Stable Isotope Tools for Tracking In Situ Degradation Processes of Military Energetic Compounds

  • Anat Bernstein
  • Faina Gelman
  • Zeev RonenEmail author
Part of the Environmental Science and Engineering book series (ESE)


Military energetic compounds are common environmental pollutants, contaminating soils and water worldwide. In situ microbial degradation of these compounds may be a positive process that reduces their concentration in the environment without any active human involvement. Nevertheless, assessing the extent of biodegradation processes in situ is challenging, and often cannot be achieved with satisfactory sensitivity using conventional methods. Isotope methods, on the other hand, may be a suitable alternative for assessing these processes. This chapter will focus on the application of compound-specific isotope analysis (CSIA) to study the fate of military energetic compounds in the subsurface, focusing on nitroaromatics, nitramines, and perchlorate. CSIA can also be used to provide an intrinsic mechanistic understanding of the transformation reactions of such compounds and this aspect will be described as well.


Enrichment Factor Reactive Position Natural Attenuation Anaerobic Biodegradation Aerobic Denitration 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



This work was supported, in part, by grant 167/2008 from the Israel Science Foundation.


  1. Abe Y, Hunkeler D (2006) Does the Rayleigh equation apply to evaluate field isotope data in contaminant hydrogeology? Environ Sci Technol 40:1588–1596CrossRefGoogle Scholar
  2. Ader M, Coleman ML, Doyle SP, Stroud M, Wakelin D (2001) Methods for the stable isotopic analysis of chlorine in chlorate and perchlorate compounds. Anal Chem 73:4946–4950CrossRefGoogle Scholar
  3. Ader M, Chaudhuri S, Coates JD, Coleman M (2008) Microbial perchlorate reduction: a precise laboratory determination of the chlorine isotope fractionation and its possible biochemical basis. Earth Planet Sci Lett 269:605–613CrossRefGoogle Scholar
  4. Aelion C, Hohener P, Hunkeler D, Aravena R (2009) Environmental isotopes in bioremediation and biodegradation. CRC Press, Boca Raton, p 450Google Scholar
  5. Ahmad F (2007) Nitrogen isotopes. Environ Sci Technol 41:1502–1502CrossRefGoogle Scholar
  6. Amaral HIF, Fernandes J, Berg M, Schwarzenbach RP, Kipfer R (2009) Assessing TNT and DNT groundwater contamination by compound-specific isotope analysis and 3H–3He groundwater dating: a case study in Portugal. Chemosphere 77:805–812CrossRefGoogle Scholar
  7. Bao H, Gu B (2004) Natural perchlorate has a unique oxygen isotope signature. Environ Sci Technol 38:5073–5077CrossRefGoogle Scholar
  8. Berg M, Bolotin J, Hofstetter TB (2007) Compound-specific nitrogen and carbon isotope analysis of nitroaromatic compounds in aqueous samples using solid-phase microextraction coupled to GC/IRMS. Anal Chem 79:2386–2393CrossRefGoogle Scholar
  9. Bernstein A (2008) Biodegradation of RDX in the Israeli coastal aquifer. Ben-Gurion University of the Negev, Beer-Sheva 95Google Scholar
  10. Bernstein A, Ronen Z (2012) Biodegradation of the explosives TNT, RDX and HMX. In: Singh, S. N., (ed) Microbial Degradation of Xenobiotics Springer-Verlag Berlin, Heidelberger Platz 3, D-14197 Berlin, Germany, pp 135-176.Google Scholar
  11. Bernstein A, Ronen Z, Adar E, Nativ R, Lowag H, Stichler W, Meckenstock RU (2008) Compound-specific isotope analysis of RDX and stable isotope fractionation during aerobic and anaerobic biodegradation. Environ Sci Technol 42:7772–7777CrossRefGoogle Scholar
  12. Bernstein A, Adar E, Ronen Z, Lowag H, Stichler W, Meckenstock RU (2010) Quantifying RDX biodegradation in groundwater using [delta] 15N isotope analysis. J Contam Hydrol 111:25–35CrossRefGoogle Scholar
  13. Bernstein A, Shouakar-Stash O, Ebert K, Laskov C, Hunkeler D, Jeannottat S, Sakaguchi-Soder K, Laaks J, Jochmann MA, Cretnik S (2011) Compound-specific chlorine isotope analysis: a comparison of gas chromatography/isotope ratio mass spectrometry and gas chromatography/quadrupole mass spectrometry methods in an interlaboratory study. Anal Chem 83:7624–7634CrossRefGoogle Scholar
