Prepared in cooperation with the 

Department of Energy Savannah River National Laboratory 

Dichloroethene and Vinyl Chloride Degradation Potential 

in Wetland Sediments at Twin Lakes and Pen Branch, 

Savannah River National Laboratory, South Carolina 

By Paul M. Bradley 

Open File 2007–1028 

U.S. Department of the Interior 

U.S. Geological Survey 
U.S. Department of the Interior 

DIRK KEMPTHORNE, Secretary 

U.S. Geological Survey 

Mark D. Myers, Director 

U.S. Geological Survey, Reston, Virginia 2007 

Revised and reprinted: 2007 

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Suggested citation: 

Bradley, P.M., 2007, DCE and VC degradation potential in wetland sediments at Twin Lakes and Pen Branch, 

Savannah River National Laboratory, South Carolina: U.S. Geological Survey Open File Report 2007-1028, 

21p. 

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Although this report is in the public domain, permission must be secured from the individual 

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ii
Contents 

Abstract................................................................................................................................................................................1 

Introduction .........................................................................................................................................................................2 

Purpose and Scope............................................................................................................................................................2 

Methods ...............................................................................................................................................................................3 

Study Sites .......................................................................................................................................................................3 

Radiochemicals...............................................................................................................................................................4 

Microcosm Studies ........................................................................................................................................................5 

Analytical Methods ........................................................................................................................................................5 

Isolation and Enrichment Methods .............................................................................................................................6 

Degradation under Oxic and Anoxic Conditions...........................................................................................................6 

DCE Degradation Under Oxic Conditions ...................................................................................................................6 

DCE Degradation Under Anoxic Conditions...............................................................................................................7 

VC Degradation Under Oxic Conditions......................................................................................................................9 

VC Degradation Under Anoxic Conditions ...............................................................................................................10 

No Evidence of Degradation in Heat-Sterilized Controls ......................................................................................11 

Microbial Isolation and Enrichment Efforts .............................................................................................................11 

References Cited ..............................................................................................................................................................12

Appendix ............................................................................................................................................................................14

Figures 

1. Location of the DOE Savannah River National Laboratory in South Carolina.. ............................4 

2. Final percentage recovery of [1,2-14C] DCE radioactivity as 14CO2 in viable sediment 

microcosms incubated under oxic conditions. Data are means of triplicate microcosms. 

Remaining 14C-radioactivity was recovered as undegraded 14C-DCE.. ...........................................7 

3. Final percentage recovery of [1,2-14C] DCE radioactivity as 14CH4, 14CO2, 14C-Ethene, 14C-Ethane, and 14C-VC in viable sediment microcosms incubated under anoxic 

conditions. Data are means of triplicate microcosms. Remaining 14C-radioactivity was 

recovered as undegraded 14C-DCE... ...............................................................................................8 

4. Final percentage recovery of [1,2-14C] VC radioactivity as 14CO2 in viable sediment 

microcosms incubated under oxic conditions. Data are means of triplicate microcosms. 

Remaining 14C-radioactivity was recovered as undegraded 14C-VC................................................9 

5. Final percentage recovery of [1,2-14C] VC radioactivity as 14CH4, 14CO2, 

14C-Ethene, and 14C-Ethane, and 14C-VC in viable sediment microcosms incubated under anoxic 

conditions. Data are means of triplicate microcosms. Remaining 14C-radioactivity was 

recovered as undegraded 14C-VC...................................................................................................10 

iii
Tables 

1. Sample collection locations and sediment descriptions.. Organic contents were 

estimated as loss on ignition. Data are means±SD for duplicate samples.. ....................................5 

A1. Final percentage accumulation of 14C-products from microbial degradation of [1,2-14C] 

DCE in viable sediment microcosms after 60 days. Data are means±SD for triplicate 

microcosms. Remaining 14C-radioactivity was recovered as undegraded 14C-DCE. No 

accumulation of 14C-products was observed in killed control or sediment free control 

microcosms....................................................................................................................................14 

A2. Final percentage accumulation of 14C-products from microbial degradation of [1,2-14C] 

VC in viable sediment microcosms after 60 days. Data are means±SD for triplicate 

microcosms. Remaining 14C-radioactivity was recovered as undegraded 14C-VC. No 

accumulation of 14C-products was observed in killed control or sediment free control 

microcosms....................................................................................................................................15 

iv
Conversion Factors 

Temperature in degrees Celsius (°C) may be converted to degrees Fahrenheit (°F) as follows: 

°F=(1.8×°C)+32 

v
Abbreviations 

oC degrees celsius 

g gram(s) 

h hour(s) 

M mole(s) per liter 

mg/L milligram(s) per liter 

mL milliliter(s) 

μCi/μmole microcuries per micromole 

μg/L microgram(s) per liter 

μm micrometer(s) 

