The main objective of this project was to develop toxicity benchmarks acceptable for ecological risk assessment (ERA) of soil contaminated with 2,4-dinitrotoluene (2,4-DNT), 2-amino-4,6-dinitrotoluene (2-ADNT), 4-amino-2,6-dinitrotoluene (4-ADNT), octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX), and nitroglycerin (NG), and for derivation of draft ecological soil screening level (Eco-SSL) values for ecologically relevant soil biota. Specific technical objectives included:

1. Determine the toxicities of 2,4-DNT, 2-ADNT, 4-ADNT, HMX, and NG to soil invertebrates and terrestrial plants in soil with high bioavailability characteristics
2. Examine the effect of various soil physical/chemical characteristics (e.g., organic matter content, pH, clay content) on 2,4-DNT toxicity to terrestrial plants and soil invertebrates
3. Determine the bioaccumulation potentials of 2,4-DNT, HMX, NG, 2,4,6-trinitrotoluene (TNT), and hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) from soil to invertebrates and terrestrial plants, respectively
4. Determine data for the effects of 2,4-DNT, 2-ADNT, 4-ADNT, and NG on the soil microbial activity endpoints and contrast these data with the toxicity benchmarks developed using standardized single-species toxicity assays
5. Develop draft Eco-SSLs for 2,4-DNT, 2-ADNT, 4-ADNT, HMX, and NG for terrestrial plants and soil invertebrates based on concentration-response relationships.

Studies were designed to develop scientifically defensible toxicity data for the derivation of plant-based and soil invertebrate-based Eco-SSL values for 2,4-DNT, 2-ADNT, 4-ADNT, HMX, and NG, weathered-and-aged individually in soil. Ecotoxicological testing was specifically designed to meet the U.S. Environmental Protection Agency (USEPA) criteria for Eco-SSL derivation. Toxicity testing was conducted with additional natural soil types to extend the range of soil physico-chemical characteristics that were hypothesized to affect the 2,4-DNT toxicity to soil organisms in order to investigate and characterize predominant soil physico-chemical parameters that can affect the bioavailability and resulting toxicity of 2,4-DNT to terrestrial plants and soil invertebrates. Assessment of soil microbial activity was included to establish data on 2,4-DNT, 2-ADNT, 4-ADNT, and NG effects on critical ecosystem-level processes such as energy and nutrient cycling. Multiple soil microbial activity endpoints—basal respiration, substrate-induced respiration, microbial biomass carbon, litter decomposition (orchard grass), and enzyme activities—were assessed to develop toxicity data for the derivation of Biological Activity-based Soil Screening Concentrations (BA-SSC) utilizing an approach similar to Eco-SSL derivation. Bioaccumulation studies were conducted to test the hypotheses that selected explosives released in soil can accumulate in soil invertebrates or terrestrial plants. Bioaccumulation factors (BAFs) were determined from the ratio of the uptake and the elimination kinetic rate constants estimated during the uptake and elimination phases of a bioaccumulation test.

Definitive studies using three plant and three soil invertebrate test species exposed in sandy loam soils established new ecotoxicological data for 2,4-DNT, 2-ADNT, 4-ADNT, HMX, and NG under conditions of very high relative bioavailability for organic chemicals in soil. These data were used to derive draft Eco-SSL concentrations for each energetic material (EM). Soil-related differences were evident in both phytotoxicity benchmarks and soil invertebrate toxicity benchmarks established in studies with 2,4-DNT. These studies identified soil organic matter and clay as the dominant properties mitigating 2,4-DNT toxicity for soil annelids (earthworms and potworms) and organic matter as the soil constituent mitigating 2,4-DNT toxicity for plants. Strong correlations were also detected for several annelid toxicity endpoints and soil cation exchange capacity. The studies showed that soil contamination with 2,4-DNT, 2-ADNT, 4-ADNT, and NG can alter the rates of biologically mediated processes in soil by either inhibiting or stimulating the soil microbial activity at the affected sites. Basal respiration and dehydrogenase activity assays were the most robust among the soil functional tests used and allowed the researchers to establish toxicity data for each of the four EM investigated. Toxicity benchmarks determined for EM effects on soil microbial activity endpoints were used for derivation of BA-SSC values. A summary of the draft Eco-SSL values for 2,4-DNT, 2-ADNT, 4-ADNT, NG, and HMX determined from the growth toxicity benchmarks for terrestrial plants (alfalfa, barnyard grass, and ryegrass) and from reproduction toxicity benchmarks for soil invertebrates (earthworm, potworm, and collembola) is presented in the table below. The BA-SSC values for the four EM are also included in this table.

This project investigated bioaccumulation (earthworms) and bioconcentration (plants) potentials for TNT, 2,4-DNT, HMX, RDX, and NG in order to determine the respective factor values (BAF and BCF) to produce data that can be used to assess the potential risks to higher trophic levels through food web transfer of energetic soil contaminants. The BAF and BCF values shown in the table below were developed for each EM over a range of non-toxic exposure concentrations.

Upon acceptance by the USEPA, the draft Eco-SSL values will allow screening of site-soil data to identify those contaminant EMs that are not of potential ecological concern and do not need to be considered in the Baseline Ecological Risk Assessment (BERA), resulting in significant cost savings during site assessments. Benchmark data and draft Eco-SSL values developed in these studies have been transitioned to the USEPA and reported to DoD constituencies. The Eco-SSLs, BA-SSCs, BAFs, and BCFs developed will provide indispensable tools for installation managers to gauge the ecotoxicological impacts of military operations that involve the use of explosives, thus ultimately promoting the sustainable use of testing and training ranges.