Combined Developmental Toxicity of Ecologically Relevant Concentrations of the PFOS Alternative F-53B and Hexavalent Chromium in Zebrafish, Danio rerio

6:2 Chlorinated polyfluoroether sulfonate (F-53B), an emerging per- and polyfluoroalkyl substance used as a perfluorooctane sulfonate substitute in electroplating, may co-occur with Cr(VI) in industrially affected aquatic environments. However, their combined developmental toxicity in vertebrates remains poorly understood. This study compared the effects of F-53B and Cr(VI) alone and in combination on zebrafish embryos and larvae exposed from 0 to 120 hpf at environmentally relevant concentrations (10 and 100 μg/L). Developmental toxicity, ocular morphology, retinal histology, locomotor behavior, and ocular-development-related gene expression were assessed. Single Cr(VI) exposure produced prominent effects on hatching, locomotor activity, retinal-layer thickness, and selected transcriptional responses related to retinoic acid signaling and ocular development. F-53B alone caused developmental and ocular alterations but generally produced weaker or more selective responses than Cr(VI). Co-exposure did not uniformly intensify the toxicity of either compound. Instead, the mixture enhanced some endpoints, including malformation, heart rate, and specific ocular or retinal alterations while showing weaker or divergent responses for other endpoints, such as locomotor activity and selected transcriptional markers. These findings indicate that F-53B/Cr(VI) co-exposure produces endpoint-dependent toxicity rather than a consistent synergistic pattern. Overall, the study highlights the importance of comparing single and combined exposures across multiple biological endpoints when assessing the developmental risks of co-occurring contaminants.