Development of a semi-automated brightfield, high-content imaging approach for quantifying morphological effects in zebrafish embryos.

Environmental pollutants often induce morphological alterations in developing organisms, yet assessments are commonly subjective, limiting reproducibility and sensitivity. We developed and validated a semi-automated brightfield high-content imaging (HCI) pipeline to quantitatively detect morphological changes in zebrafish embryos. Using FishInspector software, we adapted image analysis for microscopy systems without automated embryo positioning, extending applicability across standard laboratory setups. To validate the approach, zebrafish embryos were exposed for 96 h to two previously characterized pollutant mixtures (PFOS + PCB126; PFOS + B[a]P + arsenate) known to cause developmental effects. The pipeline sensitively quantified phenotypes, including reduced swim bladder and shortened body length. These endpoints reflect developmental delay, highlighting the method's ability to capture mechanistically relevant effects. Such changes may reduce physiological performance and behavior, ultimately impacting fish populations. While earlier subjective scoring identified some similar alterations, our findings underscore the advantages of quantitative, semi-automated morphology assessment. The method improves reproducibility, enables standardized comparisons across studies, and increases sensitivity to detecting subtle morphological effects. By integrating brightfield imaging with semi-automated analysis, this approach broadens the toxicological toolbox for developmental hazard assessment and mixture toxicity research.