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

Subscriber access provided by Grand Valley State | University

Article

Iron and electron shuttle mediated (bio)degradation of 2,4-dinitroanisole (DNAN) Jolanta Niedzwiecka, Scott Drew, Mark A. Schlautman, Kayleigh Millerick, Erin Grubbs, Nishanth Tharayil, and Kevin T. Finneran Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b02433 • Publication Date (Web): 29 Aug 2017 Downloaded from http://pubs.acs.org on August 30, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Environmental Science & Technology is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 33

Environmental Science & Technology

1

Iron and electron shuttle mediated (bio)degradation

2

of 2,4-dinitroanisole (DNAN)

3

Jolanta B. Niedźwiecka#†, Scott R. Drew , Mark A. Schlautman#, Kayleigh A. Millerick#&‡, Erin

4

Grubbs#, Nishanth Tharayil§, and Kevin T. Finneran*#

5

#

6

Anderson, SC, 29625, Geosyntec Consultants, Ewing, NJ 08628, &Department of Civil and

7

Environmental Engineering, University of Illinois at Urbana-Champaign, Urbana, IL, 61801,

8

§

9

Biosystems Research Complex, Clemson, SC 29634.

10

Y

Environmental Engineering and Earth Sciences, Clemson University, 168 Rich Laboratory, Y

School of Agriculture, Forestry, and Environmental Sciences, Clemson University, 218

KEYWORDS. Insensitive munitions, 2,4-dinitroanisole, explosives, reductive degradation

11

12

ABSTRACT. The Department of Defense has developed explosives with the insensitive

13

munition 2,4-dinitroanisole (DNAN), to prevent accidental detonations during training and

14

operations. Understanding the fate and transport of DNAN is necessary to assess the risk it may

15

represent to groundwater once the new ordnance is routinely produced and used. Experiments

16

with ferrous iron or anthrahydroquinone-2,6-disulfonate (AH2QDS) were conducted from pH 6.0

17

to 9.0 with initial DNAN concentrations of 100µM. DNAN was degraded by 1.2mM Fe(II) at pH

18

7, 8, and 9, and rates increased with increasing pH. Greater than 90% of the initial 100µM

1

ACS Paragon Plus Environment


19

DNAN was reduced within ten minutes at pH 9, and all DNAN was reduced within one hour.

20

AH2QDS reduced DNAN at all pH values tested. Cells of Geobacter metallireducens were added

21

in the presence and absence of Fe(III) and/or anthraquinone-2,6-disulfonate (AQDS), and DNAN 41

22

was also reduced in all cell suspensions. Cells reduced the compound directly, but both AQDS

23

and Fe(III) increased the reaction rate, via the production of AH2QDS and/or Fe(II). DNAN was

24

degraded via two intermediates: 2-methoxy-5-nitroaniline and 4-methoxy-3-nitroaniline, and the

25

amine product 2,4-diaminoanisole. These data suggest that an effective strategy can be

26

developed for DNAN attenuation based on combined biological-abiotic reactions mediated by

27

Fe(III)-reducing microorganisms.

28 29

Introduction

30

The Department of Defense (DoD) has developed a number of explosive formulations that

31

contain cyclic nitramines (e.g. hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX)), but that have been

32

updated with so-called insensitive munitions (IM) including 2,4-dinitroanisole (DNAN). PAX-

33

21 and IMX-101 are two of the “next generation” explosive formulations that have already

34

entered production and distribution.1 DNAN is a replacement for 2,4,6-trinitrotoluene (TNT),

35

which is considered more sensitive to shock and therefore less stable in modern warfare

36

environments.2–4 The use of DNAN in melt-cast formulations resulted in increased stability and

37

improved safety standards for explosive transport and storage.

38

Little is directly known about environmental fate of DNAN despite a significant amount of

39

available data on alternate nitroaromatic compounds and cyclic nitramines; however, several

40

studies suggest that it can be toxic to microorganisms,5–7 plants,5 and amphibians.8 Mammalian

2


Page 2 of 33

Deleted: it

Page 3 of 33


42

cells can metabolize DNAN to 2,4-dinitrophenol (DNP),9 which is acutely toxic.10,11 There are

43

few data for DNAN toxicity to humans but it is listed as a chemical hazard.6 DNAN has not

44

been detected in groundwater at military installations. However, given the increased production

45

for new explosives formulations it is important to understand attenuation mechanisms for both

46

natural and engineered environments.

