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We investigated the toxicity of selenium (Se) to the soil invertebrates Folsomia candida (Collembola) and Enchytraeus crypticus (potworm). Studies were designed to generate ecotoxicological benchmarks for developing ecological soil screening levels (Eco-SSLs) for risk assessments of contaminated soils. For the present studies, we selected Sassafras sandy loam, an aerobic upland soil with soil characteristics (low levels of clay and organic matter, soil pH adjusted from 5.2 to 7.1) that support high relative bioavailability of the anionic Se species that is typically found in aerobic soil. The Se was amended into soil as sodium selenate, subjected to weathering and aging using 21 d of alternating cycles of air-drying/rehydration to 60% of the water-holding capacity of the Sassafras sandy loam soil, under ambient greenhouse conditions. Effective concentrations at 20 and 50% (EC20 and EC50) levels for production of juveniles (reproduction) were 4.7 and 10.9 mg of Se/kg of soil (dry mass basis), respectively, for Collembola, and 4.4 and 6.2 mg/kg, respectively, for the potworms. The data enabled the derivation of toxicity benchmarks, contributing to the development of a soil invertebrate-based Eco-SSL of 4.1 mg/kg for Se. Environ Toxicol Chem 2018;37:846-853. Published 2017 Wiley Periodicals Inc. on behalf of SETAC. This article is a US government work and, as such, is in the public domain in the United States of America.

We investigated the toxicity of selenium (Se) to the soil invertebrates Folsomia candida (Collembola) and Enchytraeus crypticus (potworm). Studies were designed to generate ecotoxicological benchmarks for developing ecological soil screening levels (Eco-SSLs) for risk assessments of contaminated soils. For the present studies, we selected Sassafras sandy loam, an aerobic upland soil with soil characteristics (low levels of clay and organic matter, soil pH adjusted from 5.2 to 7.1) that support high relative bioavailability of the anionic Se species that is typically found in aerobic soil. The Se was amended into soil as sodium selenate, subjected to weathering and aging using 21 d of alternating cycles of air-drying/rehydration to 60% of the water-holding capacity of the Sassafras sandy loam soil, under ambient greenhouse conditions. Effective concentrations at 20 and 50% (EC20 and EC50) levels for production of juveniles (reproduction) were 4.7 and 10.9 mg of Se/kg of soil (dry mass basis), respectively, for Collembola, and 4.4 and 6.2 mg/kg, respectively, for the potworms. The data enabled the derivation of toxicity benchmarks, contributing to the development of a soil invertebrate-based Eco-SSL of 4.1 mg/kg for Se. Environ Toxicol Chem 2018;37:846-853. Published 2017 Wiley Periodicals Inc. on behalf of SETAC. This article is a US government work and, as such, is in the public domain in the United States of America.

We investigated the effects of nitramine explosive CL-20 (China Lake compound 20) on the indigenous soil invertebrate community in Sassafras sandy loam (SSL) soil using a 12-week soil microcosm assay. Freshly collected SSL soil was amended with CL-20 to prepare multiple treatment concentrations ranging from 0 (acetone control) to 10,300 mg kg−1. The selected concentration range of CL-20 adequately assessed the concentration–response relationships for total microarthropods, and for individual microarthropod groups. The overall composition of microarthropod community in SSL soil was not affected by exposure to CL-20, based on the number of taxonomic groups present in the individual treatments after 12 weeks. However, community structure analysis revealed greater sensitivity to CL-20 by predatory mesostigmatid mites. Microarthropod and nematode communities showed contrasting sensitivities to CL-20 in SSL soil. Total numbers of nematodes were either unaffected or significantly (p

Summary A microcosm technique was used to determine the ecotoxicity of the chemical warfare agent HD (mustard) to the indigenous soil microinvertebrate communities. HD was thoroughly incorporated into Sassafras sandy loam (SSL) soil (4.9% OM) and an oak-beech forest silt loam soil (FS, 16% OM) at nominal HD concentrations ranging from 6 to 1076 mg kg−1. After a 7-day incubation period, microarthropods were extracted from soils using a high-gradient extractor and sorted to Acari suborders Prostigmata, Mesostigmata, and Oribatida, and the insect order Collembola. Nematodes were extracted using Baermann funnels and were sorted into bacterivore, herbivore, fungivore and omnivore/predator trophic groups. Microarthropods were more sensitive to HD in both soil types compared with nematodes. The EC50 values for total numbers of microarthropods in SSL and FS were similar (65 and 71 mg kg−1, respectively). The EC50 values for total numbers of nematodes were 130 and 235 mg kg−1, respectively. Toxicity of HD to nematodes was significantly greater in SSL soil compared to FS, based on 95% confidence intervals. Results show that community-level assessment of chemical toxicity in soil using a microcosm assay is sufficiently robust and can provide the means for validating the ecotoxicity data from standardized laboratory single-species toxicity tests.