μM micromoles per liter 

μmole micromole 

nM nanomoles per liter 

nmole nanomole 

DCE cis-dichloroethene 

PCE perchloroethene 

PSI pounds per square inch 

SD standard deviation 

TCE trichloroethene 

VC vinyl chloride 

vi
Dichloroethene and Vinyl Chloride Degradation 

Potential in Wetland Sediments at Twin Lakes and 

Pen Branch, Savannah River National Laboratory, 

South Carolina 

By Paul M. Bradley 

Abstract 

A series of 14C-radiotracer-based 

microcosm experiments was conducted to 

assess the mechanisms and products of 

degradation of dichloroethene (DCE) and 

vinyl chloride (VC) in wetland sediments at 

the Department of Energy (DOE) Savannah 

River National Laboratory. This project 

investigated the potential for biotic and 

abiotic DCE and VC degradation in wetland 

sediments from the Twin Lakes area of the 

C-BRP investigative unit and from the 

portion of Pen Branch located directly down 

gradient from the CMP investigative unit. 

Substantial degradation of [1,2-14C] 

DCE and [1,2-14C] VC to 14CO2 was 

observed in all viable sediment microcosms 

prepared under oxic conditions. These 

results indicate that microbial mineralization 

processes, involving direct oxidation or 

cometabolic oxidation, are the primary 

mechanisms of DCE and VC biodegradation 

in Twin Lake and Pen Branch sediments 

under oxic conditions. 

Substantial degradation of [1,2-14C] 

DCE and [1,2-14C] VC was observed in all 

viable sediment microcosms incubated 

under anoxic conditions. Production of 

14CO2 was observed in all sediment 

microcosms under anoxic conditions. In 

general, the accumulation of mineralization 

products (14CO2 and 14CH4) was comparable 

to the accumulation of those reduced 

daughter products (14C-VC, 14C-ethene or 

14C-ethane) traditionally identified with 

chloroethene reductive dechlorination. 

These results indicate that microbial 

mineralization processes can be an 

important component of DCE and VC 

degradation in Twin Lake and Pen Branch 

sediments under anoxic conditions. These 

results demonstrate that an evaluation of the 

efficiency of in situ DCE and VC 

biodegradation in Twin Lakes and Pen 

Branch that is based solely on the observed 

accumulation of reduced daughter products 

may underestimate substantially the total 

extent of contaminant biodegradation and, 

thus, the contribution of biodegradation to 

overall contaminant attenuation. 

No evidence of abiotic degradation 

of [1,2-14C] DCE or [1,2-14C] VC was 

observed in heat-sterilized control 

treatments in this study under oxic or anoxic 

conditions. Efforts to enrich and isolate 

microorganisms involved in the 

mineralization of [1,2-14C] cis-DCE and/or 

[1,2-14C] VC were unsuccessful. 
2 

Introduction 

 Recent evidence (Bradley and others, 

variously dated) indicates that 

biodegradation of chloroethene 

contaminants in ground-water and surfacewater environments can yield two general 

types of degradation products: reduced 

daughter products associated with reductive 

degradation processes or mineralization 

products. The primary reductive 

chloroethene degradation mechanism 

reported in saturated environments is 

reductive dechlorination. This process 

involves consecutive hydrogen substitution 

reactions yielding sequentially less 

chlorinated daughter products (TCE, DCE 

and VC) and ultimately ethene. The polychlorinated ethenes, PCE and TCE, are 

highly oxidized compounds that readily 

undergo reduction reactions under anoxic 

conditions. Reductive dechlorination is 

common in anoxic groundwater and surfacewater systems and appears to be the primary 

mechanism for in situ biotransformaton of 

the parent compounds, PCE and TCE. The 

efficiency of chloroethene reductive 

dechlorination, however, appears to decrease 

with decreasing chlorine number. 

Significant reductive dechlorination of 

chloroethene contaminants to the nonchlorinated product, ethene, has been 

reported (Bradley 2003 for review), but is 

often limited in situ due to low electron 

donor supply, high electron donor 

competition, the presence of alternative 

terminal electron acceptors for facultative 

chlororespiring microorgansims, the 

potential absence or low activity of DCE 

and VC dechlorinating microorganisms, and 

the presence of inhibitory substances 

including more oxidized chloroethene 

compounds. In contrast, the potential for 

chloroethene oxidation to mineralized 

products increases with decreasing chlorine 

number and microbial mineralization to CO2

can be a significant component of in situ VC 

and DCE natural attenuation under oxic and 

anoxic conditions. The end-product(s) of 

microbial chloroethene mineralization, 

primarily CO2 (or CO2 and CH4 under 

acetotrophic methanogenic conditions), 

however, are not unique to chloroethene 

mineralization and, consequently, are not 

diagnostic of this process. Laboratory 

microcosm evaluations remain the best 

approach for assessing the in situ potential 

for microbial mineralization of chloroethene 

contaminants under oxic or anoxic 

conditions. 

