Isotopic evidence and quantification assessment of in situ RDX biodegradation in the deep unsaturated zone

Soil Biology & Biochemistry 42 (2010) 1253e1262

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Isotopic evidence and quantification assessment of in situ RDX biodegradation in the deep unsaturated zone S. Sagi-Ben Moshe a, Z. Ronen b, *, O. Dahan b, A. Bernstein b,1, N. Weisbrod b, F. Gelman c, E. Adar b a

Dept. of Soil & Water Sciences, The Hebrew University of Jerusalem, P.O. Box 12, Rehovot 76100, Israel Dept. of Environmental Hydrology & Microbiology, Zuckerberg Institute for Water Research, Ben-Gurion University of the Negev, Sede Boqer 84990, Israel c Geological Survey of Israel, 30 Malkhey Israel, Jerusalem 95501, Israel b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 6 September 2009 Received in revised form 17 April 2010 Accepted 19 April 2010 Available online 5 May 2010

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is an explosive compound whose extensive use has resulted in significant contamination of soils and groundwater worldwide. We studied its in situ biodegradation along the unsaturated zone beneath an explosives wastewater lagoon using compoundspecific isotope analysis (CSIA) of RDX in the unsaturated zone, together with biodegradation slurry experiments under anaerobic conditions. We found the highest degradation potential of RDX and its nitroso derivatives in the upper part of the soil profile while in the lower parts, RDX-degradation potential was lower and the nitroso derivatives tended to persist. This was also observed in the field, as reflected by the isotopic composition of RDX along the profile. We also found a correlation between biodegradation potential and clay content: biodegradation was further enhanced in layers characterized by high-clay content or in those influenced by the high-clay layers. In addition, in the presence of high organic matter content, further enhancement of biodegradation was observed. We obtained different isotopic enrichment factors (3) for RDX biodegradation in different sections of the unsaturated profile and suggest that different degradation pathways exist simultaneously in situ, in variable proportions. Using the range of enrichment factors, we were able to assess the biodegradation extent of RDX at different sampling points along the profile, which ranged between 30 and 99.4%. The novel application of CSIA together with slurry experiments provides better insight into degradation processes that are otherwise difficult to detect and assess. Ó 2010 Elsevier Ltd. All rights reserved.

This work is dedicated to the memory of Ronit Nativ who passed away on October 31st, 2006. Keywords: RDX Biodegradation Contamination Compound-specific isotope analysis Unsaturated zone

1. Introduction Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is an explosive that has been used extensively over the years, resulting in significant contamination of soils and groundwater, especially adjacent to explosives-manufacturing plants and in military training areas (Pennington and Brannon, 2002). Due to its toxicity, the fate of this compound in natural environments and water resources is of great interest. Volatilization of RDX is negligible, as reflected by its low vapor pressures and Henry’s constants (Townsend and Myers, 1996). In practice, sorption of RDX is often considered to be negligible due to its low sorption affinity to soil minerals and soil organic matter

* Corresponding author. Tel.: þ972 7 659 6895; fax: þ972 8 659 6909. E-mail address: [email protected] (Z. Ronen). 1 Current address: Helmholtz Zentrum München, German Research Center for Environmental Health, Institute of Groundwater Ecology, Ingolstädter Landstr. 1, 85764 Neuherberg, Germany. 0038-0717/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.soilbio.2010.04.011

(Sheremata et al., 2001; Pennington and Brannon, 2002). On the other hand, biodegradation of RDX appears to be a major mechanism attenuating the pollutant in contaminated soil and water. Different biodegradation pathways of RDX have been studied, and it has been shown to occur under both aerobic and anaerobic conditions (Hawari et al., 2000a), with the anaerobic process being significantly faster. Under anaerobic degradation, sequential reduction of RDX is often documented, with the sequential formation of mono-, diand trinitroso derivatives: hexahydro-1-nitroso-3,5-dinitro-1,3,5triazine (MNX), hexahydro-1,3-dinitroso-5-nitro-1,3,5-triazine (DNX), and hexahydro-1,3,5-trinitroso-1,3,5-triazine (TNX). The nitroso compounds can further transform to unstable hydroxylamine derivatives, followed by ring cleavage (McCormick et al., 1981; Hawari et al., 2000a; Crocker et al., 2006). Alternatively, RDX can be cleaved enzymatically, with the formation of methylenedinitramine (MEDINA) and bis(hydroxymethyl)nitramine (Hawari et al., 2000b). Aerobic degradation of RDX proceeds via a denitration reaction, in which the nitroso derivatives are not detected. This pathway

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leads to the formation of 4-nitro-2,4-diazabutanal (NDAB) (Fournier et al., 2002, 2004; Bhushan et al., 2003), which can be further mineralized (Fournier et al., 2004). It has also been shown that Escherichia coli expressing the XplA/B genes responsible for aerobic RDX degradation degrade RDX through MEDINA when incubated anaerobically (Jackson et al., 2007). In Israel, RDX contamination of soil and groundwater has been detected near explosives-manufacturing plants located above the central part of the Israeli coastal aquifer (Nativ and Adar, 2005). Natural attenuation has been proposed as a strategy to deal with this contamination: it is a cost-efficient method for the remediation of contaminated sites, used as a sole remediation process or combined with other processes, which should be accompanied by monitoring (Mulligan and Yong, 2004). Though the potential for RDX biodegradation in soils has been detected (Nejidat et al., 2008; Ronen et al., 2008; Sagi-Ben Moshe et al., 2009), evidence for in situ occurrence in the field is lacking and its extent is unknown. Attempts to quantify biodegradation extents in the environment involve unique difficulties. As precise knowledge of the amount and timing of contaminant release to the environment is often unavailable, it is difficult to establish complete mass balance for calculating the extent of biodegradation. Monitoring the concentrations of the degradation products may also be insufficient, since the investigated compound may follow various different degradation pathways and therefore form different degradation products. These products may be completely mineralized and therefore undetectable. We faced both difficulties when attempting to quantify RDX-biodegradation extent along the contaminated soil profile studied here. This soil profile, which retains high quantities of RDX, endangers the underlying aquifer. Assessing the extent of biodegradation and identifying factors that may promote this process have therefore been established as important goals. A relatively new methodology for quantifying biodegradation in a complex environment is the use of compound-specific isotope analysis (CSIA). This method takes advantage of the isotopic enrichment that commonly accompanies enzymatic processes. Since a chemical bond formed by a lighter isotope is weaker than that formed by a heavier isotope, it is more rapidly cleaved (Meckenstock et al., 2004). This leads to isotopic enrichment as degradation proceeds of the residual compound which has not yet been degraded. The extent of this enrichment is typical for the degradation pathway, and is characterized by a specific enrichment factor (Meckenstock et al., 2004; Schmidt et al., 2004; Elsner et al., 2005). Isotopic enrichment of a pollutant at a contaminated field site can therefore be used to verify the occurrence of degradation in the subsurface, and using an experimentally determined enrichment factor, the extent of biodegradation can be quantified. This concept has been successfully applied in the last decade to assess biodegradation of various environmental contaminants, e.g., benzene, toluene, and o-xylene (Mak et al., 2006), methyl tertiary butyl ether (MTBE) (McKelvie et al., 2007), and chlorinated hydrocarbons (Morrill et al., 2005). This concept has also been applied to study RDX biodegradation in the contaminated groundwater underneath the studied research site (Bernstein et al., 2009), but has never been applied to the unsaturated, RDXcontaminated soil profile. Because the unsaturated soil can continuously contribute to the contamination of the underlying groundwater, it is important to assess the extent of in situ biodegradation in the soil. This can lead to effective management of the site and reduce the cost of remediation. Therefore, the objective of this research was to study the natural attenuation of RDX along the deep unsaturated zone using both biodegradation experiments and isotopic tools.

