A series of chronic toxicity tests was conducted exposing three aquatic species to iron (Fe) in laboratory freshwaters. The test organisms included the green algae Raphidocelis subcapitata, the cladoceran Ceriodaphnia dubia, and the fathead minnow Pimephales promelas. They were exposed to Fe (as Fe (III) sulfate) in waters under varying pH (5.9-8.5), hardness (10.3-255 mg/L CaCO3 ), and dissolved organic carbon (DOC; 0.3-10.9 mg/L) conditions. Measured total Fe was used for calculations of biological effect concentrations because dissolved Fe was only a fraction of nominal and did not consistently increase as total Fe increased. This was indicative of the high concentrations of Fe required to elicit a biological response and that Fe species that did not pass through a 0.20- or 0.45-µm filter (dissolved fraction) contributed to Fe toxicity. The concentrations frequently exceeded the solubility limits of Fe(III) under circumneutral pH conditions relevant to most natural surface waters. Chronic toxicity endpoints (10% effect concentrations [EC10s]) ranged from 442 to 9607 µg total Fe/L for R. subcapitata growth, from 383 to 15 947 µg total Fe/L for C. dubia reproduction, and from 192 to 58,308 µg total Fe/L for P. promelas growth. Toxicity to R. subcapitata was variably influenced by all three water quality parameters, but especially DOC. Toxicity to C. dubia was influenced by DOC, less so by hardness, but not by pH. Toxicity to P. promelas was variable, but greatest under low hardness, low pH, and low DOC conditions. These data were used to develop an Fe-specific, bioavailability-based multiple linear regression model as part of a companion publication. Environ Toxicol Chem 2023;42:1371-1385. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.