Refining culturing and effluent testing methods for the mayfly, Neocloeon triangulifer

For decades, toxicity tests with aquatic invertebrates have been con-ducted and yet a small number of model organisms are routinely used. Test organisms are usually easily cultured in the laboratory, have rapid life-cycles, exhibit sensitivity to a variety of pollutants with repro-ducible results, and are generally available year-round. The USEPA effluent testing program uses short-term chronic freshwater tests (4d to 8d) with cladocerans (Cladocera, Ceriodaphnia dubia), green algae (Sphaeropleales, Raphidocelis subcapitata) and fish (Cypriniformes, Pimephales promelas). EPA also has standardized Hyalella azteca(Amphipoda) and Chironomus dilutus (Diptera) test methods for sedi-ments; yet EPA’s effluent and ambient testing manuals do not provide acute or short-term test methods for H. azteca, C. dilutus or mayflies (Ephemeroptera). Because mayflies have been shown to be among the most sensitive species to major ions, metals, and pesticides, we have focused on effluent method development for the mayfly, Neocloeon triangulifer, a parthenogenetic species with a short life cycle (~30d at 25C). While methods for conducting acute 4d and chronic (~25-30d) toxicity tests with this mayfly have been published, a need exists to extend and standardize the methodology for applicable methods for testing in short-term exposures (e.g., 7d or 10d). Studies began with identifying an optimal starting age, test duration, and optimal sub-lethal endpoint for whole effluent toxicity testing. We found that chronic values from tests using < 24-h-old organisms were ~4-fold more sensitive than those using 7-d-old organisms. Survival was never a more sensitive endpoint than either calculated weight or biomass. Efforts to refine the various aspects of diatom culture technique on food quality and therefore mayfly growth are underway and optimizing the diet for these organisms may be critical for achieving consistently high growth rates with low intra-treatment variability. We also determined the influence of amendments to culture water and food, and temperature on sensitivity to reference contaminants and effluents. Results of the study should provide data needed to guide the development of a toxicity test method to support NPDES permit decision-making.