<p>Investigating the characterization of toxicity from new perspective requires considering the multiple interactions between different toxicants and moving beyond the traditional indices. Therefore, in this study, it is aimed to address these shortcomings by investigating the toxicological effects of nickel (Ni), cobalt (Co) and iron (Fe) both individually and in binary and ternary combinations by focusing on the inhibition of bioluminescence of <i>Aliivibrio fischeri</i> and <i>Daphnia magna</i> as the test organisms. The toxicity of the metals was categorized according to the index methods: Combination Index (CI), Toxicity Unit (TU), Mixture Toxicity Index (MTI) and modelling techniques: Concentration Addition (CA) and Independent Action (IA) models for prediction the EC₅₀ values. Consequently, toxicity showed antagonistic effects at lower levels within the binary and ternary mixtures of Ni, Co, and Fe, while a synergistic toxicity was determined by the Ni-Co mixture, particularly at higher concentrations. These findings highlight that the CI can be used as a comprehensive tool to accurately assess chemical interactions in environmental toxicology. Particularly noteworthy is the effectiveness of the CI-isobologram equation as a suitable approach to assess the toxicological effects of metal interactions on <i>A. fischeri</i>.</p>