Iron and Electron Shuttle Mediated (Bio)degradation of 2,4

Article pubs.acs.org/est

Iron and Electron Shuttle Mediated (Bio)degradation of 2,4Dinitroanisole (DNAN) Jolanta B. Niedźwiecka,†,⊥ Scott R. Drew,‡ Mark A. Schlautman,† Kayleigh A. Millerick,†,§,# Erin Grubbs,† Nishanth Tharayil,∥ and Kevin T. Finneran*,† †

Environmental Engineering and Earth Sciences, Clemson University, 168 Rich Laboratory, Anderson, South Carolina 29625, United States ‡ Geosyntec Consultants, Ewing, New Jersey 08628, United States § Department of Civil and Environmental Engineering, University of Illinois at Urbana−Champaign, Urbana, Illinois 61801, United States ∥ School of Agriculture, Forestry, and Environmental Sciences, Clemson University, 218 Biosystems Research Complex, Clemson, South Carolina 29634, United States S Supporting Information *

ABSTRACT: The Department of Defense has developed explosives with the insensitive munition 2,4-dinitroanisole (DNAN), to prevent accidental detonations during training and operations. Understanding the fate and transport of DNAN is necessary to assess the risk it may represent to groundwater once the new ordnance is routinely produced and used. Experiments with ferrous iron or anthrahydroquinone-2,6disulfonate (AH2QDS) were conducted from pH 6.0 to 9.0 with initial DNAN concentrations of 100 μM. DNAN was degraded by 1.2 mM Fe(II) at pH 7, 8, and 9, and rates increased with increasing pH. Greater than 90% of the initial 100 μM DNAN was reduced within 10 min at pH 9, and all DNAN was reduced within 1 h. AH2QDS reduced DNAN at all pH values tested. Cells of Geobacter metallireducens were added in the presence and absence of Fe(III) and/or anthraquinone-2,6-disulfonate (AQDS), and DNAN was also reduced in all cell suspensions. Cells reduced the compound directly, but both AQDS and Fe(III) increased the reaction rate, via the production of AH2QDS and/or Fe(II). DNAN was degraded via two intermediates: 2-methoxy-5-nitroaniline and 4-methoxy-3-nitroaniline, to the amine product 2,4-diaminoanisole. These data suggest that an effective strategy can be developed for DNAN attenuation based on combined biological-abiotic reactions mediated by Fe(III)-reducing microorganisms.

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dinitrophenol (DNP),9 which is acutely toxic.10,11 There are few data for DNAN toxicity to humans but it is listed as a chemical hazard.6 DNAN has not been detected in groundwater at military installations. However, given the increased production for new explosives formulations it is important to understand attenuation mechanisms for both natural and engineered environments. DNAN has been reduced in anoxic soils by abiotic reactions to form azo-dimers, which most likely sorbed to natural organic matter.12 The same study demonstrated that DNAN in oxic soils was mostly removed through adsorption followed by slow chemical reactions, with little transformation.12 Follow up reports indicated that while DNAN reversibly sorbed to oxic

INTRODUCTION The Department of Defense (DoD) has developed a number of explosive formulations that contain cyclic nitramines (e.g., hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX)), but that have been updated with so-called insensitive munitions (IM) including 2,4-dinitroanisole (DNAN). PAX-21 and IMX-101 are two of the “next generation” explosive formulations that have already entered production and distribution.1 DNAN is a replacement for 2,4,6-trinitrotoluene (TNT), which is considered more sensitive to shock and therefore less stable in modern warfare environments.2−4 The use of DNAN in meltcast formulations resulted in increased stability and improved safety standards for explosive transport and storage. Little is known about environmental fate of DNAN despite a significant amount of available data on alternate nitroaromatic compounds and cyclic nitramines; however, several studies suggest that it can be toxic to microorganisms,5−7 plants,5 and amphibians.8 Mammalian cells can metabolize DNAN to 2,4© 2017 American Chemical Society

Received: Revised: Accepted: Published: 10729

May 11, 2017 August 23, 2017 August 29, 2017 August 29, 2017 DOI: 10.1021/acs.est.7b02433 Environ. Sci. Technol. 2017, 51, 10729−10735