The U.S. Environmental Protection Agency is developing Ecological Soil Screening Level (Eco-SSL) benchmarks for ecological risk assessment (ERA) of contaminants at Superfund sites. Eco-SSLs are developed from published values whenever sufficient quantity and quality of data exist. Because insufficient information was available to generate an Eco-SSL for Mn, standardized toxicity testing was undertaken to fill the data gaps. Tests included the earthworm (Eisenia fetida) cocoon production test, the enchytraeid (Enchytraeus crypticus) reproduction test, and the collembolan (Folsomia candida) reproduction test, all conducted in Sassafras sandy loam soil that supports a relatively high bioavailability of metals. Weathering and aging of manganese-amended soil were carried out to more closely simulate exposure effects at Superfund sites on soil invertebrates. Data were analyzed by nonlinear regression to determine EC20 and EC50 values based on concentration-response relationships. The toxicity order for manganese in Sassafras sandy loam was E. crypticus>E. fetida>F. candida, with EC20 values of 116, 629, and 1209 mg kg(-1), respectively. The Eco-SSL requirement for the testing of multiple representative species is well justified. All study results will be submitted to the Eco-SSL Task Group for quality control review prior to inclusion in the Eco-SSL database.

Summary Proteins are known to respond rapidly when an organism is exposed to a toxicant and this response is consistent and unique to the xenobiotics. Induced and repressed proteins together create a specific signature that can be used to identify a toxic agent and its concentration in the environmental matrix. The resulting protein expression signature for each agent will include a small number of the most potent proteins, which can be used to detect the presence and determine the concentration of a toxicant. We exposed the earthworm Eisenia fetida to a range of concentrations of chemical warfare agents (CWA) HD (mustard) and VX (nerve agent) in a natural sandy loam soil. Results demonstrated specificity of protein signatures in response to HD and VX exposure in soil. This study also showed that protein patterns were specific for different concentrations of HD or VX in otherwise clean agent-amended soil. Several proteins were induced in all HD or VX concentrations tested suggesting the potential of identifying the key proteins for use in detection of HD or VX presence in soil regardless of their concentrations. The robustness of the proteome-based response to chemical exposure by soil biota needs to be further investigated by including determinations of protein patterns for chemicals in different soil types and in the presence of additional chemical stressors using variety of soil invertebrate models.

Munitions manufacturing, disposal, testing, training and other operations at military sites have produced elevated levels of explosives and related materials in soil. Insufficient data were available on the toxicity of the explosives, RDX and HMX to soil invertebrates, necessitating toxicity testing. The U.S. Environmental Protection Agency (USEPA) in conjunction with stakeholders, is developing Ecological Soil Screening Level (Eco-SSL) benchmarks for ecological risk assessment (ERA) of contaminants at Superfund sites to identify those contaminants in soil that warrant additional evaluation in a baseline ERA, and to eliminate those that do not. Eco-SSLs are developed from literature values whenever sufficient quantity and quality of data exist. Tests were conducted under conditions preferred for Eco-SSL benchmarks, using a sandy loam soil that supports relatively high bioavailability of test compounds. Earthworm (Eisenia fetida) toxicity was assessed using a standardized earthworm reproduction test in freshly amended soil and weathered/aged amended soil. RDX or HMX had no effect on adult survival. Cocoon production EC20 values for RDX were 1.2 and 19 mg kg-1 in freshly amended and weathered/aged soils, respectively. Juvenile production EC20 values were 1.6 and 4.8 mg kg-1 in freshly amended and weathered/aged soils, respectively. Cocoon production and juvenile production EC20 values for HMX were 2.7 and 0.4 mg kg-1 in freshly amended soil. Both cocoon production and juvenile production in weathered/aged HMX treated soils were not significantly different (p > 0.05) from control soils. Results of these toxicity studies will be submitted to the Eco-SSL Task Group and will be included in the Eco-SSL database for Eco-SSL derivation.Munitions manufacturing, disposal, testing, training and other operations at military sites have produced elevated levels of explosives and related materials in soil. Insufficient data were available on the toxicity of the explosives, RDX and HMX to soil invertebrates, necessitating toxicity testing. The U.S. Environmental Protection Agency (USEPA) in conjunction with stakeholders, is developing Ecological Soil Screening Level (Eco-SSL) benchmarks for ecological risk assessment (ERA) of contaminants at Superfund sites to identify those contaminants in soil that warrant additional evaluation in a baseline ERA, and to eliminate those that do not. Eco-SSLs are developed from literature values whenever sufficient quantity and quality of data exist. Tests were conducted under conditions preferred for Eco-SSL benchmarks, using a sandy loam soil that supports relatively high bioavailability of test compounds. Earthworm (Eisenia fetida) toxicity was assessed using a standardized earthworm reproduction test in freshly amended soil and weathered/aged amended soil. RDX or HMX had no effect on adult survival. Cocoon production EC20 values for RDX were 1.2 and 19 mg kg-1 in freshly amended and weathered/aged soils, respectively. Juvenile production EC20 values were 1.6 and 4.8 mg kg-1 in freshly amended and weathered/aged soils, respectively. Cocoon production and juvenile production EC20 values for HMX were 2.7 and 0.4 mg kg-1 in freshly amended soil. Both cocoon production and juvenile production in weathered/aged HMX treated soils were not significantly different (p > 0.05) from control soils. Results of these toxicity studies will be submitted to the Eco-SSL Task Group and will be included in the Eco-SSL database for Eco-SSL derivation.

