Ecotoxicological Evaluation of a Bench-Scale Bioslurry Treating Explosives-Spiked Soil

The ecotoxicological effects of four bioslurry reactors treating 2,4,6-trinitotoluene (TNT)- and 1,3,5-trinitro-1,3,5-triazacyclohexane (RDX)-spiked soil were evaluated. A control bioslurry reactor was used to assess the endogenous toxicity of the bioslurry operation conditions. A battery of ecotoxicity tests was used: Microtox, green algae growth inhibition, bacterial genotoxicity and mutagenicity, and earthworm mortality and growth inhibition. Bioslurry soluble and solid phases were separated by centrifugation in order to identify toxicity and possible toxicants associated with each phase. Microtox toxicity values were initially very high in both bioslurry reactors spiked with TNT, in relation with TNT concentration. Initial toxicity was also detected by algal growth inhibition, earthworm lethality, genotoxicity and mutagenicity tests. An endogenous toxicity was detected in the control bioreactor using the Microtox and the SOS Chromotest. The soluble phase of the control bioslurry was genotoxic, suggesting that some potentially genotoxic agents were induced in the bioslurry samples. At the end of the bioremediation treatment, data showed that toxicity was reduced using all of the bioassays, except for earthworm lethality and growth inhibition tests in both RDX-spiked bioslurries. This study demonstrates the usefulness of a battery of toxicity tests to monitor bioremediation processes.     

Fate of explosives and their metabolites in bioslurrytreatment processes

Microcosm tests simulating bioslurry reactors with 40% soil content, containing high concentrations of TNT and/or RDX, and spiked with either [14C]-TNT or [14C]-RDX were conducted to investigate the fate of explosives and their metabolites in bioslurry treatment processes. RDX is recalcitrant to indigenous microorganisms in soil and activated sludge under aerobic conditions. However, soil indigenous microorganisms alone were able to mineralize 15% of RDX to CO2 under anaerobic condition, and supplementation of municipal anaerobic sludge as an exogenous source of microorganisms significantly enhanced the RDX mineralization to 60%. RDX mineralizing activity of microorganisms in soil and sludge was significantly inhibited by the presence of TNT. TNT mineralization was poor (< 2%) and was not markedly improved by the supplement of aerobic or anaerobic sludge. Partitioning studies of [14C]-TNT in the microcosms revealed that the removal of TNT during the bioslurry process was due mainly to the transformation of TNT and irreversible binding of TNT metabolites onto soil matrix. In the case of RDX under anaerobic conditions, a significant portion (35%) of original radioactivity was also incorporated into the biomass and bound to the soil matrix.     

Use of Trinitrobenzene as a Nitrogen Source by Pseudomonas vesicularis Isolated from Soil

An aerobic Gram-negative bacterium identified as Pseudomonas vesicularis was isolated from soil contaminated with 2,4,6-trinitrotoluene (TNT) and 1,3,5-trinitrobenzene (TNB). This bacterium used TNB as the sole source of nitrogen. The TNB was metabolized within 80 h of incubation. The major metabolites produced were dinitroaniline, dinitrobenzene (DNB), nitroaniline, nitrobenzene (NB), and ammonia. The concentrations of DNB and NB produced in the culture medium were nearly stoichiometric. The ammonia concentration in the culture medium increased during the course of incubation. The end product of TNB metabolism was NB, which did not undergo further degradation even after long incubation time. This bacterium could be used in a syntrophic culture system with other NB-degrading bacteria to remove TNB completely from soil and water at contaminated sites. Received: 25 July 1996 / Accepted: 10 September 1996     

Use of a Salmonella microsuspension bioassay to detect the mutagenicity of munitions compounds at low concentrations

