Submicron-size-dependent toxicity of microplastic–antibiotic mixtures in Chlorella pyrenoidosa

As an emerging contaminant, the co-presence of microplastics (MPs) of multiple sizes can exacerbate ecological risks. This study investigated the individual and combined toxicity of three-sized MPs (0.2, 0.3, 0.4 μm) and three macrolide antibiotics on Chlorella pyrenoidosa. In a single exposure scenario, 0.4 μm polystyrene (PS) and carboxyl-modified polystyrene (CPS) demonstrated greater toxicity than the 0.2 μm size, while amino-modified polystyrene (APS) showed an inverse size-dependent effect. At environmentally relevant concentrations (0.0005-0.001 mg/L), most particle-size-related differences in growth inhibition were small and statistically nonsignificant. More pronounced but non-monotonic size effects emerged at 1 mg L-1 and varied with MP type and exposure duration. Notably, 0.4 μm MPs significantly suppressed superoxide dismutase activity, and the correlation between total antioxidant capacity and malondialdehyde shifted from negative to positive at high concentrations, indicating antioxidant defense failure. Binary growth inhibition was strongly dependent on antibiotic identity, MP type, particle size, and exposure concentration. Short-term exposure (96 hr) at high levels of MPs showed toxicity order: 0.4 μm > 0.2 μm > 0.3 μm, though differences diminished over time. Compared to antibiotics alone, MP-antibiotic mixtures generally showed reduced toxicity due to MP adsorption lowering bioavailable antibiotic concentrations. The 0.3-0.4 μm APS combination increased chlorophyll a inhibition by 59.3% compared to the 0.2-0.3 μm group, attributed to enhanced sedimentation and interfacial contact affecting photosynthesis. This study elucidates how MP particle size dictates the evolution of combined toxicity by concurrently regulating sedimentation, adsorption, and pollutant slow-release kinetics, providing a critical new perspective for assessing the risks of MP-antibiotic co-pollution in realistic environments.