  14. Bernstein A, Gelman F, Ronen Z (2013) Insight on RDX degradation mechanism by Rhodococcus strains using 13C and 15N kinetic isotope effects. Environ Sci Technol 47:479–484Google Scholar
  15. Bhushan B, Trott S, Spain JC, Halasz A, Paquet L, Hawari J (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. Appl Environ Microbiol 69:1347–1351CrossRefGoogle Scholar
  16. Bockelmann A, Zamfirescu D, Ptak T, Grathwohl P, Teutsch G (2003) Quantification of mass fluxes and natural attenuation rates at an industrial site with a limited monitoring network: a case study. J Contam Hydrol 60:97–121CrossRefGoogle Scholar
  17. Böhlke JK, Sturchio NC, Gu B, Horita J, Brown GM, Jackson WA, Batista J, Hatzinger PB (2005) Perchlorate isotope forensics. Anal Chem 77:7838–7842CrossRefGoogle Scholar
  18. Böhlke JK, Hatzinger PB, Sturchio NC, Gu B, Abbene I, Mroczkowski SJ (2009) Atacama perchlorate as an agricultural contaminant in groundwater: isotopic and chronologic evidence from Long Island, New York. Environ Sci Technol 43:5619–5625CrossRefGoogle Scholar
  19. Bombach P, Richnow HH, Kästner M, Fischer A (2010) Current approaches for the assessment of in situ biodegradation. Appl Microbiol Biotechnol 86:839–852CrossRefGoogle Scholar
  20. Bordeleau G, Savard MM, Martel R, Ampleman G, Thiboutot S (2008) Determination of the origin of groundwater nitrate at an air weapons range using the dual isotope approach. J Contam Hydrol 98:97–105CrossRefGoogle Scholar
  21. Braeckevelt M, Fischer A, Kästner M (2012) Field applicability of compound specific isotope analysis (CSIA) for characterization and quantification of in situ contaminant degradation in aquifers. Appl Microbiol Biotechnol 94:1401–10421Google Scholar
  22. Cichocka D, Imfeld G, Richnow HH, Nijenhuis I (2008) Variability in microbial carbon isotope fractionation of tetra-and trichloroethene upon reductive dechlorination. Chemosphere 71:639–648CrossRefGoogle Scholar
  23. Cincinelli A, Pieri F, Zhang Y, Seed M, Jones KC (2012) Compound specific isotope analysis (CSIA) for chlorine and bromine: a review of techniques and applications to elucidate environmental sources and processes. Environ Pollut 169:112–127CrossRefGoogle Scholar
  24. Coates JD, Achenbach LA (2004) Microbial perchlorate reduction: rocket-fuelled metabolism. Nature Rev Microbiol 2:569–580CrossRefGoogle Scholar
  25. Coffin RB, Miyares PH, Kelley CA, Cifuentes LA, Reynolds CM (2001) Stable carbon and nitrogen isotope analysis of TNT: two-dimensional source identification. Environ Toxicol Chem 20:2676–2680CrossRefGoogle Scholar
  26. Coleman ML, Ader M, Chaudhuri S, Coates JD (2003) Microbial isotopic fractionation of perchlorate chlorine. Appl Environ Microbiol 69:4997–5000CrossRefGoogle Scholar
  27. DiGnazio FJ, Krothe NC, Baedke SJ, Spalding RF (1998) delta15N of nitrate derived from explosive sources in a karst aquifer beneath the ammunition burning ground, crane naval surface warfare center, Indiana, USA. J Hydrol 206:164–175CrossRefGoogle Scholar
  28. Elsner M (2010) Stable isotope fractionation to investigate natural transformation mechanisms of organic contaminants: principles, prospects and limitations. J Environ Monitor 12:2005–2031CrossRefGoogle Scholar
  29. Elsner M, Zwank L, Hunkeler D, Schwarzenbach RP (2005) A new concept linking observable stable isotope fractionation to transformation pathways of organic pollutants. Environ Sci Technol 39:6896–6916CrossRefGoogle Scholar
  30. Elsner M, Jochmann M, Hofstetter T, Hunkeler D, Bernstein A, Schmidt T, Schimmelmann A (2012) Current challenges in compound-specific stable isotope analysis of environmental organic contaminants. Anal Bioanal Chem 403:2471–2491CrossRefGoogle Scholar
  31. 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. Appl Environ Microbiol 68:166–172CrossRefGoogle Scholar
  32. Gal H (2010) Perchlorate fate in the unsaturated zone. In faculty of agriculture. Hebrew University of Jerusalem, RehovotGoogle Scholar
