Roman G. Kuperman

Reproduction and survival of Eisenia fetida in a sandy loam soil amended with the nitro-heterocyclic explosives RDX and HMX

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 EC 2 0 values for RDX were 1.2 and 19 mg kg - 1 in freshly amended and weathered/aged soils, respectively. Juvenile production EC 2 0 values were 1.6 and 4.8 mg kg - 1 in freshly amended and weathered/aged soils, respectively. Cocoon production and juvenile production EC 2 0 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-SSLTask Group and will be included in the Eco-SSL database for Eco-SSL derivation.

Toxicity benchmarks for antimony, barium, and beryllium determined using reproduction endpoints for Folsomia candida, Eisenia fetida, and Enchytraeus crypticus

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 Eiseniafetida, 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.

Toxicity and uptake of cyclic nitramine explosives in ryegrass Lolium perenne

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX), and 2,4,6,8,10,12-hexanitro-2,4,6,8,10,12-hexaazaisowurtzitane (CL-20) are cyclic nitramines used as explosives. Their ecotoxicities have been characterized incompletely and little is known about their accumulation potential in soil organisms. We assessed the toxicity and uptake of these explosives in perennial ryegrass Lolium perenne L. exposed in a Sassafras sandy loam (SSL) or in a sandy soil (DRDC, CL-20 only) containing contrasting clay contents (11% and 0.3%, respectively). A 21-d exposure to RDX, HMX or CL-20 in either soil had no adverse effects on ryegrass growth. RDX and HMX were translocated to ryegrass shoots, with bioconcentration factors (BCF) of up to 15 and 11, respectively. In contrast, CL-20 was taken up by the roots (BCF up to 19) with no translocation to the shoots. These studies showed that RDX, HMX, and CL-20 can accumulate in plants and may potentially pose a risk of biomagnification across the food chain.

Survival and reproduction of Enchytraeus crypticus (Oligochaeta, Enchytraeidae) in a natural sandy loam soil amended with the nitro-heterocyclic explosives RDX and HMX

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.

SURVIVAL AND REPRODUCTION OF ENCHYTRAEID WORMS, OLIGOCHAETA, IN DIFFERENT SOIL TYPES AMENDED WITH ENERGETIC CYCLIC NITRAMINES

Hexanitrohexaazaisowurtzitane (CL-20), a new polycyclic polynitramine, has the same functional nitramine groups (N-NO2) as the widely used energetic chemicals hexahydro-1,3,5-trinitro-1,3,5-triazacyclohexane (royal demolition explosive [RDX]) and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (high-melting explosive [HMX]). Potential impacts of CL-20 as an emerging contaminant must be assessed before its use. The effects of CL-20, RDX, or HMX on adult survival and juvenile production by potworms Enchytraeus albidus and Enchytraeus crypticus were studied in three soil types, including Sassafras sandy loam (1.2% organic matter [OM], 11% clay, pH 5.5), an agricultural soil (42% OM, 1% clay, pH 8.2), and a composite agricultural-forest soil (23% OM, 2% clay, pH 7.9) by using ISO method 16387 (International Standard Organization, Geneva, Switzerland). Results showed that CL-20 was toxic to E. crypticus with median lethal concentration values for adult survival ranging from 0.1 to 0.7 mg/kg dry mass (DM) when using the three tested soils. In addition, CL-20 adversely affected juvenile production by both species in all soils tested, with median effective concentration (EC50) values ranging from 0.08 to 0.62 mg/kg DM. Enchytraeus crypticus and E. albidus were similarly sensitive to CL-20 exposure in the composite agricultural-forest soil, which supported reproduction by both species and enabled comparisons. Correlation analysis showed weak or no relationship overall among the soil properties and reproduction toxicity endpoints. Neither RDX nor HMX affected (p > 0.05) adult survival of either species below 658 and 918 mg/kg DM, respectively, indicating that CL-20 is more toxic to enchytraeids than RDX or HMX. Examination of data shows that CL-20 should be considered as a potential reproductive toxicant to soil invertebrates, and that safeguards should be considered to minimize the potential for release of CL-20 into the environment.