Purpose and Scope 

Recent research (Bradley and others, 

variously dated) indicates that the potential 

for chloroethene biodegradation is 

particularly pronounced in wetland 

environments where spatial and temporal 

variations in the hydrologic and 

geochemical character of wetland sediments 

often lead to highly dynamic microbial 

communities with a wide range of metabolic 

capabilities and the demonstrated potential 

for both reductive dechlorination and 

mineralization. Because the reduced 

daughter products, DCE and VC, regularly 

increase in importance in the down gradient 

portion of anoxic chloroethene plumes and 

because wetlands and surface-water bodies 

are common down gradient receptors, an 

evaluation of the potentially important 

biodegradation mechanisms, including oxic 

and anoxic mineralization mechanisms, is 

particularly important in potential wetland 

and surface-water receptors. 

This report describes the relative 

contribution (relative to reductive 

dechlorination) of oxic and anoxic microbial 

mineralization processes to the natural 
3

attenuation of DCE and VC in wetland 

sediments from the Twin Lakes area of the 

C-BRP investigative unit and from the reach 

of Pen Branch located directly down 

gradient from the CMP investigative unit. 

The information presented in this report is 

intended to support ongoing efforts to 

evaluate the contribution of in situ microbial 

degradation to natural attenuation of 

chloroethene contaminants at the C-BRP 

and CMP investigative units at the Savannah 

River National Lab. In addition, this report 

describes the potential for abiotic 

degradation of DCE and VC in Twin Lakes 

and Pen Branch sediments in heat-sterilized, 

abiotic control microcosms. Finally, this 

report describes the effort to enrich and/or 

isolate chloroethene mineralizing 

microorganisms from viable, sediment 

treatments that demonstrated significant 14CVC or 14C-DCE mineralization. Specific 

objectives of this investigation were: 

Conduct a microcosm investigation 

using 14C-DCE and 14C-VC to 

evaluate the mechanisms and 

products of DCE and VC 

biodegradation in sediments from 

Twin Lakes and Pen Branch. 

Determine the contribution of 

mineralization processes, relative to 

processes leading to the 

accumulation of reductive 

dechlorination daughter products, to 

the natural attenuation capacity of 

DCE and VC in these two wetland 

systems. 

Conduct a microcosm investigation 

using 14C-DCE and 14C-VC to 

evaluate the potential for abiotic 

degradation of DCE and VC in heatsterilized sediment microcosms from 

Twin Lakes and Pen Branch. 

Initiate an effort to isolate and enrich 

defined cultures that are capable of 

anoxic mineralization of [1,2-14C] 

DCE and/or [1,2-14C] VC. Isolation 

and enrichment of microbial 

communities, however, is resource 

intensive and offers a low probability 

of success in the short-term. 

Methods 

This section presents details of the 

study sites, sediment collection locations 

and the experimental approach. 

Study Sites 

 Sediments for conducting microcosm 

studies were collected from the DOE 

Savannah River National Laboratory, 

located at the western border of South 

Carolina (figure 1). 

Wetland sediments were collected in 

September 2004 from the Twin Lakes area 

of the C-BRP investigative unit. Twin Lakes 

sediments were collected from the small 

stream that drains through the center of the 

Twin Lakes wetland prior to discharge into 

Four Mile Branch. Twin Lakes sediments 

were collected next to and identified by the 

ground-water monitoring wells; CRP-42, 

CRP-45, CRP-46 and CRP-49. 

 Wetland sediments were collected in 

September 2004 from the reach of Pen 

Branch that is located directly down gradient 

from the CMP investigative unit. Sediments 

were collected from the CMP side of Pen 

Branch in the area of potential discharge of 

chloroethene-contaminated ground water. 

Pen Branch sediments were collected next to 

and identified by the ground-water 

monitoring wells; CMP-39, CMP-40 and 

CMP-41. 

 Qualitative physical descriptions of 

the collected sediments are presented in 

table 1. Sediment organic contents for the 

collected sediments were estimated based on 

mass loss on ignition at 550 oC and are 

presented in table 1. 
4 

Figure 1. Location of the DOE Savannah River National Laboratory in South Carolina. 

Radiochemicals 

The potential for chloroethene 

biodegradation in Twin Lakes and Pen Branch 

sediments was investigated using uniformly 

labeled [1,2-14C] DCE (4 μCi/μmole; Moravek 

Biochemicals, Brea, California) and [1,2-14C] 

VC (1.6 μCi/μmole; Perkin Elmer Life 

Sciences, Boston, Massachusetts). The 

radiochemical purity of the [1,2-14C] 

chloroethene stocks was evaluated in the U.S. 