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Environmental Science & Technology

described.24 HPLC grade methanol was obtained from VWR. All other chemicals were of reagent grade quality or higher. Abiotic Transformation Study. Sixty (60) mL to 125 mL experimental bottles were buffered with 30 mM 4-morpholineethanesulfonic acid (MES) at pH 6.0, 30 mM 4-(2hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) at pH 7.0 and 8.0, and 30 mM 2-(cyclonexylamino)ethanesulfonic acid (CHES) at pH 9.0. All bottles were degassed with ultrahigh-purity nitrogen. One hundred (100) μM DNAN and 1.5 mM Fe(II) were added to the experimental bottles. Samples were taken multiple times over 24 h and quenched immediately with 200 mM ethylenediaminetetraacetic acid (EDTA) in what were operationally defined as “shorter term” suspensions. “Longer term” suspensions were run up to 96 h, and contained 1.2 mM Fe(II) (which was still stoichiometric relative to DNAN). Ferrous iron concentration was measured as described below. AH 2 QDS was added to samples via 0.2 μm polytetrafluoroethylene (PTFE) filters to remove residual palladium. Microbial Growth and Experiments. Geobacter metallireducens strain GS-15 (ATCC 53774) was maintained using ferric citrate media and 20 mM acetate as electron donor.24,25 A gas mixture of N2:CO2, 80:20 (v/v) was used to sparge ferric citrate media and flush the headspace. All gases were passed through a hot, reduced copper column to remove trace oxygen. Twenty (20) mL culture tubes were sealed with a butyl rubber stopper and aluminum crimp to maintain anoxic conditions, and all bottles were autoclaved for 20 min at 120 °C prior to inoculation. In resting cell suspension experiments,24,25 G. metallireducens was grown to late exponential growth phase in a 1 L bottle, harvested and centrifuged at 3800g for 20 min to form a dense bacterial pellet. Each pellet was resuspended in 35 mL bicarbonate buffer while constantly flushed with N2:CO2, 80:20 (v/v). Cells washed with the buffer were centrifuged once more at 3000g for 20 min. Final biomass was resuspended in 4 mL of bicarbonate buffer and added immediately to experimental tubes at 2% (v/v). Culture tubes contained 10 mL of 30 mM bicarbonate buffer under anoxic conditions that were established using the same gas mixture and procedure as described for ferric citrate media. Electron acceptors incubated with cells included AQDS (0.5 mM), poorly crystalline Fe(III) hydroxide (FeGel) (1.5 mM), and ferric citrate (1.5 mM), all amended from anoxic stock solutions. To initiate the reaction, 0.2 mL of resting cell mass was added to experimental tubes after amendment with 100 μM DNAN. Samples were collected periodically via anoxic syringe and needle and filtered prior to analysis. Analytical Methods. Liquid samples were analyzed for DNAN, MENA, iMENA, and DAAN using a high-performance liquid chromatograph (HPLC; Dionex UltiMate 3000). Samples from initial experiments were analyzed only for DNAN using a Dionex Acclaim 120 C18 5 μm 120 Å (4.6 × 250 mm) column. The eluent was a mixture of acetonitrile:water 50:50 (v/v), run at 1 mL/min. The injection volume was 50 μL of sample, and the analytical wavelength was 296 nm. At these conditions, DNAN eluted at 9.3 min. Detection of DNAN together with its transformation products was achieved with Dionex Acclaim Explosive E1 5 μM 120 Å (4.6 × 250 mm) column. A mixture of neat methanol and 2 mM ammonium acetate at pH 5, 60:40 (v/v) was run at 1 mL/min. Elution times were 7.7, 5.4, 4.1, and 3.6 min for DNAN, MENA, iMENA, and DAAN respectively. The UV

