Comparative Developmental Toxicity of Roxithromycin in <scp>Danio rerio</scp> and <scp>Xenopus laevis</scp>

Antibiotics are among the most frequently detected pharmaceuticals in aquatic environments, raising concern about their effects on nontarget organisms. Roxithromycin (ROX), a semisynthetic macrolide widely used in human medicine, occurs in wastewater and surface waters, yet its developmental and biochemical impacts on aquatic vertebrates remain poorly understood. Here, we comparatively assessed the sublethal toxicity of ROX in zebrafish (Danio rerio) and frog (Xenopus laevis) embryos using standardized assays. Embryos were exposed for 96 h to nominal concentrations of 88-1000 mg/L. No lethality occurred in either species. However, zebrafish exhibited high malformation rates (46%-92%), dominated by pericardial edema and spinal curvature, and a marked reduction in heart rate within 24 h. In contrast, X. laevis displayed lower and non-dose-dependent malformations (≤ 19%), mainly gut and abdominal edema. Biochemical markers revealed distinct species-specific responses: In zebrafish, only glutathione (GSH) increased at the highest concentration, whereas in X. laevis, glutathione S-transferase (GST) and GSH increased at all concentrations, glutathione reductase (GR) was inhibited, and catalase (CAT) decreased at the highest concentration. UV-Vis analyses confirmed that ROX remained mostly undissolved, with acid-conditional values representing analytical upper bound estimates rather than true dissolved concentrations. Accordingly, exposures are interpreted as suspensions with limited dissolved fractions. These findings show that zebrafish are more prone to cardiac and morphological disruption, while X. laevis exhibits stronger biochemical stress responses. Overall, the study highlights the ecological relevance of macrolide antibiotics and supports the use of cross-species testing in environmental risk assessment of pharmaceuticals in aquatic ecosystems.