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Final Report (Posted 3/18) FINAL REPORT Interaction of Microbial & Abiotic Processes in Soil Leading to the (Bio)Conversion and Ultimate Attenuation of New Insensitive Munitions Compounds SERDP Project ER-2221 DECEMBER 2016 Jim A. Field Jon Chorover Reyes Sierra-Alvarez Leif Abrell University of Arizona, Tucson, AZ John Coffey II CH2M HILL Distribution Statement A Page Intentionally Left Blank This report was prepared under contract to the Department of Defense Strategic Environmental Research and Development Program (SERDP). The publication of this report does not indicate endorsement by the Department of Defense, nor should the contents be construed as reflecting the official policy or position of the Department of Defense. Reference herein to any specific commercial product, process, or service by trade name, trademark, manufacturer, or otherwise, does not necessarily constitute or imply its endorsement, recommendation, or favoring by the Department of Defense. Page Intentionally Left Blank Form Approved REPORT DOCUMENTATION PAGE OMB No. 0704-0188 1. REPORT DATE (DD-MM-YYYY) 2. REPORT TYPE 3. DATES COVERED (From - To) 30-12-2016 FinalReport Feb9th 2012- Feb 8th 2017 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER Interaction of Microbial & Abiotic Processes in Soil Leading to the (Bio)Conversion W912HQ-12-C-0021 and Ultimate Attenuation of New Insensitive Munitions Compounds 5b. GRANT NUMBER SERDP ER-2221 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) 5d. PROJECT NUMBER Jim A Field ER-2221 Jon Chorover 5e. TASK NUMBER Reyes Sierra-Alvarez Leif Abrell 5f. WORK UNIT NUMBER John Coffey II 7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) 8. PERFORMING ORGANIZATION REPORT University of Arizona CH2 MHILL University of Arizona 888 N Euclid, Room 9191 South 104 Jamaica Street Tucson, AZ 85719 Englewood, CO 10. SPONSOR/MONITOR’S ACRONYM(S) 9. SPONSORING / MONITORING AGENCY NAME(S) AND ADDRESS(ES) SERDP Strategic Environmental Research and Development Program 11. SPONSOR/MONITOR’S REPORT 4800 Mark Center Drive, Suite 17D03 NUMBER(S) ER-2221 Alexandria, VA 22350-3605 12. DISTRIBUTION / AVAILABILITY STATEMENT Approved for public release; distribution is unlimited after embargo ending on June 1st, 2017 13. SUPPLEMENTARY NOTES 14. ABSTRACT Relatively little is known on insensitive munitions compounds, 2,4-dinitroanisole (DNAN) and 3-nitro-1,2,4-triazol-5-one (NTO), perform in the environment. The objectives of this study were to evaluate the interaction of abiotic and biotic factors contributing to the attenuation of IMCs in the soil leading to the formation of environmentally safe end-points. Anaerobic conditions favored the biological and/or chemical reduction of the nitro groups leading to the formation of amines intermediates. The amines retained toxicity thus futher conversion was required. The amines formed from DNAN eventually became covalently incorporated into soil organic matter. The amine from NTO became mineralized by soil bacteria or manganese dioxide to carbon dioxide and inorganic nitrogen. 15. SUBJECT TERMS Insensitive munitions compounds,; 2,4-dinitroanisole (DNAN); 3-nitro-1,2,4-triazol-5-one (NTO); explosives; nitroaromatic compounds (NACs); nitroheterocyclic compounds; aromatic amines; 3-amino-1,2,4-triazol-5-one (ATO); 2-methoxy-5-nitroaniline (MENA); 2,amino-4-nitroanisole; 2,5-diaminosnidole (DAAN); biodegradation; biotransformation, bioconversion, bacteria; soil; transformation 16. SECURITY CLASSIFICATION OF: 17. LIMITATION 18. NUMBER 19a. NAME OF OF ABSTRACT OF PAGES RESPONSIBLE UNCLASS PERSON 204 Jim Field a. REPORT b. ABSTRACT c. THIS PAGE 19b. TELEPHONE NUMBER (include UNCLASS UNCLASS UNCLASS UNCLASS area code) 520-621- 0704 Page Intentionally Left Blank Table of Contents ABSTRACT 1 1. OBJECTIVES 3 2. BACKGROUND 4 2.1. Background on Insensitive Munitions Compounds ..........................................4 2.2. Background on Adsorption of Insensitive Munitions Compounds by Soil Components .........................................................................................................................5 2.3. Background on Biodegradation and Biotransformation of IMC ......................6 2.3.1. Biodegradation and biotransformation of DNAN.................................7 2.3.2. Biodegradation and biotransformation of NTO ....................................8 2.3.3. Irreversible covalent incorporation of aromatic amines into humus ....8 2.4. Ecotoxicity of DNAN and NTO .......................................................................9 3. MATERIAL AND METHODS 11 3.1. Materials .........................................................................................................11 3.1.1. Chemicals ............................................................................................11 3.1.2. Soils and