soils, its degradation product, 2,4-diaminoanisole (DAAN), was irreversibly sorbed and therefore it could be immobilized from the environment.13 Nocardioides sp. JS1661 was able to mineralize DNAN and use it as sole carbon source in soil during aerobic metabolism.14 DNAN has been degraded aerobically by several enrichment cultures and the suggested product was DNP.15 Four anaerobic bacteria reduced DNAN in resting cell suspensions; however, the specific metabolites were unclear.13 One report demonstrated DNAN uptake by several grass species in phytoremediation of explosives-contaminated soil.16 Similarly, Penicillium sp. KH1 and Rhizobium lichtii isolated from willow trees were able to transform DNAN although no ring-cleavage products were identified.17,18 Other studies focused on DNAN degradation in wastewater streams using anaerobic fluidized-bed bioreactors,19 transformation of Fe/Cu bimetallic particles,20 zerovalent iron,7 and cometabolic degradation by Bacillus cells in artificially contaminated, oxic soil microcosms.21 In these cases, DNAN was reduced through 2-methoxy-5-nitroaniline (MENA) to DAAN by subsequent reduction of two nitro groups to amines. Pseudomonas sp., strain FK357, and Rhodococcus imtechensis, strain RKJ300, were able to aerobically degrade DNAN via intermediate DNP, which was utilized by strain RKJ300 as carbon source.22 These studies did not report degradation under the Fe(III)-reducing conditions that have been previously reported for the cyclic nitramines, which may become the model for in situ remediation of DNAN and other IM compounds. All reports referenced above indicate some level of DNAN transformation or sorption, or both. However, the knowledge gaps are in (a) specific mechanisms that will promote degradation in anoxic aquifer material, and (b) how to accelerate the rate and extent of complete reduction to DAAN by direct or indirect microbial DNAN reduction. The objective of this study was to quantify the rate and extent of DNAN degradation by mixed biological-abiotic reactions with ferrous iron and hydroquinones. The purified quinone-hydroquinone is a surrogate for naturally occurring humic acids, and has been reported in previous data for the cyclic nitramine RDX. We used past RDX studies as the model for how DNAN may (bio)degrade.23−25 Geobacter metallireducens, strain GS-15, was used as the Fe(III)-reducing microorganism to simulate microbial metabolism in situ. The data presented below demonstrate the reaction kinetics, transformation products, and the role of different electron shuttling molecules (quinones and/or iron), all of which influenced DNAN degradation.

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MATERIALS AND METHODS Chemicals. DNAN was obtained from Alfa Aesar. MENA and 4-methoxy-3-nitroaniline (iMENA) were provided by Sigma-Aldrich. DAAN was provided by Fluka. The stock solutions of DNAN, MENA, iMENA, and DAAN were prepared by dissolving analytes in methanol. Ferrous chloride was provided by Sigma-Aldrich. Iron(II) stock solutions were prepared in an anoxic glovebox, which contained an atmosphere of 95% N2 and 5% H2. The ferrous iron stock was made by dissolving 450 mM of ferrous chloride in 0.5 M hydrochloric acid at pH 2.5. Anthraquinone-2,6-disulfonate (AQDS) was obtained from Sigma-Aldrich. Anthrahydroquinone-2,6-disulfonate (AH2QDS) solution was prepared by chemical reduction of 30 mM AQDS dissolved in 30 mM bicarbonate buffer by sparging the solution with H2/CO2 (80:20 (v/v)) and palladium catalyst pellets, as previously 10730

