In cooperation with the U.S. Army Garrison, Aberdeen Proving Ground Environmental Conservation and Restoration Division Aberdeen Proving Ground, Maryland Anaerobic Degradation of 1,1,2,2-Tetrachloroethane and Association with Microbial Communities in a Freshwater Tidal Wetland, Aberdeen Proving Ground, Maryland: Laboratory Experiments and Comparisons to Field Data Water-Resources Investigations Report 02–4157 U.S. Department of the Interior U.S. Geological Survey U.S. Department of the Interior U.S. Geological Survey Anaerobic Degradation of 1,1,2,2-Tetrachloroethane and Association with Microbial Communities in a Freshwater Tidal Wetland, Aberdeen Proving Ground, Maryland: Laboratory Experiments and Comparisons to Field Data By Michelle M. Lorah,Mary A. Voytek, Julie D. Kirshtein, and Elizabeth J. (Phillips) Jones Water-Resources Investigations Report 02–4157 In cooperation with the U.S. Army Garrison, Aberdeen Proving Ground Environmental Conservation and Restoration Division Aberdeen Proving Ground, Maryland The contents of this report have been approved for public release and unlimited distribution by the U.S. Army-- clearance number 4269-A-6 Baltimore, Maryland 2003 U.S. Department of the Interior GALE A. NORTON, Secretary U.S. Geological Survey Charles G. Groat, Director The use of trade, product, or firm names in this report is for descriptive purposes only and does not imply endorsement by the U.S. Government. For additional information contact: District Chief U.S. Geological Survey 8987 Yellow Brick Road Baltimore, MD 21237 Copies may be purchased from: U.S. Geological Survey Branch of Information Services Box 25286 Denver, CO 80225-0286 CONTENTS Abstract .................................................................................................................................................................................1 Introduction ...........................................................................................................................................................................2 Purpose and scope ........................................................................................................................................................6 Description of study area ..............................................................................................................................................6 Background on anaerobic degradation pathways .........................................................................................................8 Acknowledgments ......................................................................................................................................................10 Methods and data analysis ...................................................................................................................................................10 Laboratory microcosm experiments ...........................................................................................................................12 Sediment and ground-water collection ..............................................................................................................12 Microcosm preparation and incubation .............................................................................................................12 Geochemical and microbial community analyses ..............................................................................................12 Calculation of degradation rates ........................................................................................................................13 Laboratory enrichment experiments ...........................................................................................................................14 Enrichment preparation and incubation .............................................................................................................14 Geochemical and microbial community analyses ..............................................................................................14 Field data collection and analyses ..............................................................................................................................15 Surficial wetland sediment samples ...................................................................................................................15 Ground-water samples .......................................................................................................................................16 Calculation of degradation rates ........................................................................................................................16 Laboratory experiments on anaerobic degradation of 1,1,2,2-tetrachloroethane and association with microbial communities .....................................................................................................................................................16 Degradation rate of 1,1,2,2-tetrachloroethane ............................................................................................................17 Daughter compound production and degradation ......................................................................................................22 Spatial variability ...............................................................................................................................................22 Spatial variability of vinyl chloride production ........................................................................................23 Spatial variability of vinyl chloride degradation .......................................................................................28 Seasonal variability ............................................................................................................................................29 Substrate type .....................................................................................................................................................30 Redox conditions ...............................................................................................................................................32 Toxicity ..............................................................................................................................................................33 Pre-exposure to contaminants ............................................................................................................................34 Microbial communities and associations with degradation pathways ........................................................................35 Degradation of 1,1,2,2-tetrachloroethane ..........................................................................................................36 Daughter compound production and degradation ..............................................................................................41 Spatial variability of vinyl chloride production ........................................................................................41 Spatial variability of vinyl chloride degradation .......................................................................................45 Seasonal variability ...................................................................................................................................47 Substrate type ............................................................................................................................................49 Comparisons to field data on anaerobic degradation of 1,1,2,2-tetrachloroethane and association with microbial communities .............................................................................................................................................50 Degradation rate of 1,1,2,2-tetrachloroethane ............................................................................................................50 Daughter compound production and degradation ......................................................................................................51 Microbial communities ...............................................................................................................................................53 Implications for natural attenuation and remediation .................................................................................................56 Summary and conclusions ...................................................................................................................................................59 References cited ..................................................................................................................................................................61 v Figures ′ ′ 1. Map showing sampling sites and locations of sections A-A and C-C in the wetland study area along the West Branch Canal Creek, Aberdeen Proving Ground, Maryland .................................4 2. Diagram showing anaerobic degradation pathways for 1,1,2,2-tetrachloroethane (TeCA)................................5 3. Section showing hydrogeology and 1,1,2,2-tetrachloroethane (TeCA) distribution in ′ ground water along section A-A , June–October 1995 ..................................................................................7 44-41. Graphs showing: 4A,B. Degradation of 1,1,2,2-tetrachloroethane in previous anaerobic microcosm experiment (30E4) with wetland sediment (site WB30), May–June 1996: (A) 1,1,2,2-tetrachloroethane (TeCA) and the sum of daughter products in the TeCA-amended live microcosms and sterile controls, and (B) Daughter products 1,2-dichloroethene (12DCE, total of the cis and trans isomers); vinyl chloride (VC); 1,1,2-trichloroethane (112TCA); and 1,2-dichloroethane (12DCA) in theTeCA-amended microcosms ...................................................................9 5A,B. Apparent first-order degradation rates of (A) 1,1,2,2-tetrachloroethane (TeCA) and (B) 1,1,2-trichloroethane (112TCA) in anaerobic microcosm experiments, 1998–2000 ..............................19 6A-C. Degradation of 1,1,2,2-tetrachloroethane (TeCA) and production of daughter compounds in anaerobic microcosm experiments: (A) 23TeCA.1, (B) 23TeCA.2, and (C) 23TeCA.3 conducted in February–April 1998 using sediment from site WB23, and amended with methanol and three different initial TeCA concentrations (noted on figures) ..................................................................................................................20 7A,B. Degradation of 1,1,2,2-tetrachloroethane (TeCA) and production of daughter compounds in anaerobic microcosms, March–April 1999, constructed with wetland sediment from (A) site WB23 and (B) site WB30 ...........................................................................21 8A,B. Degradation of 1,1,2,2-tetrachloroethane (TeCA) and production of daughter compounds in anaerobic microcosms, July–August 1999, constructed with wetland sediment from (A) site WB23 and (B) site WB30 ...........................................................................21 9A,B. Degradation of 1,1,2,2-tetrachloroethane (TeCA) and production of daughter compounds in anaerobic microcosms, October–November 2000, constructed with wetland sediment from (A) site WB23 and (B) site WB30 ..................................................................22 10,10. Mass balance between initial (day 1) concentration of the parent compound 1,1,2,2-tetrachloroethane (TeCA) and the sum of daughter compounds produced in the live anaerobic microcosms, 1998–2000 .............................................................................22 11A,B. Daughter product distribution in anaerobic microcosms amended with 1,1,2,2-tetrachloroethane (TeCA) and constructed with wetland sediment from sites WB23 and WB30 in microcosm experiments in (A) March–April 1999 and (B) October–November 