Past production and handling of munitions has resulted in soil contamination at various military facilities. Depending on the concentrations present, these soils pose both a reactivity and toxicity hazard and the potential for groundwater contamination. Many munitions-related chemicals have been examined for mutagenicity in the Ames test, but because the metabolites may be present in low environmental concentrations, a more sensitive method is needed to elucidate the associated mutagenicity. RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine), TNT (2,4,6-trinitrotoluene), tetryl (N-methyl-N-2,4,6-tetranitroaniline), TNB (1,3,5-trinitrobenzene) and metabolites were examined for mutagenicity in a microsuspension modification of the Salmonella histidine reversion assay with and without metabolic activation. TNB and tetryl were positive in TA98 (32.5, 5.2revertants/nmole) and TA100 (7.4, 9.5revertants/nmole) without metabolic activation and were more potent than TNT (TA98, 0.3revertants/nmole; TA100, 2.4revertants/nmole). With the exception of the tetranitroazoxytoluene derivatives, TNT metabolites were less mutagenic than TNT. RDX and two metabolites were negative in both strains, however, hexahydro-1,3,5-trinitroso-1,3,5-triazine was positive in TA100 with and without S9. Microsuspension bioassay results tend to correlate well with published Ames test data, however, there are discrepancies among the published data sets and the microsuspension assay results.     

Phytodetoxification of TNT by transgenic plants expressing a bacterial nitroreductase.

There is major international concern over the wide-scale contamination of soil and associated ground water by persistent explosives residues. 2,4,6-Trinitrotoluene (TNT) is one of the most recalcitrant and toxic of all the military explosives. The lack of affordable and effective cleanup technologies for explosives contamination requires the development of better processes. Significant effort has recently been directed toward the use of plants to extract and detoxify TNT. To explore the possibility of overcoming the high phytotoxic effects of TNT, we expressed bacterial nitroreductase in tobacco plants. Nitroreductase catalyzes the reduction of TNT to hydroxyaminodinitrotoluene (HADNT), which is subsequently reduced to aminodinitrotoluene derivatives (ADNTs). Transgenic plants expressing nitroreductase show a striking increase in ability to tolerate, take up, and detoxify TNT. Our work suggests that expression of nitroreductase (NR) in plants suitable for phytoremediation could facilitate the effective cleanup of sites contaminated with high levels of explosives.     

Cytotoxicity of nitroaromatic explosives and their biodegradation products in mice splenocytes: implications for their immunotoxicity

Nitroaromatic explosives like 2,4,6-trinitrotoluene (TNT) and 2,4,6-trinitrophenyl-N-methyl-nitramine (tetryl) comprise an important group of toxic environmental pollutants, whose toxicity is mainly attributed to the flavoenzyme electrontransferase-catalyzed redox cycling of their free radicals (oxidative stress) and DT-diaphorase [NAD(P)H:quinone oxidoreductase, NQO1, EC 1. 6.99.2]-catalyzed formation of alkylating nitroso and/or hydroxylamine metabolites. Because of the incomprehensive data on the immunotoxic effects of nitroaromatic explosives, we have studied the structure-cytotoxicity relationships in the action of tetryl, TNT as well as its amino and hydroxylamino metabolites, and related nitroaromatic compounds towards mouse splenocyte cells. The protective effects of desferrioxamine and the antioxidant N,N'-diphenyl-p-phenylene diamine against the cytotoxicity of TNT and other nitroaromatics showed that the oxidative stress-type cytotoxicity mechanism takes place. In addition, the cytotoxicity of nitroaromatics is also partly prevented by an inhibitor of NQO1, dicumarol. The cytotoxicity of the amino metabolites of TNT is also partly prevented by alpha-naphthoflavone and isoniazide, which points to the involvement of cytochromes P-450 in their activation. In general the cytotoxicity of nitroaromatics in splenocytes increases with an increase in their single-electron reduction potential, E1(7). This points to the prevailing mechanism of the oxidative stress-type cytotoxicity. The obtained structure-activity relationship and the studies of other mammalian cell lines showed that the immunotoxic potential of nitroaromatic explosives may decrease in the order tetryl > or = TNT > or = hydroxylamino metabolites of TNT > amino and diamino metabolites of TNT.     