47

DNAN has been reduced in anoxic soils by abiotic reactions to form azo-dimers, which most

48

likely sorbed to natural organic matter.12 The same study demonstrated that DNAN in oxic soils

49

was mostly removed through adsorption followed by slow chemical reactions, with little

50

transformation.12 Follow up reports indicated that while DNAN reversibly sorbed to oxic soils,

51

its degradation product, 2,4-diaminoanisole (DAAN), was irreversibly sorbed and therefore it

52

could be immobilized from the environment.13 Nocardioides sp. JS1661 was able to mineralize

53

DNAN and use it as sole carbon source in soil during aerobic metabolism.14 DNAN has been

54

degraded aerobically by several enrichment cultures and the suggested product was DNP.15 Four

55

anaerobic bacteria reduced DNAN in resting cell suspensions; however, the specific metabolites

56

were unclear.13 One report demonstrated DNAN uptake by several grass species in

57

phytoremediation of explosives-contaminated soil.16 Similarly, Penicillium sp. KH1 and

58

Rhizobium lichtii isolated from willow trees were able to transform DNAN although no ring-

59

cleavage products were identified.17,18 Other studies focused on DNAN degradation in

60

wastewater streams using anaerobic fluidized-bed bioreactors,19 transformation of Fe/Cu

61

bimetallic particles,20 zero valent iron,7 and co-metabolic degradation by Bacillus cells in

62

artificially contaminated, oxic soil microcosms.21 In these cases, DNAN was reduced through 2-

63

methoxy-5-nitroaniline (MENA) to 2,4-diaminoanisole (DAAN) by subsequent reduction of two

64

nitro groups to amines. Pseudomonas sp., strain FK357, and Rhodococcus imtechensis, strain

3



65

RKJ300, were able to aerobically degrade DNAN via intermediate DNP, which was utilized by

66

strain RKJ300 as carbon source.22 These studies did not report degradation under the Fe(III)-

67

reducing conditions that have been previously reported for the cyclic nitramines, which may

68

become the model for in situ remediation of DNAN and other IM compounds. All reports

69

referenced above indicate some level of DNAN transformation or sorption, or both. However,

70

the knowledge gaps are in: a) specific mechanisms that will promote degradation in anoxic

71

aquifer material, and b) how to accelerate the rate and extent of complete reduction to DAAN by

72

direct or indirect microbial DNAN reduction.

73

The objective of this study was to quantify the rate and extent of DNAN degradation by mixed

74

biological-abiotic reactions with ferrous iron and hydroquinones. The purified quinone-

75

hydroquinone is a surrogate for naturally occurring humic acids, and has been reported in

76

previous data for the cyclic nitramine RDX. We used past RDX studies as the model for how

77

DNAN may (bio)degrade.23–25 Geobacter metallireducens, strain GS-15, was used as the Fe(III)-

78

reducing microorganism to simulate microbial metabolism in situ. The data presented below

79

demonstrate the reaction kinetics, transformation products, and the role of different electron

80

shuttling molecules (quinones and/or iron), all of which influenced DNAN degradation.

81 82

Materials and Methods

83

Chemicals. DNAN was obtained from Alfa Aesar. MENA and 4-methoxy-3-nitroaniline

84

(iMENA) were provided by Sigma Aldrich. DAAN was provided by Fluka. The stock solutions

85

of DNAN, MENA, iMENA, and DAAN were prepared by dissolving analytes in methanol.

86

Ferrous chloride was provided by Sigma Aldrich. Iron(II) stock solutions were prepared in an

4


Page 4 of 33

Page 5 of 33


87

anoxic glove box, which contained an atmosphere of 95 % N2 and 5 % H2. The ferrous iron

88

stock was made by dissolving 450 mM of ferrous chloride in 0.5 M hydrochloric acid at pH 2.5.

89

Anthraquinone-2,6-disulfonate (AQDS) were obtained from Sigma Aldrich.

90

Anthrahydroquinone-2,6-disulfonate (AH2QDS) solution was prepared by chemical reduction of

91

30 mM AQDS dissolved in 30 mM bicarbonate buffer by sparging the solution with H2/CO2

92

(80:20 (vol/vol)) and palladium catalyst pellets, as previously described.24 HPLC grade methanol

93

was obtained from VWR. All other chemicals were of reagent grade quality or higher.

94

Abiotic Transformation Study. Sixty (60) mL to 125 mL experimental bottles were buffered

95

with 30 mM 4-morpholineethanesulfonic acid (MES) at pH 6.0, 30 mM 4-(2-

96

hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) at pH 7.0 and 8.0, and 30 mM 2-

97

(cyclonexylamino)ethanesulfonic acid (CHES) at pH 9.0. All bottles were degassed with

98

ultrahigh-purity nitrogen. One hundred (100) µM of DNAN and 1.5 mM of Fe(II) were added to

99

the experimental bottles. Samples were taken multiple times over 24 hours and quenched

109 110

Deleted: ml

Deleted: ml

100

immediately with 200 mM ethylenediaminetetraacetic acid (EDTA) in what were operationally

101

defined as “shorter term” suspensions. “Longer term” suspensions were run up to 96 hours, and

102

contained 1.2 mM Fe(II) (which was still stoichiometric relative to DNAN). Ferrous iron

103

concentration was measured as described below. AH2QDS was added to samples via 0.2 µm

104

polytetrafluoroethylene (PTFE) filter to remove residual palladium.

