced Zn accumulation in organisms under co-exposure, whereas 6PPD-Q bioaccumulation declined at elevated Zn levels, indicating that internal Zn burden was more consistently associated with organism-level impairment than measured internal 6PPD-Q concentration under the tested conditions. Integrated biochemical, histological, and multi-omics analyses showed that co-exposure was associated with enhanced oxidative stress, altered energy metabolism, impaired neuroimmune function, and destabilized gut microbial structure and predicted metabolic potential. Complementary multi-omics analyses revealed coordinated alterations in antioxidant defense, detoxification capacity, and mitochondrial metabolism under high-Zn co-exposure, consistent with a reduced physiological tolerance to chemical stress. Together, these findings support the interpretation that zinc accumulation likely represents a primary contributor to co-exposure toxicity, while 6PPD-quinone may amplify adverse outcomes by constraining organismal stress-buffering capacity. These results highlight the need to consider metal-organic interactions and differential toxic contributions in mixture-based environmental risk assessment of tire-derived contaminants.