Detection of 2,4,6-trinitrotoluene in seawater using a reversed-displacement immunosensor

Reported in this study are the experimental design and results of an immunosensor for the detection of the explosive, 2,4,6-trinitrotoluene (TNT) in seawater using a reversed-displacement format. This reversed-displacement immunosensor methodology has successfully measured TNT in seawater by direct injection, eliminating the need for preconcentration or pretreatment of samples. A microcolumn containing an Affi-Gel resin derivatized with a 2,4,6-trinitrobenzene (TNB) moiety and a fluorophore-labeled anti-TNT antibody composed the immunoassay reactive chamber. Fluorophore-labeled anti-TNT antibody was incubated with the modified Affi-Gel resin until binding equilibrium was reached. Under a constant flow, samples containing TNT were introduced into the flow stream displacing the fluorophore-labeled TNT antibody. Limits of detection were 2.5ng/mL or part-per-billion (ppb) for TNT in saline buffer and 25ppb in seawater with an analysis time of 10 min. Two anti-TNT antibodies with differing binding affinities were compared in the reversed-displacement assay format, and a correlation between affinity and detection limits was observed. Furthermore, we have demonstrated that the reversed-displacement format can be used to screen seawater samples containing TNT, remains effective after dozens of cycles, and provides significant fluorescence response before regeneration is required.     

Influence of 2,4,6-trinitrotoluene (TNT) concentration on the degradation of TNT in explosive-contaminated soils by the white rot fungus Phanerochaete chrysosporium.

The ability of Phanerochaete chrysosporium to bioremediate TNT (2,4,6-trinitrotoluene) in a soil containing 12,000 ppm of TNT and the explosives RDX (hexahydro-1,3,5-trinitro-1,3,5- triazine; 3,000 ppm) and HMX (octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine; 300 ppm) was investigated. The fungus did not grow in malt extract broth containing more than 0.02% (wt/vol; 24 ppm of TNT) soil. Pure TNT or explosives extracted from the soil were degraded by P. chrysosporium spore-inoculated cultures at TNT concentrations of up to 20 ppm. Mycelium-inoculated cultures degraded 100 ppm of TNT, but further growth was inhibited above 20 ppm. In malt extract broth, spore-inoculated cultures mineralized 10% of added [14C]TNT (5 ppm) in 27 days at 37 degrees C. No mineralization occurred during [14C]TNT biotransformation by mycelium-inoculated cultures, although the TNT was transformed.     

Influence of 2,4,6-trinitrotoluene (TNT) concentration on the degradation of TNT in explosive-contaminated soils by the white rot fungus Phanerochaete chrysosporium.

The ability of Phanerochaete chrysosporium to bioremediate TNT (2,4,6-trinitrotoluene) in a soil containing 12,000 ppm of TNT and the explosives RDX (hexahydro-1,3,5-trinitro-1,3,5- triazine; 3,000 ppm) and HMX (octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine; 300 ppm) was investigated. The fungus did not grow in malt extract broth containing more than 0.02% (wt/vol; 24 ppm of TNT) soil. Pure TNT or explosives extracted from the soil were degraded by P. chrysosporium spore-inoculated cultures at TNT concentrations of up to 20 ppm. Mycelium-inoculated cultures degraded 100 ppm of TNT, but further growth was inhibited above 20 ppm. In malt extract broth, spore-inoculated cultures mineralized 10% of added [14C]TNT (5 ppm) in 27 days at 37 degrees C. No mineralization occurred during [14C]TNT biotransformation by mycelium-inoculated cultures, although the TNT was transformed.     

Composting of explosives and propellant contaminated soils under thermophilic and mesophilic conditions

Summary Composting was investigated as a bioremediation technology for clean-up of sediments contaminated with explosives and propellants. Two field demonstrations were conducted, the first using 2,4,6-trinitrotoluene (TNT), octahydro-1,3,5,7-tetranitro-1,3,5,7-tetraazocine (HMX), hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), and N-methyl-N,2,4,6-tetranitroaniline (tetryl) contaminated sediment, and the second using nitrocellulose (NC) contaminated soil. Tests were conducted in thermophilic and mesophilic aerated static piles. Extractable TNT was reduced from 11840 mg/kg to 3 mg/kg, and NC from 13090 mg/kg to 16 mg/kg under thermophilic conditions. Under mesophilic conditions, TNT was reduced from 11 190 mg/kg to 50 mg/kg. The thermophilic and mesophilic half-lives were 11.9 and 21.9 days for TNT, 17.3 and 30.1 days for RDX, and 22.8 and 42.0 days for HMX, respectively. Known nitroaromatic transformation products increased in concentration over the first several weeks of the test period, but decreased to low concentrations thereafter.     

