David L. Freedman
Professor and Chair, Department of Environmental Engineering and Earth Sciences
David L. Freedman is a professor in the Department of Environmental Engineering and Earth Sciences at Clemson University; he also serves as the Department Chair. He received his Ph.D. in Environmental Engineering from Cornell University, where he worked with Jim Gossett on organohalide respiration of chlorinated ethenes and organohalide fermentation of dichloromethane. He started his academic career at the University of Illinois and moved to Clemson University in 1996. Dr. Freedman has received research support from SERDP, ESTCP, EPA, US DOE, US Army Construction Engineering Research Laboratory, US Air Force Office of Scientific Research, Westinghouse Savannah River Corporation, Battelle, and numerous consulting firms. He is currently the PI on a SERDP project (ER-2622) to evaluate rates of abiotic and biotic degradation of chlorinated ethenes within fractured rock at three Department of Defense sites. He is co-PI on an ESTCP project (ER-201730) that includes natural attenuation of 1,4-dioxane.
Assessing Biodegradation of 1,4-Dioxane Using a 14C Assay
Documenting the occurrence of 1,4-dioxane biodegradation in situ is challenging. One of the more significant hurdles is demonstrating that a decrease in concentration along a flow path is a consequence of contaminant destruction. Under aerobic conditions, the principal products of 1,4-dioxane biodegradation (CO2 and biomass) are difficult to discern from other sources, including naturally occurring organic matter. The overall objective of this study was to develop an assay utilizing 14C-labeled 1,4-dioxane that can definitively identify the rate at which degradation products accumulate. It is part of a larger on-going ESTCP project, ER-201730, which seeks to develop a quantitative framework for evaluating natural attenuation of 1,1,1-trichloroethane, 1,1-dichloroethane, and 1,1-dichloroethene, along with 1,4-dioxane. The 14C assay was adapted from a protocol to assess aerobic cooxidation of trichloroethene, developed with support from ESTCP Project ER-201584. The first step involves collection of groundwater samples in 160 mL serum bottles in the field, which are then sealed with Teflon-faced butyl rubber septa and shipped on ice to the laboratory. The bottles are warmed to room temperature overnight. Purified 14C-1,4-dioxane is then injected into the bottles, increasing the background concentration of 1,4-dioxane by approximately 165 µg/L. Filter sterilized groundwater from the same wells is used as a control to measure the rate of 14C product formation due to processes such as autoradiolysis. Purification of the 14C-1,4-dioxane is achieved by passing a stock solution dissolved in butanol through an Aminex HPX-87H HPLC column and trapping the mobile phase at the time when 1,4-dioxane elutes, separating it from the butanol and potential 14C-labeled contaminants. At approximately weekly intervals, for six weeks, samples from the serum bottle are removed and subjected to sparging with N2 under acidic conditions to drive off 14CO2, which is then trapped in an NaOH solution. The acid-sparged sample is then neutralized, filtered (0.2 µm), and passed through a solid phase extraction cartridge to remove 1,4-dioxane, allowing for detection of 14C-labeled soluble, non-volatile biodegradation products such as organic acids.
The assay was validated with cultures of 1,4-dioxane degrading microbes, including Pseudonocardia dioxivorans CB1190 and a mixed culture of propanotrophs (ENV487), in both laboratory media and groundwater. Thus far, groundwater samples from four sites have been evaluated. At two of the sites, there was no evidence of 1,4-dioxane biodegradation in the samples tested. Chlorinated volatile aliphatics were present but at low enough levels to suggest that they were not responsible for the lack of biodegradation activity. At a third site, statistically significant accumulation of 14C products above the filter sterilized controls occurred in samples from three of five wells. The pseudo first order rate constants for these wells are 0.017, 0.022, and 0.11 yr-1, corresponding to half-lives of 42, 31, and 6.6, years, respectively. The concentrations of 1,4-dioxane in these samples prior to adding the labeled compound were 254, 0, and 95 µg/L, respectively. Chlorinated aliphatic levels were below 29 µg/L and no 1,1-dichloroethene was detected. Biodegradation activity was not detected in samples from two other wells. For the fourth site, samples from one of the four wells tested exhibited a statistically significant accumulation of 14C products. Nevertheless, the rate was low (0.0068 yr-1), corresponding to a half-life of 102 years. For this well, the concentration of 1,4-dioxane was 349 µg/L. Overall, the results indicate that it is feasible to quantify 1,4-dioxane biodegradation in groundwater samples, even at very low rates. As data is collected from additional sites, it will afford an opportunity to correlate the rate of degradation measured with the 14C assay to other methods, including ones based on changes in ?13C enrichment and qPCR quantification of genes associated with 1,4-dioxane biodegradation.