2. Materials and methods 2.1. Site description The contaminated site is located near explosives-manufacturing plants above the coastal plain of Israel. From 1964 to 1982, and then to a much lesser extent until the year 2000, untreated industrial wastewater characterized by relatively high concentrations of RDX were disposed of in two unlined wastewater ponds and nearby creeks as part of manufacturing operations. Since then, the effluent ponds have not received any industrial discharge. Nevertheless, the ponds have been subjected to rain water and local runoff accumulation during the winter rainy seasons (average of 500 mm y1), which could promote further transport of RDX along the soil profile. Largescale site characterization and monitoring operations at the site revealed high concentrations of RDX, octahydro-1,3,5,7-tetranitro1,3,5,7-tetrazocine (HMX), 2,4,6-trinitrotoluene (TNT) and their degradation products underneath these effluent ponds, mainly in the upper soil layer (up to 2 m below the surface). However, the contamination by these compounds was not unique to the upper layers, and lower concentrations, mainly of RDX were found along the ca. 45-m thick vadose zone in this area (Nativ and Adar, 2005). Moreover, high concentrations of RDX were found in the underlying groundwater in an w1.35-km long contamination plume with peak concentrations of up to 2100 mg l1 (Nativ and Adar, 2005). The vadose zone at the site is comprised of sand, loam and clay layers and the water table is situated 45 m below land surface (Fig. 1). A dry drilling method, using flight augers for drilling and a bucket auger for sampling, was used to collect sediment samples from the entire 46-m vadose zone. Sediment samples (w2 kg) were collected into pre-cleaned glass jars and kept on ice. Samples were collected at 0.2-m intervals in the upper 3 m and at 1-m intervals along the rest of the vadose zone to 45 m. The last sample from 46 m was taken from the saturated zone below the pond. This sampling was carried out in March 2008, 8 years after the ponds had ceased to be operational. To determine the explosives concentrations along the profile, soil samples were extracted in methanol using Accelerated Solvent Extraction (ASE-200, Dionex Corporation, Sunnyvale, CA) according to the method of Ronen et al. (2008). The extraction efficiency was tested and found to be 106.86  10.36, 97.83  4.6 and 106.46  8.45% for HMX, RDX and TNT, respectively (n ¼ 4, average  SD). Extracts were analyzed for explosive concentrations by HPLC (Agilent 1100 series, Palo AltoCA) (see Section 2.5). Water content in the soil samples was determined gravimetrically by comparing sample weight relative to its weight after drying for 24 h at 105  C. Particle size was analyzed by sieving and hydrometer method for sand, silt and clay content (Sheldrick and Wang, 1993). Organic material was determined using the Walkley and Black methods (Nelson and Sommers, 1996). 2.2. Chemicals RDX (>95% purity, Sagi-Ben Moshe et al., 2009) was used for batch biodegradation experiments. Analytical standard for RDX was purchased from Supelco (Bellefonte, PA). Analytical standards for MNX, DNX, TNX, as well as NDAB and MEDINA, were prepared from solid powder purchased from SRI International (Menlo Park, CA). TNT and HMX standards for HPLC were prepared from solid powder (>95% pure, Sagi-Ben Moshe et al., 2009). Methanol and acetone were HPLC-grade, and all other chemicals were reagent-grade. 2.3. Biodegradation experiment The biodegradation experiments had two objectives: (1) to compare the anaerobic RDX-biodegradation potential by the

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15.5-21.5

Unconsolidated fine yellow sand

achieve anaerobic conditions while the internal air in the headspace was flushed out. Then, aqueous samples were taken to determine the initial RDX concentrations. The pH value in the slurry was 7.1  0.3. The biodegradation experiment was performed in triplicates, each triplicate having sediments from a distinct soil layer. A control bottle with sterile sediments was used to assess the significance of any abiotic processes. Sediments for sterile controls were autoclaved twice at 121  C for 30 min on two successive days. The bottles with the slurry were incubated statically in the dark at 23  C and were sampled on a daily basis for concentration analysis. Samples at ca. 0, 50, 75, 90 and 95% degradation were also taken for isotope analysis. The sample volume for isotopic analysis was determined according to RDX concentration, assuring a minimal mass of 300 mg RDX in each sample. This implied that the volume of the first samples was on the scale of a few milliliters, whereas the volume of the last samples was on the scale of a few hundred milliliters. After each sampling, nitrogen was re-purged for 10 min and nitrogen headspace was left in order to maintain anaerobic conditions.

