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The acetonitrile-sonication extraction method (US EPA SW-846 Method 8330) and aquatic-based toxicity tests were used on laboratory and field samples, to characterize the ecotoxicity of soils contaminated with energetic substances. Spiked soil studies indicated that 2,4,6-trinitrotoluene (TNT)-dependent soil toxicity could be measured in organic extracts and aqueous leachates using the 15-min Microtox (Vibrio fischeri, IC50=0.27 to 0.94 mg TNT/liter incubation medium) and 96-h Selenastrum capricornutumgrowth inhibition (IC50=0.62 to 1.14 mg/liter) toxicity tests. Analyses of leachates of composite soil samples [containing TNT and some TNT metabolites, 1,3,5-trinitro-1,3,5-triazacyclohexane (RDX), and 1,3,5,7-tetranitro-1,3,5,7-tetrazacyclooctane (HMX)] from an explosives manufacturing facility, indicated toxicities similar to those found in the TNT-spiked soil studies and pure TNT in solution, and suggested that TNT was the major toxicant. Using TNT as a model toxicant in soils having different moisture contents (20% vs dry) and textures (sandy vs clayey–sandy) but similar organic matter content (3–4%), multi-factorial analyses of Microtox test data revealed that these soil factors significantly influenced the TNT extractability from soil and subsequent toxicity measurements. Taken together, data indicate that the modified Method 8330 may be used in conjunction with ecotoxicity tests to reflect the toxic potential of soils contaminated with energetic substances.

The acetonitrile–sonication extraction method (US EPA Method 8330) associated with aquatic-based toxicity tests was examined to study the ecotoxicity of energetic substances in soil. Three studies were carried out: (1) toxicological characterization of different energetic substances to select a representative toxicant and to validate the choice of bioassays; (2) choice of an appropriate solvent to transfer acetonitrile extracts to the bioassay incubation media; and (3) optimization of Method 8330 using soil samples spiked with the toxicant. Initial studies indicated that pure 2,4,6-trinitrotoluene (TNT) was toxic toVibrio fischeri[Microtox; IC50(15 min) of 4.2 μM], whereas RDX was less toxic (IC20=181 μM) and HMX was not toxic up to its limit of water solubility (<22 μM). Selected pure TNT metabolites were less toxic than TNT. Similar results were found using the 96-hSelenastrum capricornutumgrowth inhibition test. The toxicity of pure TNT in different solvents (acetonitrile, acetone, and DMSO) and that from Method 8330-extracted TNT-spiked soil samples were compared to TNT dissolved in water. Data indicated that DMSO was the most appropriate solvent to transfer the acetonitrile extracts. A modified Method 8330 may be used in conjunction with bioassays and chemical analyses to examine the ecotoxicity of soils contaminated with energetic substances.