The US Environmental Protection Agency's short-term freshwater effluent test methods include a fish (Pimephales promelas), a cladoceran (Ceriodaphnia dubia), and a green alga (Raphidocelis subcapitata). There is a recognized need for additional taxa to accompany the three standard species for effluent testing. An appropriate additional taxon is unionid mussels because mussels are widely distributed, live burrowed in sediment and filter particles from the water column for food, and exhibit high sensitivity to a variety of contaminants. Multiple studies were conducted to develop a relevant and robust short-term test method for mussels. We first evaluated the comparative sensitivity of two mussel species (Villosa constricta and Lampsilis siliquoidea) and two standard species (P. promelas and C. dubia) using two mock effluents prepared by mixing ammonia and five metals (cadmium, copper, nickel, lead, and zinc) or a field-collected effluent in 7-day exposures. Both mussel species were equally or more sensitive (more than two-fold) to effluents compared with the standard species. Next, we refined the mussel test method by first determining the best feeding rate of a commercial algal mixture for three age groups (1, 2, and 3 weeks old) of L. siliquoidea in a 7-day feeding experiment, and then used the derived optimal feeding rates to assess the sensitivity of the three ages of juveniles in a 7-day reference toxicant (sodium chloride [NaCl]) test. Juvenile mussels grew substantially (30%–52% length increase) when the 1- or 2-week-old mussels were fed 2 ml twice daily and the 3-week-old mussels were fed 3 ml twice daily. The 25% inhibition concentrations (IC25s) for NaCl were similar (314–520 mg Cl/L) among the three age groups, indicating that an age range of 1- to 3-week-old mussels can be used for a 7-day test. Finally, using the refined test method, we conducted an interlaboratory study among 13 laboratories to evaluate the performance of a 7-day NaCl test with L. siliquoidea. Eleven laboratories successfully completed the test, with more than 80% control survival and reliable growth data. The IC25s ranged from 296 to 1076 mg Cl/L, with a low (34%) coefficient of variation, indicating that the proposed method for L. siliquoidea has acceptable precision. Environ Toxicol Chem 2021;40:3392–3409. © 2021 SETAC