Summary Munition manufacturing, disposal, testing, training and other operations at military sites produced elevated levels of explosives and related materials in soil. The effects of these persistent and highly mobile in the environment energetic materials on soil biota have not been sufficiently investigated. The U.S. Environmental Protection Agency (USEPA) in conjunction with stakeholders, is developing Ecological Soil Screening Level (Eco-SSL) benchmarks for ecological risk assessment (ERA) of contaminants at Superfund sites to identify those contaminants in soil that warrant additional evaluation in a Baseline ERA, and to eliminate those that do not. Eco-SSLs are developed from literature values whenever sufficient quantity and quality of data exist. Insufficient data were available on the toxicity of energetic compounds, RDX and HMX, to soil invertebrates, necessitating toxicity testing. Tests were conducted under conditions preferred for Eco-SSL benchmarks, using a Sassafras sandy loam soil that supports relatively high bioavailability of test compounds. Toxicity testing was performed using enchytraeid reproduction test (ISO/16387 2001) measuring adult survival and juvenile production by the potworm Enchytraeus crypticus in freshly amended soil and weathered/aged amended soil. RDX or HMX had no effect on adult survival in the definitive tests in all treatment concentrations. Juvenile production EC 20 values were 3,715 and 8,797 mg kg –1 RDX in freshly amended and weathered/aged amended soils, respectively. Juvenile E. crypticus production was not adversely affected by exposure to HMX in freshly amended and weathered/aged amended soils. Juvenile production was stimulated in freshly amended soil up to 21,750 mg kg –1 HMX. Results of these toxicity studies will be submitted to the Eco-SSL Task Group for quality control review, and pending approval will be included in the Eco-SSL database for Eco-SSL derivation.

Proteins are known to respond rapidly when an organism is exposed to a toxicant and this response is consistent and unique to the xenobiotics. Induced and repressed proteins together create a specific signature that can be used to identify a toxic agent and its concentration in the environmental matrix. The resulting protein expression signature for each agent will include a small number of the most potent proteins, which can be used to detect the presence and determine the concentration of a toxicant.We exposed the earthworm Eisenia fetida to a range of concentrations of chemical warfare agents (CWA) HD (mustard) and VX (nerve agent) in a natural sandy loam soil. Results demonstrated specificity of protein signatures in response to HD and VX exposure in soil. This study also showed that protein patterns were specific for different concentrations of HD or VX in otherwise clean agent-amended soil. Several proteins were induced in all HD or VX concentrations tested suggesting the potential of identifying the key proteins for use in detection of HD orVX presence in soil regardless of their concentrations. The robustness of the proteome-based response to chemical exposure by soil biota needs to be further investigated by including determinations of protein patterns for chemicals in different soil types and in the presence of additional chemical stressors using variety of soil invertebrate models.

Summary Munitions manufacturing, disposal, testing, training and other operations at military sites have produced elevated levels of explosives and related materials in soil. Insufficient data were available on the toxicity of the explosives, RDX and HMX to soil invertebrates, necessitating toxicity testing. The U.S. Environmental Protection Agency (USEPA) in conjunction with stakeholders, is developing Ecological Soil Screening Level (Eco-SSL) benchmarks for ecological risk assessment (ERA) of contaminants at Superfund sites to identify those contaminants in soil that warrant additional evaluation in a baseline ERA, and to eliminate those that do not. Eco-SSLs are developed from literature values whenever sufficient quantity and quality of data exist. Tests were conducted under conditions preferred for Eco-SSL benchmarks, using a sandy loam soil that supports relatively high bioavailability of test compounds. Earthworm (Eisenia fetida) toxicity was assessed using a standardized earthworm reproduction test in freshly amended soil and weathered/aged amended soil. RDX or HMX had no effect on adult survival. Cocoon production EC20 values for RDX were 1.2 and 19 mg kg–1 in freshly amended and weathered/aged soils, respectively. Juvenile production EC20 values were 1.6 and 4.8 mg kg–1 in freshly amended and weathered/aged soils, respectively. Cocoon production and juvenile production EC20 values for HMX were 2.7 and 0.4 mg kg–1 in freshly amended soil. Both cocoon production and juvenile production in weathered/aged HMX treated soils were not significantly different (p > 0.05) from control soils. Results of these toxicity studies will be submitted to the Eco-SSL Task Group and will be included in the Eco-SSL database for Eco-SSL derivation.

