Interaction of Microbial and Abiotic Processes in Soil Leading to the (Bio)Conversion and Ultimate Attenuation of New Insensitive Munitions Compounds

Objective

Relatively little is known about the environmental behavior of two new insensitive munitions compounds (IMCs), 3-nitro-1,2,4-triazole-5-one (NTO) and 2,4-dinitroanisol (DNAN). Bacteria are known that can degrade and biotransform nitroaromatic compounds, and biotransformations catalyzing the reduction of nitro-groups or demethylation of methoxy groups create reactive intermediates that are subject to oxidative coupling and formation of bound residue. In order to better understand the environmental fate of the new IMCs, the objective of this project is to study the interaction of biological and abiotic processes in the soil that collectively contribute to their conversion and attenuation under a range of soil conditions. This project has expanded our understanding of the potential risk for the accumulation of toxic intermediates, the potential for soil to attenuate the risk, as well as provide a scientific basis to predict the long-term fate of the IMCs to either mineralized products, bound residue in humus, or tightly adsorbed species based on soil composition and biogeochemical conditions.

 

Technical Approach

We undertook this project by evaluating the environmental fate of IMCs as a function of soil type collected from seven sites. The fates to be monitored included IMC sorption onto soil and (bio)transformation yielding intermediate compounds, mineralized products, and humus-bound residue. Based on the fates observed, we selected soils for further study to elucidate the main mechanisms of biodegradation and sorption/abiotic transformation. This information will be combined to test whether small structural modifications catalyzed by abiotic and biotic reactions will impact the ultimate fate of IMCs. The consequences of the findings are being assessed by evaluating the microbial and ecological toxicity of IMC-contaminated soils, IMCs, and their (bio)transformation products. An understanding of which mechanisms lead to the conversion of IMCs to safe end products can be used to predict which soil conditions are suitable for attenuating IMCs and to apply measures for improving attenuation.

The conversion processes are monitored using state-of-the art analytical techniques. The microbial diversity of IMC-degrading organisms is characterized by creating clone libraries. Selected pure cultures that benefit from IMC degradation have been isolated in order to identify putative genes involved in biodegradation. The reaction of IMCs and intermediates with reactive soil particles is being studied using batch and column experiments to gauge the importance of abiotic processes in IMC attenuation.

Benefits

Land management and bioremediation will be aided by the knowledge of soil conditions conducive to attenuation of IMCs. The identity of intermediates can be used to design environmental monitoring protocols. The identification of dominant microorganisms and genes involved in biodegradation can be translated into PCR reactions to monitor IMC sites for biodegradation potential. The elucidation of important secondary minerals involved in the abiotic conversion of IMCs can translate in practice to monitoring key constituents in soil that predict abiotic conversion.

Madeira, C.L., Jog, K.V., Vanover, E.T., Brooks, M.D., Taylor, D.K., Sierra-Alvarez, R., Waidner, L.A., Spain, J.C., Krzmarzick, M.J., Field, J.A. 2019. Microbial Enrichment Culture Responsible for the Complete Oxidative Biodegradation of 3-Amino-1,2,4-triazol-5-one (ATO), the Reduced Daughter Product of the Insensitive Munitions Compound 3-Nitro-1,2,4-triazol-5-one (NTO). Environ. Sci. Technol. 53: 12648-12656.

Khatiwada, R., Root, R., Abrell, L., Sierra-Alvarez, R., Field, J., Chorover, J. 2018. Abiotic reduction of insensitive munition compounds by sulfate green rust. Env. Chem. 15: 259-266.

Khatiwada, R., Root, R., Abrell, L., Sierra-Alvarez, R., Field, J., Chorover, J. 2018. Adsorption and oxidation of 3-nitro-1,2,4-triazole-5-one (NTO) and its transformation product (3-amino-1,2,4-triazole-5-one, ATO) at ferrihydrite and birnessite surfaces. Environmental Pollution 240: 200-208.

Khatiwada, R., Olivares, C., Abrell, L., Root, R., Sierra-Alvarez, R., Field, J,A., Chorover, J. 2018. Oxidation of Reduced Daughter Products from 2,4-dinitroanisole (DNAN) by Mn(IV) and Fe(III) Oxides. Chemosphere 201: 790-798.

Jagadish, B., Hurley, N.R. and E. A. Mash. 2017. Solvent-free nitration of electron-rich arenes. Synthetic Communications 47:2395-2398.

Olivares, C. Madiera, C., R. Sierra-Alvarez, Kadoya, W., Abrell, L., Chorover, J., Field, J. 2017 Environmental fate of ¹⁴C radiolabeled 2,4-dinitroanisole (DNAN) in soil microcosms. Environmental Science & Technology 51:13327–13334.

Olivares, C. I., J. Wang, C. D. Silva Luna, J. A. Field, L. Abrell, and R. Sierra-Alvarez. 2016. Continuous treatment of the insensitive munitions compound N-methyl-p-nitro aniline (MNA) in an upflow anaerobic sludge blanket (UASB) bioreactor. Chemosphere 144:1116-1122.

