Application of a multiple lines of evidence approach to document natural attenuation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) in groundwater.

This study utilized innovative analyses to develop multiple lines of evidence for natural attenuation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) in groundwater at the U.S. Department of Energy's Pantex Plant. RDX, as well as the degradation product 4-nitro-2,4-diazabutanal (NDAB; produced by aerobic biodegradation or alkaline hydrolysis) were detected in a large portion of the plume, with lower concentrations of the nitroso-containing metabolites produced during anaerobic biodegradation. 16S metagenomic sequencing detected the presence of bacteria known to aerobically degrade RDX (e.g., Gordonia, Rhodococcus) and NDAB (Methylobacterium), as well as the known anoxic RDX degrader Pseudomonas fluorescens I-C. Proteomic analysis detected both the aerobic RDX degradative enzyme XplA, and the anoxic RDX degradative enzyme XenB. Groundwater enrichment cultures supplied with low concentrations of labile carbon confirmed the potential of the extant groundwater community to aerobically degrade RDX and produce NDAB. Compound-specific isotope analysis (CSIA) of RDX collected at the site showed fractionation of nitrogen isotopes with δN values ranging from approximately -5‰ to +9‰, providing additional evidence of RDX degradation. Taken together, these results provide evidence of in situ RDX degradation in the Pantex Plant groundwater. Furthermore, they demonstrate the benefit of multiple lines of evidence in supporting natural attenuation assessments, especially with the application of innovative isotopic and -omic technologies.