Geological Survey laboratory by direct injection 

radiometric detection gas chromatography 

(GC/RD) and found to be greater than 98 

percent pure. Authentic H14CO3

-

 (Sigma 

Biochemicals, St. Louis, Missouri) and 14CH4

(Perkin Elmer Life Sciences, Boston, 

Massachusetts) were used as radiolabeled 

standards for calibration and methods 

development. Each had radiochemical purities 

greater than 98 percent. 
5

Site Sample Location Description

Table 1. Sample collection locations and sediment descriptions. Organic contents were 

estimated as loss on ignition. Data are means±SD for duplicate samples. [%, percent] 

Microcosm Studies 

In general, sediment microcosms were 

composed of 10 mL serum vials with 10±0.5 g 

of saturated sediment and an atmosphere of 

nitrogen (anoxic treatments) or ambient air 

(oxic treatments). Triplicate experimental 

(viable) microcosms were prepared for each 

sediment treatment. Duplicate autoclaved 

control microcosms and a single sediment-free 

control microcosm were prepared for each 

sediment treatment and autoclaved three times 

for 1 h at 15 PSI and 121 oC. All anoxic 

microcosm treatments were pre-incubated in the 

dark at room temperature for 7 days prior to the 

addition of 14C-substrates, in order to ensure 

strictly anoxic conditions. Microcosms were 

amended with [1,2-14C] DCE or [1,2-14C] VC to 

yield initial dissolved substrate concentrations 

of 280 μg/L DCE or 310 μg/L VC, respectively. 

Following the addition of 14C-substrates, 

microcosms were incubated in the dark at room 

temperature (approximately 23 oC) for 60 days. 

Analytical Methods 

Headspace concentrations of O2, CH4, 14CH4

, CO2, 14CO2, ethene, 14C-ethene, ethane, 14C-ethane, VC, and 14C-VC were assessed by 

analyzing 1.0 ml of the microcosm headspace 

using packed column gas chromatography with 

sequential radiometric detection and thermal 

conductivity detection. The headspace sample 

volumes were replaced with pure oxygen (oxic 

treatments) or nitrogen (anoxic treatments). 

Dissolved phase concentrations of 14C-analytes 

were estimated based on experimentally 

determined Henry’s partition coefficients 

(Bradley and others, 2000b). Because inorganic 

carbon is present as dissolved CO2 and HCO3

-

 at 

the circum-neutral pH values observed in the 

collected sediments, a dimension less partition 

coefficient for the distribution of inorganic 

carbon between the headspace and the dissolved 

phase was determined by injecting H14CO3

-

 into 

the dissolved phase of triplicate autoclaved 

sediment microcosms (prepared as described for 

the degradation study), allowing the 

microcosms to equilibrate for 24 h, and 

measuring the 14CO2 radioactivity in the 

headspace using radiometric detection gas 

chromatography. The radiometric detector was 

calibrated by liquid scintillation counting using 

H14CO3

-

. 

Organic Content 

(% dry weight)

CRP-42 organic silt/clay 10 0 

CRP-45 organic silt/clay 23 1 

CRP-46 organic silt/clay 21 1 

C-BRP 

(Twin Lakes)

CRP-49 sand 0.2 0.1 

CMP-39 sand/gravel 0.1 0.0 

CMP-40 organic sand 2 1 

CMP 

(Pen Branch)

CMP-41 organic silt/clay 15 1
6 

Isolation and Enrichment Methods 

Isolation of the microorganisms that are 

responsible for anoxic chloroethene 

mineralization is preferred in order to clarify the 

mechanisms underlying the mineralization 

process and to provide molecular targets for the 

identification of the responsible organisms in 

situ. Isolation and enrichment of microbial 

communities, however, is resource intensive 

and offers little probability of success in the 

short-term. The approximate 15 year period 

between the initial field evidence for 

chloroethene reductive dechlorination (See for 

review: Bradley, 2003, McCarty and Semprini, 

1994) and the ultimate isolation of a 

microorganism capable of complete 

chloroethene reductive dechlorination to ethene 

(Dehalococcoides ethenogenes) (Maymó-Gatell 

and others, 1997) indicated, from the outset, 

that identification of anoxic chloroethene 

mineralizing microorganisms, in all probability, 

would be a lengthy process that would exceed 

the DOE project time constraints. Nevertheless, 

in light of the demonstrated capability of several 

sediments collected from Twin Lakes and Pen 

Branch to catalyze substantial mineralization of 

[1,2-14C] DCE and/or [1,2-14C] VC, an effort to 

enrich and/or isolate microorganisms capable of 

chloroethene mineralziation was initiated. 

Efforts to isolate and/or identify cultures 

capable of DCE and/or VC mineralization 

involved two general approaches: (1) serial 

dilution of active Twin Lakes and Pen Branch 

sediment slurries using heat-sterilized water 

(both filtered and unfiltered treatments were 

assessed) collected from Twin Lakes and Pen 

Branch as the growth media and [1,2-14C] DCE 

or [1,2-14C] VC as the growth substrates, and (2) 

serial transfer of selected inocula using [1,2-14C] 

DCE or [1,2-14C] VC as the growth substrate 

and various artificial growth media. Cultures 

tested using the second approach included the 

active sediment slurries as well as a number of 

fermentative, sulfate-reducing and ironreducing cultures that had demonstrated 

capacities for contaminant degradation and that 

were commercially available from American 

Type Culture Collection (ATCC; Manassas, 

Virginia). Artificial media used for the second 

approach included minimal salt media as well as 

preparations recommended for and specific to 

the purchased ATCC cultures. The selection 

criteria for the isolation effort was the persistent 

ability to oxidize [1,2-14C] DCE or [1,2-14C] VC 

in consecutive culture transfers. 