Olivares, C. I., L. Abrell, R. Khatiwada, J. Chorover, R. Sierra-Alvarez, and J. A. Field. 2016. (Bio)transformation of 2,4-dinitroanisole (DNAN) in Soils. J. Haz. Materials 34:214-221.

Krzmarzick, M., R. Khatiwada, C. Olivares, L. Abrell, R. Sierra-Alvarez, J. Chorover, and J. A. Field. 2015. Biotransformation and Degradation of the Insensitive Munitions Compound, 3-nitro-1,2,4-triazol-5-one (NTO), by Soil Bacterial Communities. Environmental Science & Technology 49:5681-5688.

Linker, B. R., R. Khatiwada, N. Perdrial, L. Abrell, R. Sierra, J. A. Field, and J. Chorover. 2015. Adsorption of novel insensitive munitions compounds at clay mineral and metal oxide surfaces. Environmental Chemistry 12:74-84.

Jagadish, B., J. A. Field, J. Chorover, R. Sierra-Alvarez, L. Abrell, and E. A. Mash. 2014. Synthesis of ¹³C and ¹⁵N labeled 2,4-dinitroanisole. Journal of Labelled Compounds and Radiopharmaceuticals 57:434-436.

Liang, J., C. Olivares, J. A. Field, and R. Sierra-Alvarez. 2013. Microbial toxicity of the insensitive munitions compound, 2,4-dinitroanisole (DNAN), and its aromatic amine metabolites. Journal of Hazardous Materials 262:281-287.

Olivares, C., J. Liang, L. Abrell, R. Sierra-Alvarez, and J. A. Field. 2013. Pathways of reductive 2,4-dinitroanisole (DNAN) biotransformation in sludge. Biotechnol. Bioeng. 110:1595-1604.

SERDP Project Graduate Student Camila Madeira

Camila's Trainee video

Camila and her award winning poster on November 3, 2015 at the Environmental Grad Blitz, organized by the University of Arizona Institute of the Environment.

 

Warren Kadoya

Guangbin Li

Past group members

Raju Khatiwada 

Chris Olivares

Jidong Liang

Sam Speet

Hector Amezquita

Carlos David Silva Luna

Mark Krzmarzick

Billy Linker

Cristina Alvarez Nieto

Stephan Cameron

 

Olivares, C.I. , Sierra-Alvarez, R., Abrell, L., Chorover, J., Field, J.A. Biotransformation and Microbial Toxicity of 2,4-Dinitroanisole (DNAN) Association of Environmental Engineering & Science Professors 2015 Conference,  Yale, CT. June 13-16, 2015.

Sierra-Alvarez, R., Westerhoff, P., Olivares, C.I , Bi, X., Field, J.A., Simonich, M., Tanguay, R.  Toxicity testing on chemical mechanical planarization nanoparticles and III-V ions using the zebrafish embryonic model. 2015 Annual Review Meeting of the Semiconductor Research Corporation Engineering Research Center. Tucson, AZ. April 1-2, 2014.

Olivares, C.I. , Sierra-Alvarez, R., Abrell, L., Chorover, J., Field, J.A. Biotransformation and Microbial Toxicity of 2,4-Dinitroanisole (DNAN). Society of Environmental Toxicology and Chemistry North America 35^TH Annual Meeting, Vancouver, B.C., Canada. Nov. 9-13, 2014.

Olivares, C.I., Sierra-Alvarez, R., Abrell, L.,  Field, J. A.  Biotransformation and microbial toxicity of 2,4-dinitroanisole. Arizona Mass Spectrometry Discussion Group. March 17, 2014.

Olivares, C.I., Sierra-Alvarez, R., Abrell, L.,  Field, J. A.  Biotransformation and microbial toxicity of 2,4-dinitroanisole. . Grad Blitz. Institute of the Environment. University of Arizona. Nov. 7, 2013.

Olivares, C.I., Sierra-Alvarez, R., Abrell, L.,  Field, J. A.  Biotransformation and microbial toxicity of 2,4-dinitroanisole. Superfund Annual Meeting 2014. Louisiana State University. Oct. 15-17, 2013.

Oral Presentations
Field, J., Sierra-Alvarez, R., Krzmarzick, M., Madeira, C., Olivares, C., Chorover J., Abrell, L. Biotransformation and biodegradation of insensitive munitions compounds in soil. Environmental Chemistry Session, Microbial and Molecular Tools to Determine the Fate and Biotransformation of Emerging Contaminants at 252nd American Chemical Society National Meeting in Philadelphia, PA, August 21-25, 2016. [Keynote talk]

Field, J.A, Sierra-Alvarez, R., Krzmarzick, M., Madeira, C.L., Olivares, C.I., Chorover, J., Abrell, L. Biotransformation pathways dictating the fate in soil of insensitive munition compounds, 2,4-dinitroanisole (DNAN) and 3-nitro-1,2,4-triazole-5-one (NTO). 2016 Society of Industrial Microbiology and Biotechnology Annual Meeting. New Orleans, LA. July 24-28, 2016 (invited talk).