DOI: 10.1021/acs.est.7b02433 Environ. Sci. Technol. 2017, 51, 10729−10735

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treatments, and are summarized in Supporting Information (SI) Table S1. The degradation products identified varied based on short versus longer term sampling. Short-term samples (under 24 h for pH 7.0 and under 1 h for pH 8.0 and 9.0) accumulated 2HA-4-NAN (Figure 2, left panels). The 2-HA-4-NAN was further transformed to MENA, and its isomer, iMENA, was analyzed but not detected (Figure 2, right panels). Reduction of the − NO2 functional group in the ortho position was more favorable under all conditions. DAAN was the terminal product we quantified at both pH 8.0 and 9.0. The intermediate 2-HA4-NAN preceded MENA at all pH values, which suggests that the reduction of − NHOH to − NH2 may be the rate-limiting step in abiotic DNAN degradation to DAAN. Figure 3 is a predicted pathway for DNAN transformation with ferrous iron as the sole reductant. There were rate differences among the experiments, depending on the pH. The initial (screening) experiments were not mixed, but all subsequent experiments were mixed on a rotary mixer for uniform distribution of reactants. The exact effect of mixing on DNAN degradation was not investigated further; however, it may be related to Fe(II) adsorption to freshly precipitated Fe(III) in mixed versus nonmixed samples. If mixing is a strong influence on rates this will impact in situ iron-mediated DNAN transformation. DNAN transformation rates will vary based on groundwater flow, as well as ferrous iron concentration. The DNAN reduction rates generally follow a previously reported Fe(II) autocatalytic oxidation pattern, in which the rates increase as more Fe(II) interacts with freshly formed Fe(III) solids.27,28 The initial 2 h lag phase at pH 7.0 in DNAN reduction could be thus explained as the time required for sufficient precipitation of Fe(III) to enhance iron oxidation and subsequently DNAN reduction, which would be limited at lower pH values and would explain the lack of DNAN transformation at pH 6.0.29 However, this is speculative based on prior Fe(II) oxidation data, and will require additional experiments to determine if this is partially the rate-controlling mechanism. Iron measurements taken at the beginning and at the end of the experiment indicated a total loss of 0.15 mM (10%) Fe(II) at pH 6.0, 0.75 mM (50%) loss at pH 7.0 and approximately 1.2 mM (80%) loss at pH 8.0 and 9.0 (data not shown), which suggests that more Fe(II) was lost than can be expected based solely on oxidation−reduction stoichiometry. The soluble electron shuttle AH2QDS reduced 100 μM DNAN within a few minutes at pH 7.0 (Figure 4); therefore, it was not tested at higher pH values. AH2QDS has been used in many laboratory studies to mimic humic substances present in soil. The main intermediate detected was 2-HA-4-NAN, similarly to the experiments with Fe(II); however, no MENA was measured after disappearance of 2-HA-4-NAN, thus further products of 2-HA-4-NAN transformation are not known at this point. Instead, approximately 10 μM iMENA was recovered, suggesting that DNAN degradation can be initiated by the reduction of either the ortho −NO2 group, or the group in para position. Mass balances were more complete when the products of DNAN degradation were monitored for several hours following complete DNAN disappearance (Table 1). Approximately 90% and 71% of DNAN was recovered as DAAN at pH 9 and 8 after 1 day, respectively, and 55% DNAN was recovered as MENA at pH 7 after 4 days. Fewer intermediates were recovered in the shorter time frame experiments, because of the shorter

detector wavelengths were set to 300 nm for DNAN, 254 nm for MENA, and 210 nm for iMENA and DAAN, or to 210 nm for all analytes. The detection limits for analytes were 0.05 μM, 0.11 μM, 0.14 μM, 5.75 μM, respectively. Blank water samples and known concentration standards were run periodically to ensure adequate quality of data. The intermediate of DNAN degradation, 2-hydroxylamino-4nitroanisole (2-HA-4-NAN), was identified using liquid chromatography−mass spectrometry (LC-MS) at the MultiUser Analytical Laboratory in the Plant and Environmental Sciences Department at Clemson University. Based on the retention time of 2-HA-4-NAN in the HPLC method, its peak was collected in a separate vial and analyzed using LC-MS by performing a positive and negative scan, as well as product ion scan at collision energies of 15, 20, and 25 V. Positive and negative scans confirmed that the analyte has a m/z ratio of 184, which corresponds to the molecular weight of 2-HA-4NAN. Additionally, product ion scan analysis confirmed that the original compound of m/z 184 splits into three fragments of m/z: 168, 138, and 108, which was consistent with the previously reported 2-HA-4-NAN fragmentation pathway.21 Fe(II) concentration was measured by Ferrozine assay.26 A sample aliquot was acidified at the collection time using 0.5 N HCl to preserve dissolved Fe(II). Then, the acidified aliquot was mixed with a Ferrozine solution and absorbance was measured at 562 nm.

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RESULTS AND DISCUSSION Abiotic Degradation by Iron or Electron Shuttles. Initial suspensions with soluble ferrous iron alone reduced DNAN at pH 7.0, 8.0, and 9.0; however, DNAN was not reduced at pH 6.0 (Figure 1). DNAN concentrations were non-

Figure 1. DNAN reduction by ferrous iron from pH 6.0 to 9.0. Experimental bottles were buffered with 30 mM MES, HEPES, and CHES buffers at pH 6.0, 7.0−8.0, and 9.0 respectively. DNAN was amended at 100 μM and initial Fe(II) concentration was 1.5 mM. Bottles were not mixed. Results are the mean of triplicate incubations; bars indicate one standard deviation.

detect within 24 h at pH 7.0, and within 2 h at pH 8.0 and 9.0. The time zero data points at pH 8 and 9 indicated that only 40 μM and 10 μM of the initial 100 μM DNAN remained when sampled after 5 min. We sampled the next series of experiments at several seconds after Fe(II) amendment, and DNAN was reduced at pH 8.0 and 9.0 in minutes to seconds (Figure 2, left panels). In subsequent experiments, initial data points were taken before addition of Fe(II) for improved DNAN accuracy. DNAN degradation rates varied among all experimental 10731

