Impacts of size-fractionation on toxicity of marine microplastics: Enhanced integrated biomarker assessment in the tropical mussels, Perna viridis.

Accumulation of microplastics (MP) in oceanic waters is eroding the health of marine biota. We investigated how size-fractionated MP influence the toxicity risks towards a tropical keystone species, Perna viridis. Tissue-specific bioaccumulation and in vivo toxicity of polystyrene (PS) particles (0.5, 5, and 50 μm) were measured upon continuous exposure for 7 days, followed by 7 days depuration. P. viridis were exposed to equivalent mass (0.6 mg/L), corresponding to 4.0-4.6 particles/mL, 4.6-7.1 × 10 particles/mL, and 1.1-4.8 × 10 particles/mL for 50 μm, 5 μm and 0.5 μm PS particles, respectively. Onset toxicity were quantified through the enhanced integrated multi-biomarker response (EIBR) model, measured by weighting of biological organisation levels of eight biomarkers: (i) molecular (i.e., DNA damage (comet), 7-ethoxy resorufin O-deethylase (EROD), Catalase (CAT), Superoxide dismutase (SOD), Ferric Reducing Antioxidant Power (FRAP)); (ii) cellular (i.e., Neutral red retention (NRR), phagocytosis); and (iii) physiological (i.e., filtration rate). Data showed slightly elevated lysosomal instability (NRR) and antioxidant defences (FRAP, SOD, CAT, EROD) in specimens exposed to nano-PS (0.5 μm) compared to micro-PS (5 and 50 μm). Immunotoxicity (phagocytosis) and genotoxicity (comet) for haemocyte cells were significantly higher in specimens exposed to nano-PS (p < 0.05). EIBR index corroborated increasing toxicity modulated by MP sizes in descending order: 0.5 μm > 5 μm > 50 μm, with nano-PS exerted significantly higher biological effects (EIBR = 19.77 ± 5.89) than the unexposed group (EIBR = 10.97 ± 2.02; p < 0.05). Symptomatic organismal depression was manifested by the depleting filtering proficiency and weakened defence against invasive Zymosan bioparticles in the phagocytosis assay. Although impaired mussels duly recovered during depuration, individuals affected by nano-PS showed immunocompetence deficiency and gill responses that were not readily reversible, which could potentially increase their vulnerability towards further environmental stressors.