21.5-27

Well consolidated brown sandy clay

2.4. Sample preparation for isotope analysis

27-28.5

Low consolidated brown clayey sand

28.5-29

Well consolidated gray sandy clay

29-35.5

Unconsolidated fine brown sand

35.5-38.5

Unconsolidated fine brown sand with

= Sample location for chemical analysis: first 3m - every 0.2 m 3m-46m - every 1 m

= Groundwater level Meters bgs

0

0.60 0.80 1.80 2.20

Depth (m)

Sediment description

0-0.6

Low consolidated brown clayey sand with

3 6

organic materials 6.50

9

0.6-0.8

Well consolidated brown clay

0.8-1.8

Unconsolidated fine white sand

1.8-2.2

Semi-consolidated sandy clay

2.2-6.5

Low consolidated fine orange clayey sand

6.5-14.5

Unconsolidated fine yellow sand

14.5-15.5

Unconsolidated fine yellow sand with

12 15

14.5 15.5

18 21

1255

little kurkar 21.5

24 27 30

27.0

29.0

33 36

35.5

kurkar 39

38.5 40.5

42 45

38.5-40.5

Semi-consolidated red-brown sandy clay

40.5-46

Low consolidated orange clayey sand

44.9 46.0

48 Fig. 1. The vadose zone lithological profile as obtained through borehole drilling and description of each soil layer. bgs: below ground surface.

indigenous microorganisms in different soil layers. Different soil layers were identified along the profile, and therefore experiments were performed in slurries from several distinct soil layers; (2) to determine the isotopic enrichment factors representing RDX biodegradation by the native microbial population present in these soil layers. Sediments for the biodegradation experiment were excavated from five depths, each representing a different soil layer: 0e0.5 m, 11 m, 22 m, 40 m and 46 m below the surface of the studied infiltration pond. RDX (24 mg), dissolved in acetone, was added to each 1-l sterilized dark serum bottle and served as the sole external nitrogen source. The acetone was then allowed to freely evaporate for 15 min before adding 1 l of nitrogen-free sterile mineral solution (Ronen et al., 1998). Filtered (0.22 mm) molasses (1 g l1 as carbon) was used as the external carbon source. After the nutrient mineral solution was added, the resultant solution was left for 24 h on a stirring magnetic plate, until equilibration of the explosives was achieved. Soil samples from the different soil layers (non-dried, 40 g, with gravimetric water content of 8e20%) were then suspended in the nitrogen-free nutrient mineral solution in the serum bottles. The bottles were sealed with a rubber stopper and then purged with filtered (0.45 mm) nitrogen (99%) for 30 min in order to

To determine the isotopic composition of RDX along the soil profile, 0.5 kg of soil from each of the field samples was suspended in 1.5 l double-distilled water and shaken on a rotary shaker for 24 h. The pH value in these aqueous solutions was 6.4  1.2. The suspended sediments were allowed to settle and the supernatants were centrifuged to further reduce suspended solids. RDX was extracted from the clear supernatant using 47-mm SDB-RPS solidphase extraction disks (Empore 3M, St Paul, MN) as described in Bernstein et al. (2008). For elution, the disks were extracted three sequential times with dichloromethane (DCM) (15 ml each time). The collected DCM solution was reduced to 0.75 ml under nitrogen at a pressure of up to 30 psi and temperature of 30  C (TurboVap Zymark, Hopkinton, MA). Samples from the biodegradation experiments were analyzed for isotopic composition as well. Samples containing a minimal mass of 300 mg RDX were centrifuged and then RDX was extracted into DCM by liquideliquid extraction following three sequential cycles. The amount of DCM used for the liquideliquid extraction depended on the volume of the aqueous sample: a DCM volume of 10 ml was added to low-volume samples (<50 ml) and was increased to up to 50 ml with high-volume samples (>1 l). The collected DCM solution was reduced to 0.75 ml under nitrogen at up to 30 psi and 30  C. The RDX in the DCM extracts from the field samples and from the biodegradation experiments was separated from other compounds using thin-layer chromatography, and collected in tin capsules for liquids (Hekatech, Wegberg, Germany) as described by Bernstein et al. (2008). 2.5. Analytical methods Concentrations of the following explosive compounds were analyzed by HPLC (Agilent 1100 series, Agilent Technologies, Inc., Santa Clara, CA) according to EPA method 8330 (EPA, 1994): TNT, 2-Am-4,6-DNT, 4-Am-2,6-DNT, RDX, MNX, DNX, TNX and HMX. Detection limit in the soil extracts and water samples was 0.05 mg l1. NDAB and MEDINA were determined by the same HPLC apparatus using a Synergi 4 m Hydro-RP column (250  4.6 mm, Phenomenex, Torrance, CA), a mobile phase consisting of 20 mM KH2PO4, pH 3.4, and a flow rate of 0.8 ml min1. NDAB was detected at 210 nm and MEDINA was analyzed at 225 nm.

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2.6. Isotope analysis

3. Results

d15N analysis was performed using a Flash EA 1112 (Thermo Finnigan, Milano, Italy) elemental analyzer (EA) interfaced to a Delta V Plus (Thermo Fisher Scientific, Bremen, Germany) isotope-ratio mass spectrometer (IRMS). Samples were combusted at 1020  C in a reactor packed with chromium oxide and silvered cobaltous/ cobaltic oxide. A reduction reactor filled with reduced copper was held at 650  C. A post-reactor gas chromatography (GC) column was kept at 50  C. International standards USGS-40, USGS-41, IAEA N-1 and IAEA N-2 were used for d15N calibration. In addition, two internal laboratory standards (urea and glycine), calibrated against international standards, were analyzed every five samples to correct for instrument drift.

3.1. The soil profile and explosives concentrations along it

2.7. Calculations of isotopic enrichment The isotope analysis provided the isotopic composition of 15N in RDX in the measured sample relative to the isotopic composition of an international standard. This isotope ratio, d, is measured in per mil units (&) and is defined as




d15 Nsample ¼

 Rsample  Rstd 1000 Rstd

(1)

where d15Nsample is the isotopic ratio of the RDX in the investigated sample, and Rsample and Rstd are the ratios of heavy to light isotopes in the investigated RDX sample and in the international standard, respectively. Rayleigh’s distillation approach was adopted to calculate the isotopic enrichment during the biodegradation process. As biodegradation proceeds, enrichment of the heavy isotopes is expected in the residual compounds. Accordingly, the relative enrichment in RDX at time t relative to time 0 can be defined as

Rsample;t ¼ Rsample;0

d15 Nt 1000

!, þ1

d15 N0 1000

! þ1

(2)

and the ratio between the degradation extent and isotopic composition of the investigated RDX sample can be expressed as