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Figure 2. Products of DNAN degradation by 1.5 mM ferrous iron at pH 7 (A), pH 8 (C), and pH 9 (E) in short time frame experiments (left panels); and by 1.2 mM ferrous iron at pH 7 (B), pH 8 (D), and pH 9 (F) in longer time frame experiments (right panels). Primary y-axis shows concentration of analytes: DNAN, MENA, iMENA, and DAAN. The secondary y-axis in plots A, C, and E shows the peak area of 2-HA-4-NAN as there are no certified standards of the intermediate 2-HA-4-NAN to compare with the amounts detected in experimental samples. In short time frame experiments (A, C, and E) bottles were buffered with 30 mM HEPES at pH 7 and 8, and with 30 mM CHES at pH 9, and they were mixed on a rotary shaker. In longer time frame experiments (B, D, and F) bottles were buffered with 30 mM HEPES for pH 7−9. Control suspensions were not shown in these plots, but DNAN (alone) did not deviate at any pH over the time frame of the experiments (shown in SI Figure S2). Results are the mean of triplicate incubations; bars indicate one standard deviation.

Figure 3. DNAN reductive degradation pathway. Complete nitro reduction of 1 mol of DNAN to 1 mol of DAAN requires 12 electrons.

Figure 4. DNAN degradation by 600 μM AH2QDS at pH 7, buffered with 30 mM HEPES. Primary y-axis shows concentration of analytes: DNAN, MENA, iMENA, and DAAN. The secondary y-axis shows the peak area of 2-HA-4-NAN. Results are the mean of triplicate incubations; bars indicate one standard deviation.

sampling period and due to the inability to directly quantify 2HA-4-NAN (no available standards). Microbially Mediated DNAN Reduction. Geobacter metallireducens, strain GS-15, reduced DNAN in the presence and absence of extracellular shuttling compounds (Figure 5). GS-15 can transfer electrons directly to contaminants or indirectly via shuttles/iron, which can undergo sequential reduction and oxidation. The electron transport system of GS15 has been well documented, and includes standard electron acceptors such as Fe(III), but also unique compounds including electron shuttles.30 DNAN was transformed to DAAN mostly

via intermediate formation of MENA; however, lower concentrations of iMENA were also detected. In amendments with cells alone (no electron donor) 100 μM DNAN was transformed to 55 μM MENA, 20 μM iMENA, and 25 μM DAAN in 30 h. The electron donor acetate was not critical for the DNAN reduction; this has been documented with resting 10732

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Table 1. Mass Balance (%) Following DNAN Reduction and Transformation Products Formation by Abiotic, Biological and Mixed Abiotic-Biological Pathways short-term mass balance (%) compound

Fe(II) alone, pH 8 (abiotic)b

Fe(II) alone, pH 9 (abiotic)c

Fe(II) alone, pH 7 (abiotic)d

Fe(II) alone, pH 8 (abiotic)e

Fe(II) alone, pH 9 (abiotic)f

8.71

0.39

0.40

0.00

0.26

0.01

27.02

6.68

1.42

53.58

12.69

1.04

3.91

1.62

0.34

0.59

0.97

3.56

63.74

91.58

40.23

9.66

5.72

76.69

92.63

C7H4N2O5 (DNAN) C7H8N2O3 (MENA) C7H8N2O3 (iMENA) C7H10N2O (DAAN) total compound C7H6N2O5 C7H8N2O3 C7H8N2O3 C7H10N2O total a

long-term mass balance (%)

Fe(II) alone, pH 7 (abiotic)a

(DNAN) (MENA) (iMENA) (DAAN)

53.58 mass balance (%)

cells alone (biological)g

cells + AQDS (mixed)g

cells + FeGel (mixed)g

cells + FeCit (mixed)g

cells + AQDS + FeGel (mixed)g

0.44 53.78 18.67 26.32 99.21

0.01 0.08 0.00 82.79 82.88

0.12 41.53 15.53 1.81 58.99

0.01 33.83 6.89 75.23 115.95

0.02 0.00 0.00 145.07 145.09

Measured at 24 h. b1 h. c2 min. d96 h. e24 h. f52 min. g30 h.