We investigated the ecotoxicity of the chemical-warfare agent (CWA) HD (Mustard) using Collembola reproduction test with numbers of adults and juveniles as measurement endpoints. Toxicity tests were conducted using soils with contrasting parameters to investigate the effects of soil properties on chemical toxicity. These included standard artificial soil (SAS; 10% OM; 6 pH), O’Neill-Hall sandy loam (OHSL; natural soil with 4.3% OM; 5.1 pH), and Sassafras sandy loam (SSL; natural soil with 2% OM; 4.9 pH). Soils were individually spiked with HD concentrations ranging from 1 to 125 mg kg –1 . Lowest observed effect concentrations (LOECs) for adult mortality were 25, 50, and 6.97 mg kg –1 for SAS, OHSL, and SSL, respectively. The LOECs for reproduction were 6.97, 6.25, and 1.9 mg kg –1 for SAS, OHSL, and SSL, respectively. HD toxicity to both adults and juveniles was greater in SSL. These results show that soil toxicity testing should not rely solely on the adult acute endpoints using artificial soils, but should include assays with reproductive endpoints using natural soils with varying physical and chemical parameters to adequately assess toxicity to test species.

We investigated the effects of individual nitrogen-based energetic materials (EMs) 2,4-dinitrotoluene (2,4-DNT), 2-amino-4,6-dinitrotoluene (2-ADNT), 4-amino-2,6-dinitrotoluene (4-ADNT), nitroglycerin (NG), and 2,4,6,8,10,12-hexanitrohexaazaisowurtzitane (CL-20) on decomposition, an essential soil process, utilizing Orchard grass (Dactylis glomerata) straw in Sassafras sandy loam (SSL) soil. Three pieces of 5-cm long internodular sections of straw were used to form a straw cluster. Pre-weighed straw clusters were placed in the soil in each 900 mL volume test container containing approximately 200 g of loosely packed EM-amended soil or carrier (acetone) control soil. After 1, 2, 3, 4, 6, and 8 months of exposure, one straw cluster was harvested from a set of randomly selected replicate containers from within each treatment to quantify the decomposition rates. The exposure concentrations of each EM in soil were analytically determined soils using the U.S. Environmental Protection Agency Method 8330A. The results showed that soil contamination with 2,4-DNT or NG can inhibit litter decomposition rates based on the EC50 values of 1122 mg/kg and 860 mg/kg, respectively. Exposure to 2-ADNT, 4-ADNT or CL-20 did not significantly affect litter decomposition in SSL soil at 10000 mg/kg. These ecotoxicological benchmarks can help identify concentrations of contaminant EM in soil that present an acceptable ecological risk for biologically-mediated processes in soil.

The authors used a refined microcosm technique to investigate the toxicity of copper, cadmium, malathion, and the polychlorinated biphenyl, to trophic groups of soil nematodes and to the microarthropod community. Comparisons of changes in nematode abundance in control soils through time indicated that day 7 was the most appropriate time to sample the microcosms after chemical application. Nematode abundance was reduced after exposure to copper at 100 {micro}g/g, with fungivore, bacterivore, and omnivore-predator nematodes being the most sensitive groups. Cadmium did not affect the nematode or microarthropod communities. Microarthropods were far more sensitive to malathion than were nematodes, and total microarthropod abundance was lower than controls at 400 {micro}g/g. Prostigmatid mites and other arthropods were the most affected groups. PCB also had a greater negative impact on microarthropods than on nematodes. Total microarthropod abundance declined at 2,500 {micro}g/g, with prostigmatid and oribatid mites exhibiting the highest susceptibility. Strong differential sensitivity between nematode and microarthropod communities indicates that both groups should be examined to fully evaluate the biological impact of chemicals on soils. The authors conclude that microcosms with field-collected soil microfaunal communities offer high resolution of the ecotoxicologic effects of chemicals in complex soil systems.

The authors investigated individual toxicities of 2,4,6-trinitrotoluene (TNT) and hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) to the potworm Enchytraeus crypticus using the enchytraeid reproduction test. Studies were designed to generate ecotoxicological benchmarks that can be used for developing ecological soil-screening levels for ecological risk assessments of contaminated soils and to identify and characterize the predominant soil physicochemical parameters that can affect the toxicities of TNT and RDX to E. crypticus. Soils, which had a wide range of physicochemical parameters, included Teller sandy loam, Sassafras sandy loam, Richfield clay loam, Kirkland clay loam, and Webster clay loam. Analyses of quantitative relationships between the toxicological benchmarks for TNT and soil property measurements identified soil organic matter content as the dominant property mitigating TNT toxicity for juvenile production by E. crypticus in freshly amended soil. Both the clay and organic matter contents of the soil modulated reproduction toxicity of TNT that was weathered and aged in soil for 3 mo. Toxicity of RDX for E. crypticus was greater in the coarse-textured sandy loam soils compared with the fine-textured clay loam soils. The present studies revealed alterations in toxicity to E. crypticus after weathering and aging TNT in soil, and these alterations were soil- and endpoint-specific.