2000 ...........................................................................23 12,12. Daughter product distribution in anaerobic microcosms amended with 1,1,2-trichloroethane (112TCA) and constructed with wetland sediment from site WB23 (March–April 1999) and site WB30 (March–April 1999 and October–November 2000) ............................................................................................................................24 13A-D. Methane and ferrous iron concentrations in 1999 and 2000 microcosms amended with 1,1,2,2-tetrachloroethane (TeCA) (and live controls, LC): (A) ferrous iron in microcosms constructed with sediment from site WB23; (B) ferrous iron in microcosms constructed with sediment from site WB30; (C) methane in microcosms constructed with sediment from site WB23; and (D) methane in microcosms constructed with sediment from site WB30 ....................................................25 vi Figures—Continued 44-41. Graphs showing–Continued 14A,B. Degradation of 1,1,2-trichloroethane (112TCA), production of daughter compounds 1,2-dichloroethane (12DCA) and vinyl chloride (VC), and production of methane in anaerobic microcosms, March–April 1999, constructed with wetland sediment from (A) site WB23 and (B) site WB30 ..............................................26 15. Degradation of 1,1,2-trichloroethane (112TCA), production of daughter compounds 1,2-dichloroethane (12DCA) and vinyl chloride (VC), and production of methane in anaerobic microcosms, October–November 2000, constructed with wetland sediment from site WB30 ....................................................................................26 16A,B. Production of vinyl chloride in enrichment cultures inoculated with WB23 or WB30 microcosm slurry, October–November 2000 and (A) amended with 1,1,2-trichloroethane and either acetate (TCA30A and TCA23A) or hydrogen (TCA30H and TCA23H), or (B) amended with 1,2-cis-dichloroethene (cDCE) or 1,2-trans-dichloroethene (tDCE) and with either acetate (cDCE30A and cDCE23A) or hydrogen (cDCE30H and cDCE23H) ............................................................................27 17,17. Percent of vinyl chloride (VC) degraded in anaerobic microcosms amended with 1,1,2,2-tetrachloroethane (TeCA) or amended with TeCA and methanol (MeOH), 1996–2000 ....................................................................................................................................28 18A,B. (A) Degradation of vinyl chloride and (B) production of methane in sediment enrichments inoculated with WB23 or WB30 microcosm slurry, October–November 2000, and amended with only vinyl chloride (“no addition”), with vinyl chloride and 2-bromoethanesulfonic acid (BES), or with vinyl chloride and amorphous ferric oxyhydroxide (FeOOH) .........................................................29 19A,B. Daughter product distribution in anaerobic microcosms constructed with wetland sediment from site WB23, February–April 1998, that were amended with methanol and with (A) 1,1,2,2-tetrachloroethane (TeCA) and (B) 1,1,2-trichloroethane (112TCA) .............................................................................................................31 20A,B. Methane concentrations in February–April 1998 anaerobic microcosms amended with methanol and (A) 1,1,2,2-tetrachloroethane (TeCA) and (B) 1,1,2-trichloroethane (112TCA)........................................................................................................32 21 ,21. Degradation of 1,1,2,2-tetrachloroethane (TeCA) and production of daughter compounds in anaerobic microcosms amended with 2-bromoethanesulfonic acid (BES), methanol, and TeCA and constructed with wetland sediment from site WB23, February–April 1998 ........................................................................................................33 22A,B. Degradation of (A) 1,1,2,2-tetrachloroethane (TeCA) and production of daughter compounds in anaerobic microcosms amended with sulfate (SO ), methanol, 4 and TeCA and constructed with wetland sediment from site WB23, February–April 1998, and (B) effect of SO and 2-bromoethanesulfonic 4 acid (BES) addition on methane and SO concentrations ...........................................................................34 4 23,23. Degradation of 1,1,2,2-tetrachloroethane (TeCA) and production of daughter compounds in anaerobic microcosms conducted in February–April 1998 using sediment from background site WB19 and amended with methanol ..........................................................................35 24A,B. (A) Degradation of vinyl chloride and (B) production of methane in sediment enrichment experiments amended with vinyl chloride and constructed with fresh, pre-exposed, and preincubated wetland sediment from sites WB23 and WB30 .................................................................................................................................36 vii Figures—Continued 44-41. Graphs showing–Continued 25A-D. Bacteria terminal-restriction fragment length polymorphism profiles in selected TeCA-amended microcosms constructed with sediment from site WB23, February–April 1998: (A) live control [no 1,1,2,2-tetrachloroethane (TeCA) added, experiment 23LC], (B) killed control for microcosms with low initial TeCA concentration [experiment killed 23TeCA.1], (C) killed control for microcosms with TeCA and sulfate amendment [experiment killed 23SO -TeCA.2], and 4 (D) live microcosm with low initial TeCA concentration [experiment 23TeCA.1] ....................................37 26A,B. Bacteria terminal-restriction fragment length polymorphism profiles in 1,1,2,2-tetrachloroethane- (TeCA-) amended microcosms constructed with sediment from site WB23 compared to site WB30, July–August 1999: (A) site WB23 (experiment 23TeCA.7/99) and (B) site WB30 (experiment 30TeCA.7/99) ..................................................................................................38 27A,B. Bacteria