Influence of Age and Exposure Pathway on Copper and Cadmium Mixture Toxicity: A Study on <scp> <i>Daphnia magna</i> </scp>

ABSTRACT Metal contamination in aquatic ecosystems poses substantial risks to freshwater organisms, with mixture effects often deviating from predictions based on single‐metal toxicity. Understanding how age‐specific sensitivities and multiple exposure pathways influence mixture effects is critical for accurate risk assessment. This study investigated the combined toxicity of copper (Cu) and cadmium (Cd) in Daphnia magna . Responses were compared between adults (20–21 days old) and neonates (less than 24 h old) under three exposure scenarios: aqueous‐only (0.25 μM Cu, 0.02 μM Cd, and their co‐exposure), dietary‐only (4.33 × 10 3 ng Cu/daphnid and 5.72 × 10 1 ng Cd/daphnid and their co‐exposure, delivered via metal‐spiked algae Raphidocelis subcapitata ), and combined aqueous‐dietary exposure. Following seven‐day exposures, physiological stress indicators (reproduction, growth, and survival), metal accumulation, biochemical and transcriptional changes were evaluated. Co‐exposure produced functionally synergistic toxic effects, with neonates showing markedly lower tolerance than adults at equivalent concentrations. Toxicity differed strongly among exposure routes, with combined aqueous–dietary exposure causing the most severe effects, including a 52% reduction in adult reproduction and 30% growth inhibition in neonates. Metal accumulation pattern varied between adults and neonates. Across both life stages, Cu–Cd co‐exposures disrupted redox homeostasis, evidenced by elevated reactive oxygen species and significant alterations in antioxidant enzyme activity. These responses were corroborated by pathway‐specific transcriptional changes, including upregulation of antioxidant, apoptosis‐related, and metal‐handling genes, alongside downregulation of development‐related genes in neonates. Overall, life stage and exposure pathway emerged as critical determinants of Cu‐Cd mixture toxicity. This emphasizes the need of incorporating age‐specific sensitivities and realistic multi‐metal, multi‐pathway exposures into freshwater risk assessment frameworks.