Ecologically relevant toxicity tests may provide the best protection of sensitive aquatic fauna, but without established culturing or test methodology for such organisms, results may be unreliable and difficult to repeat. Further, field-collected organisms may not be feasible for routine testing purposes, as often required for permitted discharges. This study examined the feasibility of testing two field-collected mayflies, Isonychia bicolor and Maccaffertium spp., over a 1-year period. Seasonal comparisons of availability indicated I. bicolor and Maccaffertium spp. were most abundant during the winter months, resulting in 31 and 49 % of total organisms collected in 2009, while summer was the most difficult time to collect either species. Initial testing in January 2009 resulted in the highest no observable effect concentration (NOEC) values for survivorship (8 g NaCl for I. bicolor and 4 and 8 g NaCl/L for Maccaffertium spp.) when tested at 9 °C. Subsequent tests conducted at 20-23 °C resulted in 7-day NOEC values substantially lower (mean = 1.44 and 1.59 g NaCl/L). Geometric means of exuviae indicated a dose-dependent response for I. bicolor exposed to NaCl, while no dose-dependent response was observed for Maccaffertium spp. with average number of molts varying from 4.93 in the 0.5 g NaCl/L concentration to 3.80 for control organisms followed by 2.24 (1 g NaCl/L). Averages again increased to 3.09 in the 2 g NaCl/L concentration, but declined in the highest concentrations (4-10 g NaCl/L). Based on the results of this feasibility study, field-collected mayflies appear to be too unpredictable in test responses, and therefore, such tests would be unreliable as stand-alone indicators of effluent toxicity.