Single and Combined Impact of Environmental Concentrations of Galaxolide (HHCB) and Tonalide (AHTN) on Melanopsis praemorsa: Evaluating Genotoxicity and Stress Response Mechanisms.

Synthetic musk compounds (SMCs), such as galaxolide (HHCB) and tonalide (AHTN), are commonly used fragrance ingredients in personal care products and are frequently detected in aquatic environments due to their persistence and bioaccumulative nature. This study aimed to evaluate the individual and combined toxic effects of HHCB (1000 ng L^-1) and AHTN (400 ng L^-1) on the freshwater gastropod Melanopsis praemorsa following a 7-day exposure. Five experimental groups were formed: control, vehicle control, HHCB, AHTN, and HHCB+AHTN. Hepatopancreatic tissues were analyzed for oxidative stress biomarkers (malondialdehyde [MDA] and antioxidant enzymes), DNA damage responses (RAD21, RAD51), heat shock proteins (sHSP17.9, HSP60, HSC70-4, HSP90), apoptotic markers (AIF3, Caspase-3), and histopathological changes. MDA levels were significantly increased in the AHTN group (~ 2-fold) and the combined group (~ 2.8-fold), indicating increased lipid peroxidation. Antioxidant responses were markedly impaired. While RAD21 expression remained unchanged, RAD51 expression was significantly increased. All heat shock protein genes showed increased expression, with the highest induction in the AHTN group (p ≤ 0.05). Similarly, AIF3 and Caspase-3 levels were elevated in all exposed groups, suggesting apoptotic activation. Histopathological changes confirmed the biochemical and molecular findings. These results indicate that environmentally relevant concentrations of HHCB and AHTN, alone and in combination, induce oxidative stress, DNA damage, and apoptosis in M. praemorsa. Co-exposure leads to more pronounced effects, suggesting potential additive or synergistic toxicity. This study aimed to evaluate the toxic effects of HHCB and tonalide (AHTN), both alone and in combination, at environmentally significant concentrations on M. praemorsa, highlighting the urgent need for regulatory strategies to reduce the ecological risks posed by these pollutants to freshwater ecosystems.