Methemoglobin formation in human erythrocytes by nitroaromatic explosives

We have examined the structure-activity relationships in methemoglobin (MetHb) formation by high explosives 2,4,6-trinitrotoluene (TNT), 2,4,6-trinitrophenyl-N-nitramine (tetryl) and 2,4,6-trinitrophenyl-N-nitraminoethylnitrate (pentryl), and a number of model nitrobenzenes. In lysed human erythrocytes the rate constants of oxyhemoglobin (OxyHb) oxidation increased with an increase in single-electron reduction potential (E(1)7) or with a decrease of the enthalpies of single-electron reduction of nitroaromatics. Tetryl and pentryl oxidized OxyHb almost 3 times faster than TNT. Although the initial rates of MetHb formation in intact erythrocytes by tetryl, pentryl, and TNT matched their order of reactivity in the oxidation of OxyHb in lysed erythrocytes, TNT was a more efficient MetHb forming agent than tetryl and pentryl during a 24-h incubation. The decreased efficiency of tetryl and pentryl was attributed to their reaction with intraerythrocyte reduced glutathione (GSH) producing 2,4,6-trinitrophenyl-Sglutathione, which acted as a less efficient OxyHb oxidizing agent.     

Origin of p-cresol in the anaerobic degradation of trinitrotoluene

p-Cresol was repeatedly detected as a trace metabolite in anaerobic slurry reactors treating 2,4,6-trinitrotoluene (TNT)-contaminated soils. This study shows that p-cresol was not a metabolite of the anaerobic degradation of TNT, by using a combination of analytical techniques and 13C-labelled TNT. Instead, p-cresol, an intermediate in the degradation pathway of some amino acids, was shown to be inhibited by TNT and its metabolites. The range and persistence of inhibition to p-cresol microbial degradation decreased with the level of amino-substitution of the derivatives. This explains why p-cresol accumulated within the TNT-treating anaerobic bioslurry, as it could not be further biodegraded in the presence of TNT.     

Fate of explosives and their metabolites in bioslurry treatment processes

Microcosm tests simulating bioslurry reactors with 40% soilcontent, containing high concentrations of TNT and/or RDX,and spiked with either [¹⁴C]-TNT or[¹⁴C]-RDX were conducted to investigate the fate ofexplosives and their metabolites in bioslurry treatment processes.RDX is recalcitrant to indigenous microorganisms in soil andactivated sludge under aerobic conditions. However, soilindigenous microorganisms alonewere able to mineralize 15% of RDX to CO₂ underanaerobic condition, and supplementation of municipal anaerobicsludge as an exogenous source of microorganismssignificantly enhanced the RDX mineralization to 60%. RDXmineralizing activity of microorganisms in soil and sludge wassignificantly inhibited by the presence of TNT. TNTmineralization was poor (< 2%) and was not markedlyimproved by the supplement ofaerobic or anaerobic sludge. Partitioning studies of[¹⁴C]-TNT in the microcosmsrevealed that the removal of TNTduring the bioslurry process was due mainly to thetransformation of TNT and irreversiblebinding of TNT metabolites onto soil matrix. In the case ofRDX under anaerobic conditions,a significant portion (35%) of original radioactivity wasalso incorporated into the biomass andbound to the soil matrix.     

Bioremediation of polynitrated aromatic compounds: plants and microbes put up a fight

Industrialization and the quest for a more comfortable lifestyle have led to increasing amounts of pollution in the environment. To address this problem, several biotechnological applications aimed at removing this pollution have been investigated. Among these pollutants are xenobiotic compounds such as polynitroaromatic compounds--recalcitrant chemicals that are degraded slowly. Whereas 2,4,6-trinitrophenol (TNP) can be mineralized and converted into carbon dioxide, nitrite and water, 2,4,6-trinitrotoluene (TNT) is more recalcitrant--although several microbes can use it as a nitrogen source. The most effective in situ biotreatments for TNT are the use of bioslurry (which can be preceded by an abiotic step) and phytoremediation. Phytoremediation can be enhanced by using transgenic plants alone or together with microbes.     