Degradation under Oxic and Anoxic 

Conditions 

The following sections present the 

results of the microcosm degradation studies 

and the enrichment efforts that were conducted 

in this investigation. 

DCE Degradation under Oxic Conditions 

Degradation of [1,2-14C] DCE was 

observed in all experimental sediment 

microcosms incubated under oxic conditions 

(figure 2, table A1). The sole product of [1,2-

14C] DCE degradation that was observed under 

oxic conditions was 14CO2. The mean 

accumulation of 14CO2 after 60 days incubation 

ranged from 35±3 percent of theoretical in 

microcosms prepared with sediments from 

CRP-46 to 60±3 percent in microcosms 

prepared with sediments from CMP-40 

treatments. The remaining microcosm 14Cradioactivity was associated with residual [1,2-

14C] DCE. The degradation of [1,2-14C] DCE 

observed in these oxic sediment treatments was 

attributable to biological activity because no 

significant 14C-product accumulation was 

observed in autoclaved control or sediment-free 

control microcosms. The results of this 

experiment indicate that microbial 

mineralization, involving direct oxidation or 

cometabolic oxidation, is the primary 

mechanism of DCE degradation in Twin Lake 

and Pen Branch sediments under oxic 

conditions. No evidence of reductive 
7

CRP-42 CRP-45 CRP-46 CRP-49 N.a.N. CMP-39 CMP-40 CMP-41

0

20

40

60

80

100 CO2 

Twin Lakes Pen Branch

14C-DCE Degradation

(Percent Theroetical)

Figure 2. Final percentage recovery of [1,2-14C] DCE radioactivity as 14CO2 in viable 

sediment microcosms incubated under oxic conditions. Data are means of triplicate 

microcosms. Remaining 14C-radioactivity was recovered as undegraded 14C-DCE.

degradation or reduced daughter products was 

observed under oxic incubation conditions. 

Wetlands and shallow surface-water 

systems are characterized by considerable 

temporal and spatial variability in the sediment 

geochemistry and in the activity of associated 

microbial redox communities. In such settings, 

oxic microbial mineralization is expected to be 

a significant contributor to in situ DCE 

attenuation. Thus, an evaluation of the 

efficiency of in situ DCE biodegradation based 

solely on the accumulation of reduced daughter 

products may underestimate substantially the 

actual extent of contaminant biodegradation and 

the overall contribution of oxic biodegradation 

to contaminant attenuation. 

DCE Degradation under Anoxic Conditions 

Substantial degradation of [1,2-14C] DCE 

also was observed in all experimental sediment 

microcosms incubated under anoxic conditions 

(figure 3, table A1). The degradation of [1,2-

14C] DCE observed in this study was attributable 

to biological activity because no significant 14Cproduct accumulation was observed in 

autoclaved control or sediment free control 

microcosms. The establishment and 

maintenance of anoxic conditions was 

confirmed in experimental, autoclaved control 

and sediment free control microcosms by 

headspace gas chromatography.
8 

CRP-42 CRP-45 CRP-46 CRP-49 N.a.N. CMP-39 CMP-40 CMP-41

0

20

40

60

80

100 CH4 

CO2 

ETHENE 

ETHANE 

VC 

Twin Lakes Pen Branch

14C-DCE Degradation

(Percent Theroetical)

Figure 3. Final percentage recovery of [1,2-14C] DCE radioactivity as 14CH4, 14CO2, 14C-Ethene, 14C-Ethane, and 14C-VC in viable sediment microcosms incubated under 

anoxic conditions. Data are means of triplicate microcosms. Remaining 14C-radioactivity 

was recovered as undegraded 14C-DCE.

Production of 14CO2 was observed in all 

sediment microcosms under anoxic conditions. 

The mean accumulation of 14CO2 after 60 days 

incubation ranged from 16±2 percent of 

theoretical in CMP-41 microcosms to 40±8 

percent in CRP-45 treatments. The sole product 

of [1,2-14C] DCE degradation in CMP-39 

sediments was 14CO2. In contrast, all other 

sediment treatments contained both reductive 

dechlorination and mineralization products. In 

this study, the accumulation of mineralization 

products (14CO2

 and 14CH4) was comparable to or 

greater than the accumulation of those products 

(

14C-VC, 14C-ethene or 14C-ethane) associated 

with reductive dechlorination. 