This study was designed to produce scientifically defensible benchmark data for the development of ecological-based soil screening levels for 2,4,6-trinitrotoluene (TNT) and hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) for terrestrial plants. The physical and chemical properties of different soils may alter the bioavailability and toxicity of TNT and RDX to plant species. We investigated individual ecotoxicities of TNT and RDX to alfalfa (Medicago sativa L.), Japanese millet (J. millet; Echinochloa crus-galli L. [Beauv.]), and perennial ryegrass (Lolium perenne L.) in five natural soils that varied in pH, organic matter, and clay contents. On the basis of EC50 values (the TNT or RDX concentrations that produce 50% reductions in selected measurement endpoints) and 95% confidence intervals for shoot fresh and dry masses (SDM), TNT weathered-and-aged (W-A) in soils significantly reduced (p 0.05) growth of alfalfa, J. millet, and perennial ryegrass. RDX W-A in soil was moderately inhibitory to growth of J. millet and perennial ryegrass in all five soils but did not affect growth of alfalfa. No statistically significant correlations between TNT toxicity and these soil properties were identified; however, statistically significant (p 0.05) inverse relationships were identified between the RDX EC50 values for perennial ryegrass SDM and soil clay content and pH.

The authors investigated individual toxicities of 2,4,6-trinitrotoluene (TNT) and hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) to the potworm Enchytraeus crypticus using the enchytraeid reproduction test. Studies were designed to generate ecotoxicological benchmarks that can be used for developing ecological soil-screening levels for ecological risk assessments of contaminated soils and to identify and characterize the predominant soil physicochemical parameters that can affect the toxicities of TNT and RDX to E. crypticus. Soils, which had a wide range of physicochemical parameters, included Teller sandy loam, Sassafras sandy loam, Richfield clay loam, Kirkland clay loam, and Webster clay loam. Analyses of quantitative relationships between the toxicological benchmarks for TNT and soil property measurements identified soil organic matter content as the dominant property mitigating TNT toxicity for juvenile production by E. crypticus in freshly amended soil. Both the clay and organic matter contents of the soil modulated reproduction toxicity of TNT that was weathered and aged in soil for 3 mo. Toxicity of RDX for E. crypticus was greater in the coarse-textured sandy loam soils compared with the fine-textured clay loam soils. The present studies revealed alterations in toxicity to E. crypticus after weathering and aging TNT in soil, and these alterations were soil- and endpoint-specific.

Environmental toxicity testing and chemical analyses of soil were performed as part of an ecological risk assessment at the Joliet Army Ammunition Plant (JAAP), Johet, Illinois Soils were collected from an area where munitions were loaded, assembled, and packed (area L7, group 1), and from an area where waste explosives were burned on unprotected soil (area L2) Control samples were collected from an adjacent field Soil toxicity was determined using early seedling growth and vigor tests, earthworm survival and growth tests, and Microtox® assays Relative toxicity of soils was determined within each area based on statistical significance (p = 0 05) of plant and earthworm growth and survival, and the effective concentration at which luminescence of the bacterium Photobacterium phosphoreum was reduced by 50% (EC50) in the Microtox assay Samples were designated as having high, moderate, or no significant toxicity Soil that had significant toxicity according to at least one test, and representative samples showing no toxicity, were analyzed for munitions via HPLC Chemical residues found in soils were 2,4,6 trinitrotoluene (TNT), 1,3,5 trinitrobenzene (TNB), 2,4 dinitrotoluene (2,4-DNT), 2,6 dinitrotoluene, 2 amino-4,6-DNT, 4-ammo-2,6 DNT, 1,3,5 trinitro 1,3,5 triazine (RDX), and octahydro-1,3,5,7 tetramtro-l,3,5,7-tetrazocme (HMX) All soils with no significant toxicity were void of these chemicals However, some soils void of munitions still showed toxicity that may have been caused by elevated levels of heavy metals Linear regressions of toxicity test results vs chemical concentrations showed that TNT and TNB accounted for most of the soil toxicity Lowest observable-effect concentrations (LOEC) of TNT were de termined from these data This study presents a simple, relatively inexpensive methodology for assessing toxicity of soils containing TNT, RDX, and other contaminants related to munitions production

The ecotoxicological benchmarks developed for terrestrial plants and soil invertebrates were compiled, reviewed, and used in this report to develop scientifically defensible terrestrial plant-based and soil invertebrate-based draft ecological soil screening levels (Eco-SSLs) for 2,4,6-trinitrotoluene (TNT) and hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX). The draft Eco-SSL values were derived using the EC20 (the 20% negative) level toxicity benchmarks for the effects of each explosive compound as a contaminant in soil and on plant growth or soil invertebrate reproduction endpoints, which were determined in standardized toxicity tests. Ecotoxicological testing was specifically designed and reviewed for these studies to meet the U.S. Environmental Protection Agency (USEPA) criteria for Eco-SSL derivation. Following acceptance by the USEPA, these draft Eco-SSL values will be used to screen site-soil data to identify the contaminants that are not of potential ecological concern and do not need to be considered in the baseline ecological risk assessment, which will result in significant cost-savings during site assessments. These Eco-SSLs will also provide an indispensable tool 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.

