Abstract
Microplastic (MP) pollution poses an increasing threat to the aquatic ecosystems, yet most existing detection methods remain largely destructive and spatially limited. Here, we present a novel, non-invasive imaging framework integrated with micro-computed tomography (µ-CT) and fluorescence microscopy to investigate ingestion, accumulation and internal distribution of fluorescently labelled polystyrene microplastics (PS-MP) in the commercially important fish species Labeo rohita. Fluorescence microscopy confirmed presence of MPs in the gastrointestinal (GI) tract, gills, muscle tissues and excreta of exposed fish, whereas, micro-computed tomography (µ-CT) facilitated non-destructive, three-dimensional whole-body imaging, allowing precise localization and visualization of internalized particles within intact organisms. A clear, progressive and exposure-dependent increase in microplastic presence was observed over the 28-day experimental period, supported by non-parametric trend and effect-size analyses, with consistently higher relative burdens under waterborne exposure compared to the dietary exposure, as revealed by complementary µ-CT and fluorescence imaging. Detection of microplastics in muscle tissue indicates systemic translocation beyond primary uptake organs, whereas their presence in excreta confirms active elimination processes. Strong spatial concordance between µ-CT and fluorescence microscopy validates the robustness of the dual-imaging approach. Collectively, this study advances microplastic ecotoxicology by establishing a scalable, high-resolution, non-invasive imaging framework for mapping and tracking microplastic fate in freshwater systems as well as organ-specific microplastic distribution and burden in freshwater fish under environmentally realistic exposure conditions.