105

Microbial Growth and Experiments. Geobacter metallireducens strain GS-15 (ATCC 53774)

106

was maintained using ferric citrate media and 20mM acetate as electron donor.24,25 A gas mixture

107

of N2:CO2, 80:20 (vol/vol) was used to sparge ferric citrate media and flush the headspace. All

108

gases were passed through a hot, reduced copper column to remove trace oxygen. Forty (40) mL 111

5


Deleted: ml


112

culture tubes were sealed with a butyl rubber stopper and aluminum crimp to maintain anoxic

113

conditions, and all bottles were autoclaved for 20 min at 120oC prior to inoculation.

114

In resting cell suspension experiments,24,25 G. metallireducens was grown to late exponential

115

growth phase in a 1 L bottle, harvested and centrifuged at 3800 x g for 20 min to form a dense

116

bacterial pellet. Each pellet was resuspended in 35 mL bicarbonate buffer while constantly

Page 6 of 33

134

Deleted: ml

117

flushed with N2:CO2, 80:20 (vol/vol). Cells washed with the buffer were centrifuged once more

118

at 3000 x g for 20 min. Final biomass was resuspended in 4 mL of bicarbonate buffer and added 135

Deleted: ml

119

immediately to experimental tubes at 2% (vol/vol).

120

Culture tubes contained 10 mL of 30 mM bicarbonate buffer under anoxic conditions that were 136

121

established using the same gas mixture and procedure as described for ferric citrate media.

122

Electron acceptors incubated with cells included AQDS (0.5 mM), poorly crystalline Fe(III)

123

hydroxide (FeGel) (1.5 mM), and ferric citrate (1.5 mM), all amended from anoxic stock

124

solutions. To initiate the reaction, 0.2 mL of resting cell mass was added to experimental tubes

125

after amendment with 100 µM DNAN. Samples were collected periodically via anoxic syringe

126

and needle and filtered prior to analysis.

127

Analytical Methods. Liquid samples were analyzed for DNAN, MENA, iMENA, and DAAN

128

using a high-performance liquid chromatograph (HPLC; Dionex UltiMate™ 3000). Samples

137

129

138 139 from initial experiments were analyzed only for DNAN using a Dionex Acclaim® 120 C18 5 µm 140

130

120 Å (4.6 x 250 mm) column. The eluent was a mixture of acetonitrile:water 50:50 (vol/vol),

131

run at 1 mL/min. The injection volume was 50 µL of sample, and the analytical wavelength was 141

132

296 nm. At these conditions, DNAN eluted at 9.3 min. Detection of DNAN together with its

133

transformation products was achieved with Dionex Acclaim® Explosive E1 5 µM 120 Å (4.6 x

6


Deleted: ml

Deleted: ml

Deleted: n Deleted: i Deleted: ® or

Deleted: ml

Page 7 of 33


142

250 mm) column. A mixture of neat methanol and 2 mM ammonium acetate at pH 5, 60:40

164

Deleted: mixed with

143

(vol/vol) was run at 1 mL/min. Elution times were 7.7 min, 5.4 min, 4.1 min, and 3.6 min for

165

Deleted: ml

144

DNAN, MENA, iMENA, and DAAN respectively. The UV detector wavelengths were set to

145

300 nm for DNAN, 254 nm for MENA, and 210 nm for iMENA and DAAN, or to 210 nm for all

146

analytes. The detection limits for analytes were 0.05 µM, 0.11 µM, 0.14 µM, 5.75 µM,

147

respectively. Blank water samples and known concentration standards were run periodically to

148

assure adequate quality of data.

149

The intermediate of DNAN degradation, 2-hydroxylamino-4-nitroanisole (2-HA-4-NAN), was

150

identified using liquid chromatography-mass spectrometry (LC-MS) at the Multi-User Analytical

151

Laboratory in the Plant and Environmental Sciences Department at Clemson University. Based

152

on the retention time of 2-HA-4-NAN in the HPLC method, its peak was collected in a separate

153

vial and analyzed using LC-MS by performing a positive and negative scan, as well as product

154

ion scan at collision energies of 15 V, 20 V, and 25 V. Positive and negative scans confirmed

155

that the analyte has a m/z ratio of 184, which corresponds to the molecular weight of 2-HA-4-

156

NAN. Additionally, product ion scan analysis confirmed that the original compound of m/z 184

157

splits into three fragments of m/z: 168, 138, and 108, which was consistent with the previously

158

reported 2-HA-4-NAN fragmentation pathway.21

159

Fe(II) concentration was measured by Ferrozine assay.26 A sample aliquot was acidified at the

160

collection time using 0.5 N HCl to preserve dissolved Fe(II). Then, the acidified aliquot was

161

mixed with a Ferrozine solution and absorbance was measured at 562 nm.