  33. Gelman F, Kotlyar A, Chiguala D, Ronen Z (2011) Precise and accurate compound-specific carbon and nitrogen isotope analysis of RDX by GC-IRMS. Intl J Environ Anal Chem 91:1392–1400CrossRefGoogle Scholar
  34. Halasz A, Manno D, Strand SE, Bruce NC, Hawari J (2010) Biodegradation of RDX and MNX with Rhodococcus sp. strain DN22: New insights into the degradation pathway. Environ Sci Technol 44:9330–9336CrossRefGoogle Scholar
  35. Hartenbach A, Hofstetter TB, Berg M, Bolotin J, Schwarzenbach RP (2006) Using nitrogen isotope fractionation to assess abiotic reduction of nitroaromatic compounds. Environ Sci Technol 40:7710–7716CrossRefGoogle Scholar
  36. Hartenbach AE, Hofstetter TB, Aeschbacher M, Sander M, Kim D, Strathmann TJ, Arnold WA, Cramer CJ, Schwarzenbach RP (2008) Variability of nitrogen isotope fractionation during the reduction of nitroaromatic compounds with dissolved reductants. Environ Sci Technol 42:8352–8359CrossRefGoogle Scholar
  37. Hatzinger PB (2011) Applications of stable isotope analysis for remediation and forensics. In: The strategic environmental research and development program (SERDP) and the environmental security technology certification program (ESTCP). Annual symposium Washington, D.CGoogle Scholar
  38. Hatzinger PB, Bohlke JK, Sturchio NC, Gu B, Heraty LJ, Borden RC (2009) Fractionation of stable isotopes in perchlorate and nitrate during in situ biodegradation in a sandy aquifer. Environ Chem 6:44–52CrossRefGoogle Scholar
  39. Hoffsommer JC, Kubose DA, Glover DJ (1977) Kinetic isotope effects and intermediate formation for the aqueous alkaline homogeneous hydrolysis of 1,3,5-triaza-1,3,5-trinitrocyclohexane (RDX). J Phys Chem 81:380–385CrossRefGoogle Scholar
  40. Hofstetter TB (2011) Tracking transformation processes of nitrogen-containing organic micropolutants in aquatic environments using multi-element isotope fractionation analysis. In: International conference ISOTOPES 2011. Gréoux-les-Bains, France, p 49Google Scholar
  41. Hofstetter TB, Berg M (2011) Assessing transformation processes of organic contaminants by compound-specific stable isotope analysis. Trends Anal Chem 30:618–627CrossRefGoogle Scholar
  42. Hofstetter TB, Neumann A, Arnold WA, Hartenbach AE, Bolotin J, Cramer CJ, Schwarzenbach RP (2008a) Substituent effects on nitrogen isotope fractionation during abiotic reduction of nitroaromatic compounds. Environ Sci Technol 42:1997–2003CrossRefGoogle Scholar
  43. Hofstetter TB, Spain JC, Nishino SF, Bolotin J, Schwarzenbach RP (2008b) Identifying competing aerobic nitrobenzene biodegradation pathways by compound-specific isotope analysis. Environ Sci Technol 42:4764–4770CrossRefGoogle Scholar
  44. Hunkeler D, N.R.M.R.L.O.o. Research, and Development (2008) A guide for assessing biodegradation and source identification of organic ground water contaminants using compound specific isotope analysis (CSIA). Office of Research and Development, National Risk Management Research Laboratory, US Environmental Protection AgencyGoogle Scholar
  45. Illman WA, Alvarez PJ (2009) Performance assessment of bioremediation and natural attenuation. Crit Rev Environ Sci Technol 39:209–270CrossRefGoogle Scholar
  46. Jackson RG, Rylott EL, Fournier D, Hawari J, Bruce NC (2007) Exploring the biochemical properties and remediation applications of the unusual explosive-degrading P450 system XplA/B. Proc Nat Acad Sci 104:16822–16827CrossRefGoogle Scholar
  47. Jackson WA, Bohlke JK, Gu B, Hatzinger PB, Sturchio NC (2010) Isotopic composition and origin of indigenous natural perchlorate and co-occurring nitrate in the Southwestern United States. Environ Sci Technol 44:4869–4876CrossRefGoogle Scholar
  48. Juhasz AL, Naidu R (2007) Explosives: fate, dynamics, and ecological impact in terrestrial and marine environments. Rev Environ Contam Toxicol 191:163–215Google Scholar
  49. Kampara M, Thullner M, Richnow HH, Harms H, Wick LY (2008) Impact of bioavailability restrictions on microbially induced stable isotope fractionation. 2. Experimental evidence. Environ Sci Technol 42:6552–6558CrossRefGoogle Scholar