terminal-restriction fragment length polymorphism profiles in 1,1,2,2-tetrachloroethane- (TeCA-) amended microcosms constructed with sediment from site WB23 compared to site WB30, March–April 1999: (A) site WB23 (experiment 23TeCA.3/99), and (B) site WB30 (experiment 30TeCA.3/99) ...........................................................................................................................39 28A,B. Bacteria terminal-restriction fragment length polymorphism profiles in 1,1,2,2-tetrachloroethane- (TeCA-) and 1,1,2-trichloroethane- (112TCA-) amended microcosms constructed with sediment from site WB30, October–November 2000: (A) site WB30, amended with TeCA (experiment 30TeCA.10/00), and (B) site WB30, amended with 112TCA (experiment 30TCA.10/00) ..........................................................................................................................40 29A,B. Terminal-restriction fragment length polymorphism (TRFLP) profiles for (A) bacteria and (B) methanogens in live control microcosms (experiment 23LC.3/99) constructed with sediment from site WB23, March–April 1999 .................................................................42 30A,B. Bacteria terminal-restriction fragment length polymorphism profiles in 1,1,2-trichloroethane- (112TCA-) amended microcosms constructed with sediment from site WB23 compared to site WB30, March–April 1999: (A) site WB23 (experiment 23TCA.3/99), and (B) site WB30 (experiment 30TCA.3/99) .................................................................................................................................................43 31A,B. Methanogen terminal-restriction fragment length polymorphism profiles in 1,1,2,2-tetrachloroethane- (TeCA-) amended microcosms constructed with sediment from site WB23 compared to site WB30, March–April 1999: (A) site WB23 (experiment 23TeCA.3/99), and (B) site WB30 (experiment 30TeCA.3/99) ...............................................................................................................................................44 32,32. Proportion of Methanosarcinaceae and the relative abundance of total methanogens in 1,1,2,2-tetrachloroethane- (TeCA-) amended microcosms constructed with WB23 and WB30 sediments, March–April 1999 (experiments 23TeCA.3/99 and 30TeCA.3/99) ...........................................................................................45 33,33. Proportion of Methanosarcinaceae in 1,1,2,2-tetrachloroethane- (TeCA-) amended microcosms constructed with WB30 sediments, with or without addition of methanol (MeOH), October–November 2000 ..............................................................46 34,34. Ferrous [Fe(II)] and ferric [Fe(III)] iron concentrations (in micromoles per gram wet weight) in wetland sediment samples that were collected by coring at sites WB23 and WB30, August 2000 .......................................................................................46 35. Bacteria terminal-restriction fragment length polymorphism profiles in sediment samples from site WB23 prior to microcosm preparation and incubation, March and August 1999 ......................................................................................................48 viii Figures—Continued 44-41. Graphs showing–Continued 36A,B. Methanogen terminal-restriction fragment length polymorphism profiles in 1,1,2,2-tetrachloroethane- (TeCA-) amended microcosms constructed with sediment from sites WB23 and WB30, July–August 1999: (A) site WB23 (experiment 23TeCA.7/99), and (B) site WB30 (experiment 30TeCA.7/99) ...........................................................................................................................49 37A,B. Natural log of 1,1,2,2-tetrachloroethane (TeCA) concentrations [(ln (C)] (normalized by chloride concentrations) and traveltime in wetland porewater (A) from piezometer siteWB30 in July–August 1995 and (B) from a multi-level sampler (WBM30) at the same site in June–July 2000 ............................................52 38A,B. Concentrations of (A) ferrous iron and (B) methane in wetland porewater collected from multi-level samplers (WBM23 and WBM30) at sites WB23 and WB30, June–July 2000 ..........................................................................................................................53 39,39. Distributions of 1,1,2,2-tetrachloroethane (TeCA) and daughter compounds in wetland porewater collected from multi-level samplers (WBM23 and WBM30) at sites WB23 and WB30, June–July 2000 ..................................................................................54 40A,B. Bacteria terminal-restriction fragment length polymorphism profiles in the ′ ′, surficial wetland sediment along (A) transect A-A and (B) transect C-C 1999 .......................................55 4 1,41. Bacteria terminal-restriction fragment length polymorphism profile in surficial wetland sediment at background site WB19, March 1999 .............................................................56 Tables 1. Treatments used in anaerobic microcosm experiments to examine 1,1,2,2-tetrachloroethane or 1,1,2-trichloroethane degradation in wetland sediments from the West Branch Canal Creek study area, Aberdeen Proving Ground, Maryland, May 1996–November 2000 ......................11 2. Treatments used in enrichment experiments with anaerobic daughter products of 1,1,2,2-tetrachloroethane (TeCA) degradation ..............................................................................................15 3. First-order rate constants and half-lives for degradation of 1,1,2,2-tetrachloroethane (TeCA) or 1,1,2-trichloroethane (112TCA) in anaerobic microcosm experiments conducted using wetland sediments, May 1996–November 2000 ................................................................18 4. Percentage of each daughter product observed in the TeCA-amended microcosms constructed with sediment from site WB23 and incubated at 5 degrees Celsius (experiment 23TeCA5.3/99) compared