Flow-dependent modulation of microplastic toxicity in grass carp: Insights from multi-level biological endpoints and machine learning

Microplastics (MPs) are emerging contaminants in freshwater systems, yet how hydrodynamic conditions modulate their biological effects remains unclear. This study investigated the accumulation, histopathology, immune response, and gut microbiota disruption in Ctenopharyngodon idella exposed to 5 µm polystyrene MPs (1000 µg/L; ∼ 1.46 × 10⁷ particles/L) under static conditions and water velocities of 1, 3, and 5 Body Lengths per second (BL/s), representing low, medium, and high flow. Water velocity alone caused notable tissue damage, including gill lesions and intestinal alterations, and significantly upregulated immune response genes, with the strongest responses observed at high flow. MPs accumulated in both gill and intestinal tissues, with highest concentration observed under high velocity flow. Combined exposure (MPs + flow) induced more severe effects, including gill necrosis, lamellar fusion, intestinal mucosal degeneration, inflammatory infiltration, and further immune gene upregulation, with IL-6 and TNF-α demonstrating the most substantial effect sizes (Cohen's d > 5). Gut microbiome evaluations indicated trends towards reduced alpha diversity and an elevation in pathogenic taxa in different groups, specifically with increased abundance of Aeromonas and Vibrio under the influence of combined stressors. Mediation analysis suggested a possible role for tissue damage in dysbiosis development, although wide confidence intervals precluded definitive conclusions. These initial findings indicate that hydrodynamic conditions alone can impact fish physiology and microbiota, and that flow can exacerbate MPs accumulation and toxicity through multiple biological pathways, emphasizing the imperative for extensive research efforts to confirm these relationships within aquatic ecological systems.