Rsample;t ln Rsample;0

! ¼ ða  1Þln

Ct C0

The general lithological profile at the site (Fig. 1) exhibits three pronounced clay layers, at depths of 0.6e0.8 m, 1.8e2.2 m and 21.5e27 m. The clay layers are composed mainly of illiteesmectite, and clay content in these layers is 29, 50 and 20%, respectively. A sandy layer with very little clay content (2%) was detected between 6.5 and 21.5 m. Other soil layers were determined as clayey sands. The explosives concentration along the unsaturated zone was normally lower than 2 mg kg1, with one exception in the 2 m sample, which contained 26.14 mg kg1 of RDX (Fig. 2). Converting those concentration to mg l1 in soil pore water showed that this sample contains undissolved RDX, as its concentration was above its solubility values (108 mg l1 was detected for RDX, whereas its solubility in water is about 40 mg l1). All other samples (51 out of 52 samples) had RDX concentrations that were below solubility limits, with maximal concentrations of ca. 40% of their solubility limits. RDX concentrations together with the water content of the soil samples implied that even during periods of extreme dryness of the soil profile, RDX is expected to remain in the aqueous phase and not precipitate. The aerobic degradation product NDAB was not detected in any of the soil samples. The anaerobic degradation products, on the other hand, were indeed detected, especially in the uppermost 2 m and at depths of 21e29 m (Fig. 2). 3.2. d15N in N-RDX

d15N values of RDX along the unsaturated zone were measured in order to acquire evidence for in situ biodegradation in the subsurface (Fig. 3). For comparison, RDX produced at this industrial site was used as a reference. The d15N composition of this reference material ranged between 9.95 and 10.78&. The d15N values of RDX along the entire unsaturated zone were enriched relative to d15N values measured in RDX produced at the industrial site. One single exception was detected in the sample taken 2 m below ground surface, in which the d15N measured value, 10.38&, was not different from the value of the RDX produced at the site.

(3) -1

3 ¼ ða  1Þ1000

(4)

MNX, DNX and TNX Concentrations (mg kg dry soil) 0.0

.2

.4

.6

.8

0

RDX TNX MNX DNX

10

Depth (m)

where C0 and Ct are the compound’s concentrations at time 0 and time t, respectively, and a is the isotopic fractionation constant. This constant can be obtained by plotting the natural logarithm of the isotopic enrichment, (Rsample,t/Rsample,0), against the natural logarithm of the remaining fraction f (or Ct/C0). The linear slope of the obtained curve is thus (a  1). Enrichment factors (3) were determined for the degradation in different soil layers, expressed as

20

30

2.8. Statistical analysis A correlation matrix was generated for the different soil parameters using Prism software (GraphPad Software, Inc. La Jolla, CA). Using the same software, we analyzed the biodegradation experiment using a non-linear regression model composed of an initial lag phase (plateau) followed by exponential decay. We compared both lag times and degradation rates among the different treatments (depths).

40

50 0.0

.5

1.0

1.5

22.0

24.0

26.0

-1

RDX Concentration (mg kg dry soil) Fig. 2. Concentrations of RDX and its degradation products in the unsaturated zone.

S. Sagi-Ben Moshe et al. / Soil Biology & Biochemistry 42 (2010) 1253e1262 0

Depth (m)

10

20

30

40

50 -10

-8

-6

-4

-2

0

2

δ15N (‰) Fig. 3. Values of d15N in RDX molecules along the unsaturated zone profile.

Degradation products (RDX+deg. products) 0.0

.2

.4

.6

.8

1.0 Meters bgs

1 δ• 15 • N (‰) dN15(%) deg. prod (RDX+deg. prod)

10

0

0.60 0.80 1.80 2.20

3 6

6.50

9 12

Depth (m)

2 20

15

30

14.5 15.5

18 21

3

21.5

24 27 30

27.0

29.0

33

4

36 39

40

35.5

38.5 40.5

42 45

5

44.9 46.0

48

50 -10

-8

-6

-4

-2

0

Correlations between the soil parameters (Table S1) and concentrations of RDX and its degradation products and d15N composition are presented in Table S2. The only significant correlations found were for degradation product concentration and d15N in RDX with soil organic carbon (p < 0.01). To explain the observed d15N composition of RDX along the profile, we divided the unsaturated zone into five sections according to soil texture, RDX isotopic composition and DPR (Fig. 4). In doing so, we observed the most significant RDX enrichment in the upper soil layer, just above the first two clayey layers. In the third clayey layer, at 21.5e27 m depth, somewhat less enrichment of RDX was observed. The d15N values in the second and fourth sections, characterized as sandy or clayey sands, were almost constant with respect to RDX isotopic composition, with a little scattering around an average value of 8.76& in the second section and an average of 7.76& in the fourth section. RDX enrichment was also observed in the sample taken from 46 m, representing the saturated zone beneath the pond (fifth profile section). 3.3. Biodegradation experiment

The most enriched value of d15N in RDX, corresponding to the highest fraction of biodegraded RDX, was detected in the upper soil layer (d15N ¼ þ0.2&). The d15N values along the rest of the profile were more depleted, ranging between 1.9& and 9.4&. To gain information on the factors that may promote anaerobic biodegradation along the profile, the ratio between the concentrations of the degradation products (MNX, DNX and TNX) and the concentrations of RDX plus its degradation products was calculated for the different soil samples. This value, referred to as the “degradation products ratio” (DPR), provides chemical evidence for the extent of biodegradation. Similar patterns could be observed in the trends of the DPR and the d15N composition of RDX along the unsaturated profile (Fig. 4). Thus, a high fraction of degradation products was found concurrently in the profile sections in which d15N -enriched RDX was observed. In addition, the RDX degradation products were mostly detected in samples that contained a highclay fraction or in samples that were under the latter’s immediate influence in terms of water content.

0

1257

2

δ•N15 • N(‰) (‰) •dN15 •15 (‰) Fig. 4. d15N of RDX and degradation products ratio (DPR) along the unsaturated zone profile. Circled numbers represent the different sections of the unsaturated zone and horizontal lines represent the borders between them.

Anaerobic biodegradation potential along the profile and isotopic enrichment during the biodegradation process were examined using five soil samples excavated from depths of 0e0.5 m, 11 m, 22 m, 40 m and 46 m (Fig. 5). These samples were selected to represent the five different sections of the unsaturated zone. Biodegradation in the clayey layers or in the layers under their immediate influence was more rapid, correlating with the d15N enrichment trend and DPR of RDX along the profile. Biodegradation started first in the sample which was excavated from 0 to 0.5 m depth (the first profile section) after only 1 d, and then continued rapidly for an additional 12 d. Degradation in the sediment sample from 22 m depth (the third profile section) started after 8 d and continued for an additional 18 d. These are also the two layers that presented the most significant isotopic enrichment in the field. The RDX biodegradation in the sample from 40 m (the fourth profile section) started after 9 d and RDX fully disappeared from the solution after an additional 17 d. Slower biodegradation was observed in the remaining, non-clayey layers: RDX biodegradation in samples from 46 m depth (the fifth profile section) started after 9 d and it fully disappeared from solution after an additional 32 d. RDX biodegradation in the sample from 11 m depth (the second profile section) started only after 33 d and continued for an additional 28 d. Sterile controls of all treatments showed no decrease in concentration (data not shown). Non-linear regression of the RDX decay data using a model consisting of a lag phase followed by exponential decay provided estimation for these two parameters (Table S3). The fitted lag phase was shortest in the surface soil treatment (6.29  0.28 days), and longest in the 11 m treatment (34.36  1.57 days). The fastest fitted degradation-rate constant was found for the 22 m slurry treatment (0.785  0.092 day1), and the slowest for the 46 m slurry treatment (0.081  0.010 day1). Mass balance of RDX and its degradation intermediates at the end of the experiment revealed different patterns in samples from different depths (Fig. 5). In slurries from 0 to 0.5 m, 40 m and 46 m depth, RDX was completely transformed to nitroso intermediates (MNX, DNX and TNX). The detection of nitroso derivatives in these slurries was concurrent with the disappearance of RDX. In the slurries from the profile’s surface, these intermediates were further degraded and completely disappeared from the solution, whereas in the slurries from the 40 m and 46 m sediments, they persisted during the entire incubation period. In contrast to the sediments from those three sections, in samples from 11 m to 22 m, complete transformation of RDX to

