Jan E. Kolakowski

Enzymatic hydrolysis of the chemical warfare agent VX and its neurotoxic analogues by organophosphorus hydrolase

Organophosphorus hydrolase (OPH) is a bacterial enzyme that hydrolyzes a variety of organophosphorus (OP) neurotoxins, including many widely used pesticides and chemical warfare agents containing P-O, P-F, P-CN and P-S bonds. It has extremely high efficiency in hydrolysis of many different phosphotriester and phosphothiolester pesticides (P-O bond) such as paraoxon (kcat3800s−1) and coumaphos (kcat = 800s−1) or phosphonate (P-F) neurotoxins such as DFP (kcat = 350s−1) and the chemical warfare agent sarin (kcat = 56s−1). In contrast, the enzyme has much lower catalytic capabilities for phosphonothioate neurotoxins such as acephate (kcat = 2.8 s−1) or the chemical warfare agent VX [O-ethyl S-(2-diisopropyl-aminoethyl) methylphosphonothioate] (kcat = 0.3s−1). This lower specificity for VX and its analogues are reflected by the specificity constants (kcat/Km values) for VX = 0.75 × 103 M−1 s−1 compared to 5.5 × 107M−1 s−1 for paraoxon. Different metal-associated forms of the enzyme demonstrated significantly ...

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.

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.

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.

Toxicities of dinitrotoluenes and trinitrobenzene freshly amended or weathered and aged in a sandy loam soil to Enchytraeus crypticus

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.

Soil properties affect the toxicities of 2,4,6-trinitrotoluene (TNT) and hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) to the enchytraeid worm Enchytraeus crypticus.

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.