Thallium (Tl) is a trace element associated with base metal mining and processing, but little is known regarding how its toxicity is influenced by water chemistry. In the present study, the 48-h median lethal concentration (LC50) of Tl to Daphnia magna was determined in a standard laboratory water, and toxicity was reassessed under conditions of varying cation (Ca2+ , K+ , Na+ ), anion (Cl- , HCO-3 ), and dissolved organic matter (DOM) concentrations. The calculated 48-h LC50 of 1.86 mg Tl/L was consistent with previous work on Tl toxicity to D. magna. At the 48-h LC50 concentration, changes in water chemistry had no statistically significant effect on mortality, although there was a trend toward lower Tl toxicity with elevated water K+ . Test waters containing 10 mM CaCl2 did not support control survival. The measurement of Tl complexation with DOM using asymmetric flow field flow fractionation confirmed the outcomes of biogeochemical speciation modeling: Tl speciation was relatively unaffected by water chemistry, and the majority of Tl remained in the ionic form across all treatments. These data indicate that Tl toxicity is largely independent of speciation, a property that will greatly simplify risk assessments for this metal in freshwaters. Environ Toxicol Chem 2019;38:2472-2479. © 2019 SETAC.