Multi-Level Toxicity of 6PPD-Q and TiO2 Nanoparticles in Mytilus coruscus: Links among Neurochemical, Circadian and Metabolic Responses.

Tire-derived 6PPD-quinone (6PPD-Q) and titanium dioxide nanoparticles (TiO2 NPs) are increasingly detected in aquatic environments, yet their combined effects on marine bivalves remain poorly understood. In this study, the thick-shell mussel Mytilus coruscus was exposed to 6PPD-Q, TiO2 NPs, and their mixtures to evaluate multi-level physiological, biochemical, histological, and molecular responses. The results demonstrated that 6PPD-Q and TiO2 NPs, particularly under high-concentration co-exposure, significantly impaired mussel physiological performance, as indicated by reduced clearance rate and O:N ratio, increased oxygen consumption and excretion rate, and pronounced gill histopathological injury. Co-exposure also caused clear energy metabolic disruption, including decreased ATP content and inhibited CS, LDH, and PK activities, together with suppressed neurotransmitter levels of DA, ACh, and GABA. In addition, the expression patterns of circadian rhythm- and metabolism-related genes, including CRY, Per2, PPARγ, ACO, and PEPCK, were markedly altered, suggesting disruption of neuro-circadian-metabolic regulation. Oxidative stress was evident in both gills and digestive gland, as reflected by altered CAT and SOD activities and elevated MDA levels. These responses were accompanied by significant modulation of inflammatory and apoptotic genes, including TNF-α, IL-17, TGF-β, Caspase-3, Caspase-8, and Bcl-2. These findings suggest that 6PPD-Q and TiO2 NPs may jointly impair mussel health by linking gill first-contact injury with metabolic dysregulation, oxidative stress, and immune-apoptotic damage.