Size-dependent toxicity cascades of microplastics in Perna viridis: Mechanistic insights into membrane damage, microbial dysbiosis, and persistent genotoxicity

Microplastics are pervasive contaminants in coastal ecosystems, but their size-dependent toxicity cascades across multiple biological levels remain poorly resolved. This study integratively assesses the 0.5-, 5-, and 50-μm microplastics toxicity in Perna viridis through multi-endpoints (cellular membrane stability, microbial homeostasis, haemolymph DNA integrity) across exposure (14-day) and depuration (7-day), revealing a size-dependent cascade. Exposure to 0.5-μm MPs triggered intracellular organelle stress, leading to oxidative stress responses marked by elevated ROS and MDA along with progressive ATP depletion. This was accompanied by downregulation of cytoskeletal genes (Actin and Tubb4b) and upregulation of the membrane repair gene preCOL-p. Concurrently, 0.5-μm MPs triggered severe intestinal dysbiosis reflected in reduced Shannon and Chao indices, enrichment of virulence genes and significant haemolymph DNA damage as measured by the Genetic Damage Index. Although membrane biomarkers recovered after depuration, irreversible DNA damage persisted exclusively in the 0.5-μm groups. GO enrichment analysis showed consistent top functions across MPs sizes but divergence in lower-ranked terms, while KEGG analysis revealed distinct size-dependent upregulation of metabolic pathways in exposed microbial communities. We propose a size-dependent cascading toxicity framework in which internalized small MPs first perturb cell membranes, then drive microbiome dysbiosis and microbiome-derived inflammatory signals, ultimately leading to sustained DNA damage.