Multi-level biomarker responses to 6-PPD-quinone and microplastics co-exposure in Mediterranean mussels: Evaluating synergistic stress and histopathological damage

Tire wear particles (TWPs) are an increasingly recognized source of microplastics and associated chemical additives in aquatic environments. Among these, 6-PPD-quinone (6-PPD-Q), a transformation product of the tire antioxidant 6PPD, has emerged as a contaminant of global concern. However, its effects in marine ecosystems, particularly under co-exposure with microplastics (MPs), remain poorly understood in filter-feeding bivalves. This study investigated the combined effects of nominal concentrations of 6-PPD-Q (1 μg/L) and MPs (polystyrene, 1 μm) in the Mediterranean mussel Mytilus galloprovincialis . Using an integrated multi-level approach, we assessed molecular, biochemical, and histopathological responses in the gills and digestive gland. While individual stressors induced moderate antioxidant responses, two-way ANOVA revealed significant interaction effects during co-exposure, characterized by oxidative exhaustion, depletion of total antioxidant capacity, and significant modulation of superoxide dismutase and glutathione peroxidase activities. These effects were accompanied by increased energetic demand and the activation of pro-apoptotic signaling. Multivariate analysis (PCA) confirmed that the interaction between 6-PPD-Q and MPs generates a distinct toxicological profile, resulting in severe structural damage to target tissues. Although internal dose measurements were not performed, our findings suggest a potential ‘Trojan Horse’ mechanism whereby microplastics may exacerbate 6-PPD-Q toxicity by enhancing internal exposure. Overall, this study demonstrates that synergistic effects between tire-derived chemicals and microplastics amplify ecological risks, highlighting the need for mixture-aware approaches in marine environmental risk assessment.