Previous reports indicate that, with the 

possible exception of DCE degradation under 

Mn(IV)-reducing conditions, the net 

mineralization of DCE involves an initial 

reduction to VC, which may or may not 

accumulate to detectable concentrations 

(Bradley and Chapelle, 1997; 1998b). The 

detection of 14C-VC in six of the seven sediment 

treatments in this study is consistent with those 

reports and indicates that reductive 

dechlorination is the primary mechanism of 

biological removal of DCE in Twin Lakes and 

Pen Branch wetland sediments. The results of 

this experiment, however, do indicate that 

mineralization may be an important component 

of DCE degradation in Twin Lake and Pen 

Branch sediments under anoxic conditions. 

Moreover, these results further illustrate that an 

evaluation of in situ DCE biodegradation in 

Twin Lakes and Pen Branch that is based solely 

on the accumulation of reduced daughter 

products may significantly underestimate the 

extent of anoxic contaminant biodegradation 

and the contribution of anoxic biodegradation to 

contaminant attenuation. 
9

VC Degradation under Oxic Conditions 

Essentially complete degradation of 

[1,2-14C] VC was observed in all experimental 

sediment microcosms incubated under oxic 

conditions (figure 4, table A2). The sole product 

of [1,2-14C] VC degradation observed under 

oxic conditions was 14CO2.The degradation of 

[1,2-14C] VC observed in this study was 

attributable to biological activity because no 

significant 14C-product accumulation was 

observed in autoclaved control or sediment-free 

control microcosms. The results of this 

experiment indicate that mineralization, 

involving direct oxidation or cometabolic 

oxidation, is the primary mechanism of VC 

degradation in Twin Lake and Pen Branch 

sediments under oxic conditions. No evidence 

of reductive degradation or reduced daughter 

products was observed under these conditions.

CRP-42 CRP-45 CRP-46 CRP-49 N.a.N. CMP-39 CMP-40 CMP-41

0

20

40

60

80

100

CO2 

Twin Lakes Pen Branch

14C-VC Degradation

(Percent Theroetical)

Figure 4. Final percentage recovery of [1,2-14C] VC radioactivity as 14CO2 in viable 

sediment microcosms incubated under oxic conditions. Data are means of triplicate 

microcosms. Remaining 14C-radioactivity was recovered as undegraded 14C-VC. 

As noted earlier for DCE, the spatial and 

temporal variability in the sediment 

geochemistry and the activity of associated 

microbial redox communities in wetland and 

shallow surface-water systems indicates that 

oxic, microbial mineralization of VC may 

contribute significantly to VC attenuation at 

Twin Lakes and Pen Branch. Thus, an 

evaluation of the efficiency of in situ VC 

biodegradation based solely on the 

accumulation of reduced daughter products may 

significantly underestimate the extent of oxic 

contaminant biodegradation and the 
10

contribution of oxic biodegradation to 

contaminant attenuation. 

VC Degradation under Anoxic Conditions 

Degradation of [1,2-14C] VC also was 

observed in all experimental sediment 

microcosms incubated under anoxic conditions 

(figure 5, table A2). The degradation of [1,2-

14C] VC observed in this study was attributed to 

biological activity because no 14C-product 

accumulation was evident in autoclaved control 

or sediment-free control microcosms. The 

establishment and maintenance of anoxic 

conditions was confirmed in experimental, 

autoclaved control and sediment-free control 

microcosms by headspace gas chromatography.

CRP-42 CRP-45 CRP-46 CRP-49 N.a.N. CMP-39 CMP-40 CMP-41

0

20

40

60

80

100 CH4 

CO2 

ETHENE 

ETHANE 

Twin Lakes Pen Branch

14C-VC Degradation

(Percent Theroetical)

Figure 5. Final percentage recovery of [1,2-14C] VC radioactivity as 14CH4, 14CO2, 14CEthene, and 14C-Ethane, and 14C-VC in viable sediment microcosms incubated under 

anoxic conditions. Data are means of triplicate microcosms. Remaining 14C-radioactivity 

was recovered as undegraded 14C-VC. 

Production of 14CO2 was observed in all 

sediment microcosms under anoxic conditions. 

The mean accumulation of 14CO2 after 60 days 

incubation ranged from 7±2 percent of 

theoretical in CRP-46 microcosms to 16±14 

percent in CRP-45 treatments. 14CO2 was the 

sole product of [1,2- 14C] VC degradation 

observed in anoxic, CMP sediment microcosms, 

despite the fact that the sediments from CMP-41 

were organic-rich. Because oxidation of 14Cethene to 14CO2 under anoxic conditions has 

been demonstrated previously, these results do 

not preclude reductive dechlorination of VC in 

Pen Branch (Bradley and Chapelle, 2002). 

Rather, these results indicate that microbial 

degradation of VC to mineralization products 

may be important in Pen Branch. 

In the CRP sediment treatments, the 

products of [1,2-14C] VC degradation reflected 

the sediment organic content. CRP-49 
11

sediments were relatively low in organic 

content, and the sole products of [1,2-14C] VC 

biodegradation detected in these microcosms 

were mineralization products (14CO2 and 14CH4). 

