Microbiota-associated intestinal injury in bivalves exposed to 6PPD-Quinone and TiO2 nanoparticles

Emerging contaminants such as 6PPD-quinone (6PPD-Q) and titanium dioxide nanoparticles (TiO 2 NPs) are increasingly detected in aquatic environments; however, their combined effects on marine bivalves remain poorly understood. In this study, we investigated intestinal responses of Mytilus coruscus following exposure to 6PPD-Q (1 and 10 μg/L), TiO 2 NPs (0.1 mg/L), individually and in combination Mix Low (1 μg/L 6PPD-Q + 0.1 mg/L TiO 2 ), Mix High (10 μg/L 6PPD-Q + 0.1 mg/L TiO 2 ), for 14 days under controlled laboratory conditions. Compared with individual treatments, co-exposure elicited more pronounced oxidative stress, as reflected by elevated superoxide dismutase (SOD), catalase (CAT), and malondialdehyde (MDA) levels. Tissue-associated concentrations ranged from 0.0023 to 0.0518 μg/g for 6PPD-Q and 0.0468–0.344 μg/g for TiO 2 , with higher accumulation observed under mixed exposure. Additionally, digestive enzyme activities, including AMS, LPS, LZM, PS, and TRS, were significantly altered, indicating impaired intestinal digestive function, particularly under co-exposure conditions. Transcriptional analyses revealed upregulation of pro-inflammatory cytokines ( TNF-α and IL-17 ), accompanied by decreased Bcl-2 expression and increased Caspase-3 and Caspase-8 levels, suggesting activation of apoptosis-related pathways. High-throughput 16S rRNA sequencing showed marked alterations in gut microbial composition, characterized by reduced Cyanobacteriota and enrichment of Proteobacteria , indicative of microbial dysbiosis. Consistent with these molecular and microbial changes, histopathological examination revealed aggravated intestinal lesions, including epithelial vacuolation, particularly in the mixture group. Collectively, these findings indicate that co-exposure to 6PPD-Q and TiO 2 NPs is associated with enhanced intestinal oxidative stress, inflammatory and apoptotic responses, and gut microbiota imbalance in M. coruscus . This study provides the first evidence linking 6PPD-Q–associated exposure scenarios with intestinal injury in marine bivalves and highlights the potential ecological risks posed by interactions between tire-derived chemicals and engineered nanoparticles in coastal ecosystems.