Shape-dependent intestinal toxicity of bioplastic PLA and azithromycin co-exposure in zebrafish driven by the gut microbiota-metabolite axis: A case study of bubble tea seal film

Abstract Polylactic acid microplastics (PLA-MPs) are ubiquitous environmental contaminants that can act as carriers for antibiotics such as azithromycin (AZI). However, the manner in which PLA-MPs shape modulates antibiotic-induced disruption of the gut microbiota remains poorly understood. In this study, we evaluated the intestinal toxicity of four PLA-MPs shapes—film, fiber, particle, and sphere—all of which were co-exposed with AZI at 1 and 10 mg/L in zebrafish ( Danio rerio ). Biochemical analysis showed that the combined exposure to particle PLA-MPs at 10 mg/L AZI (Particle_10AZI) induced the most severe oxidative stress and digestive dysfunction. Co-exposure also caused locomotor hyperactivity and reduced anxiety-like behavior, with the strongest effects observed in the Particle_10AZI group. Histopathological analysis suggested that combined exposure to sphere PLA-MPs at 10 mg/L AZI (Sphere_10AZI) induced pronounced intestinal atrophy, whereas Particle_10AZI elicited a mixed response involving both tissue compensation and injury. PLA-MP shape further determined distinct patterns of gut microbial disruption: combined exposure to film PLA-MPs at 10 mg/L AZI (Film_10AZI) and Particle_10AZI reduced both richness and diversity, whereas the combined exposure of fiber PLA-MPs at 10 mg/L AZI (Fiber_10AZI) and Sphere_10AZI reduced richness and increased diversity. Particle_10AZI induced the most severe dysbiosis, characterized by enrichment of Pseudomonadota (particularly Gemmobacter ) and depletion of Bacteroidota (particularly Flavobacterium ). Metabolomic profiling further confirmed shape-dependent metabolic disturbances. Correlation analysis revealed that pro-inflammatory Pseudomonadota were positively associated with elevated AZI levels and a pro-fibrotic metabolite, whereas beneficial Bacteroidota were associated with the anti-inflammatory metabolite docosahexaenoic acid. Collectively, these findings suggested that AZI could determine the overall direction of toxicity, whereas PLA-MP shape was linked to the intensity and specificity of toxic effects, potentially through the gut microbiota–metabolite axis. This study highlights the importance of incorporating MP shape in toxicity assessments and wastewater management strategies.