Article pubs.acs.org/est
Using Compound-Specific Isotope Analysis to Assess Biodegradation of Nitroaromatic Explosives in the Subsurface Reto S. Wijker,†,‡ Jakov Bolotin,† Shirley F. Nishino,§ Jim C. Spain,§ and Thomas B. Hofstetter*,†,‡ †
Eawag, Swiss Federal Institute of Aquatic Science and Technology, CH-8600 Dübendorf, Switzerland Institute of Biogeochemistry and Pollutant Dynamics (IBP), ETH Zürich, Zürich, Switzerland § School of Civil and Environmental Engineering, Georgia Institute of Technology, Atlanta, Georgia 30332, United States ‡
S Supporting Information *
ABSTRACT: Assessing the fate of nitroaromatic explosives in the subsurface is challenging because contaminants are present in different phases (e.g., bound to soil or sediment matrix or as solid-phase residues) and transformation takes place via several potentially competing pathways over time-scales of decades. We developed a procedure for compound-specific analysis of stable C, N, and H isotopes in nitroaromatic compounds (NACs) and characterized biodegradation of 2,4,6-trinitrotoluene (TNT) and two dinitrotoluene isomers (2,4-DNT and 2,6-DNT) in subsurface material of a contaminated site. The type and relative contribution of reductive and oxidative pathways to the degradation of the three contaminants was inferred from the combined evaluation of C, N, and H isotope fractionation. Indicative trends of Δδ15N vs Δδ13C and Δδ2H vs Δδ13C were obtained from laboratory model systems for biodegradation pathways initiated via (i) dioxygenation, (ii) reduction, and (iii) CH3-group oxidation. The combined evaluation of NAC isotope fractionation in subsurface materials and in laboratory experiments suggests that in the field, 86−89% of 2,4DNT transformation was due to dioxygenation while TNT was mostly reduced and 2,6-DNT reacted via a combination of reduction and CH3-group oxidation. Based on historic information on site operation, our data imply biodegradation of 2,4-DNT with half-lives of up to 9−17 years compared to 18−34 years for cometabolic transformation of TNT and 2,6-DNT.
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INTRODUCTION Contaminations of soil, sediment, and groundwater with nitroaromatic compounds (NACs) such as the explosive 2,4,6-trinitrotoluene (TNT) and its mono- and dinitrotoluene precursors and manufacturing byproducts are a widespread problem at military training installations, abandoned production facilities, and munition disposal sites.1−3 Even though many soil microbes have been found that evolved the ability to degrade NACs,1,4,5 soil and subsurface pollution still persists. Owing to the toxicity of NACs,6,7 soil contaminations pose a significant threat to human health and the environment.8 However, assessing the extent and pathways of NAC transformation in soil is very challenging. Because NACs are often spilled in significant quantities, they are present in different phases in soil, that is as solid/crystalline NAC residues9−11 and as NACs sorbed to soil organic matter, as well as bound to inorganic soil constituents.12 Moreover, NAC transformation can occur by different, sometimes competing, reaction pathways over years and decades leading to both complete mineralization and products that exceed the toxicity of the parent compounds.1,13 An interpretation of NAC concentration dynamics in time and space, especially for assessing (bio)degradation thus needs to be based on a thorough quantification of multiple and interdependent phase© 2013 American Chemical Society
transfer and transformation processes over long time scales. Because such information typically requires labor- and costintensive monitoring networks, alternative approaches based on compound-specific isotope analysis (CSIA) have been proposed14−16 that can reveal the predominant biotransformation pathway based on information obtained from the contaminants found in situ. Indeed, a series of laboratory studies have shown that abiotic NAC reduction and biodegradation by pure cultures can lead to typical C and N isotope fractionation trends that are indicative for the pathway of transformation.14−19 Such observations are due to apparent kinetic isotope effects (AKIE) at the reacting bonds, which are specific for the initial steps of transformation.20−22 Oxidative biodegradation pathways such as dioxygenation (Scheme 1a) are typically initiated by C hybridization changes at the aromatic ring followed by dioxygen addition.17,23 The corresponding 13C-AKIEs are primary, Special Issue: Rene Schwarzenbach Tribute Received: Revised: Accepted: Published: 6872
December 18, 2012 March 21, 2013 April 3, 2013 April 3, 2013 dx.doi.org/10.1021/es3051845 | Environ. Sci. Technol. 2013, 47, 6872−6883
Environmental Science & Technology
Article
Scheme 1. Oxidative and Reductive (Bio)Transformation Pathways of Nitroaromatic Compounds: (a) Dioxygenation, (b) Mono-oxygenation, (c) CH3-Group Oxidation, (d) Partial Reduction, (e) Co-Metabolic and Abiotic Reduction, and (f) Hydride Transfer
