Quantifying Organ-Specific Toxicity of Dietary Heavy Metals in Zebrafish Using a Histopathological Scoring Index

This study investigates sublethal effects of eight dietary heavy metals—copper (Cu), zinc (Zn), iron (Fe), cobalt (Co), chromium (Cr), aluminum (Al), manganese (Mn), and molybdenum (Mo)—on juvenile zebrafish (Danio rerio). Over a three-month period, fish were fed diets supplemented with each metal at two concentrations (1 mg/kg and 5 mg/kg), resulting in 16 exposure groups plus a control. Histological sections of gills, intestine, axial muscle, liver, kidney, and brain were evaluated across 49 histopathological parameters. A Metal Impact Index (MI) was used to quantify organ-specific toxicity. Both tested concentrations induced significant alterations in multiple organs, with severity varying by metal and tissue. Gill tissues exhibited the highest MI values, with Cu, Al, and Cr causing epithelial hyperplasia, lamellar degeneration, and pillar cell collapse. Cr exposure led to significant intestinal damage, while Fe caused the most prominent muscle pathology. Liver lesions (vacuolation, necrosis) increased with Cu, Cr, and Fe, and kidney damage (tubular degeneration) was evident in Cr- and Fe-treated groups. Brain tissue showed no significant MI variation. Hierarchical clustering based on cumulative MI scores grouped Cu, Cr, and Fe as high-toxicity metals, particularly in gills, liver, and kidney, while Mn, Zn, Al, and Mo formed lower-toxicity clusters. The MI approach provides a standardized framework for detecting early organ-specific toxicity and may support aquaculture health monitoring, environmental risk assessment, and fish welfare management in metal-contaminated ecosystems.