A microcosm technique is presented that uses community and trophic-level analysis of soil nematodes and microarthropods to determine the effects of chemicals on soil systems. Forest soil was treated with either copper (0, 100, 200, 400, and 600 mg g−1), p-nitrophenol (0, 20, 40, 80, and 160 mg g−1), or trinitrotoluene (0, 25, 50, 100, and 200 mg g−1). Nematodes were sorted into bacterivore, fungivore, herbivore, and omnivore-predator trophic groups, and a hatchling category. Microarthropods were sorted to the acarine suborders Prostigmata, Mesostigmata, and Oribatida; the insectan order Collembola; and a miscellaneous group. Omnivore-predator nematodes and mesostigmatid and oribatid mites were the groups most sensitive to copper and were significantly reduced at levels as low as 100 mg g−1 copper. Total nematode and microarthropod numbers declined above 200 mg g−1 copper. Trophic structure analysis suggested that high sensitivity of nematode predators to intermediate levels of copper reduced predation on herbivore nematodes and resulted in greater numbers of nematodes compared to controls. p-Nitrophenol was very toxic to the nematode community, and all trophic groups were significantly reduced above 20 mg g−1. However, there was no effect of p-nitrophenol on microarthropods. Trinitrotoluene had no significant negative effect on total abundance of either group of soil fauna, but oribatids were significantly reduced at 200 mg g−1. Our results demonstrated that soil nematodes and microarthropods were sensitive indicators of environmental contaminants and that trophic-structure and community analysis has the potential to detect more subtle indirect effects of chemicals on soil food-web structure. We conclude that microcosms with field communities of soil microfauna offer high resolution of the ecotoxicological effects of chemicals in complex soil systems.

The controlled environment soil‐core microcosm unit (CESMU) methods embody a collection of techniques that began with soil sampling in the field and continued throughout the laboratory investigation of chemical fate, migration, and transformation in site‐specific soils; it was a cost‐effective investigative methodology that could be used to screen chemical materials before initiating high‐cost environmental field studies. Intact soil cores were collected in the field using a hydraulically controlled probe, delivering intact soil‐cores with minimal disturbance directly into high‐density polyethylene pipe (10.3‐cm ID). The inert polyethylene pipe was an effective hydrophobic barrier that remained an integral part of the soil‐core column, obviating subsequent transfers of soil. In the laboratory, each soil column was fitted with a porous ceramic plate and a polyethylene endcap containing fittings for teflon tubing, so that a tension could be applied at the bottom of each soil column (30–35 kPa) to m...

The use of lead in military and civilian small arms projectiles is widely acknowledged to have resulted in high soil lead concentrations at many small arms ranges. These ranges are often adjacent to wildlife habitat or have become habitat when no longer used. To assess the potential toxicity of lead to terrestrial amphibians in contaminated areas, we exposed 100 red-backed salamanders (Plethodon cinereus) to either a control soil or one of four soil treatments amended with lead acetate for 28 days. Analytical mean soil concentrations were 14 (control), 553, 1700, 4700, and 9167 mg Pb/kg soil dry weight. An additional 60 salamanders were also exposed for 28 days to one of six field-collected soil samples from a small arms range and a skeet range. The field soil concentrations ranged from 11 (background) to 16,967 mg Pb/kg soil dry weight. Food consisted of uncontaminated flightless Drosophila melanogaster. Salamander survival was reduced in amended soil treatments of 4700 and 9167 mg/kg by 15% and 80%, respectively. Inappetence was observed at 4700 and 9167 mg/kg and growth decreased in the 9167 mg/kg treatment. Total white blood cells decreased 32% at 4700 mg/kg compared to controls and were 22% lower in the 9167 mg/kg treatment. In contrast, survival was 100% for all field-collected soils with no hematological effects. At 16,967 mg/kg there was evidence of soil avoidance and decreased growth. These data suggest marked differences in toxicity and bioavailability of the lead-amended soil in contrast to the field-collected soil containing lead.