162 163

Results and Discussion

7



166

Abiotic degradation by iron or electron shuttles. Initial suspensions with soluble ferrous iron

167

alone reduced DNAN at pH 7.0, 8.0, and 9.0; however, DNAN was not reduced at pH 6.0

168

(Figure 1). DNAN concentrations were non-detect within 24 hours at pH 7.0, and within 2 hours

169

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

170

of the initial 100 µM DNAN remained when sampled after 5 minutes. We sampled the next

171

series of experiments at several seconds after Fe(II) amendment, and DNAN was reduced at pH

172

8.0 and 9.0 in minutes to seconds (Figure 2, left panels). In subsequent experiments, initial data

173

points were taken before addition of Fe(II) for improved DNAN accuracy. DNAN degradation

174

rates varied amongst all experimental treatments, and are summarized in Supporting Information

175

Table S1.

176

The degradation products identified varied based on short versus longer term sampling. Short

177

term samples (under 24 hours for pH 7.0 and under 1 hour for pH 8.0 and 9.0) accumulated 2-

178

hydroxylamino-4-nitroaniline (2-HA-4-NAN) (Figure 2, left panels). The 2-HA-4-NAN was

179

further transformed to 2-methoxy-5-nitroaniline (MENA), and its isomer, 3-methoxy-4-

180

nitroaniline (iMENA), was analyzed but not detected (Figure 2, right panels). Reduction of the –

181

NO2 functional group in the ortho position was more favorable under all conditions.

182

Diaminoanisole (DAAN) was the terminal product we quantified at both pH 8.0 and 9.0. The

183

intermediate 2-HA-4-NAN preceded MENA at all pH values, which suggests that the reduction

184

of –NHOH to –NH2 may be the rate-limiting step in abiotic DNAN degradation to DAAN.

185

Figure 3 is a predicted pathway for DNAN transformation with ferrous iron as the sole reductant.

186

There were rate differences amongst the experiments, depending on the pH. The initial

187

(screening) experiments were not mixed, but all subsequent experiments were mixed on a rotary

188

mixer for uniform distribution of reactants. The exact effect of mixing on DNAN degradation

8


Page 8 of 33

Page 9 of 33


189

was not investigated further; however, it may be related to Fe(II) adsorption to freshly

190

precipitated Fe(III) in mixed versus non-mixed samples. If mixing is a strong influence on rates

191

this will impact in situ iron-mediated DNAN transformation. DNAN transformation rates will

192

vary based on groundwater flow, as well as ferrous iron concentration.

193

The DNAN reduction rates generally follow a previously reported Fe(II) autocatalytic oxidation

194

pattern, in which the rates increase as more Fe(II) interacts with freshly-formed Fe(III) solids.27,28

195

The initial 2 hr lag phase in DNAN reduction could be thus explained as the time required for

196

sufficient precipitation of Fe(III) to enhance iron oxidation and subsequently DNAN reduction,

197

which would be limited at lower pH values and would explain the lack of DNAN transformation

198

at pH 6.0.29 However, this is speculative based on prior Fe(II) oxidation data, and will require

199

additional experiments to determine if this is partially the rate-controlling mechanism. Iron

200

measurements taken at the beginning and at the end of the experiment showed a total loss of 0.15

201

mM (10%) Fe(II) at pH 6.0, 0.75 mM (50%) loss at pH 7.0 and approximately 1.2 mM (80%)

202

loss at pH 8.0 and 9.0 (data not shown), which suggests that more Fe(II) was lost than can be

203

expected based solely on oxidation-reduction stoichiometry.

204

The soluble electron shuttle AH2QDS reduced 100 µM DNAN within a few minutes at pH 7.0

205

(Figure 4); therefore, it was not tested at higher pH values. AH2QDS has been used in many

206

laboratory studies to mimic humic substances present in soil. The main intermediate detected

207

was 2-HA-4-NAN, similarly to the experiments with Fe(II); however, no MENA was measured

208

after disappearance of 2-HA-4-NAN, thus further products of 2-HA-4-NAN transformation are

209

not known at this point. Instead, approximately 10 µM iMENA was recovered, suggesting that

210

DNAN degradation can be initiated by the reduction of either the ortho –NO2 group or the group

211

in para position.

9



212

Mass balances were more complete when the products of DNAN degradation were monitored for

213

several hours following complete DNAN disappearance (Table 1). Approximately 90 % and 71

214

% of DNAN was recovered as DAAN at pH 9 and 8 after 1 day, respectively, and 55 % DNAN

215

was recovered as MENA at pH 7 after 4 days. Fewer intermediates were recovered in the shorter

216

timeframe experiments, because of the shorter sampling period and due to the inability to

217

directly quantify 2-HA-4-NAN (no available standards).

