Recent studies have shown that the insensitive nitoaromatic explosive 2,4,-dinitroanisole (DNAN) and the insensitive nitramine explosive 3-nitro-1,2,4-triazol-5-one (NTO) are each susceptible to biotic and abiotic transformation by various mechanisms. In many instances, the degradation of these compounds in soils, groundwaters, and marine environments is difficult to assess because the degradation products produced are either unknown, common in nature (e.g., NO2-, N2O, NO3-), or are labile and/or difficult to measure. Moreover, abiotic and biotic processes may occur simultaneously, making them difficult to distinguish or apportion based solely on analyses of concentrations. The development and application of compound-specific stable isotope analysis (CSIA) methods for DNAN and NTO is therefore a necessary step to allow the extent of in situ degradation to be quantified and to potentially provide insights into degradation mechanisms. 

This project is being conducted in two phases. During Phase I, the key objectives were (1) to develop and validate CSIA methods for carbon (C) and nitrogen (N) in DNAN and NTO; and (2) to use these new CSIA methods and other analytical and modeling tools to quantify kinetic isotope effects in C and N during the biotic and abiotic transformations of these insensitive munition compounds. Laboratory experiments were performed to examine a variety of commonly occurring biotic and abiotic transformations, and the measured isotope effects provided new diagnostic measurement tools that may allow these transformations to be more clearly assessed and quantified in natural environments.

During Phase II, methods will be developed and applied for compound-specific oxygen isotope ratio (¹⁸O/¹⁶O) analysis of DNAN and NTO, and the solvent 1,4-dioxane for the purpose of obtaining improved understanding of the biotic and abiotic transformation mechanisms of these compounds in the environment. CSIA of oxygen isotope ratios in organic pollutants has been largely overlooked because of its daunting technical challenges. A capability for routine analysis of oxygen isotope ratios in munitions compounds and their transformation products, as well as other common chemicals of concern will provide a crucial new tool for deciphering the transformation pathways that may naturally attenuate these compounds. The importance of oxygen isotope analysis is based on the fact that many of the principal transformation pathways for these compounds in aqueous systems, as well as those for other nitroaromatics and nitramines, involve the rate-determining cleavage or formation of covalent oxygen bonds with carbon, nitrogen, or hydrogen (e.g., via abiotic and biotic hydrolysis, hydroxylation, nitro-reduction, oxygenation, and photoreduction or photo-oxidation by reactive oxygen and nitrogen species). All such reactions are likely to cause relatively large primary kinetic isotope effects in oxygen which could be diagnostic for the transformation pathways that are not clearly resolved by carbon and nitrogen isotope analysis. 

There are four general tasks to be pursued during Phase II:

1. The development of methods for accurate and precise CSIA of oxygen in DNAN, NTO, and 1,4-dioxane using GC-IRMS and Orbitrap-based instruments.
2. Applications of oxygen CSIA measurements to evaluate kinetic isotope effects for oxygen during abiotic and biotic transformations that are common in the environment. In addition, we anticipate that carbon and oxygen isotope ratios will have forensic value for tracing sources of 1,4-dioxane.
3. Evaluation of oxygen isotope exchange rates between water and insensitive munitions and 1,4-dioxane. Oxygen exchange with water or with solvents used in sample processing could complicate measurement and application of oxygen isotope measurements and kinetic isotope effects. 
4. Theoretical calculations will be performed, using density-functional theory combined with larger-scale molecular mechanics calculations to estimate isotope fractionation factors in model aqueous systems based on the differences in reaction energetics associated with position-specific isotopic substitutions.

Available GC-IRMS methodology for CSIA of oxygen isotope ratios in organic compounds has a number of technical challenges. Specific analytical hurdles include: extraneous sources of oxygen within the high-temperature reaction tube, such as water and nickel oxide; isobaric interferences of N₂ with CO at m/z 28-30; and production of CO₂ along with CO in the high-temperature reaction tube. These challenges are not insurmountable, and successful measurements of oxygen isotope ratios in vanillin, MTBE, and ethyl acetate have been published. Nonetheless, further refinement of GC-IRMS methods for oxygen CSIA is urgently needed, and will be pursued in this research using the GC-IRMS setup in our laboratory. Evaluation of the performance of Orbitrap-based high-resolution Fourier-Transform mass spectrometry for this purpose will also be examined. Orbitrap-based instruments have the advantage of being able to measure isotope ratios of intact molecules, opening the possibility of simultaneous C, N, and O isotope ratio measurements. Recent publications have demonstrated the potential of Orbitrap-based mass spectrometers for multi-element isotope analysis of intact molecules and ions, including position-specific isotope analysis with selective fragmentation.