Copper (Cu) has widespread military use in munitions and small arms, particularly as a protective jacket for lead projectiles. The distribution of Cu at many US military sites is substantial and sites of contamination include habitats in and around military storage facilities, manufacturing, load and packing plants, open burning/open detonation areas, and firing ranges. Some of these areas include habitat for amphibian species, which generally lack toxicity data for risk assessment purposes. In an effort to ascertain Cu concentrations in soil that are toxic to terrestrial amphibians, 100 red-backed salamanders (Plethodon cinereus) were randomly sorted by weight, assigned to either a control soil or one of four treatments amended with copper acetate in soil, and exposed for 28 days. Analytical mean soil concentrations were 18, 283, 803, 1333, and 2700 mg Cu/kg soil dry weight. Food consisted of uncontaminated flightless Drosophila melanogaster. Survival was reduced in salamanders exposed to 1333 and 2700 mg/kg by 55% and 100%, respectively. Mortality/morbidity occurred within the first 4 days of exposure. These data suggest that a Cu soil concentration of and exceeding 1333.3 +/- 120.2 mg/kg results in reduced survival, whereas hematology analyses suggest that a concentration of and exceeding 803.3 +/- 98.4 mg/kg might result in reduced total white blood cell count. No effects were observed at 283.3 +/- 36.7 mg/kg.

Energetic materials are employed in a wide range of commercial and military activities and often are released into the environment. Scientifically based ecological soil-screening levels (Eco-SSLs) are needed to identify contaminant explosive levels in soil that present an acceptable ecological risk. Insufficient information for 2,4,6-trinitrotoluene (TNT) to generate Eco-SSLs for soil invertebrates necessitated toxicity testing. We adapted the standardized Enchytraeid Reproduction Test and selected Enchytraeus crypticus for these studies. Tests were conducted in Sassafras sandy loam soil, which supports relatively high bioavailability of TNT. Weathering and aging procedures for TNT amended to test soil were incorporated into the study design to produce toxicity data that better reflect the soil exposure conditions in the field compared with toxicity in freshly amended soils. This included exposing hydrated TNT-amended soils in open glass containers in the greenhouse to alternating wetting and drying cycles. Definitive tests showed that toxicity for E. crypticus adult survival and juvenile production was increased significantly in weathered and aged soil treatments compared with toxicity in freshly amended soil based on 95% confidence intervals. The median effect concentration and 20% effective concentration for reproduction were 98 and 77 mg/kg, respectively, for TNT freshly amended into soil and 48 and 37 mg/kg, respectively, for weathered and aged TNT soil treatments. These findings of increased toxicity to E. crypticus in weathered and aged TNT soil treatments compared with exposures in freshly amended soils show that future investigations should include a weathering and aging component to generate toxicity data that provide more complete information on ecotoxicological effects of energetic contaminants in soil.

Munition manufacturing, disposal, testing, training and other operations at military sites produced elevated levels of explosives and related materials in soil. The effects of these persistent and highly mobile in the environment energetic materials on soil biota have not been sufficiently investigated. The U.S. Environmental Protection Agency (USEPA) in conjunction with stakeholders, is developing Ecological Soil Screening Level (Eco-SSL) benchmarks for ecological risk assessment (ERA) of contaminants at Superfund sites to identify those contaminants in soil that warrant additional evaluation in a Baseline ERA, and to eliminate those that do not. Eco-SSLs are developed from literature values whenever sufficient quantity and quality of data exist. Insufficient data were available on the toxicity of energetic compounds, RDX and HMX, to soil invertebrates, necessitating toxicity testing. Tests were conducted under conditions preferred for Eco-SSL benchmarks, using a Sassafras sandy loam soil that supports relatively high bioavailability of test compounds. Toxicity testing was performed using enchytraeid reproduction test (ISO/16387 2001) measuring adult survival and juvenile production by the potworm Enchytraeus crypticus in freshly amended soil and weathered/aged amended soil. RDX or HMX had no effect on adult survival in the definitive tests in all treatment concentrations. Juvenile production EC 2 0 values were 3,715 and 8,797 mg kg - 1 RDX in freshly amended and weathered/aged amended soils, respectively. Juvenile E. crypticus production was not adversely affected by exposure to HMX in freshly amended and weathered/aged amended soils. Juvenile production was stimulated in freshly amended soil up to 21,750 mg kg - 1 HMX. Results of these toxicity studies will be submitted to the Eco-SSL Task Group for quality control review, and pending approval will be included in the Eco-SSL database for Eco-SSL derivation.

The U.S. Environmental Protection Agency is developing Ecological Soil Screening Level (Eco-SSL) benchmarks for ecological risk assessment of contaminants at Superfund sites. Benchmarks for invertebrates Were developed from existing literature. Insufficient information for Ba, Be, Mn, and Sb to generate Eco-SSLs necessitated standardized toxicity testing to fill the data gaps. We used the Enchytraeid Reproduction Test (ERT) with Enchytraeus crypticus in this study. This test was selected on the bases of its ability to measure chemical toxicity to ecologically relevant test species during chronic assay, and its inclusion of at least one reproductive component among the measurement endpoints. Tests were conducted in Sassafras sandy loam soil, which supports relatively high bioavailability of metals. Aging/weathering procedures for amended treatment soil were incorporated into the study to better reflect the "real world" exposure conditions. The toxicity order based on juvenile production was Be> Mn> Sb> Ba with EC20 values of 45, 116, 194, and 585 mg kg-1, respectively. These results show that ERT is a robust and sensitive assay for toxicity assessments and is appropriate for the Eco-SSL development.

