The assessment of quantitative intestinal morphometry and multi-organ histopathology to reveal sublethal pollution...

This study investigated morphometric and histopathological alterations in brown trout (Salmo trutta, n = 60) along a pollution gradient in the Krka River from a reference river source (KRS) to a wastewater-impacted site (KRK) and a downstream site in a national park (KNP). Semi-quantitative scoring of the liver and kidney was used as a multi-organ context associated with intestinal responses. Intestinal morphometry showed reduced fold dimensions, altered epithelial structure, modified goblet-cell density and mucus layer thickness and changes in enterocyte nuclear morphology at the downstream sites. Among the analysed parameters, fold dimensions, supranuclear space and goblet-cell-related metrics showed particularly consistent responses along the pollution gradient. These alterations were most pronounced at KRK, while fish from KNP often showed intermediate responses consistent with a partial karst self-purification process. Morphometric changes were generally stronger in spring, whereas histopathological scores of intestines, liver and kidney were higher in autumn, suggesting cumulative tissue injury and seasonal physiological stress. Importantly, intestinal morphometry detected early structural changes, while multi-organ histopathology revealed later-stage damage, demonstrating complementary sensitivity of these approaches. Overall, the integration of quantitative intestinal morphometry with multi-organ histopathological scoring represents a useful tool for biomonitoring freshwater ecosystems exposed to mixed anthropogenic stressors.