Phase I Results

Results from Phase I of this project are described in the Phase I Final Report. Salient results include:

1. Demonstration of accurate and precise methods for CSIA of carbon and nitrogen isotope ratios in DNAN and NTO, along with some of their important transformation products. This task also refined sampling and extraction/purification methods needed for optimal analytical results.
2. Measurement of carbon and nitrogen kinetic isotope effects during alkaline hydrolysis of DNAN, and abiotic and biotic transformations of DNAN and NTO. The abiotic transformations of DNAN and NTO included reduction by 9,10-anthraquinone-2-disulfonate and a hematite-aqueous Fe²⁺ redox couple, as well as photolytic transformations under UV-A and UV-C irradiation. The biotic transformations involved pure cultures and consortia under aerobic and anoxic conditions, all exhibiting oxygen-independent nitroreduction pathways.
3. Calculations of reaction energetics and position-specific equilibrium isotope fractionation factors for carbon and nitrogen in DNAN during alkaline hydrolysis.

Phase I of this project achieved its objectives of the development and application of methods for CSIA of carbon and nitrogen in DNAN and NTO. The new analytical methods developed were used to measure isotope enrichment factors and enrichment factor ratios of carbon and nitrogen for different biotic and abiotic transformation pathways of DNAN and NTO. Phase II of this project will augment the results of Phase I with new methods and applications for CSIA of oxygen isotopes in DNAN and NTO, as well as C, N, and O isotopes in 1,4-dioxane. During Phase II, improved quantum mechanical calculations for reaction energetics and isotope fractionation pertinent to environmental transformations of the target compounds will also be explored.

The overall results of this project will provide new diagnostic measurements and predictive tools that enable transformation mechanisms of DNAN, NTO, and 1,4-dioxane to be more clearly evaluated, along with quantitative assessments of the extents of transformation of DNAN, NTO, and 1,4-dioxane on ranges and other impacted field sites. (Anticipated Phase II Completion - 2025)

Fuller, M., R. Rezes, P. Hedman, J. Jones, N.C. Sturchio, and P. Hatzinger. 2020. Biotransformation of the Insensitive Munition Constituents 3-Nitro-1,2,4-triazol-5-one (NTO) and 2,4-Dinitroanisole (DNAN) by Aerobic Methane-Oxidizing Consortia and Pure Cultures. Journal of Hazardous Materials 407:24341. doi.org/10.1016/j.jhazmat.2020.124341.

Wang, C., L.J. Heraty, A.F. Wallace, C. Liu, X. Li, G.P. McGovern, J. Horita, M.E. Fuller, P.B. Hatzinger, and N.C. Sturchio. 2021. Position-Specific Isotope Effects During Alkaline Hydrolysis of 2,4- Dinitroanisole Resolved by Compound-Specific Isotope Analysis, 13C NMR, and Density-Functional Theory. Chemosphere, 280:130625. doi.org/10.1016/j.chemosphere.2021.130625.

Wang, C., M.E. Fuller, L.J. Heraty, P.B. Hatzinger, and N.C. Sturchio. 2021. Photocatalytic mechanisms of 2,4-dinitroanisole Degradation in Water Deciphered by C and N Dual-Element Isotope Fractionation. Journal of Hazardous Materials, 411:125109. doi.org/10.1016/j.jhazmat.2021.125109.

Wang C.,L.J. Heraty, H. Li, M.E. Fuller, P.B. Hatzinger, and N.C. Sturchio. 2021. Method for Derivatization and Isotopic Analysis of the Insensitive Munition Compound 3-nitro-1,2,4-triazol-5-one (NTO). Journal of Hazardous Materials Letters, 2:100044. doi.org/10.1016/j.hazl.2021.100044.

Wang, C.L., A.F. Wallace, L.J. Heraty, H. Qi, and N.C. Sturchio. 2020. Alkaline Hydrolysis Pathway of 2,4-Dinitroanisole Verified by 18O Tracer Experiment. Journal of Hazardous Materials, 396:122627. doi.org/10.1016/j.jhazmat.2020.122627.