Transformation of 2,4,6-trinitrotoluene (TNT) by <Emphasis Type="Italic">Raoultella</Emphasis> <Emphasis Type="Italic">terrigena</Emphasis>

Manufacture of nitroorganic explosives generates toxic wastes leading to contamination of soils and waters, especially groundwater. For that reason bacteria living in environments highly contaminated with 2,4,6-trinitrotoluene (TNT) and other nitroorganic compounds were investigated for their capacity for TNT degradation. One isolate, Raoultella terrigena strain HB, removed TNT at concentrations between 10 and 100 mg l⁻¹ completely from culture supernatants under optimum aerobic conditions within several hours. Only low concentrations of nutrient supplements were needed for the cometabolic transformation process. Radioactivity measurements with ring-labelled ¹⁴C–TNT detected about 10–20% of the initial radioactivity in the culture supernatant and the residual 80–90% as water-insoluble organic compounds in the cellular pellet. HPLC analysis identified aminodinitrotoluenes (2-ADNT, 4-ADNT) and diaminonitrotoluenes (2,4-DANT) as the metabolites which remained soluble in the culture medium and azoxy-dimers as the main products in the cell extracts. Hence, the new isolate could be useful for the removal of TNT from contaminated waters.     

In Situ Biodegradation of High Explosives in Soils: Field Demonstration

The first field pilot-scale demonstration of a technology for in situ remediation of vadose zone soils contaminated with high explosives (HEs) has been performed at the Department of Energy's Pantex Plant. The HEs of concern at the demonstration site were hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) and the 2,4,6-trinitrotoluene (TNT) metabolite 1,3,5-trinitrobenzene (TNB). Concentrations ranged from 70 ppm, above the (prior to 1999) risk reduction clean-up criteria of 2.6 and 0.51 ppm, respectively. The shallow (<10?m depth) soils at the site could not be excavated due to the presence of buried utilities. Based on previous laboratory studies, it was found that the contaminated soils had indigenous microbial populations that could be stimulated to degrade the RDX and TNB anaerobically. A 5-spot well pattern with injection at the central well and extraction at the four outer wells (each 4.6?m from the injection well) was used to flood the target vadose zone soils with nitrogen gas with the intent of stimulating the activity of the HE degraders. The system was monitored periodically for gas composition as well as HE concentrations and microbial activity in retrievable soil samples. After 295 days of in situ treatment, the average target HE concentrations were approximately one-third lower than the initial site averages. Operation of the pilot-scale treatment system continues.     

Enhanced transformation of tnt by tobacco plants expressing a bacterial nitroreductase

The manufacture, disposal, and detonation of explosives have resulted in the pollution of large tracts of land and groundwater. Historically, 2,4,6-trinitrotoluene (TNT) is the most widely used military explosive and is toxic to biological systems and recalcitrant to degradation. To examine the feasibility of enhancing the ability of plants to detoxify the explosive TNT, we created transgenic tobacco (Nicotiana tabacum) constitutively expressing the nsfI nitroreductase gene from Enterobacter cloacae. The product of TNT reduction by the nitroreductase was found to be 4-hydroxylamino-2,6-dinitrotoluene (4-HADNT). Characterization of the transgenic lines in sterile, aqueous conditions amended with TNT demonstrated that these plants were able to remove all of the TNT from the medium at an initial concentration of 0.5 mM (113 mg L(-1)) TNT. In contrast, growth was suppressed in wild-type plants at 0.1 mM (23 mg L(-1)). Following uptake, transgenic seedlings transformed TNT predominantly to 4-HADNT and its high levels appeared to correlate with enhanced tolerance and transformation of TNT. Transformation products of TNT were subsequently conjugated to plant macromolecules to a greater degree in transgenic tobacco, indicating enhanced detoxification compared to the wild type.     