  50. Krummen M, Hilkert AW, Juchelka D, Duhr A, Schlüter H-J, Pesch R (2004) A new concept for isotope ratio monitoring liquid chromatography/mass spectrometry. Rapid Commun Mass Spectrom 18:2260–2266CrossRefGoogle Scholar
  51. Landis R (2000) Section 11 in: In situ permeable reactive barriers: application and deployment, U.S. EPA training manual EPA/542/B-00/001Google Scholar
  52. Lima DRS, Bezerra MLS, Neves EB, Moreira FR (2011) Impact of ammunition and military explosives on human health and the environment. Rev Environ Health 26:101–110CrossRefGoogle Scholar
  53. Madsen EL (1998) Epistemology of environmental microbiology. Environ Sci Technol 32:429–439CrossRefGoogle Scholar
  54. Meckenstock RU, Morasch B, Griebler C, Richnow HH (2004) Stable isotope fractionation analysis as a tool to monitor biodegradation in contaminated acquifers. J Contam Hydrol 75:215–255CrossRefGoogle Scholar
  55. Meyer AH, Penning H, Lowag H, Elsner M (2008) Precise and accurate compound specific carbon and nitrogen isotope analysis of atrazine: critical role of combustion oven conditions. Environ Sci Technol 42:7757–7763CrossRefGoogle Scholar
  56. Meyer AH, Penning H, Elsner M (2009) C and N isotope fractionation suggests similar mechanisms of microbial atrazine transformation despite involvement of different enzymes (AtzA and TrzN). Environ Sci Technol 43:8079–8085CrossRefGoogle Scholar
  57. Miyares PH, Reynolds CM, Pennington JC, Coffin RB, Jenkins TF (1999) Using stable isotopes of carbon and nitrogen as in situ tracers for monitoring the natural attenuation of explosives. In: DTIC documentGoogle Scholar
  58. Neta H, Bernstein A, Gelman F, Ronen Z (2012) Isotopic fractionation of nitrogen during anaerobic metabolism of HMX (octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine). In: Remediation technologies and their integration in water management Institut d’Estudis Catalans, BarcelonaGoogle Scholar
  59. Nijenhuis I, Andert J, Beck K, Kästner M, Diekert G, Richnow HH (2005) Stable isotope fractionation of tetrachloroethene during reductive dechlorination by Sulfurospirillum multivorans and Desulfitobacterium sp. strain PCE-S and abiotic reactions with cyanocobalamin. Appl Environ Microbiol 71:3413–3419CrossRefGoogle Scholar
  60. Penning H, Elsner M (2007) Intramolecular carbon and nitrogen isotope analysis by quantitative dry fragmentation of the phenylurea herbicide isoproturon in a combined injector/capillary reactor prior to GC separation. Anal Chem 79:8399–8405CrossRefGoogle Scholar
  61. Penning H, Sørensen SR, Meyer AH, Aamand J, Elsner M (2010) C, N, and H isotope fractionation of the herbicide isoproturon reflects different microbial transformation pathways. Environ Sci Technol 44:2372–2378CrossRefGoogle Scholar
  62. Pennington JC, Brannon JM, Gunnison D, Harrelson D, Zakikhani M, Miyares P, Jenkins TF, Clarke J, Hayes C, Ringleberg D (2001) Monitored natural attenuation of explosives. Soil Sediment Contam 10:45–70CrossRefGoogle Scholar
  63. Pierrini G, Doyle S, Champod C, Taroni F, Wakelin D, Lock C (2007) Evaluation of preliminary isotopic analysis (13C and 15N) of explosives a likelihood ratio approach to assess the links between semtex samples. Forensic Sci Int 167:43–48CrossRefGoogle Scholar
  64. Reinnicke S, Bernstein A, Elsner M (2010) Small and reproducible isotope effects during methylation with trimethylsulfonium hydroxide (TMSH): a convenient derivatization method for isotope analysis of negatively charged molecules. Anal Chem 82:2013–2019CrossRefGoogle Scholar
  65. Reinnicke S, Simonsen A, Sørensen SR, Aamand J, Elsner M (2012) C and N isotope fractionation during biodegradation of the pesticide metabolite 2, 6-dichlorobenzamide (BAM): potential for environmental assessments. Environ Sci Technol 46:1447–1454CrossRefGoogle Scholar
  66. Rügner H, Finkel M, Kaschl A, Bittens M (2006) Application of monitored natural attenuation in contaminated land management-a review and recommended approach for Europe. Environ Sci Pol 9:568–576CrossRefGoogle Scholar
