Abstract
The extensive use of caramel colourants in processed foods has led to increasing production volumes, yet information regarding their biological effects in aquatic organisms remains limited. While safety assessments have largely focused on dietary exposure, comparative data on ammonia-based caramel classes under waterborne conditions are scarce. The present study investigated the acute effects of Ammonia Caramel (Caramel III) and Sulfite-Ammonia Caramel (Caramel IV) in the Mediterranean mussel Mytilus galloprovincialis, a widely used marine sentinel species. Mussels were exposed for 96 h to 0.05, 0.5, and 5 mg/L of each caramel class. Concentrations were selected based on available ecotoxicological information for Caramel IV and to allow comparative evaluation of Caramel III under controlled aquatic conditions. Histopathological alterations and oxidative stress biomarkers were assessed in gill and digestive gland tissues. Both caramel classes induced concentration-dependent and tissue-specific responses. Gill tissue exhibited epithelial deformation, hyperplasia, lamellar fusion, hemocyte infiltration, and lipofuscin accumulation, whereas the digestive gland showed progressive tubular disorganisation, luminal dilation, vacuolisation, and epithelial atrophy. Antioxidant enzymes (SOD, CAT, GST) were activated in parallel with increased lipid peroxidation, indicating that compensatory antioxidant responses were insufficient to fully prevent oxidative damage. Multivariate analyses confirmed integrated biochemical shifts and clear separation between control and exposed groups. Across most endpoints, Caramel III elicited stronger structural and biochemical alterations than Caramel IV at comparable concentrations. These findings highlight differential biological responses between ammonia-based caramel classes and provide new ecotoxicological evidence for Caramel III in a marine invertebrate model.