Figure 5. DNAN degradation in the cell suspension of GS-15 alone (A), with acetate (B), with acetate and AQDS (C), with acetate and poorly crystalline Fe(III) (FeGel) (D), with acetate, AQDS, and FeGel (E), with acetate and soluble Fe(III) (FeCit) (F). Experimental conditions: 100 μM DNAN, 1 mM acetate, 0.5 mM AQDS, 1.5 mM Fe(III), buffered with 30 mM bicarbonate at pH 7. Additionally, panels D-F show Fe(II) generated in the incubations. Results are the mean of triplicate incubations; bars indicate one standard deviation.

cell suspensions and has been attributed to endogenous respiration because of the high biomass.22 AQDS accelerated DNAN reduction by GS-15 in the presence or absence of poorly crystalline Fe(III), with complete reduction of both nitro groups and formation of DAAN; the other intermediates only transiently accumulated. Reduction with poorly crystalline Fe(III) alone (referred to as FeGel in figures) was slower than soluble ferric citrate (FeCit). This was expected based on soluble electron acceptors being reduced faster than insoluble compounds as they are more easily

accessed by microorganisms. This is important for iron-based degradation studies, since soluble Fe(III) forms tend to overestimate transformation rates for contaminants. DAAN eventually disappeared in the treatments with FeGel alone. It is possible that DAAN sorbs to Fe(III) solids after DNAN reduction. The carbon mass balances were not closed in treatments with cells with AQDS alone, or cells with FeGel alone. In both cases, this suggests the formation of unidentified degradation products, given that mass balances for all other treatments 10733

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were closed. In treatments with Fe(III)-citrate or combined AQDS plus FeGel, the carbon mass balances were higher than 100%. We believe this was due to analytical error in both cases−perhaps the influence of either the quinone, hydroquinone, or citrate on the aqueous phase analyses. Overall, the combined biological-chemical DNAN degradation was more complete than either the strictly abiotic experiments with ferrous iron or AH2QDS, or the strictly biological conditions with cells alone, assuming all other conditions were similar. This is similar to what was previously reported for cyclic nitramines, and is the most likely scenario for either enhanced in situ bioremediation, or natural attenuation, as both microbial and chemical processes will be functioning simultaneously in situ. This study presents the first direct evidence for DNAN degradation mediated by ferrous iron, electron shuttles, or some combination of those with Fe(III)-reducing biomass. Previous data with cyclic nitramines demonstrated that RDX was unreactive with dissolved Fe(II) and that it required Fe(II) adsorbed to magnetite28 or Fe(II) complexed with organic ligands31 to promote degradation. The reactions between DNAN and dissolved Fe(II) were different; soluble ferrous iron alone mediated the reaction. Future studies can investigate the production of freshly precipitated iron solids that will form more reactive iron complexes, which may promote simultaneous DNAN and RDX reduction when both explosives are combined in a solution. Microbially reduced extracellular electron shuttles can effectively reduce not only DNAN but also RDX and HMX,23 which are commonly used in IM formulations. Under appropriate conditions the biologicalabiotic reactions may contribute to natural attenuation of both explosives and IM. These findings are the basis for future remediation strategies at sites where several explosives are present in the soil and groundwater, and where active Fe(III)reducing microorganisms can mediate electron transfer to contaminants. The fate of the terminal amine product, DAAN, is unknown. DAAN has been reported to form dimers.12,13 The size of the dimers contributes to their low solubility in water, which would result in their precipitation and immobilization− an important attenuation mechanism even if DAAN is not further transformed.12,13

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K.A.M.: Civil, Environmental, and Construction Engineering, Texas Tech University, Box 41023, 911 Boston Avenue, Lubbock, Texas 79409, United States. Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Kelly Nevin of the University of Massachusetts at Amherst for the original Geobacter metallireducens culture. This work was supported by the Department of Defense Strategic Environmental Research and Development Program (SERDP), project number ER-2222.

