Department Environmental Chemistry

Methods are needed to verify that attenuation of energetic compounds, such as trinitrotoluene (TNT) and hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), in groundwater is occurring, and it must also be possible to verify that strategies to enhance degradation processes are having the desired effects. This document provides a primer on sample collection, processing, and laboratory methods for compound specific isotope analysis (CSIA)methods for nitro explosives. Data evaluation, analysis, and interpretation for both single and dual isotope fractionation is presented. Two case studies are used to demonstrate the procedures for using CSIA to support the identification of the relevant reaction process and calculate the extent of which reaction, versus dilution or sorption, is leading to decreases in concentration.

Ferrous iron-bearing minerals are important reductants in the contaminated subsurface, but their availability for the reduction of anthropogenic pollutants is often limited by competition with other electron acceptors including microorganisms and poor accessibility to Fe(II) in complex hydrogeologic settings. The supply of external electron donors through in situ chemical reduction (ISCR) has been proposed as one remediation approach, but the quantification of pollutant transformation is complicated by the perturbations introduced to the subsurface by ISCR. Here, we evaluate the application of compound specific isotope analysis (CSIA) for monitoring the reduction of 2,4-dinitroanisole (DNAN), a component of insensitive munitions formulations, by mineral-bound Fe(II) generated through ISCR of subsurface material from two field sites. Electron balances from laboratory experiments in batch and column reactors showed that 3.6% to 11% of the total Fe in the sediments was available for the reduction of DNAN and its partially reduced intermediates after dithionite treatment. The extent of DNAN reduction was successfully quantified from its N isotope fractionation measured in the column effluent based on the derivation of a N isotope enrichment factor, ϵ_N, derived from a comprehensive series of isotope fractionation experiments with numerous Fe(II)-bearing minerals as well as dithionite-reduced subsurface materials. Our observations illustrate the utility of CSIA as a robust approach to evaluate the success of in situ remediation through abiotic contaminant reduction.

Subsurface contamination with the explosive hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) at ordnance production and testing sites is a problem because of the persistence, mobility, and toxicity of RDX and the formation of toxic products under anoxic conditions. While the utility of compound-specific isotope analysis for inferring natural attenuation pathways from stable isotope ratios has been demonstrated, the stable isotope fractionation for RDX reduction by iron-bearing minerals remains unknown. Here, we evaluated N and C isotope fractionation of RDX during reduction by Fe(II) associated with Fe minerals and natural sediments and applied N isotope ratios to the assessment of mineral-catalyzed RDX reduction in a contaminant plume and in sediment columns treated by in situ chemical reduction. Laboratory studies revealed that RDX was reduced to nitroso compounds without denitration and the concomitant ring cleavage. Fe(II)/iron oxide mineral-catalyzed reactions exhibited N isotope enrichment factors, ^ϵ_N, between -6.3±0.3‰ and -8.2±0.2‰, corresponding to an apparent ¹⁵N kinetic isotope effect of 1.04-1.05. The observed variations of the δ¹⁵N of ∼15‰ in RDX from groundwater samples suggested an extent of reductive transformation of 85% at an ammunition plant. Conversely, we observed masking of N isotope fractionation after RDX reduction in laboratory flow-through systems, which was presumably due to limited accessibility to reactive Fe(II).

The emerging use of 2,4-dinitroanisole (DNAN) in insensitive munitions formulations has caused concern about future contamination of subsurface environments, generating significant interest in understanding and identifying its transformation processes. Here we characterized the C and N isotope fractionation associated with abiotic and biological DNAN hydrolysis through alkaline hydrolysis at high pH as well as enzymatic hydrolysis by Nocardioides sp. JS1661 and partially purified DNAN O-demethylase. Whereas both reactions generated 2,4-dinitrophenol (DNP), compound-specific isotope analysis (CSIA) of DNAN and DNP revealed that these reactions occur by different mechanisms. Alkaline hydrolysis was associated with apparent ¹³C and ¹⁵N kinetic isotope effects (¹³C-AKIE and ¹⁵N-AKIE) of 1.044 ± 0.003 and 1.0027 ± 0.0004, respectively, reflecting the previously postulated nucleophilic aromatic substitution mechanism. Conversely, enzyme-catalyzed DNAN hydrolysis exhibited a ¹³C-AKIE of 1.027 ± 0.005 and a ¹⁵N-AKIE of 1.0032 ± 0.0003. On the basis of these AKIE values and the C and N isotope fractionation of DNP, our results imply that enzymatic O-demethylation of DNAN occurs through a nucleophilic substitution reaction at the aliphatic C of the methoxy group. This work provides a basis for the assessment of DNAN transformation by CSIA, as the C and N isotope fractionation patterns observed in this work are distinct from other hypothesized degradation pathways.