Tests were conducted to determine if methyl salicylate (MS), a component of HL simulant, was responsible for the toxicity exhibited by cucumbers and earthworms. The HL simulant without methyl salicylate (HLMS) and MS were tested for their toxicity to cucumbers and earthworms. The HLMS was tested at 0, 1,000, 5,000, 1 0,000, and 25,000 mg/kg by weight; MS was tested at 0, 50, 100, 200, 350, and 500 mg/kg by weight. The HLMS in soil produced no lethal or sublethal effects on cucumber plants at 1,000, 5,000, and 10,000 mg/kg levels, but the HLMS in soil produced sublethal effects at the 25,000 mg/kg level. Earthworms treated with HLMS had a survival rate of 100% for all treatment levels. The MS produced sublethal effects on cucumbers at the 350 and 500 mg/kg levels. Earthworms in soil dosed with MS showed an increasing weight los with increasing concentrations. Survival rates were 100% at the 0-200 mg/kg levels, 87% at 350 mg/kg, and 0% at the 500 mg/kg level. An earlier CRDEC study (CRDEC TR-257, March 1991), HL simulant containing 130 mg/kg of MS produced lethal and sublethal effects on earthworms and cucumbers. The study results indicated that is responsible for the effects exhibited by cucumbers and earthworms.

Graphite flakes were tested for their toxicity to corn and cucumber. The flakes were tested at 0 (control), 0.05, 0.10, 0.50% concentration by weight. No lethal or sublethal effects on corn plants were produced. The two-way analysis of variance (ANOVA) for plant heights showed no significant (p < 0.05) difference between control plants and those grown at other concentrations. The ANOVA showed no significant (p < 0.05) difference in the mean dry weights of corn plants. The ANOVA of mean plant heights for cucumbers indicated a significant (p

The U.S. Environmental Protection Agency is developing ecological soil screening levels (Eco-SSLs) for the ecological risk assessment of contaminants at Superfund sites. The Eco-SSLs for several soil contaminants have been developed from toxicity benchmarks for soil invertebrates in the existing literature. Insufficient information to generate Eco-SSLs for Sb, Ba, and Be necessitated toxicity testing to fill the data gaps. We used standardized toxicity tests with the earthworm Eisenia fetida, enchytraeid Enchytraeus crypticus, and collembolan Folsomia candida in the present study. These tests were selected on the basis of their ability to measure chemical toxicity to ecologically relevant test species during chronic assays and their inclusion of at least one reproduction component among the measurement endpoints. Tests were conducted in Sassafras Sandy Loam soil, which supports relatively high bioavailability of metals. Weathering and aging procedures for metals in amended soil were incorporated into these studies to better reflect exposure conditions in the field. The relative toxicity of metals to the soil invertebrates tested was Be > Sb > Ba based on the median effective concentration values for reproduction. These studies produced toxicological data that can contribute to the development of Eco-SSLs for Sb, Ba, and Be for soil invertebrates.

Scientifically based ecological soil-screening levels are needed to identify concentrations of contaminant energetic materials (EMs) in soil that present an acceptable ecological risk at a wide range of military installations. Insufficient information regarding the toxicity of 2,4-dinitrotoluene (2,4-DNT), 2,6-dinitrotoluene (2,6-DNT), and 1,3,5-trinitrobenzene (TNB) to soil invertebrates necessitated toxicity testing. We adapted the standardized Enchytraeid Reproduction Test (International Standardization Organization 16387:2003) and selected Enchytraeus crypticus for these studies. Tests were conducted in Sassafras sandy loam soil, which supports relatively high bioavailability of nitroaromatic EMs. Weathering and aging procedures for EMs amended to test soil were incorporated into the study design to produce toxicity data that better reflect the soil exposure conditions in the field compared with toxicity in freshly amended soils. This included exposing hydrated, EM-amended soils in open glass containers in the greenhouse to alternating wetting and drying cycles. Definitive tests established that the order of EM toxicity to E. crypticus based on the median effect concentration values for juvenile production in either freshly amended or weathered and aged treatments was (from the greatest to least toxicity) TNB > 2,4-DNT > 2,6-DNT. Toxicity to E. crypticus juvenile production was significantly increased in 2,6-DNT weathered and aged soil treatments compared with toxicity in freshly amended soil, based on 95% confidence intervals. This result shows that future investigations should include a weathering and aging component to generate toxicity data that provide more complete information regarding ecotoxicological effects of energetic contaminants in soil.