In ecological risk assessment, acute to chronic ratio (ACR) uncertainty factors are routinely applied to acute mortality benchmarks to estimate chronic toxicity thresholds. To investigate variability of aquatic ACRs, we first compiled and compared 56 and 150 pairs of acute and subchronic/chronic growth/reproductive toxicity data for fishes (Pimephales promelas (53), Danio rerio (2), and Oryzias latipes (1)) and the crustacean Daphnia magna, respectively, for 172 chemicals with different modes of action (MOA). We found that there were only significant relationships between P. promelas acute median lethal concentrations and growth lowest-observed effect concentrations for class 1 (nonpolar narcosis) chemicals, though significant relationships were demonstrated for D. magna to all Verhaar et al. MOA classes (Class 1: nonpolar narcosis, Class 2: polar narcosis, Class 3: reactive chemicals, and Class 4: AChE inhibitors and estrogenics). Probabilistic ecological hazard assessment using chemical toxicity distributions was subsequently employed for each MOA class to estimate acute and chronic thresholds, respectively, to identify MOA and species specific ecological thresholds of toxicological concern. Finally, novel MOA and species specific ACRs using both chemical toxicity distribution comparison and individual ACR probability distribution approaches were identified using representative MOA and chemical categories. Our data-driven approaches and newly identified ACR values represent robust alternatives to application of default ACR values, and can also support future research and risk assessment and management activities for other chemical classes when toxicity information is limited for chemicals with specific MOAs within invertebrates and fish.