inoculum ..............................................................................11 3.1.3. Humin purification ..............................................................................11 3.2. Analytical Methods .........................................................................................12 3.2.1. HPLC-DAD ........................................................................................12 3.2.2. UHPLC-Q-ToF-MS ............................................................................12 3.2.3. Ion chromatography ............................................................................13 3.2.4. Methane analysis by GC-FID .............................................................13 3.2.5. Nitrogen and carbon dioxide analysis by GC-TCD ............................13 3.2.6. Ultraviolet-visible (UV-Vis) analyses coupled to zebrafish toxicity ..13 3.2.7. Solid phase characterization of the soils .............................................13 3.2.8. 14C - fractionation ...............................................................................14 3.3 Bioassays..........................................................................................................15 3.3.1. Inocula and basal medium ..................................................................15 i 3.3.2. Biotransformation assays .............................................................................16 3.3.3. Inhibition Bioassays ............................................................................17 3.4. NTO/ATO Column Experiments ....................................................................20 3.5. Adsorption Studies ..........................................................................................22 3.5.1. Mineral synthesis and clay preparation. ..............................................22 3.5.2. Batch sorption-desorption method ......................................................22 3.6. Oxidation of IMCs and IMC-Daughter Products by Minerals .......................23 3.7. Clone Library Methods to Characterize an ATO-Biodegrading Enrichment Culture 25 3.7.1. MR DNA primers and methods ..........................................................25 3.7.2. Conventional clone library ..................................................................25 4. RESULTS AND DISCUSSION 27 4.1. Adsorption of IMCs by Mineral Components of the Soil ...............................27 4.1.1. Results: SR-XRD: Synthesis products and clay characterization .......27 4.1.2. Results: adsorption isotherms .............................................................27 4.1.3. Results: Oxidation of MENA by acid birnessite .................................33 4.1.4. Discussion: Adsorption trends for Na+ and K+ smectite ...................33 4.1.5. Discussion: Adsorption to metal oxides .............................................36 4.1.6. Discussion: Adsorption reversibility...................................................36 4.1.7. Discussion: Preliminary Indications of Oxidation of MENA and ATO by Acid Birnessite..............................................................................................37 4.1.8. Conclusions adsorption work ..............................................................37 4.2. Reaction of DNAN and Daughter Products with Birnessite and Ferrihydritre39 4.2.1. Results: Reaction of DNA, MENA and DAAN with ferrihydrite ......39 4.2.2. Results: Reaction of DNA, MENA and DAAN with birnessite .........40 4.2.3. Results: Extent of mineralization ........................................................41 4.2.4. Transformation of birnessite based on X-ray diffraction (XRD) and X-ray photoelectron spectroscopy (XPS). ......................................................42 4.2.5. Discussion DNAN and daughter product reaction with birnessite and ferrihydrite. ..............................................................................................................44 4.3. Reaction of NTO and Daughter Products with Birnessite and Ferrihydrite ...47 4.3.1. Results: Oxidation assessment of NTO and ATO by birnessite .........47 ii 4.3.2. Results: Adsorption of IMCs to ferrihydrite: ......................................51 4.3.4. Discussion: Oxidation assessment of NTO and ATO by birnessite ...53 4.3.5. Discussion: Adsorptive loss from solution with ferrihydrite ..............55 4.3.6. Conclusion: Reaction of NTO and Daughter Products with Birnessite and Ferrihydrite ..........................................................................................55 4.4. Reduction of IMCs with Green Rust ..............................................................56 4.4.1. Results and discussion: reduction with green rust ..............................56 4.4.2 Conclusion: reduction with green rust .................................................59
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