218

Microbially mediated DNAN reduction. Geobacter metallireducens, strain GS-15, reduced

219

DNAN in the presence and absence of extracellular shuttling compounds (Figure 5). GS-15 can

220

transfer electrons directly to contaminants or indirectly via shuttles/iron, which can undergo

221

sequential reduction and oxidation. The electron transport system of GS-15 has been well

222

documented, and includes standard electron acceptors such as Fe(III), but unique compounds

223

including electron shuttles.30 DNAN was transformed to DAAN mostly via intermediate

224

formation of MENA; however, lower concentrations of iMENA were also detected. In

225

amendments with cells alone (no electron donor) 100 µM DNAN was transformed to 55 µM

226

MENA, 20 µM iMENA, and 25 µM DAAN in 30 hours. The electron donor acetate was not

227

critical for the DNAN reduction; this has been documented with resting cell suspensions and has

228

been attributed to endogenous respiration because of the high biomass.22

229

AQDS accelerated DNAN reduction by GS-15 in the presence or absence of poorly crystalline

230

Fe(III), with complete reduction of both nitro groups and formation of DAAN; the other

231

intermediates only transiently accumulated. Reduction with poorly crystalline Fe(III) alone

232

(referred to as FeGel in figures) was slower than soluble ferric citrate (FeCit). This was expected

233

based on soluble electron acceptors being reduced faster than insoluble compounds as they are

234

more easily accessed by microorganisms. This is important for iron-based degradation studies,

10


Page 10 of 33

Page 11 of 33


235

since soluble Fe(III) forms tend to overestimate transformation rates for contaminants. DAAN

236

eventually disappeared in the treatments with FeGel alone. It is possible that DAAN sorbs to

237

Fe(III) solids after DNAN reduction.

238

The carbon mass balances were not closed in treatments with cells with AQDS alone, or cells

239

with FeGel alone. In both cases, this suggests the formation of unidentified degradation products,

240

given that mass balances for all other treatments were closed. In treatments with Fe(III)-citrate or

241

combined AQDS plus FeGel, the carbon mass balances were higher than 100%. We believe this

242

was due to analytical error in both cases – perhaps the influence of either the quinone,

243

hydroquinone, or citrate on the aqueous phase analyses. Overall, the combined biological-

244

chemical DNAN degradation was more complete than either the strictly abiotic experiments with

245

ferrous iron or AH2QDS, or the strictly biological conditions with cells alone, assuming all other

246

conditions were similar. This is similar to what was previously reported for cyclic nitramines,

247

and is the most likely scenario for either enhanced in situ bioremediation, or natural attenuation,

248

as both microbial and chemical processes will be functioning simultaneously in situ.

249

This study presents the first direct evidence for DNAN degradation mediated by ferrous iron,

250

electron shuttles, or some combination of those with Fe(III)-reducing biomass. Previous data

251

with cyclic nitramines demonstrated that RDX was unreactive with dissolved Fe(II) and that it

252

required Fe(II) adsorbed to magnetite28 or Fe(II) complexed with organic ligands31 to promote

253

degradation. The reactions between DNAN and dissolved Fe(II) were different; soluble ferrous

254

iron alone mediated the reaction. Future studies can investigate the production of freshly

255

precipitated iron solids that will form more reactive iron complexes, which may promote

256

simultaneous DNAN and RDX reduction when both explosives are combined in a solution.

257

Microbially reduced extracellular electron shuttles can effectively reduce not only DNAN but

11



258

also RDX and HMX,23 which are commonly used in IM formulations. Under appropriate

259

conditions the biological-abiotic reactions may contribute to natural attenuation of both

260

explosives and IM. These findings are the basis for future remediation strategies at sites where

261

several explosives are present in the soil and groundwater, and where active Fe(III)-reducing

262

microorganisms can mediate electron transfer to contaminants. The fate of the terminal amine

263

product, DAAN, is unknown. DAAN has been reported to form dimers.12,13 The size of the

264

dimers contributes to their low solubility in water, which would result in their precipitation and

265

immobilization – an important attenuation mechanism even if DAAN is not further

266

transformed.12,13

268

267

12


Page 12 of 33

Deleted:

Page 13 of 33

269


FIGURES

270 271

Figure 1. DNAN reduction by ferrous iron from pH 6.0 to 9.0. Experimental bottles were

272

buffered with 30mM MES, HEPES, and CHES buffers at pH 6.0, 7.0-8.0, and 9.0 respectively.

273

DNAN was amended at 100 µM and initial Fe(II) concentration was 1.5 mM. Bottles were not

274

mixed. Results are the mean of triplicate incubations; bars indicate one standard deviation.