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(1) Fung, V.; Newland, S. A modernized IM melt pour explosive manufacturing facility at Holston Army Ammunition Plant. Insensitive Munitions Energy Mater. Technol. Symp. 2015, No. No. May, 1−10. (2) Trzciński, W. A.; Cudziło, S.; Dyjak, S.; Nita, M. A comparison of the sensitivity and performance characteristics of melt-pour explosives with TNT and DNAN binder. Cent. Eur. J. Energy Mater. 2014, 11 (3), 443−455. (3) Fung, V.; Morris, J.; Price, D.; Tucker, N.; Carrillo, A.; Leclaire, E. Holston Army Ammunition Plant 2010 Insensitive Munitions & Energetic Material Technology Symposium IM Melt-Pour Formulations Development 2010, 1−16. (4) Davies, P. J.; Provatas, A. Characterisation of 2,4-Dinitroanisole: An Ingredient for use in Low Sensitivity Melt Cast Formulations 2006, No. No. Im, 21. (5) Dodard, S. G.; Sarrazin, M.; Hawari, J.; Paquet, L.; Ampleman, G.; Thiboutot, S.; Sunahara, G. I. Ecotoxicological assessment of a high energetic and insensitive munitions compound: 2,4-Dinitroanisole (DNAN). J. Hazard. Mater. 2013, 262, 143−150. (6) Liang, J.; Olivares, C.; Field, J. A.; Sierra-Alvarez, R. Microbial toxicity of the insensitive munitions compound, 2,4-dinitroanisole (DNAN), and its aromatic amine metabolites. J. Hazard. Mater. 2013, 262, 281−287. (7) Ahn, S. C.; Cha, D. K.; Kim, B. J.; Oh, S. Y. Detoxification of PAX-21 ammunitions wastewater by zero-valent iron for microbial reduction of perchlorate. J. Hazard. Mater. 2011, 192 (2), 909−914. (8) Stanley, J. K.; Lotufo, G. R.; Biedenbach, J. M.; Chappell, P.; Gust, K. A. Toxicity of the conventional energetics TNT and RDX relative to new insensitive munitions constituents DNAN and NTO in Rana Pipiens tadpoles. Environ. Toxicol. Chem. 2015, 34 (4), 873−879. (9) Hoyt, N.; Brunell, M.; Kroeck, K.; Hable, M.; Crouse, L.; O’Neill, A.; Bannon, D. I. Biomarkers of oral exposure to 3-nitro-1,2,4-triazol5-one (NTO) and 2,4-dinitroanisole (DNAN) in blood and urine of rhesus macaques (Macaca mulatta). Biomarkers 2013, 18 (7), 587− 594. (10) Brecken-Folse, J. A.; Mayer, F. L.; Pedigo, L. E.; Marking, L. L. Acute toxicity of 4-nitrophenol, 2,4-dinitrophenol, terbufos and trichlorfon to grass shrimp (Palaemonetes spp.) and sheepshead minnows (Cyprinodon variegatus) as affected by salinity and temperature. Environ. Toxicol. Chem. 1994, 13 (1), 67−77. (11) Grundlingh, J.; Dargan, P. I.; El-Zanfaly, M.; Wood, D. M. 2,4Dinitrophenol (DNP): a weight loss agent with significant acute toxicity and risk of death. J. Med. Toxicol. 2011, 7, 205−212. (12) Olivares, C. I.; Abrell, L.; Khatiwada, R.; Chorover, J.; SierraAlvarez, R.; Field, J. A. (Bio)transformation of 2,4-dinitroanisole (DNAN) in soils. J. Hazard. Mater. 2016, 304, 214−221. (13) Hawari, J.; Monteil-Rivera, F.; Perreault, N. N.; Halasz, A.; Paquet, L.; Radovic-Hrapovic, Z.; Deschamps, S.; Thiboutot, S.; Ampleman, G. Environmental fate of 2,4-dinitroanisole (DNAN) and its reduced products. Chemosphere 2015, 119, 16−23.

ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.est.7b02433. Reaction equations, degradation rates and additional experimental data that supports findings presented in this article (PDF)

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*Phone: 864-656-4143; e-mail: [email protected]. ORCID

Mark A. Schlautman: 0000-0001-6522-4345 Nishanth Tharayil: 0000-0001-6866-0804 Kevin T. Finneran: 0000-0003-4685-6958 Present Addresses ⊥

J.B.N.: Faculty of Advanced Technologies and Chemistry, Military University of Technology, Gen. Sylwestra Kaliskiego 2 str, 00−908 Warsaw, Poland. 10734

DOI: 10.1021/acs.est.7b02433 Environ. Sci. Technol. 2017, 51, 10729−10735

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DOI: 10.1021/acs.est.7b02433 Environ. Sci. Technol. 2017, 51, 10729−10735