In contrast, treatments containing organic-rich 

CRP sediments accumulated both reductive 

dechlorination and mineralization products. In 

these cases, [1,2-14C] VC biodegradation yielded 

either predominantly reductive dechlorination 

daughter products (CRP-42) or comparable 

accumulation of reductive dechlorination and 

mineralization products (CRP-45 and CRP-46). 

Because the accumulation of 14CO2 that 

was observed in Twin Lake and Pen Branch 

microcosms under anoxic conditions may be the 

result of a net microbial mineralization of [1,2- 14C] VC involving an initial reduction to 14Cethene followed by oxidation to 14CO2

 (Bradley 

and Chapelle, 2002), the results of this study do 

not necessarily reflect the full contribution of 

reductive dechlorination processes to the 

degradation of VC in these sediments. Indeed, 

the fact that [1,2-14C] VC biodegradation under 

anoxic conditions (table A2) was less efficient 

than anoxic [1,2-14C] DCE biodegradation 

indicates that an initial step, such as reduction to 

ethene (Bradley and Chapelle, 2002), may limit 

the net oxidation of VC to CO2 in these 

sediments. The results of this experiment, 

however, do indicate that microbial 

mineralization may be an important component 

of VC biodegradation in Twin Lake and Pen 

Branch sediments under anoxic conditions, and 

indicate that anoxic mineralization should be 

considered when evaluating the efficiency of in 

situ VC biodegradation in Twin Lakes and Pen 

Branch systems. 

No Evidence of Degradation in HeatSterilized Controls 

In this study, no loss of [1,2-14C] DCE or 

[1,2-14C] VC and no production of 14Cdegradation products were observed in heatsterilized control microcosms containing 

sediments from Twin Lakes or Pen Branch. 

Thus, the degradation of [1,2-14C] DCE and 

[1,2-14C] VC that was observed in this study was 

consistent with and attributed to biologically 

driven degradation processes. The possibility 

remains, however, that abiotic processes may 

have contributed to the observed degradation of 

[1,2-14C] DCE and [1,2-14C] VC if the 

mechanism(s) involved in abiotic degradation 

were significantly inhibited by the heatsterilization conditions employed in this study, 

autoclaving three times for 1 h at 121 oC and 15 

PSI. 

Microbial Isolation and Enrichment Efforts 

Efforts to isolate or enrich 

microorganisms capable of mineralizing [1,2- 14C] DCE or [1,2-14C] VC have not been 

successful as of the time of this writing. 

Sediment-slurry inocula that demonstrated a 

significant potential to mineralize [1,2-14C] DCE 

or [1,2-14C] VC failed to retain their 

mineralization capacity through repeated 

transfers and/or serial dilutions. In all cases, 

[1,2-14C] DCE and/or [1,2-14C] VC 

mineralization activity was extinguished at 

dilutions greater than 105

. In all cases, transfers 

of culture dilutions less than 105 failed to grow 

and/or retain their mineralization activity. As 

noted earlier, isolation and enrichment of 

microbial communities is a resource intensive 

and lengthy endeavor. The approximate 15 year 

period between the initial field evidence for 

chloroethene reductive dechlorination and the 

ultimate isolation of a microorganism capable 

of complete chloroethene reductive 

dechlorination to ethene (Dehalococcoides 

ethenogenes) indicated, from the outset, that 

identification of anoxic chloroethene 

mineralizing microorganisms, in all probability, 

would be a lengthy process that would exceed 

the DOE project time constraints.
12

References Cited 

Bradley, P. M., and F. H. Chapelle. 1996. 

Anaerobic mineralization of vinyl chloride in 

Fe(III)-reducing, aquifer sediments. Environ. 

Sci. Technol. 30:2084-2086. 

Bradley, P. M., and F. H. Chapelle. 1997. 

Kinetics of DCE and VC mineralization under 

methanogenic and Fe(III)-reducing 

conditions. Environ. Sci. Technol. 31:2692-

2696. 

Bradley, P. M., and F. H. Chapelle. 1998a. 

Effect of contaminant concentration on 

aerobic microbial mineralization of DCE and 

VC in stream-bed sediments. Environ. Sci. 

Technol. 32:553-557. 

Bradley, P. M., and F. H. Chapelle. 1998b. 

Microbial mineralization of VC and DCE 

under different terminal electron accepting 

conditions. Anaerobe. 4:81-87. Bradley, P. M., and F. H. Chapelle. 2000b. 

Acetogenic microbial degradation of vinyl 

chloride. Environ. Sci. Technol. 34, 2761-

2763. 

Bradley, P. M., F. H. Chapelle, and D. R. 

Lovley. 1998a. Humic acids as electron 

acceptors for anaerobic microbial oxidation of 

vinyl chloride and dichloroethene. Appl. 

Environ. Microbiol. 64:3102-3105. 

Bradley, P. M., and F. H. Chapelle. 2002. 

Microbial mineralization of ethene under 

sulfate reducing conditions. Bioremed. J. 6, 1-

Bradley, P. M., F. H. Chapelle, and J. T. 8. 