275

13



276

Page 14 of 33

284

277

Figure 2. Products of DNAN degradation by 1.5 mM ferrous iron at pH 7 (A), pH 8 (C), and pH

278

9 (E) in short timeframe experiments (left panels); and by 1.2mM ferrous iron at pH 7 (B), pH 8

279

(D), and pH 9 (F) in longer timeframe experiments (right panels). Primary y-axis shows

280

concentration of analytes: DNAN, MENA, iMENA, and DAAN. The secondary y-axis in plots

281

A, C, and E shows the peak area of 2-HA-4-NAN as there are no certified standards of the

282

intermediate 2-HA-4-NAN to compare with the amounts detected in experimental samples. In

283

short timeframe experiments (A, C, and E) bottles were buffered with 30 mM HEPES at pH 7

14


Deleted:

Page 15 of 33


285

and 8, and with 30 mM CHES at pH 9, and they were mixed on a rotary shaker. In longer

286

timeframe experiments (B, D, and F) bottles were buffered with 30 mM HEPES for pH 7-9.

287

Control suspensions were not shown in these plots, but DNAN (alone) did not deviate at any pH

288

over the timeframe of the experiments (shown in Supporting Information Figure S2). Results are

289

the mean of triplicate incubations; bars indicate one standard deviation.

290

291 292 293

Figure 3. DNAN reductive degradation pathway. Complete nitro reduction of 1 mol of DNAN to

294

1 mol of DAAN requires 12 electrons.

295 296

15



297 298

Figure 4. DNAN degradation by 600 µM AH2QDS at pH 7, buffered with 30 mM HEPES.

299

Primary y-axis shows concentration of analytes: DNAN, MENA, iMENA, and DAAN. The

300

secondary y-axis shows the peak area of 2-HA-4-NAN. Results are the mean of triplicate

301

incubations; bars indicate one standard deviation.

16


Page 16 of 33

Page 17 of 33


302 303

Figure 5. DNAN degradation in the cell suspension of GS-15 alone (A), with acetate (B), with acetate and AQDS (C), with acetate

304

and poorly crystalline Fe(III) (FeGel) (D), with acetate, AQDS, and FeGel (E), with acetate and soluble Fe(III) (FeCit) (F).

305

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.

17



306

Additionally, panels D-F show Fe(II) generated in the incubations. Results are the mean of triplicate incubations; bars indicate one

307

standard deviation.

Page 18 of 33

18


Page 19 of 33

308


TABLES.

309

19



Page 20 of 33

310

20


Page 21 of 33


311

ASSOCIATED CONTENT

312

Supporting Information includes reaction equations, degradation rates and additional

313

experimental data that supports findings presented in this article.

314

AUTHOR INFORMATION

315

Corresponding Author

316

*Phone: 864-656-4143, e-mail: [email protected]

317

Present Addresses

318

†

319

Sylwestra Kaliskiego 2 str, 00-908 Warsaw, Poland

320

‡

321

Boston Avenue, Lubbock, TX 79409

322

Author Contributions

323

The manuscript was written through contributions of all authors. All authors have given approval

324

to the final version of the manuscript.

325

Funding Sources

326

This work was supported by the Department of Defense Strategic Environmental Research and

327

Development Program (SERDP), project number ER-2222.

328

ACKNOWLEDGMENTS

329

We thank Kelly Nevin of the University of Massachusetts at Amherst for the original Geobacter

330

metallireducens culture.

Faculty of Advanced Technologies and Chemistry, Military University of Technology, Gen.

Civil, Environmental, and Construction Engineering, Texas Tech University, Box 41023, 911

21



331

REFERENCES

332

(1)

Fung, V.; Newland S. A modernized IM melt pour explosive manufacturing facility at

333

Holston Army Ammunition Plant. Insensitive Munitions Energ. Mater. Technol. Symp.

334

2015, No. May, 1–10.

335

(2)

Trzciński, W. A.; Cudziło, S.; Dyjak, S.; Nita, M. A comparison of the sensitivity and

336

performance characteristics of melt-pour explosives with TNT and DNAN binder. Cent.

337

Eur. J. Energ. Mater. 2014, 11 (3), 443–455.

338

(3)

Fung, V.; Morris, J.; Price, D.; Tucker, N.; Carrillo, A.; Leclaire, E. Holston Army

339

Ammunition Plant 2010 Insensitive Munitions & Energetic Material Technology

340

Symposium IM Melt-Pour Formulations Development. 2010, 1–16.

341

(4)

342 343

Davies, P. J.; Provatas, A. Characterisation of 2,4-Dinitroanisole: An Ingredient for use in Low Sensitivity Melt Cast Formulations. 2006, No. Im, 21.

(5)

Dodard, S. G.; Sarrazin, M.; Hawari, J.; Paquet, L.; Ampleman, G.; Thiboutot, S.;

344

Sunahara, G. I. Ecotoxicological assessment of a high energetic and insensitive munitions

345

compound: 2,4-Dinitroanisole (DNAN). J. Hazard. Mater. 2013, 262, 143–150.

346

(6)

Liang, J.; Olivares, C.; Field, J. A.; Sierra-Alvarez, R. Microbial toxicity of the insensitive

347

munitions compound, 2,4-dinitroanisole (DNAN), and its aromatic amine metabolites. J.