whereas 15N-AKIEs are secondary which leads to a stronger fractionation of C isotopes compared to N. Kinetic isotope effects pertinent to reduction arise from N−O bond cleavage in reactions leading to aminophenol intermediates during biodegradation (Scheme 1d) and to the corresponding substituted anilines in abiotic processes 24 (Scheme 1e). In contrast to oxidative biodegradation, reduction is subject to substantial N isotope fractionation while that for C is only secondary.14,15,17,18,25,26 As shown for the enzymatic and abiotic NAC reduction (Scheme 1d and e), AKIE-values of the same degradation pathway can vary slightly due to kinetic constraints.14,17,18 However, the correlated isotope fractionation trends, for example for C and N (i.e., Δδ15N vs Δδ13C) are identical and thus represent a robust proxy for detecting reductive transformation. Moreover, evaluation of isotope fractionation of several elements is also likely to reveal combinations of oxidative and reductive NAC biodegradation pathways.17 While multielement isotope fractionation analysis has frequently been used to assess contaminant degradation,27 field studies on explosives are scarce28 and none have been reported for (bio)degradation of nitroaromatic contaminants. To date, application of CSIA to sites contaminated with nitroaromatic explosives has been compromised because NACs are largely bound to the organic and mineral subsurface matrix and analytical procedures for extraction and isotopic analyses of NACs from such materials do not exist. In addition, it is unclear whether isotope fractionation observed in the field can be distinguished unequivocally from isotope signatures of different production batches. Finally, information about the isotope effects of the various metabolic and cometabolic transformation pathways of NACs such as mono-oxidation, CH3−group oxidation, or hydride-Meisenheimer complex formation (Scheme 1b, c, and f) is currently lacking. It thus remains unknown whether these reaction pathways are associated with any unique isotope fractionation patterns that would enable their identification. The goal of the present study was to establish CSIA for assessing biodegradation of nitroaromatic contaminants in subsurface materials including soils and sediments. To this end, we (i) developed an analytical protocol for the extraction, purification, and enrichment of seven typical mono-, di-, and trinitrotoluenes from contaminated subsurface material for CSIA by gas chromatography coupled to isotope ratio mass spectrometry. (ii) Applying this procedure, we analyzed C, N,
and H isotope signatures of two dinitrotoluene isomers (2,4DNT and 2,6-DNT) and C and N isotopes of TNT in a subsurface profile at a contaminated site in Switzerland, where contamination existed for several decades. (iii) NAC (bio)degradation assessment was carried out based on the characterization of N vs C and H vs C isotope fractionation at the field site. To this end, the pathways of NAC transformation were inferred from the comparison of field data with isotope fractionation patterns observed in laboratory model systems for biodegradation of mono- and dinitrotoluene via dioxygenation, reduction, and CH3-group oxidation.
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EXPERIMENTAL SECTION A complete list of all used chemicals including purities and suppliers can be found in the Supporting Information (SI). Sampling Site. Subsurface samples were obtained at different locations of a former explosives factory in Switzerland and included (a) uncontaminated soil for analytical method development and (b) contaminated subsurface material, i.e., perturbed soil and sediment, at a former chemical storage place for biodegradation assessment. At the storage place, mixtures of DNT isomers and TNT were unloaded from trucks, heated, fluidized, and filled into one of three tanks in a storage room. Mixtures consisted of 90% DNT and 10% TNT from 1935 to 1970 and of only DNT isomers thereafter. After 1980, special trucks filled the tanks directly until operations ceased in 2001. Regardless of the unloading method, leakages led to substantial subsurface contamination with DNT and TNT as well as with dozens of other NACs. Sampling at this site was carried out with a digger and included seven samples from depths of 0.5− 1.0, 1.5, 2, 4, 5, and 6 m. The subsurface, which was perturbed to depth of 1.5 m due to construction activities over the years, consisted of a first 0.6-m brownish layer of sandy gravel containing stones (average grain size 2 cm) followed by 0.9 m of different sandy gravel layers that could be distinguished by color, grain size (1−2 cm), degree of oil, and NAC contamination. At 1.5 m depth a solid, yellow-brown piece of DNT-containing TNT (55 cm diameter) was found that was covered with black crust. Between 1.5 and 6 m depth, the subsurface consisted of creek sediments (sandy gravel) and stones. The groundwater table was located at a depth of 6.7 m. A sample from the former storage room was taken in front of former storage tanks to obtain information about the original isotopic compositions of the contaminants. Additional samples for the evaluation of isotope fractionation associated with the 6873
dx.doi.org/10.1021/es3051845 | Environ. Sci. Technol. 2013, 47, 6872−6883
Environmental Science & Technology
Article
isotope fractionation D (ΔδhE4→5, eq 2), 1 L of Milli-Q water was spiked with the seven NACs and the solution was treated as described above. Filtration and water removal from dichloromethane during step E (ΔδhE5→6, eq 3 was investigated through addition of the Na2SO4 to 2 mL of dichloromethane containing the analytes followed by filtration. Evaporationrelated effects on the isotope signatures in step F (ΔδhE6→7, eq 4) were evaluated through the evaporation of 20 mL of dichloromethane to 100 μL.
synthesis of explosives were obtained from another contaminated spot 100 m from the storage place. Sample Preparation. A seven-step procedure was developed to extract NACs from soil samples, purify extracts, and determine the compound’s stable C, N, and H isotope compositions. The procedure is shown schematically at the bottom of Table S1. Approximately 1 kg of soil was dried at 25 °C in a drying cabinet to a constant weight, ground with a jaw crusher and rotor mill, sieved to