Lime Treatment of Explosives-Contaminated Soil from Munitions Plants and Firing Ranges

Microcosms were prepared using soils from munitions plants and active firing ranges and treated with hydrated lime. The presence of particulate explosives and co-contaminants, and the concentration of soil total organic carbon (TOC) on the alkaline hydrolysis reaction were studied. Trinitrobenzene (TNB) and dinitrobenzene (DNB) were sensitive to alkaline hydrolysis under these experimental conditions. The TNT metabolites, 2A- and 4A-DNT, were also removed, although more slowly than the parent compound, and the reaction required a higher pH (>12). RDX retention in the soil was proportional to the TOC content. The degradation intermediates of the alkaline hydrolysis reaction partitioned in the soil matrix in a manner similar to the parent. Solid particles of explosives are also degraded by alkaline hydrolysis. RDX and HMX exhibited 74 and 57% removal, respectively, in 21 days. TNT, as whole and broken grains, showed 83 and 99.9% removal in 21 days, respectively. The propellants, 2,4- and 2,6-DNT, were insensitive to alkaline hydrolysis. Alkaline hydrolysis is an inexpensive and effective means of reducing the varied explosives contamination.     

Cytotoxic and genotoxic effects of energetic compounds on bacterial and mammalian cells in vitro.

The mutagenicity and toxicity of energetic compounds such as 2,4, 6-trinitrotoluene (TNT), 1,3,5-trinitrobenzene (TNB), hexahydro-1,3, 5-trinitro-1,3,5-triazine (RDX) and octahydro-1,3,5,7-tetranitro-1,3, 5,7-tetrazocine (HMX), and of amino/nitro derivatives of toluene were investigated in vitro. Mutagenicity was evaluated with the Salmonella fluctuation test (FT) and the V79 Chinese hamster lung cell mutagenicity assay. Cytotoxicity was evaluated using V79 and TK6 human lymphoblastic cells. For the TK6 and V79 assays, TNB and 2, 4,6-triaminotoluene were more toxic than TNT, whereas RDX and HMX were without effect at their maximal aqueous solubility limits. The primary TNT metabolites (2-amino-4,6-dinitrotoluene, 4-amino-2, 6-dinitrotoluene, 2,4-diamino-6-nitrotoluene and 2, 6-diamino-4-nitrotoluene) were generally less cytotoxic than the parent compound. The FT results indicated that TNB, TNT and all the tested primary TNT metabolites were mutagenic. Except for the cases of 4-amino-2,6-dinitrotoluene and 2,4-diamino-6-nitrotoluene in the TA98 strain, addition of rat liver S9 resulted in either no effect, or decreased activity. None of the tested compounds were mutagenic for the V79 mammalian cells with or without S9 metabolic activation. Thus, the FT assay was more sensitive to the genotoxic effects of energetic compounds than was the V79 test, suggesting that the FT might be a better screening tool for the presence of these explosives. The lack of mutagenicity of pure substances for V79 cells under the conditions used in this study does not preclude that genotoxicity could actually exist in other mammalian cells. In view of earlier reports and this study, mutagenicity testing of environmental samples should be considered as part of the hazard assessment of sites contaminated by TNT and related products.     

Field Demonstration of Acetone Pretreatment and Composting of Particulate-TNT-Contaminated Soil

Solid fragments of explosives in soil are common in explosives testing and training areas. In this study we initially sieved the upper 6 in of contaminated soil through a 3-mm mesh, and found 2,4,6-trinitrotoluene (TNT) fragments. These contributed to an estimated concentration of 1.7 kg per cubic yard soil, or for 2000 ppm TNT in the soil. Most of the fragments ranged 4 mm to 10 mm diameter in size, but explosives particles weighing up to 56 g (about 4 cm diameter) were frequently observed. An acetone pretreatment/composting system was then demonstrated at field scale. The amount of acetone required for a TNT-dissolving slurry process was controlled by the viscosity of the soil/acetone mix rather than the TNT dissolution rate. The amount needed was estimated at about 55 gallons acetone per cubic yard soil. Smaller, 5- to 10-mm-diameter fragments went into solution in less than 15 min at a mixer speed of 36 rpm, with a minimum of 2 g TNT going into solution per 30 min for the larger chunks. The slurries were then mixed with compost starting materials and composted in a vented 1 yd³ container. After 34 days incubation time TNT was below the site-specific regulatory threshold of 44 ppm. TNT metabolites and acetone were also below their regulatory thresholds established for the site.