Oil and gas extraction in the Prairie Pothole Region (PPR) of the northern USA has resulted in elevated chloride concentrations in ground and surface water due to widespread contamination with highly saline produced water, or brine. The toxicity of chloride is poorly understood in the high hardness waters characteristic of the region. We evaluated the toxicity of chloride to two endemic species, Daphnia magna (water flea) and Lemna gibba (duckweed), exposed in field-collected waters (hardness ~ 3000 mg/L as CaCO₃) and reconstituted waters (hardness 370 mg/L as CaCO₃) intended to mimic PPR background waters. We also investigated the role of chloride in the toxicity of water reconstituted to mimic legacy brine-contaminated wetlands, using two populations of native Pseudacris maculata (Boreal Chorus Frog). Chloride toxicity was similar in field-collected and reconstituted waters for both D. magna (LC50s 3070–3788 mg Cl⁻¹/L) and L. gibba (IC50s 2441–2887). Although hardness can ameliorate chloride toxicity at low to high hardness, we did not observe additional protection as hardness increased from 370 to ~ 3000 mg/L. In P. maculata exposures, chloride did not fully explain toxicity. Chloride sensitivity also differed between populations, with mortality at 2000 mg Cl⁻/L in one population but not the other, and population-specific growth responses. Overall, these results (1) document toxicity to native species at chloride concentrations occurring in the PPR, (2) indicate that very high hardness in the region’s waters may not provide additional protection against chloride and (3) highlight challenges of brine investigations, including whether surrogate study populations are representative of local populations.