A critical process of natural attenuation is contaminant degradation. It is difficult to demonstrate degradation of 1,4-dioxane, which is often present at chlorinated solvent sites but is considered recalcitrant in the subsurface. This research project aimed to develop a method to provide direct evidence for the intrinsic degradation of 1,4-dioxane at low concentrations in groundwater. Ultimately, the goal of this project was to develop a cost-effective and readily implementable “field method” to assess the natural and enhanced biodegradation of 1,4-dioxane in groundwater. Specifically, this project sought to extend the applicability of compound-specific isotope analysis (CSIA) to low 1,4-dioxane concentrations in groundwater so that CSIA can be used to document in situ biodegradation of 1,4-dioxane at Department of Defense (DoD) sites. The specific objectives of this project were to:

1. Develop a reliable method for performing CSIA on low levels of 1,4-dioxane in groundwater;
2. Assess whether CSIA can be used to document the cometabolic degradation of 1,4-dioxane; and
3. Assess the use of CSIA as a tool to evaluate the degradation of 1,4-dioxane and chlorinated volatile organic compounds (CVOCs) at DoD sites with different groundwater conditions.

The project was conducted in two phases. Phase I efforts were focused on objectives 1 and 2, while In Phase II, activities focused on addressing the third objective in order to expand the applicability of the method and drive efficient technology transfer.

Phase I of the research involved three major components. First, the method to concentrate dilute 1,4-dioxane was developed by adding a small quantity of synthetic carbonaceous sorbent to the water sample containing 1,4-dioxane. The dried solid sorbent was then subjected to thermal desorption to recover the 1,4-dioxane into a gas chromatograph for separation, conversion to carbon dioxide or hydrogen gas, and mass separation with isotope ratio mass spectrometry.

Microcosm studies were used to determine enrichment factors. The propane-grown cells of Mycobacterium sp. 1A degraded 1,4-dioxane by an aerobic cometabolic process. Carbon and hydrogen isotope ratios of 1,4-dioxane were analyzed in samples collected from the microcosms at different times during degradation using the newly-developed method.

Groundwater samples were collected from four separate DoD sites with low 1,4-dioxane concentrations with different co-contaminants in various aquifer conditions. At McClellan Air Force Base, a biostimulation pilot test for aerobic cometabolic 1,4-dioxane degradation was investigated. Carbon and hydrogen isotope ratios of 1,4-dioxane were analyzed in samples collected from the pilot test monitoring wells during different operational phases of the test. At the Cape Canaveral Air Force Station, two different sites were investigated. One had undergone a variety of remediation methods and is currently managed by monitored natural attenuation; a remedy at the second site had not yet been implemented when samples were collected. Groundwater samples from Vandenberg Air Force Base Site 24, which had undergone biosparging and bioaugmentation, were collected from two different aquifer zones and analyzed using the method developed herein.

In Phase II, two tasks were completed. Under Task 1, the stable carbon and hydrogen isotope ratios of 1,4-dioxane from ten different commercially available samples were analyzed to expand the isotopic database. In addition to analyzing different 1,4-dioxane suppliers, a simple evaporation experiment was performed to assess whether the isotopic composition of 1,4-dioxane changes when neat 1,4-dioxane undergoes evaporation. As part of Task 2, six case studies were completed at DoD sites while two were completed at non-DoD sites. An average of six samples were collected from each site for CSIA of 1,4-dioxane. When combined with the four Phase I case studies, a total of 12 sites have been sampled for this project; 68 groundwater samples have been analyzed for δ13 C, and 54 have been analyzed for δ2 H, not including duplicate and replicate analyses and samples with insufficient mass for isotopic characterization.

Phase I Results: It was determined that 0.5 grams of the synthetic carbonaceous sorbent, when added to a 40 mL vial containing aqueous 1,4-dioxane in the 10 to 100 µg/L range, could adsorb more than 99 percent of the 1,4-dioxane from solution. The 1,4-dioxane was successfully recovered from the dried solid sorbent by thermal desorption into a gas chromatograph with isotope ratio mass spectrometry. The method was successfully applied to samples at concentrations in the 1 µg/L range.