S. Sagi-Ben Moshe et al. / Soil Biology & Biochemistry 42 (2010) 1253e1262

-1 Concentrations (μmol l )

160

160

0-0.5 m

140

RDX MNX DNX TNX

120 100 80 60 40 20

Concentrations (μmol l-1)

1258

0

11 m

140

RDX MNX DNX TNX

120 100 80 60 40 20 0

0

10

20

30

40

50

60

0

10

20

Time (days)

40

50

60

160

22 m

140

RDX MNX DNX TNX

120 100 80 60 40 20 0

Concentrations (μmol l-1)

-1 Concentrations (μmol l )

160

40 m

140

RDX MNX DNX TNX

120 100 80 60 40 20 0

0

10

20

30

40

50

60

160

46 m

140

0

10

20

30

40

50

60

Time (days)

Time (days) Concentrations (μmol l-1)

30

Time (days)

RDX MNX DNX TNX

120 100 80 60 40 20 0 0

10

20

30

40

50

60

Time (days) Fig. 5. Biodegradation of RDX in samples from several depths representing the different sections of the unsaturated profile. Data points of representative experiments are means of triplicates  SD.

nitroso derivatives was not observed: the nitroso derivatives did not surpass concentrations of 13 and 55%, respectively, of the initial RDX, despite the fact that complete degradation of RDX was observed. The biodegradation experiments were further used to obtain enrichment factors representative of anaerobic biodegradation along the profile. Isotopic analyses were carried out for RDX samples at ca. 0, 50, 75, 90 and 95% biodegradation. From this analysis, the isotopic enrichment factors were calculated for each of the five soil samples representing the different sections of the unsaturated zone (Fig. 6). Differences were found between the enrichment factors calculated for the microbial consortia in slurries from different sections along the deep unsaturated profile, suggesting the occurrence of different degradation pathways. 3.4. Assessing RDX degradation extent in the unsaturated zone The extent of RDX biodegradation along the unsaturated profile can be assessed by combining the d15N composition of RDX found in the field and the isotopic fractionation constants obtained from the biodegradation experiment (Equation (3)). To assess biodegradation

extents, an initial isotopic composition (R0) must be defined. The

d15N composition of RDX measured in the 2 m sample, which probably contained undissolved RDX particles that remained undegraded, was equal to the d15N composition of the reference RDX produced at the industrial site (10.38&). We therefore used the isotopic composition observed in this sample as the Rsample,0 value, i.e., the isotopic composition of non-degraded RDX. Biodegradation is expressed in percent of the material originally present, calculated as B ¼ (1  f)  100, where f is the fraction remaining, as calculated in Equation (3). Since RDX molecules are mobile and can be transported to deeper sections of the unsaturated profile, it is likely that their degradation occurs in different layers by different microbial populations. Therefore, we calculated the range of RDX-degradation extents in each soil sample using the highest and lowest isotopic fractionation factors obtained from the biodegradation experiments (Fig. 7), under the assumption that this range represents the range of possible enrichment factors for RDX biodegradation along the profile. In accordance to d15N composition, the most degraded RDX (Fig. 4), which was found in the uppermost section of the soil profile, showed the most pronounced extents of estimated

S. Sagi-Ben Moshe et al. / Soil Biology & Biochemistry 42 (2010) 1253e1262

.018

.018

0-0.5 m

.016

y=-0.0022x R2=0.8893

11 m

.016

y=-0.0021x R2=0.7554

.014

.012

ln (R/R0)

ln (R/R0)

.014 .010 .008 .006 .004

.012 .010 .008 .006 .004

.002

.002

0.000

0.000

0

-1

-2

-3

-4

-5

0

-1

ln (C/C0) .018

.018

.010

-4

-5

.008 .006

y=-0.0028x R2=0.8983

.014

ln (R/R0)

y=-0.0037x R2=0.6356

.012

-3

40 m

.016

.014

-2

ln (C/C0)

22 m

.016

ln (R/R0)

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.012 .010 .008 .006 .004

.004 .002

.002

0.000

0.000

0

-1

-2

-3

-4

-5

0

ln (C/C0) .018

-1

-2

-3

-4

-5

ln (C/C0)

46 m

.016

ln (R/R0)

.014 y=-0.0035x R2=0.6785

.012 .010 .008 .006 .004 .002 0.000

0

-1

-2

-3

-4

-5

ln (C/C0) Fig. 6. Enrichment in d15N composition of RDX during biodegradation in samples from several depths representing the different sections of the unsaturated zone.

degradation (by isotopic analysis), ranging between 35 and 99.4% (Fig. 7). Estimates of RDX degradation in the second section of the profile ranged mostly from 30 to 70%. From the third section downwards through the fourth section, estimates for RDX-degradation ranged mostly from 45 to 80%. Estimates of RDX degradation in the fifth section of the profile, representing the saturated zone, ranged from 61 to 86%. 4. Discussion

Fig. 7. RDX biodegradation percentages along the unsaturated zone, calculated using equation (3) and the highest and lowest enrichment factors. Circled numbers represent the different sections of the unsaturated zone and horizontal lines represent the borders between them.