  67. Sagi-Ben Moshe S, Ronen Z, Dahan O, Bernstein A, Weisbrod N, Gelman F, Adar E (2010) Isotopic evidence and quantification assessment of in situ RDX biodegradation in the deep unsaturated zone. Soil Biol Biochem 42:1253–1262CrossRefGoogle Scholar
  68. Schmidt TC, Jochmann MA (2012) Origin and fate of organic compounds in water: characterization by compound-specific stable isotope mass spectrometry. Annual Review of Analytical Chemistry 5Google Scholar
  69. Schmidt TC, Zwank L, Elsner M, Berg M, Meckenstock RU, Haderlein SB (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. Anal Bioanal Chem 378:283–300CrossRefGoogle Scholar
  70. Sturchio NC, Hatzinger PB, Arkins MD, Suh C, Heraty LJ (2003) Chlorine isotope fractionation during microbial reduction of perchlorate. Environ Sci Technol 37:3859–3863CrossRefGoogle Scholar
  71. Sturchio NC, Böhlke JK, Beloso AD Jr, Streger SH, Heraty LJ, Hatzinger PB (2007) Oxygen and chlorine isotopic fractionation during perchlorate biodegradation: laboratory results and implications for forensics and natural attenuation studies. Environ Sci Technol 41:2796–2802CrossRefGoogle Scholar
  72. Sturchio NC, Hoaglund JR III, Marroquin RJ, Beloso AD Jr, Heraty LJ, Bortz SE, Patterson TL (2012) Isotopic mapping of groundwater perchlorate plumes. Ground Water 50:94–102CrossRefGoogle Scholar
  73. Thullner M, Kampara M, Richnow HH, Harms H, Wick LY (2008) Impact of bioavailability restrictions on microbially induced stable isotope fractionation. 1. Theoretical calculation. Environ Sci Technol 42:6544–6551CrossRefGoogle Scholar
  74. Thullner M, Centler F, Richnow H–H, Fischer A (2012) Quantification of organic pollutant degradation in contaminated aquifers using compound specific stable isotope analysis—review of recent developments. Organ Geochem 42:1440–1460CrossRefGoogle Scholar
  75. Tobler NB, Hofstetter TB, Schwarzenbach RP (2007) Assessing iron-mediated oxidation of toluene and reduction of nitroaromatic contaminants in anoxic environments using compound-specific isotope analysis. Environ Sci Technol 41:7773–7780CrossRefGoogle Scholar
  76. Urbansky ET (2002) Perchlorate as an environmental contaminant. Environ Sci Pollut Res 9:187–192CrossRefGoogle Scholar
  77. Van Breukelen BM (2007) Quantifying the degradation and dilution contribution to natural attenuation of contaminants by means of an open system Rayleigh equation. Environ Sci Technol 41:4980–4985CrossRefGoogle Scholar
  78. Van Breukelen BM, Prommer H (2008) Beyond the rayleigh equation: reactive transport modeling of isotope fractionation effects to improve quantification of biodegradation. Environ Sci Technol 42:2457–2463CrossRefGoogle Scholar
  79. Widory D, Minet J-J, Barbe-Leborgne M (2009) Sourcing explosives: a multi-isotope approach. Sci Amp; Justice 49:62–72Google Scholar
  80. Wiedemeier TH, Wilson JT, Hansen JE, Chapelle FH, Swanson MA (1996) Technical protocol for evaluating natural attenuation of chlorinated solvents in groundwater. Revision 1. In: DTIC documentGoogle Scholar
  81. Wilson RD, Thornton SF, Mackay DM (2004) Challenges in monitoring the natural attenuation of spatially variable plumes. Biodegradation 15:359–369CrossRefGoogle Scholar
  82. Zwank L, Berg M, Elsner M, Schmidt TC, Schwarzenbach RP, Haderlein SB (2005) New evaluation scheme for two-dimensional isotope analysis to decipher biodegradation processes: application to groundwater contamination by MTBE. Environ Sci Technol 39:1018–1029CrossRefGoogle Scholar

Copyright information

© Springer International Publishing Switzerland 2014

Authors and Affiliations

  1. 1.Institute for Soil, Water and Environmental SciencesAgricultural Research Organization, Volcani CenterBet DaganIsrael
  2. 2.Geological Survey of IsraelJerusalemIsrael
  3. 3.Zuckerberg Institute for Water Research, Department of Environmental Hydrology and MicrobiologyBen-Gurion University of the NegevNegevIsrael

Personalised recommendations