Genotoxicity of 2,4- and 2,6-dinitrotoluene as measured by the Tradescantia micronucleus (Trad-MCN) bioassay

The phytogenotoxicity of 2,4-dinitrotoluene (2,4-DNT) and 2,6-dinitrotoluene (2,6-DNT) was assessed using the Tradescantia micronucleus (Trad-MCN) bioassay. Tradescantia cuttings bearing young inflorescences were exposed for 6h to 2,4- or 2,6-DNT amended water solutions up to their respective solubilities. The nominal concentrations were 0, 1.9, 3.8, 7.5, 15, 30, 60, 100, 150, 200mg/l of 2,4-DNT, and 0, 7.5, 15, 30, 60, 90, 120, 180mg/l of 2,6-DNT. Each treatment was repeated three or four times. Chemical concentrations in test solutions were analyzed prior to and after the exposure. Cadmium chloride (0-20mM) was used as the positive control. Micronuclei (MCN) were scored in the tetrad-stage pollen mother cells. The MCN frequency (%), i.e. the number of micronuclei scored in 100 tetrads, was the measurement endpoint. Results indicated that both 2,4-DNT and 2,6-DNT were genotoxic with the minimum effective dose (MED) of 30 and 135mg/l, respectively. Longer exposure (30h) without recovery time at 150mg/l of 2,4-DNT and 180mg/l of 2,6-DNT did not induce significantly higher MCN frequencies.

Weathering and aging of 2,4,6-trinitrotoluene in soil increases toxicity to potworm Enchytraeus crypticus

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.

Accumulation of hexahydro-1,3,5-trinitro-1,3,5-triazine by the earthworm Eisenia andrei in a sandy loam soil†

The heterocyclic polynitramine hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is a highly energetic compound found as a soil contaminant at some defense installations. Although RDX is not lethal to soil invertebrates at concentrations up to 10,000 mg/kg, it decreases earthworm cocoon formation and juvenile production at environmentally relevant concentrations found at contaminated sites. Very little is known about the uptake of RDX in earthworms and the potential risks for food-chain transfer of RDX in the environment. Toxicokinetic studies were conducted to quantify the bioaccumulation factors (BAFs) using adult earthworms (Eisenia andrei) exposed for up to 14 d to sublethal concentrations of nonlabeled RDX or [14C]RDX in a Sassafras sandy loam soil. High-performance liquid chromatography of acetonitrile extracts of tissue and soil samples indicated that nonlabeled RDX can be accumulated by the earthworm in a concentration- and time-dependent manner. The BAF, expressed as the earthworm tissue to soil concentration ratio, decreased from 6.7 to 0.1 when the nominal soil RDX concentrations were increased from 1 to 10,000 mg/kg. Tissue concentrations were comparable in earthworms exposed to nonlabeled RDX or [14C]RDX. The RDX bioaccumulation also was estimated using the kinetically derived model (BAFK), based on the ratio of the uptake to elimination rate constants. The established BAFK of 3.6 for [14C]RDX uptake was consistent with the results for nonlabeled RDX. Radioactivity also was present in the tissue residues of [14C]RDX-exposed earthworms following acetonitrile extraction, suggesting the formation of nonextractable [14C]RDX metabolites associated with tissue macromolecules. These findings demonstrated a net accumulation of RDX in the earthworm and the potential for food-chain transfer of RDX to higher-trophic-level receptors.

A proteome-based assessment of the earthworm Eisenia fetida: response to chemical warfare agents in a sandy loam soil

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.

State of the science and the way forward for the ecotoxicological assessment of contaminated land

Over the past two decades, soil ecotoxicologists have made strides in utilizing the basic concepts and advancements in soil zoology and ecology. They have applied the existing tools, and developed new ones to investigate how chemical contamination can affect soil ecosystems, including the degradation or destruction of soil quality and habitats or the diminishment of belowground biodiversity. Soil ecotoxicologists are applying a suite of standard protocols, originally developed as laboratory tests with single chemicals (e.g., pesticides), and further enhancing both the approaches and protocols for the assessment of contaminated lands. However, ecological relevance of some approaches remains unresolved. The authors discuss the main challenges for a coherent ecotoxicological assessment of soil ecosystems amid contaminated lands, and provide recommendations on how to integrate the effects of physical and chemical soil properties, the variations in the diversity of soil invertebrates, and the interactions among organisms of various trophic levels. The review examines new international approaches and test methods using examples from three continents (in particular research conducted in Brazil), and provides recommendations for improving ecological relevance of ecotoxicological investigations of contaminated lands.