Freshwater mussels are one of the most imperiled groups of animals in the world and are among the most sensitive species to a variety of chemicals. However, little is known about the sensitivity of freshwater mussels to wastewater effluents. The objectives of the present study were to (1) assess the toxicity of a permitted effluent, which entered the Deep Fork River, Oklahoma (USA), to a unionid mussel (Lampsilis siliquoidea) and to two standard test species (cladoceran Ceriodaphnia dubia; and fathead minnow Pimephales promelas) in short-term 7-day effluent tests; (2) evaluate the relative sensitivities of the three species to potassium (K), an elevated major ion in the effluent, using 7-day toxicity tests with KCl spiked into a Deep Fork River upstream reference water; (3) determine the potential influences of background water characteristics on the acute K toxicity to the mussel (96-h exposures) and cladoceran (48-h exposure) in four reconstituted waters that mimicked the hardness and ionic composition ranges of the Deep Fork River; and (4) determine the potential influence of temperature on acute K toxicity to the mussel. The effluent was found to be toxic to mussels and cladocerans, and it contained elevated concentrations of major cations and anions relative to the upstream Deep Fork River reference water. The K concentration in the effluent was 48-fold greater than in the upstream water. Compared with the standard species, the mussel was more than 4-fold more sensitive to the effluent in the 7-day effluent tests and more than 8-fold more sensitive to K in the 7-day K toxicity tests. The acute K toxicity to the mussel decreased by a factor of 2 when the water hardness was increased from soft (42 mg/L as CaCO₃) to very hard (314 mg/L as CaCO₃), whereas the acute K toxicity to the cladoceran remained almost the same as hardness increased from 84 to 307 mg/L as CaCO₃. Acute K toxicity to the mussel at 23 °C was similar to the toxicity at an elevated temperature of 28 °C. The overall results indicate that the two standard test species may not represent the sensitivity of the tested mussel to both the effluent and K, and the toxicity of K was influenced by the hardness in test waters, but by a limited magnitude.