Olivares, C.I., Abrell, L., Simonich, M., Chorover, J., Sierra-Alvarez, R., Tanguay, R.L., Field, J.A.  Coupling biotransformation of 2,4-dinitroanisole (DNAN) in anaerobic soil microcosms to a multidimensional toxicity assay using zebrafish embryos.  2015 Superfund Research Program Annual Meeting. San Juan, PR. Nov. 19, 2015.

Olivares, C.I., Abrell, L., Simonich, M., Chorover,J., Sierra-Alvarez, R., Tanguay, R.L., Field, J.A.  Coupling biotransformation of 2,4-dinitroanisole (DNAN) in anaerobic soil microcosms to a multidimensional toxicity assay using zebrafish embryos.  Society of Environmental Toxicology and Chemistry North America 36th Annual Meeting. Salt Lake City, UT., Nov. 1-5, 2015.

Olivares, C.I. , Abrell, L., Sierra-Alvarez, R., Chorover, J., Field, J.A. Characterization of products of 2,4-dinitroanisole (DNAN) microbial biotransformation and their inhibitory impact to microorganisms. 250^th American Chemical Society National Meeting. Boston, MA. Aug. 16, 2015.

Olivares, C.I. , Madeira, C.L., Abrell, L., Sierra-Alvarez, R., Chorover, J., Field, J.A. Environmental fate of 14C- ring labeled 2,4-dinitroanisole (DNAN) in anaerobic saturated soils. 250th American Chemical Society National Meeting. Boston, MA. Aug. 18, 2015.

Speet, S., Madeira, C.L., Cameron, S., Abrell, L., Chorover, J., Sierra-Alvarez, R., Field, J.A. Degradation and fate of 3-nitro-1,2,4-triazol-5-one in batch studie sin an anaerobic column. Earth Week 2015. University of Arizona.  April 19, 2015.

Khatiwada, R., Abrell, L., Chorover, J., Sierra-Alvarez, R., Field, J.A. Abiotic transformation of insensitive munitions compounds by birnessite. Earth Week 2015. University of Arizona.  April 19, 2015.

Olivares, C.I., Khatiwada, R., Sierra-Alvarez, R., Abrell, L., Chorover, J., Field, J. A.  Biotransformation of 2,4-dinitroanisole (DNAN) in soils. CH2MHILL Chemistry conference call. Oct. 23, 2014. (webinar)

Field, J. A. R. Sierra, C. Olivares, S. Cameron, M. Krzmarzick, H. Amezquita Garcia, C. Alvarez Nieto, L. Abrell, J. Chorover, R. Khatiwada and John Coffey II. Biotransformation of Insensitive Munition Compounds in Soil Microbial Cultures. JANNAF Interagency Propulsion Committee. 61^st JANNAF Propulsion Meeting (JPM), 42^nd Structures and Mechanical Behavior (SMBS), 38^th Propellant and Explosives Development and Characterization (PEDCS), 29^th Rocket Nozzle Technology (RNTS), 27^th Safety and Environmental Protection (SEPS), Charleston, SC 19 – 22 May 2014.

Olivares, C.I., Abrell, L., Chorover, J., Sierra-Alvarez, R., Field, J. A. In vivo ecotoxicity of 2,4-dinitroanisole (DNAN) biotransformation products using zebrafish (Danio rerio) embryos. Graduate Research Symposium. Chemical and Environmental Engineering. University of Arizona.  Sept. 25, 2014.

Olivares, C.I., Abrell, L., Sierra-Alvarez, R., Field, J. A.  Coupling fate and toxicity: Biotransformation and microbial toxicity of 2,4-dinitroanisole (DNAN) in anaerobic conditions. Earth Week 2014. University of Arizona.  April 10, 2014.

Olivares, C.I., Liang, J., Abrell, L., Sierra-Alvarez, R., Field, J.A. Bomb-eating microbes: Reductive biotransformation of 2,4-dinitroanisole and microbial toxicity. Soil, Water, and Environmental Science Student Plenary talk.  Earth Week 2013. University of Arizona.  April 12, 2013.

Olivares, C.I., Sierra-Alvarez, R., Abrell, L.,  Field, J. A.  Biotransformation and microbial toxicity of 2,4-dinitroanisole. Grad Blitz. Institute of the Environment. University of Arizona. Nov. 9, 2012. 

Proceedings

Field, J. A., R. Sierra, C. Olivares, S. Cameron, M. Krzmarzick, H. Amezquita Garcia, L. Abrell, J. Chorover,  R. Khatiwada  and J. Coffey II. 2014. Biotransformation of insensitive munition components in soil microbial cultures.. In: M. S. Johnson (ed), JANNAF Workshop Proceedings – Fate, Transport and Effects of Insensitive Munitions: Issues and Recent Data, Environmental Restoration Report. pp. 67-81.

Leif joined the Strategic Workshop on Research & Demonstration Needs for Management of Munitions Constituents  from July 28-29, 2015 at the Washington Navy Yard in Washington D.C.