Freshwater ecosystems are increasingly exposed to complex cocktails of chemicals, including pharmaceuticals, pesticides, and metals derived from human activity. Traditional environmental risk assessment remains largely based on single-chemical testing, which fails to capture the combined and potentially synergistic effects of these contaminants under realistic exposure conditions. In this study, the freshwater sentinel species Daphnia magna were used to investigate the biological impacts of a six-component xenobiotic mixture comprising three common pharmaceuticals (propranolol, diltiazem, metformin), two metals (nickel and lithium), and one herbicide (glyphosate) under standardised laboratory conditions. Acute and chronic toxicity were assessed using toxicity curves and survival plots, respectively, while sub-lethal endpoints, including filtration rate, growth, and key enzymatic activities, were evaluated to detect early biological responses. The mixture exhibited greater toxicity than individual compounds, indicating non-additive interactions. Beyond mortality, mixture induced concentration-dependent response across all endpoints. There was a clear decline in growth over 7, 14 and 21 days of exposure, while enzymes and feeding rate changed dose-dependently. This study highlights the limitations of conventional single-chemical risk assessment in predicting the ecological risks of xenobiotic mixtures. Building on these laboratory findings, ongoing work aims to evaluate how natural river water matrices modulate mixture toxicity and whether laboratory-derived biomarkers retain predictive value under more environmentally realistic exposure conditions. This integrative approach supports the development of daphnid-based New Approach Methodologies (NAMs) for improved early warning strategies in freshwater monitoring.