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A review of the limited reliable toxicity test results for exposure of aquatic organisms resulted in data from studies that included freshwater species representing fish, invertebrates, algae, and a snail. No reliable marine data were available. Short-term (acute) exposures of NTA to freshwater invertebrates ranged from 80 to 115 mg/L NTA for Gammarus pseudolimnaeus. Long-term NTA exposure to Gammarus pseudolimnaeus indicated a NOEC for survival to be 9.3 mg/L NTA. However, it should be noted that control survival for this test was only 56%. A freshwater snail (Helisoma trivolis) was exposed to NTA for 120 days in a multigenerational test where the NOEC was found to be 12.5 mg/L NTA for growth in the F1 generation. The Arthur et al. (1974) study is used in a weight of evidence approach in support of the toxicity of NTA to aquatic invertebrates. Results of this study were in the same range as the snail study.

Fish species were generally found to be equally sensitive to short-term NTA exposure as invertebrate organisms. Short-term survival LC50values were103 -125 mg/L NTA for Pimephales promelas. However, effects on reproduction and survival from long-term NTA exposure to freshwater fish were found to be generally higher than invertebrates. Other organisms tested included NTA exposure to algae (Scenedesmus subspicatus) which resulted in 72 hr NOEC of 1.43 mg/L. This study indicates that algae may be the most sensitive aquatic freshwater organism to NTA exposure. Navicula seminulum were exposed for 5 days to NTA resulting in EC50 for growth of 143 -507 mg/L, depending on a static or flow-through test system.

It has been suggested that the toxicity of NTA salts to aquatic organisms can be affected by Ca, Mg and metal ions, to a lesser degree, in natural waters. The EU Risk Assessment Report for Trisodium Nitrilotriacetate (2005) ("EURAR 2005") suggests that, in general, the Ca, Mg, and trace metal ions occurring in natural waters may actually complex with NTA salts, making the NTA salts unavailable biologically. The EURAR 2005 Report states that the available aquatic toxicity tests for NTA may not be relevant for environmental conditions. Uncomplexed NTA salts may only be available in conditions where the NTA concentration is over the stoichiometric limit. This implies that as hardness increases, as a measure of Ca and Mg ions, the toxic potential of NTA is likely to decrease due to the increased binding capacity of the natural water ions. EURAR 2005 summarizes aquatic toxicity studies where the effect of hardness on toxicity has been investigated. In addition, Biesinger et al. (1974) exposed Daphnia magna to Na3NTA in Lake Superior water of different hardness concentrations for three weeks. The three week LC50 results were 145, 405, and 650 mg/L Na3NTA when tested in Lake Superior water with hardness of 45, 130, and 220 mg/L, respectively. As a conservative approach, the PNECaquatic will utilize the lowest long-term result from available reliable data, even though is it anticipated that laboratory tests may overestimate aquatic toxicity.