The aim of this research was to assess the extent of in situ RDX biodegradation along the unsaturated zone beneath wastewater ponds from which RDX percolated from 1964 to 2000. Using CSIA, we obtained evidence for RDX biodegradation along the deep soil profile, without the need for information on RDX historical inputs or transport at the site. Modeling and assessing RDX biodegradation along the soil profile based only on changes in concentration demands precise knowledge of RDX inputs and water transport along the profile, knowledge which is normally lacking when long time periods are being considered. In addition, it is evident from this case study that the presence of certain degradation products

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and the absence of others cannot lead to definitive conclusions about the degradation pathway. The presence of the nitroso derivatives and the absence of NDAB along the profile may indicate that aerobic biodegradation of RDX does not occur there and that anaerobic biodegradation dominates, but aerobic biodegradation cannot be excluded. Previous works identified a bacterial strain that degrades RDX aerobically in the surface soil at this site (Rhodococcus strain YH1 (Nejidat et al., 2008) and Gordonia YY1 (Ronen et al., 2008)). It is possible that aerobic biodegradation indeed occurs, but to a much lesser extent, as anaerobic biodegradation of RDX has been documented in several studies to be much faster than its aerobic biodegradation (Hawari, 2000; Ringelberg et al., 2003). Alternatively, it is possible that aerobic biodegradation is more pronounced than might be expected from the degradation products and that the relatively stable aerobic product, NDAB, has simply undergone further degradation (Fournier et al., 2005). d15N values of RDX along the entire unsaturated zone were enriched relative to d15N values measured in RDX produced at the industrial site. One single exception was detected in a sample taken 2 m below ground surface, in which the measured d15N value, 10.38&, was equal to that of RDX produced at the site. As already mentioned, the concentration of RDX in this sample indicated that it contains a large fraction of undissolved RDX. Thus, the non-enriched values in this sample, which were similar to those of non-degraded RDX produced at the site, can be explained by the fact that biodegradation occurs in the dissolved phase and therefore the undissolved fraction retains its original isotopic composition. It is possible that biodegradation in this dissolved fraction was negligible compared to the amount of undissolved RDX, and therefore did not affect the bulk isotopic composition of the sample as a whole. Precipitation and dissolution may also be accompanied by isotopic fractionation. However, the RDX quantities in the soil were sufficiently low, with the dissolved fraction not exceeding 40% of RDX solubility, to exclude the possibility of these processes playing a role in the isotopic composition of RDX along the profile, with only the aforementioned single exceptional point at a depth of 2 m. The studied profile has faced many changes over the years. The current RDX profile was formed over more than four decades, from the start of the percolation ponds’ operation. Its characteristics are influenced by several parameters that have not been constant over the years. In particular, it is influenced by the discharge pattern of effluents which, since the year 2000, no longer exists. Many important parameters that dictated transport along the soil profile cannot be reconstructed, for example water content, redox potential and carbon content. We hypothesize that anaerobic biodegradation plays an important role in the current RDX profile and occurs mainly in two active layers. These layers are the first and third profile sections, which are characterized as clayey layers (third section) or layers that are hydraulically influenced by the clayey layers (first section) (Fig. 4). The first layer also contains high quantities of natural organic matter originating from the vegetation that covers the ponds today (1.53% organic carbon was observed in the first 0.5 m compared to 0.011e0.056% in other parts of the soil profile). This high content of organic matter is also expected to promote biodegradation. The laboratory batch experiment and d15N analysis of RDX along the profile provided further support for this hypothesis, as did the Pearson correlation coefficients which showed a significant positive correlation for soil organic carbon with degradation product concentration and d15N in RDX. RDX degradation in the upper soil layer occurs rapidly, promoted by the fact that this layer contains high quantities of natural organic matter as well as two clayey layers, found in the lower part of this section, which reduce water drainage and can lead to the formation of the negative redox potential required to promote anaerobic

degradation of RDX. This layer contained the most enriched RDX and the highest biodegradation potential in the slurry experiment. On the other hand, the second section showed relatively dilute d15N values and lower degradation rates in the slurry experiments. The relatively dilute d15N value could represent the average of enriched RDX that was degraded and transported from the first section and undegraded RDX, which was quickly transported away from the uppermost layers, possibly in channels formed by roots or other heterogeneities. The clayey layer of the third section was identified as another highly active layer, in which suitable environmental conditions can develop to enhance anaerobic biodegradation (i.e., high water content in clay leading to formation of the required redox potential). Indeed, we observed relatively rapid biodegradation in the slurry experiments as well as enrichment in d15N values. Interestingly, some retardation was observed in the DPR pattern relative to that of the enriched RDX molecules underneath this third clayey layer. This can be explained by the fact that the nitroso compounds are more likely to be adsorbed on the clay particles whereas RDX itself shows low affinity to soil (Haderlein et al., 1996; Sheremata et al., 2001; Pennington and Brannon, 2002). Therefore, RDX degradation products may be undergoing attenuation in the third clayey section while enriched RDX can be observed somewhat deeper in the profile. The d15N value in the fourth section together with the relatively slow degradation rate in the slurry experiments indicated that this layer is not highly active either. The d15N values of RDX in this layer represent the enriched RDX that was transported through the first three sections rather than the biodegradation occurring there. Thus, in this fourth section, no additional pronounced shifts in isotopic composition were observed. Rapid RDX degradation was again observed with the arrival to the saturated zone (fifth section). Mass balance of RDX and its degradation intermediates at the end of the slurry experiment revealed different pattern in slurries from different depths (Fig. 5). All RDX molecules in the slurries from 0 to 0.5, 40 and 46 m completely degraded to nitroso intermediates (MNX, DNX and TNX). In the slurry of the surface sediments, these intermediates further degraded and completely disappeared from the solution whereas in the sediment samples from 40 m to 46 m, they persisted throughout the incubation period. These results agree with an earlier report of rapid degradation of nitroso derivatives in the surface soil as well as their persistence in the deeper layers (Ronen et al., 2008). On the other hand, in slurries with sediments from 11 m to 22 m, nitroso derivatives did not reach concentrations above 13 and 55%, respectively, of the initial RDX, despite complete degradation of the latter. The lack of significant accumulation of nitroso derivatives suggests that RDX degradation in these slurries does not follow the reductive pathway exclusively. The possibility that this lack of mass balance was a consequence of sorption is unlikely, as abiotic controls did not indicate measurable amounts of sorbed RDX, and as the treatment with 0- to 0.5-m deep soilsdsoils that are characterized by high quantities of organic matterdpresented a complete mass balance. It is possible that another anaerobic degradation process was occurring, as constructed by Hawari et al. (2000b), involving simultaneous enzymatic cleavage of the inner NeCeN bonds leading to ring cleavage, the formation of MEDINA and bis (hydroxymethyl)nitramine, and mineralization. However, we did not obtain any chemical evidence of MEDINA in the samples. As MEDINA is an unstable intermediate (Hawari et al., 2000b), it may have decomposed prior to the analysis. The unique degradation patterns at different depths were further evidenced by fitting the data to the model curves with initial lag and exponential decay. This model exhibited R2 values from 0.82 for the sample from 46 m to 0.99 for the 22-m sample (Table S3). Short lag times and fast degradation rates are indicative of a microbial population that has adapted to RDX degradation.