to those incubated at 19 degrees Celsius in March–April 1999 (experiment 23TeCA.3/99) and July–August 1999 (experiment 23TeCA.7/99) ...............................................................................................................................................30 5. Degradation rates for 1,1,2,2-tetrachloroethane (TeCA) and trichloroethene (TCE) estimated from field data in July–August 1995 and June–July 2000 along upward flowpaths in the wetland sediments, using TeCA and TCE concentrations normalized with chloride and an estimated linear flow velocity of 0.9 meters per year ..................................................51 6. Abundance of Methanosarcinaceae as a percentage of the total methanogen community in surface grab samples of wetland sediment, March 1999 ..........................................................................56 ix Conversion Factors, Vertical Datum, and Abbreviations _____________________________________________________________________________________________________________________________________ Multiply By To obtain _____________________________________________________________________________________________________________________________________ Length centimeter (cm) 0.3937 inch millimeter (mm) 0.03937 inch meter (m) 3.281 foot meter (m) 1.094 yard Volume liter (L) 33.82 ounce, fluid liter (L) 2.113 pint liter (L) 1.057 quart liter (L) 0.2642 gallon liter (L) 61.02 cubic inch Flow rate meter per year (m/yr) 3.281 foot per year Mass gram (g) 0.03527 ounce, avoirdupois kilogram (kg) 2.205 pound, avoirdupois ____________________________________________________________________________________________________________________________________ ° ° Temperatures in degrees Celsius ( C) may be converted to degrees Fahrenheit ( F) as follows: ° ° F = (1.8 x C) + 32 Vertical Datum: In this report, “sea level” refers to the National Geodetic Vertical Datum of 1929—a geodetic datum derived from a general adjustment of the first-order level nets of the United States and Canada, formerly called Sea Level Datum of 1929. Concentrations of chemical constituents in water are given either in milligrams per liter (mg/L), micrograms per liter (µg/L), millimoles per liter (mmol/L), or micromoles per liter (µmol/L). x Anaerobic Degradation of 1,1,2,2-Tetrachloroethane and Association with Microbial Communities in a Freshwater Tidal Wetland, Aberdeen Proving Ground, Maryland: Laboratory Experiments and Comparisons to Field Data By Michelle M. Lorah, Mary A. Voytek, Julie D. Kirshtein, and Elizabeth J. (Phillips) Jones Abstract Defining biodegradation rates and processes is at 19 degrees Celsius, whereas lower rate con- a critical part of assessing the feasibility of moni- stants of 0±0.03 and 0.06±0.03 per day were tored natural attenuation as a remediation method obtained in July–August 1999 microcosms incu- for ground water containing organic contaminants. bated at 19 degrees Celsius. Microbial community During 1998–2001, the U.S. Geological Survey profiles showed that low microbial biomass and conducted a microbial study at a freshwater tidal microbial diversity in the summer, possibly due to wetland along the West Branch Canal Creek, competition for nutrients by the wetland vegeta- Aberdeen Proving Ground, Maryland, as part of an tion, could account for these unexpectedly low investigation of natural attenuation of chlorinated degradation rates. In microcosms incubated at volatile organic compounds (VOCs) in the wet- 5 degrees Celsius, about 50 percent of the initial land sediments. Geochemical analyses and molec- TeCA in solution was converted to daughter prod- ular biology techniques were used to investigate ucts within a 35-day incubation period, indicating factors controlling anaerobic degradation of that biodegradation in the wetland sediments can 1,1,2,2-tetrachloroethane (TeCA), and to charac- continue during cold winter temperatures. terize the microbial communities that potentially Initial pathways of TeCA degradation were the are important in its degradation. Rapid TeCA and same in the wetland sediment microcosms regard- daughter product degradation observed in labora- less of the season or sediment collection site, the tory experiments and estimated with field data reduction-oxidation conditions, and the previous confirm that natural attenuation is a feasible reme- exposure of the sediment to contamination. diation method at this site. The diverse microbial Immediate and simultaneous dichloroelimination community that seems to be involved in TeCA and hydrogenolysis, producing 1,2-dichloro- degradation in the wetland sediments varies with ethene (12DCE) and 1,1,2-trichloroethane changing spatial and seasonal conditions, allowing (112TCA), respectively, were the initial TeCA continued effective natural attenuation throughout degradation pathways in all live microcosm exper- the year. iments. The production and degradation of vinyl Rates of TeCA degradation in anaerobic micro- chloride (VC), which is the most toxic of the cosm experiments conducted with wetland sedi- TeCA daughter compounds, was affected by spa- ment collected from two different sites (WB23 and tial and seasonal variability, reduction-oxidation WB30) and during three different seasons condition, and pre-exposure of the wetland sedi- (March–April 1999, July–August 1999, and ment. TeCA-amended microcosms constructed October–November 2000) showed little spatial with WB30 sediment showed approximately twice variability but high seasonal variability. Initial as much VC production as those constructed with first-order degradation rate constants for TeCA WB23 sediment. Results of 112TCA-amended ranged from 0.10±0.01 to 0.16±0.05 per day (half- microcosms indicated that the greater production lives of 4.3 to 6.9 days) for March–April 1999 and of VC in the WB30 sediment resulted from a October–November 2000 microcosms incubated greater predominance of the 112TCA dichloro- Abstract 1