348

Hazard. Mater. 2013, 262, 281–287.

349

(7)

Ahn, S. C.; Cha, D. K.; Kim, B. J.; Oh, S. Y. Detoxification of PAX-21 ammunitions

350

wastewater by zero-valent iron for microbial reduction of perchlorate. J. Hazard. Mater.

351

2011, 192 (2), 909–914.

352 353

(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

22


Page 22 of 33

Page 23 of 33


354

DNAN and NTO in Rana Pipiens tadpoles. Environ. Toxicol. Chem. 2015, 34 (4), 873–

355

879.

356

(9)

Hoyt, N.; Brunell, M.; Kroeck, K.; Hable, M.; Crouse, L.; O’Neill, A.; Bannon, D. I.

357

Biomarkers of oral exposure to 3-nitro-1,2,4-triazol-5-one (NTO) and 2,4-dinitroanisole

358

(DNAN) in blood and urine of rhesus macaques ( Macaca mulatta ). Biomarkers 2013, 18

359

(7), 587–594.

360

(10)

Brecken-Folse, J. A.; Mayer, F. L.; Pedigo, L. E.; Marking, L. L. Acute toxicity of 4-

361

nitrophenol, 2,4-dinitrophenol, terbufos and trichlorfon to grass shrimp (Palaemonetes

362

spp.) and sheepshead minnows (Cyprinodon variegatus) as affected by salinity and

363

temperature. Environ. Toxicol. Chem. 1994, 13 (1), 67–77.

364

(11)

Grundlingh, J.; Dargan, P. I.; El-Zanfaly, M.; Wood, D. M. 2,4-Dinitrophenol (DNP): a

365

weight loss agent with significant acute toxicity and risk of death. Journal of Medical

366

Toxicology. 2011, pp 205–212.

367

(12)

Olivares, C. I.; Abrell, L.; Khatiwada, R.; Chorover, J.; Sierra-Alvarez, R.; Field, J. A.

368

(Bio)transformation of 2,4-dinitroanisole (DNAN) in soils. J. Hazard. Mater. 2016, 304,

369

214–221.

370

(13)

Hawari, J.; Monteil-Rivera, F.; Perreault, N. N.; Halasz, A.; Paquet, L.; Radovic-

371

Hrapovic, Z.; Deschamps, S.; Thiboutot, S.; Ampleman, G. Environmental fate of 2,4-

372

dinitroanisole (DNAN) and its reduced products. Chemosphere 2015, 119, 16–23.

373

(14)

Fida, T. T.; Palamuru, S.; Pandey, G.; Spain, J. C. Aerobic biodegradation of 2,4-

374

dinitroanisole by Nocardioides sp. strain JS1661. Appl. Environ. Microbiol. 2014, 80 (24),

375

7725–7731.

376

(15)

Richard, T.; Weidhaas, J. Biodegradation of IMX-101 explosive formulation constituents:

23



377

2,4-Dinitroanisole (DNAN), 3-nitro-1,2,4-triazol-5-one (NTO), and nitroguanidine. J.

378

Hazard. Mater. 2014, 280, 372–379.

379

(16)

Richard, T.; Weidhaas, J. Dissolution, sorption, and phytoremediation of IMX-101

380

explosive formulation constituents: 2,4-dinitroanisole (DNAN), 3-nitro-1,2,4-triazol-5-one

381

(NTO), and nitroguanidine. J. Hazard. Mater. 2014, 280, 561–569.

382

(17)

383 384

Schroer, H. W.; Langenfeld, K. L.; Li, X.; Lehmler, H. J.; Just, C. L. Biotransformation of 2,4-dinitroanisole by a fungal Penicillium sp. Biodegradation 2017, 28 (1), 95–109.

(18)

Schroer, H. W.; Langenfeld, K. L.; Li, X.; Lehmler, H. J.; Just, C. L. Stable isotope-

385

enabled pathway elucidation of 2,4-dinitroanisole metabolized by Rhizobium lichtii.

386

Environ. Sci. Technol. Lett. 2015, 2 (12), 362–366.

387

(19)

Platten, W. E.; Bailey, D.; Suidan, M. T.; Maloney, S. W. Biological transformation

388

pathways of 2,4-dinitro anisole and N-methyl paranitro aniline in anaerobic fluidized-bed

389

bioreactors. Chemosphere 2010, 81 (9), 1131–1136.

390

(20)

Koutsospyros, A.; Pavlov, J.; Fawcett, J.; Strickland, D.; Smolinski, B.; Braida, W.

391

Degradation of high energetic and insensitive munitions compounds by Fe/Cu bimetal

392

reduction. J. Hazard. Mater. 2012, 219–220, 75–81.