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The biodegradation experiments were further used to obtain enrichment factors representative of anaerobic biodegradation along the profile. There were differences between the enrichment factors calculated for the microbial consortia in slurries from different sections along the deep unsaturated profile; these factors ranged between 2.1  0.4& and 3.7  0.8&. Bernstein et al. (2008) conducted batch biodegradation experiments to analyze stable isotope fractionation in aerobic and anaerobic cultures, and found enrichment factors of 2.1  0.1& and 5.0  0.3& for aerobic and anaerobic degradation, respectively. The enrichments factors calculated in our experiment, using indigenous microorganisms from several selected soil layers, were within this range. The extent of RDX biodegradation along the unsaturated profile was assessed by combining the d15N composition of RDX found in the field and the isotopic fractionation constants obtained from the biodegradation experiment (Equation (3)). We calculated the range of RDX degradation extents in each soil sample using the highest and lowest isotopic fractionation factors obtained from the biodegradation experiment (Fig. 7). Since we could not exclude the occurrence of aerobic biodegradation as well, especially in the surface layer (Ronen et al., 2008), even though chemical evidence for the existence of this pathway was lacking (i.e., absence of the degradation product NDAB), by setting a range of possible enrichment factors of 2.1 to 3.7, we assumed that the possibility of this pathway was covered as well: the isotopic enrichment found in a previous work for the aerobic strain YH1, which was isolated from this contaminated site, was 2.1  0.1 (Bernstein et al., 2008). Hence, we suggest that this wide conservative range of enrichment factors represents the spectrum of possible enrichment factors along the profile. Calculation of RDX-biodegradation extent estimated a high percentage of degradation along the unsaturated profile. The most pronounced degradation extent (by isotopic analysis) was estimated in the uppermost section of the soil profile, reaching up to 99.4%. Estimates for RDX degradation in the rest of the profile ranged between 30 and 80%, with the higher degradation percentages assessed from the third section downwards. Using CSIA, we were able to gain qualitative evidence for, and a quantitative assessment of the occurrence of biodegradation along a deep soil profile, without previous knowledge of pollutant input and transport in the soil. Moreover, application of the CSIA concept to the unsaturated zone gave us better insight into vertical variations in biodegradation activities. Our observations imply that in situ biodegradation can be considered a significant mechanism for RDX attenuation in the profile. To the best of our knowledge, this is the first attempt to quantify the biodegradation of contaminants, RDX in particular, in an unsaturated zone using CSIA. The observed variability in biodegradation potential in different soil layers of the profile is of importance when testing the applicability of natural attenuation as a remediation strategy. It appears that the ability to completely degrade RDX, as well as its nitroso derivatives, is present in the upper part of the profile. In the lower parts of the profile, although RDX has the potential to be completely degraded, the nitroso derivatives tend to persist. The ability to reduce RDX to nitroso derivatives is widespread among soil bacteria (Crocker et al., 2006). Further degradation of the nitroso products, however, requires more reduced conditions and strict anaerobic bacteria. These conditions may occur in the upper parts of soil profiles that receive relatively high quantities of natural organic matter from the surface. The formation and accumulation of nitroso products should not be neglected when considering the applicability of natural attenuation: they are toxic (Zhang et al., 2006), and their accumulation is therefore undesirable. Enrichment factors for the degradation of different compounds are often highly variable, with different values being characteristic of different degradation mechanisms (Schmidt et al., 2004). When

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different processes cannot be distinguished, it may be useful to use a range of factors that represents different microbial processes (e.g., aerobic and anaerobic biodegradation). Here we found different isotopic fractionation factors for the different sections of the unsaturated profile and we suggest that they are related to different degradation pathways that exist simultaneously in varying proportions. Finally, the application of CSIA to solve biodegradation unknowns in groundwater is now relatively well accepted. In the unsaturated zone, however, this concept has not been extensively applied, and more work is needed to construct adequate protocols and considerations for this methodology. However, our study shows the potential for this concept’s use in this environment as well. Acknowledgments We would like to acknowledge the Israel Water Authority as well as Israel Science Foundation (167/08) for funding this research and Ms. Natalia Bondarenko for her technical assistance. Appendix. Supplementary material Supplementary data associated with this article can be found in the online version at doi:10.1016/j.soilbio.2010.04.011. References Bernstein, A., Adar, E., Ronen, Z., Lowag, H., Stichler, W., Meckenstock, R.U., 2009. Quantifying RDX biodegradation in groundwater using d15N isotope analysis. Journal of Contaminant Hydrology. doi:10.1016/j.jconhyd.2009.10.010. Bernstein, A., Ronen, Z., Adar, E., Nativ, R., Lowag, H., Stichler, W., Meckenstock, R.U., 2008. Compound-specific isotope analysis of RDX and stable isotope fractionation during aerobic and anaerobic biodegradation. Environmental Science and Technology 42, 7772e7777. Bhushan, B., Trott, S., Spain, J.C., Halasz, A., Paquet, L., Hawari, M., 2003. Biotransformation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) by a rabbit liver cytochrome p450: insight into the mechanism of RDX biodegradation by Rhodococcus sp strain DN22. Applied and Environmental Microbiology 69, 1347e1351. Crocker, F.H., Indest, K.J., Fredrickson, H.L., 2006. Biodegradation of the cyclic nitramine explosives RDX, HMX, and CL-20. Applied Microbiology and Biotechnology 73, 274e290. Elsner, M., Zwank, L., Hunkeler, D., Schwarzenbach, R.P., 2005. A new concept linking observable stable isotope fractionation to transformation pathways of organic pollutants. Environmental Science and Technology 39, 6896e6916. EPA (U.S. Environmental Protection Agency), 1994. Nitroaromatics and nitramines by HPLC. Second update SW-846 Method 8330. Office of Solid Waste and Emergency Response, Washington, DC. Fournier, D., Halasz, A., Spain, J., Fiurasek, P., Hawari, J., 2002. Determination of key metabolites during biodegradation of hexahydro-1,3,5-trinitro-1,3,5-triazine with Rhodococcus sp strain DN22. Applied and Environmental Microbiology 68, 166e172. Fournier, D., Halasz, A., Spain, J., Spanggord, R.J., Bottaro, F.C., Hawari, J., 2004. Biodegradation of the hexahydro-1,3,5-trinitro-1,3,5-triazine ring cleavage product 4-nitro-2,4-diazabutanal by Phanerochaete chrysosporium. Applied and Environmental Microbiology 70, 1123e1128. Fournier, D., Trott, S., Hawari, J., Spain, J., 2005. Metabolism of the aliphatic nitramine 4-nitro-2,4-diazabutanal by Methylobacterium sp strain JS178. Applied and Environmental Microbiology 71, 4199e4202. Haderlein, S.B., Weissmahr, K.W., Schwarzenbach, R.P., 1996. Specific adsorption of nitroaromatic explosives and pesticides to clay minerals. Environmental Science and Technology 30, 612e622. Hawari, J., 2000. Biodegradation of RDX and HMX: from basic research to field application. In: Spain, J.C., Hughes, J.B., Knackmuss, H.-J. (Eds.), Biodegradation of Nitroaromatic Compounds and Explosives. Lewis Publishers, Boca Raton, pp. 277e310. Hawari, J., Beaudet, S., Halasz, A., Thiboutot, S., Ampleman, G., 2000a. Microbial degradation of explosives: biotransformation versus mineralization. Applied Microbiology and Biotechnology 54, 605e618. Hawari, J., Halasz, A., Sheremata, T., Beaudet, S., Groom, C., Paquet, L., Rhofir, C., Ampleman, G., Thiboutot, S., 2000b. Characterization of metabolites during biodegradation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) with municipal anaerobic sludge. Applied and Environmental Microbiology 66, 2652e2657. Jackson, R.G., Rylott, E.L., Fournier, D., Hawari, J., Bruce, N.C., 2007. Exploring the biochemical properties and remediation applications of the unusual explosivedegrading P450 system XpIA/B. Proceedings of the National Academy of Sciences of the United States of America 104, 16822e16827.