Wilson. 1998b. Field and laboratory evidence 

for intrinsic biodegradation of vinyl chloride 

contamination in a Fe(III)-reducing aquifer. J. 

Contam. Hydrol. 31:111-127. 

Bradley, P.M. 2003. History and ecology of 

chloroethene biodegradation: A Review. 

Bioremed. J. 7, 1-29. 

Bradley, P. M., J. E. Landmeyer, and R. S. 

Dinicola. 1998c. Anaerobic oxidation of [1,2-

Maymó-Gatell, X., Y.-T. Chien, J.M. Gossett, 

and S.H. Zinder. 1997. Isolation of a 

bacterium that reductively dechlorinates 

tetrachloroethene to ethene. Science. 276, 

1568-1571. 

14C] dichloroethene under Mn(IV)-reducing 

conditions. Appl. Environ. Microbiol. 

64:1560-1562. 

Bradley, P. M., and F. H. Chapelle. 1999a. 

Methane as a product of chloroethene 

biodegradation under methanogenic 

conditions. Environ. Sci. Technol. 33, 653-

656. 

McCarty, P.L., and L. Semprini. 1994. Groundwater treatment for chlorinated solvents. In: 

R.D. Norris et al. (Eds.), Handbook of 

Bioremediation. Lewis Publishers, Boca 

Raton, FL. Pp. 87-116. 

Bradley, P. M., and F. H. Chapelle. 1999b. Role 

for acetotrophic methanogens in 

methanogenic biodegradation of vinyl 

chloride. Environ. Sci. Technol. 33, 3473-

3476. 

Bradley, P. M., and F. H. Chapelle. 2000a. 

Aerobic microbial mineralization of 

dichloroethene as sole carbon substrate. 

Environ. Sci. Technol. 34, 221-223. 
13

Manuscript approved for publication on January 26, 2007. 

Final graphics prepared by P.M. Bradley. 

Photocomposition and design by P.M. Bradley. 

For more information concerning the research in this 

report, contact: 

 Paul M. Bradley, U.S. Geological Survey, Stephenson 

Center, Suite 129, 720 Gracern Road, Columbia, SC 

29210. 

 pbradley@usgs.gov.
14

Appendix 

Table A1. Final percentage accumulation of 14C-products from microbial degradation of [1,2-

14C] DCE in viable sediment microcosms after 60 days. Data are means±SD for triplicate 

microcosms. Remaining 14C-radioactivity was recovered as undegraded 14C-DCE. No 

accumulation of 14C-products was observed in killed control or sediment free control 

microcosms. [%, percent] 

Site Sediment Treatment Product Accumulation (% Theoretical) 

14CH414CO214C-Ethene 14C-Ethane 14C-VC

C-BRP Oxic NDaCRP-42 50 2 ND ND ND 

CRP-45 ND 52 6 ND ND ND 

CRP-46 ND 35 3 ND ND ND 

CRP-49 ND 54 12 ND ND ND 

CMP CMP-39b ND 58 11 ND ND ND 

CMP-40 ND 60 3 ND ND ND 

CMP-41 ND 46 4 ND ND ND 

C-BRP CRP-42 Anoxic ND 19 10 19 26 ND 17 5 

CRP-45 ND 40 8 3 0 ND 36 10 

CRP-46 7 7 20 6 9 5 ND 20 8 

CRP-49 6 3 36 6 2 3 ND 2 3 

CMP CMP-39 ND 20 2 ND ND ND 

CMP-40 ND 18 2 ND ND 47 2 

CMP-41 ND 16 2 ND ND 2 3 
15

Table A2. Final percentage accumulation of 14C-products from microbial degradation of 

[1,2-14C] VC in viable sediment microcosms after 60 days. Data are means±SD for 

triplicate microcosms. Remaining 14C-radioactivity was recovered as undegraded 14C-VC. 

No accumulation of 14C-products was observed in killed control or sediment free control 

microcosms. [%, percent] 

Site Sediment Treatment Product Accumulation (% Theoretical) 

14CH414CO214C-Ethene 14C-Ethane 

C-BRP CRP-42 Oxic NDa

100 1 ND ND 

CRP-45 ND 100 3 ND ND 

CRP-46 ND 100 2 ND ND 

CRP-49 ND 100 0 ND ND 

CMP CMP-39 ND 98 3 ND ND 

 CMP-40 ND 93 12 ND ND 

CMP-41 ND 78 7 ND ND 

C-BRP CRP-42 Anoxic ND 8 0 30 21 ND 

CRP-45 ND 16 14 11 4 5 8 

CRP-46 4 6 7 2 13 7 ND 

CRP-49 4 5 13 0 ND ND 

CMP CMP-39 ND 10 0 ND ND 

CMP-40 ND 14 1 ND ND 

CMP-41 ND 10 1 ND ND 

a

 Not detected. Minimum detection limits were 1%, 2%, 1%, and 1% for 14CH4, 14CO2, 14C-ethene, and 14Cethane, respectively.