Hexahydro‐1,3,5‐trinitro‐1,3,5‐triazine (RDX) is a common soil contaminant at current and former military facilities, and also impacts groundwater and drinking water at numerous locations. RDX contamination often occurs over expansive areas, making in situ or ex situ treatment technologies difficult to implement. One potential alternative for managing RDX sites is monitored natural attenuation (MNA), in which contaminants are controlled by natural processes, including biodegradation. However, one limitation of this approach for RDX is that biodegradation rates can be relatively slow under field conditions, making accurate rate measurements difficult. Compound‐specific isotope analysis (CSIA) may overcome this limitation, because it allows measurements of slow degradation rates by measuring changes in the ratios of the stable isotopes of present in RDX, specifically the ratios of ¹⁵N/¹⁴N and ¹³C/¹²C.

The objective of this project was to validate a CSIA method to confirm and constrain rates of aerobic and anaerobic biodegradation of RDX at field sites. If successful, this method can be used by DoD to provide critical data to support MNA as a remedy for RDX in groundwater, and also to confirm the effectiveness of in situ enhanced aerobic or anaerobic bioremediation. The stable isotopic composition of NO₃^- and NO₂^- were also measured when these anions co‐occurred with RDX, to evaluate whether these potential degradation products from RDX could be used to further demonstrate MNA in the field.

A CSIA method was developed that utilizes gas‐chromatography coupled to isotope‐ratio mass‐spectrometry (GC‐IRMS) to quantify C and N isotope ratios in RDX. In summary, RDX collected from groundwater is concentrated via solid‐phase extraction (SPE) either in the field using a column developed during this project (primarily for wells with low RDX concentrations) or in the laboratory. The RDX is then eluted from the SPE columns into acetonitrile, concentrated, and analyzed for δ¹⁵N and δ¹³C in RDX using GC‐IRMS. To evaluate the use of CSIA to document anaerobic biodegradation of RDX, δ¹⁵N and δ¹³C in RDX were measured in a series of wells along a groundwater flow path at Dahlgren Naval Surface Warfare Center, MD (Dahlgren NSWC) before and after injection of emulsified oil into a biobarrier to promote RDX biodegradation. To evaluate aerobic biodegradation of RDX via CSIA, δ¹⁵N in RDX was measured in groundwater samples collected both (1) during a series of push‐pull tests in which a culture capable of aerobically degrading RDX was bioaugmented into an aquifer at Umatilla Chemical Depot, OR (UCMD) along with a low dose of carbon substrate, and (2) from the bulk aerobic aquifer at Dahlgren NSWC along the flowpath of two RDX plumes. δ¹³C values were not measured for RDX during these field tests because previous studies with pure cultures indicated no measurable fractionation of C during aerobic RDX biodegradation.

This study demonstrated that δ¹⁵N and δ¹⁸O in NO₂^- and/or NO₃^- can be a useful marker of aerobic or anaerobic RDX biodegradation, provided that the amounts of these anions generated from RDX are not overwhelmed by those generated from other sources. Further, the combination of RDX and NO₂^-/NO₃^- stable isotope analyses can be used to confirm natural degradation processes, particularly under anaerobic conditions.

Stable isotope analysis of N and O in nitrate is currently estimated at $149 per sample (for 6 to 20 samples) from the USGS Reston, VA Stable Isotope Laboratory and C and N isotope analysis in RDX at $500 per sample by the University of Delaware EIGL Laboratory of Dr. Neil Sturchio, which is currently the only laboratory performing this method on a per sample basis. The estimated total cost for sampling and analysis of 20 wells to support a natural attenuation evaluation of RDX was just over $21,000.

The CSIA technology is applicable for documenting the biological degradation of RDX in groundwater by both aerobic and anaerobic mechanisms. However, when RDX is degraded aerobically via the typical denitration pathway, the extent of N fractionation is expected to be low (ɛ= ~ ‐2.4 ‰) and C is not expected to fractionate measurably based on pure culture studies. Thus, for the method to be useful for field samples, losses of RDX in groundwater either over distance (e.g., along a groundwater flowpath) or time (e.g., in an individual well) must be substantial, on the order of 80% or higher from initial concentrations. In many instances, and given the observed variability in this measurement, it is unlikely that aerobic biodegradation of RDX in the field will be definitively proven by N isotope fractionation. It is recommended that additional lines of evidence of RDX biodegradation under aerobic conditions be assessed along with N isotope analysis of RDX, including (1) measurements of NDAB as a possible degradation intermediate; (2) molecular analysis of aquifer samples for the presence of xplA/xplB genes, which encode key enzymes involved in aerobic RDX biodegradation; (3) analysis of N and O stable isotopes in NO₂^- and/or NO₃^- that co‐occur with RDX (particularly if initial RDX concentrations are in the mg/L range or higher); (4) laboratory microcosms or columns incubated aerobically to document RDX biodegradation under controlled conditions; and (5) application of stable isotope probing (SIP) in laboratory microcosms or mesocosms to identify organisms that aerobically degrade RDX. The combination of one or more of these techniques in conjunction with N stable isotope analysis of RDX at a field site is recommended to clearly document aerobic RDX biodegradation or confirm the absence of this process.

When RDX is biodegraded via anaerobic mechanisms, C and N stable isotopes are both applicable to document this process, due to the relatively large fractionation factors measured in culture studies (ɛ= ~ ‐4.7 ‰ for C and ɛ= ~ ‐9.9 ‰ for N). Dual isotope plots can be used to confirm biodegradation, as was done for Dahlgren NSWC field samples downgradient of an emulsified oil biobarrier. Many of the general lines of evidence previously suggested for evaluating aerobic RDX biodegradation are also applicable for anaerobic biodegradation, including (1) evaluation of degradation intermediates, but in this case MNX, DNX and TNX rather than NDAB; (2) analysis of N and O stable isotopes in NO₂^- and/or NO₃^- that co‐occur with RDX; (3) laboratory microcosms or columns incubated anaerobically; and (4) application of SIP in laboratory microcosms or mesocosms to identify anaerobic RDX degraders. As previously noted for evaluating aerobic biodegradation, a combination of one or more of these techniques in conjunction with C and N stable isotope analyses of RDX is recommended to document anaerobic RDX biodegradation.

Fuller, M.E., L. Heraty, C.W. Condee, S. Vainberg, N.C. Sturchio, J.K. Bohlke, and P.B. Hatzinger. 2016. Relating Carbon and Nitrogen Isotope Effects to Reaction Mechanisms During Aerobic and Anaerobic Degradation of Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) by Pure Bacterial cultures. Applied and Environmental Microbiology, 82(11):3297-3309.