393

(21)

Perreault, N. N.; Manno, D.; Halasz, A.; Thiboutot, S.; Ampleman, G.; Hawari, J. Aerobic

394

biotransformation of 2,4-dinitroanisole in soil and soil Bacillus sp. Biodegradation 2012,

395

23 (2), 287–295.

396

(22)

Khan, F.; Pal, D.; Ghosh, A.; Cameotra, S. S. Degradation of 2,4-dinitroanisole (DNAN)

397

by metabolic cooperative activity of Pseudomonas sp. strain FK357and Rhodococcus

398

imtechensis strain RKJ300. Chemosphere 2013, 93 (11), 2883–2888.

399

(23)

Kwon, M. J.; Finneran, K. T. Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) and

24


Page 24 of 33

Page 25 of 33


400

Octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) biodegradation kinetics amongst

401

several Fe(III)-reducing genera. Soil Sediment Contam. 2008, 17, 189–203.

402

(24)

Kwon, M. J.; Finneran, K. T. Biotransformation products and mineralization potential for

403

hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) in abiotic versus biological degradation

404

pathways with anthraquinone-2,6-disulfonate (AQDS) and Geobacter metallireducens.

405

Biodegradation 2008, 19 (5), 705–715.

406

(25)

Kwon, M. J.; Finneran, K. T. Microbially mediated biodegradation of hexahydro-1,3,5-

407

trinitro-1,3,5- triazine by extracellular electron shuttling compounds. Appl. Environ.

408

Microbiol. 2006, 72 (9), 5933–5941.

409

(26)

410 411

sediments. Appl. Environ. Microbiol. 1987, 53 (7), 1536–1540. (27)

412 413

Lovley, D. R.; Phillips, E. J. P. Rapid assay for microbially reducible ferric iron in aquatic

Sung, W.; Morgan, J. J. Kinetics and product of ferrous iron oxygenation in aqueous systems. Environ. Sci. Technol. 1980, 14 (5), 561–568.

(28)

Gregory, K. B.; Larese-Casanova, P.; Parkin, G. F.; Scherer, M. M. Abiotic

414

transformation of hexahydro-1,3,5-trinitro-1,3,5-triazine by FeII bound to magnetite.

415

Environ. Sci. Technol. 2004, 38 (5), 1408–1414.

416

(29)

Morgan, B.; Lahav, O. The effect of pH on the kinetics of spontaneous Fe(II) oxidation by

417

O2 in aqueous solution - basic principles and a simple heuristic description. Chemosphere

418

2007, 68 (11), 2080–2084.

419

(30)

Smith, J. A.; Lovley, D. R.; Tremblay, P. L. Outer cell surface components essential for

420

Fe(III) oxide reduction by Geobacter metallireducens. Appl. Environ. Microbiol. 2013, 79

421

(3), 901–907.

422

(31)

Kim, D.; Strathmann, T. J. Role of organically complexed iron(II) species in the reductive

25



423

transformation of RDX in anoxic environments. Environ. Sci. Technol. 2007, 41 (4),

424

1257–1264.

425

26


Page 26 of 33

Page 27 of 33


Figure 1. DNAN reduction by ferrous iron from pH 6.0 to 9.0. Experimental bottles were buffered with 30mM 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. 82x59mm (300 x 300 DPI)



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 timeframe experiments (left panels); and by 1.2mM ferrous iron at pH 7 (B), pH 8 (D), and pH 9 (F) in longer timeframe 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 timeframe 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 timeframe 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 timeframe of the experiments (shown in Supporting Information Figure S2). Results are the mean of triplicate incubations; bars indicate one standard deviation. 177x157mm (96 x 96 DPI)


Page 28 of 33

Page 29 of 33


Figure 3. DNAN reductive degradation pathway. Complete nitro reduction of 1 mol of DNAN to 1 mol of DAAN requires 12 electrons. 82x56mm (300 x 300 DPI)



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 2HA-4-NAN. Results are the mean of triplicate incubations; bars indicate one standard deviation. 82x53mm (300 x 300 DPI)


Page 30 of 33

Page 31 of 33


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. 228x121mm (300 x 300 DPI)



Page 32 of 33

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

Short term mass balance (%) Compound C7H6N2O5 (DNAN) C7H8N2O3 (MENA) C7H8N2O3 (iMENA) C7H10N2O (DAAN) total

Long term mass balance (%)

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

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 27.02 3.91 0.59 40.23

0.39 6.68 1.62 0.97 9.66

0.40 1.42 0.34 3.56 5.72

0.00 53.58 53.58

0.26 12.69 63.74 76.69

0.01 1.04 91.58 92.63

Mass balance (%) Compound C7H6N2O5 (DNAN) C7H8N2O3 (MENA) C7H8N2O3 (iMENA) C7H10N2O (DAAN) total

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 a24 h, b1 h, c2 min, d96 h, e24 h, f52 min, g30 h.


Page 33 of 33


TOC Art 254x190mm (72 x 72 DPI)


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

Recommend Documents