Adverse outcome pathways of titanium dioxide nanoparticles and marine heatwaves on byssal performance in mussels

Adverse outcome pathways (AOPs) describe toxicity across levels of biological organization by measurable key events (KEs), thus providing a structured framework for predicting population-level effects. Here, mussels were exposed to different temperature conditions-22 °C and marine heatwave (MHW), in combination with nano-titanium dioxide (nano-TiO₂) at 0, 25, and 250 μg/L. High-concentration nano-TiO₂ and MHW exposure promoted the secretion of byssal threads and enhanced the adhesion to the substrate. However, they also caused severe histopathological damage in the foot and reduced the condition index (CI), indicating serious organism-level adverse outcomes (AOs). Accumulation of lipid peroxidation (LPO) products and redox imbalance suggested that MHW and nano-TiO₂ induced oxidative stress, leading to increased cell apoptosis (CA), reduced cell viability (CV), and consequent tissue damage in the foot. Nano-TiO₂ and MHW exposure significantly upregulated adhesion-related genes (Mfp-3/5/6) as well as immune- and cytotoxicity-related genes (Caspase-3, TLR-4, Myd88, and TNF-α). In contrast, Mfp-1/2/4 and pre-collagen proteins (preCOL-D/P/NG) were significantly downregulated, resulting in poor extensibility of byssal threads. Using the AOP framework, this study links nano-TiO₂ toxicity and MHW-induced stress to molecular disruptions, organism-level adverse outcomes, and altered mussel byssal defenses. These findings highlight that the interaction between MHW and nanoparticles may compromise mussel attachment and survival, with potential implications for the stability of coastal benthic ecosystems under future climate change scenarios.