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Mak, K.S., Griebler, C., Meckenstock, R.U., Liedl, R., Peter, A., 2006. Combined application of conservative transport modelling and compound-specific carbon isotope analyses to assess in situ attenuation of benzene, toluene, and o-xylene. Journal of Contaminant Hydrology 88, 306e320. McCormick, N.G., Cornell, J.H., Kaplan, A.M.,1981. Biodegradation of hexahydro-1,3,5trinitro-1,3,5-triazine. Applied and Environmental Microbiology 42, 817e823. McKelvie, J.R., Mackay, D.M., de Sieyes, N.R., Lacrampe-Couloume, G., Lollar, B.S., 2007. Quantifying MTBE biodegradation in the Vandenberg Air Force Base ethanol release study using stable carbon isotopes. Journal of Contaminant Hydrology 94, 157e165. Meckenstock, R.U., Morasch, B., Griebler, C., Richnow, H.H., 2004. Stable isotope fractionation analysis as a tool to monitor biodegradation in contaminated acquifers. Journal of Contaminant Hydrology 75, 215e255. Morrill, P.L., Lacrampe-Couloume, G., Slater, G.F., Sleep, B.E., Edwards, E.A., McMaster, M.L., Major, D.W., Lollar, B.S., 2005. Quantifying chlorinated ethene degradation during reductive dechlorination at Kelly AFB using stable carbon isotopes. Journal of Contaminant Hydrology 76, 279e293. Mulligan, C.N., Yong, R.N., 2004. Natural attenuation of contaminated soils. Environment International 30, 587e601. Nativ, R., Adar, E.M., 2005. Soil and Groundwater Contamination in the Ramat Hasharon Area. Annual scientific report submitted to the Water Authority. Ministry of National Infrastructures, Tel Aviv. Nejidat, A., Kafka, L., Tekoah, Y., Ronen, Z., 2008. Effect of organic and inorganic nitrogenous compounds on RDX degradation and cytochrome P-450 expression in Rhodococcus strain YH1. Biodegradation 19, 313e320. Nelson, D.W., Sommers, L.E., 1996. Total carbon, organic carbon and organic matter. In: Sparks, D.L. (Ed.), Methods of Soil Analysis, Part 3SSSA Book Ser, third ed., vol. 5. SSSA, Madison, WI, pp. 961e1010. Pennington, J.C., Brannon, J.M., 2002. Environmental fate of explosives. Thermochimica Acta 384, 163e172.

Ringelberg, D.B., Reynolds, C.M., Walsh, M.E., Jenkins, T.F., 2003. RDX loss in a surface soil under saturated and well drained conditions. Journal of Environmental Quality 32, 1244e1249. Ronen, Z., Brenner, A., Abeliovich, A., 1998. Biodegradation of RDX-contaminated wastes in a nitrogen-deficient environment. Water Science and Technology 38, 219e224. Ronen, Z., Yanovich, Y., Goldin, R., Adar, E., 2008. Metabolism of the explosive hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) in a contaminated vadose zone. Chemosphere 73, 1492e1498. Sagi-Ben Moshe, S., Ronen, Z., Dahan, O., Weisbrod, N., Groisman, L., Adar, E., Nativ, R., 2009. Sequential biodegradation of TNT, RDX and HMX in a mixture. Environmental Pollution 157, 2231e2238. Schmidt, T.C., Zwank, L., Elsner, M., Berg, M., Meckenstock, R.U., Haderlein, S.B., 2004. Compound-specific stable isotope analysis of organic contaminants in natural environments: a critical review of the state of the art, prospects, and future challenges. Analytical and Bioanalytical Chemistry 378, 283e300. Sheldrick, B.H., Wang, C., 1993. Particle-size distribution. In: Carter, M.R. (Ed.), Soil Sampling and Methods of Analysis. Canadian Society of Soil Science, Lewis Publishers, Ann Arbor, MI, pp. 499e511. Sheremata, T.W., Halasz, A., Paquet, L., Thiboutot, S., Ampleman, G., Hawari, J., 2001. The fate of the cyclic nitramine explosive RDX in natural soil. Environmental Science and Technology 35, 1037e1040. Townsend, D.M., Myers, T.E., 1996. Recent Developments in Formulating Model Descriptors for Subsurface Transformation and Sorption of TNT, RDX, and HMX. Technical Report IRRP_1, U.S. Army Corps of Engineers, Waterways Experiment Station, Vicksburg, MS. NTIS No. AD A305 176. Zhang, B.H., Kendall, R.J., Anderson, T.A., 2006. Toxicity of the explosive metabolites hexahydro-1,3,5-trinitroso-1,3,5-triazine (TNX) and hexahydro-1-nitroso-3,5dinitro-1,3,5-triazine (MNX) to the earthworm Eisenia fetida. Chemosphere 64, 86e95.