In the microcosms, an enrichment of heavier carbon and hydrogen isotopes was observed during 1,4-dioxane degradation. The enrichment trend closely followed the Rayleigh-type isotopic enrichment trend that is characteristic of degradation. Enrichment factors for carbon and hydrogen were determined to be approximately -1.98 and -25.6‰, respectively.

At McClellan AFB, an enrichment of heavier carbon and hydrogen isotopes was observed in samples from wells collected within the biostimulation zone compared to those outside of the biostimulation zone. The carbon and hydrogen isotopic composition of 1,4-dioxane in samples from wells outside of the biostimulation zone appeared to vary over time by approximately 5 and 50‰, respectively.

Because the source isotopic composition of 1,4-dioxane was not characterized at any of the sites, it is difficult to conclusively show that biodegradation had occurred with CSIA. However, enrichment of carbon and hydrogen isotope ratios beyond values reported for neat 1,4-dioxane was observed in certain samples. The results of the Phase I studies can be found in the Phase I Final Report.

Phase II Results: For the 10 samples of manufactured 1,4-dioxane, the δ13C values ranged from -33.6 to -28.8‰ (median: -32.2‰) and δ2 H ranged from -60 to -17‰ (median: -36‰). The majority of δ13C and δ2 H values for 1,4-dioxane measured in groundwater samples were within the range determined for manufactured (i.e. undegraded source) 1,4-dioxane. Samples from some field sites had δ13C or δ2 H values lower than the lowest values reported for neat 1,4-dioxane, indicating that the isotopic composition of manufactured 1,4-dioxane samples analyzed in this study does not represent the full range that can be expected at groundwater sites, and therefore conclusions regarding biodegradation of 1,4-dioxane based on the extent of enrichment beyond the “undegraded source” isotopic composition are subject to higher uncertainty.

Dual isotope plots can provide more robust qualitative indications of biodegradation and can provide insights into the degradation conditions by comparing the dual isotope slope to those reported for different laboratory-controlled degradation reactions. Dual isotope trends consistent with biodegradation of 1,4-dioxane were found at four of the 12 sites studied over the course of this project; biostimulation of 1,4-dioxane degradation was occurring at two of these sites.

The case studies show that CSIA can provide direct field-based evidence for 1,4-dioxane biodegradation in groundwater and, through the use of dual isotope plots, CSIA results can be compared to published enrichment trends to provide insights into the degradation conditions. On the other hand, at two of the sites where the dual isotope trend aligned with biodegradation, the magnitude of isotopic enrichment was smaller than observed in laboratory studies, perhaps due to blending of 1,4-dioxane from degradation zones with undegraded 1,4-dioxane in the screen interval of monitoring wells. Variability in isotopic composition of 1,4-dioxane at a site and/or increased analytical uncertainty at low concentrations of 1,4-dioxane can introduce uncertainty into the interpretation of field-based dual-isotope trends. Multiple lines of evidence are recommended for the interpretation of CSIA results.  The results of the Phase II studies can be found in the Phase II Final Report.

The method for documenting degradation of 1,4-dioxane would promote regulatory and stakeholder concurrence for natural attenuation of 1,4-dioxane at field sites. It could ultimately translate to using CSIA to measure degradation rates for 1,4-dioxane at field sites, and this would be an important evaluation tool for supporting cost-benefit analyses of different methods for enhancing attenuation at field sites. The results of this work could have widespread applicability and promote follow-on field-based studies that would further improve understanding of degradation processes at low concentrations of 1,4-dioxane. (Project Completion - 2020)

Bennett, P., M. Hyman, C. Smith, H. El Mugammar, M.-Y. Chu, M. Nickelsen, and R. Aravena. 2018. Enrichment with Carbon-13 and Deuterium During Monooxygenase-mediated Biodegradation of 1,4-Dioxane. Environmental Science & Technology Letters, 5(3):148-153.