In this study, the toxicity of hexavalent chromium Cr­(VI) reduced by citric acid in ice was measured using representative aquatic model invertebrates (i.e., rotifer, water flea, amphipod, and polychaete) and a vertebrate (zebrafish) by analyzing short- and/or long-term endpoints that are frequently applied to each animal. Cr­(VI) reduction in the presence of citric acid was markedly enhanced in the ice phase compared to that in an aqueous solution through the freeze concentration effect. The highly concentrated Cr­(VI) and citric acid in ice grain boundaries were also confirmed using in situ cryogenic confocal Raman spectroscopy. Overall, exposure to Cr­(VI) resulted in higher acute and/or chronic effects on aquatic animals, such as drastic mortality, growth inhibition, and decrease in offspring number, whereas the animals were increasingly tolerant to Cr­(VI) that was reduced in the ice phase. Sublethal concentrations of Cr­(VI) significantly decreased the antioxidant capacity in the aquatic animals. However, when the same concentrations of Cr­(VI) were reduced in ice, these treatments showed no modulation or increase in the antioxidant defense system. Taken together, our results suggest that Cr­(VI) reduction into Cr­(III) was successfully achieved in ice and that this methodology can decrease the actual toxicity of Cr­(VI) in aquatic animals.