&EPA
United States
Environmental Protection
Agency
Evaluation of the Role of
Dehalococcoides
Organisms in the Natural
Attenuation of Chlorinated
Ethylenes in Ground Water
LANDHLL J
(SWMU-5J
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EPA/600/R-06/029
July 2006
Evaluation of the Role of Dehalococcoides
Organisms in the Natural Attenuation of
Chlorinated Ethylenes in Ground Water
Xiaoxia Lu
National Research Council Post Doctoral Associate
tenable at the
U.S. Environmental Protection Agency
Office of Research and Development
National Risk Management Laboratory
Ada, Oklahoma 74820
Donald H. Kampbell, and John T. Wilson
U.S. Environmental Protection Agency
Office of Research and Development
National Risk Management Laboratory
Ada, Oklahoma 74820
Support from the U.S. Air Force Center for
Environmental Excellence through
Interagency Agreement # RW-57939566
Project Officer
John T. Wilson
Ground Water and Ecosystems Restoration Division
National Risk Management Research Laboratory
Ada, Oklahoma 74820
National Risk Management Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, Ohio 45268
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Notice
The U.S. Environmental Protection Agency through its Office of Research
and Development funded the research described here. This work was
conducted under in-house Task 3674, Monitored Natural Attenuation of
Chlorinated Solvents, and in association with and with support from the
U.S. Air Force Center for Environmental Excellence through Interagency
Agreement # RW-57939566, Identification of Processes that Control Natural
Attenuation at Chlorinated Solvent Spill Sites. Mention of trade names and
commercial products does not constitute endorsement or recommendation
for use.
All research projects making conclusions and recommendations based on
environmentally related measurements and funded by the U.S. Environ-
mental Protection Agency are required to participate in the Agency Quality
Assurance Program. This project was conducted under a Quality Assurance
Plan prepared for Task 3674, Monitored Natural Attenuation of Chlorinated
Solvents. Work performed by U.S. EPA employees or by the U.S. EPA
on-site analytical contractor followed procedures specified in these plans
without exception. Information on the plan and documentation of the quality
assurance activities and results is available from John T Wilson.
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Foreword
The U.S. Environmental Protection Agency is charged by Congress with protecting the Nation's land, air,
and water resources. Under a mandate of national environmental laws, the Agency strives to formulate
and implement actions leading to a compatible balance between human activities and the ability of natural
systems to support and nurture life. To meet this mandate, EPA's research program is providing data
and technical support for solving environmental problems today and building a science knowledge base
necessary to manage our ecological resources wisely, understand how pollutants affect our health, and
prevent or reduce environmental risks in the future.
The National Risk Management Research Laboratory is the Agency's center for investigation of technologi-
cal and management approaches for preventing and reducing risks from pollution that threatens human
health and the environment. The focus of the Laboratory's research program is on methods and their
cost-effectiveness for prevention and control of pollution to air, land, water, and subsurface resources;
protection of water quality in public water systems; remediation of contaminated sites, sediments and
ground water; prevention and control of indoor air pollution; and restoration of ecosystems. NRMRL
collaborates with both public and private sector partners to foster technologies that reduce the cost of
compliance and to anticipate emerging problems. NRMRLs research provides solutions to environmental
problems by: developing and promoting technologies that protect and improve the environment; advanc-
ing scientific and engineering information to support regulatory and policy decisions; and providing the
technical support and information transfer to ensure implementation of environmental regulations and
strategies at the national, state, and community levels.
This publication has been produced as part of the Laboratory's strategic long-term research plan. It is
published and made available by EPA's Office of Research and Development to assist the user community
and to link researchers with their clients.
Chlorinated solvents such as tetrachloroethylene and trichloroethylene are an important category of con-
taminants in ground water at hazardous waste sites. Frequently, these compounds are subject to natural
anaerobic biodegradation in ground water. During anaerobic biodegradation they undergo a sequential
biological reductive dechlorination to produce c/s-dichloroethylene, then vinyl chloride, and finally ethylene
or ethane. Although c/s-dichloroethylene is less hazardous than trichloroethylene or tetrachloroethylene,
vinyl chloride is more hazardous. In contrast, ethylene or ethane is not hazardous to humans. If the bio-
logical reductive dechlorination is complete, with ethylene or ethane as the final product, then monitored
natural attenuation can be used a remedy for the ground water contamination.
In recent years, bacteria that can dechlorinate dichloroethylene to ethylene or ethane have been isolated
and characterized. All the strains that can dechlorinate vinyl chloride to ethylene or ethane belong to the
genus Dehalococcoides. A biochemical assay for DNA specific to the genus Dehalococcoides is commer-
cially available. This report provides technical recommendations on the interpretation of the biochemical
assay and on the contribution of bacteria in the Dehalococcoides group to monitored natural attenuation
of chlorinated solvents in ground water.
Stepnen G. Schmelling, Director^
Ground Water and Ecosysteras Restoration Division
National Risk Management Research Laboratory
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Contents
Foreword iii
Figures vii
Tables ix
Acknowledgments xi
Abstract xiii
Section 1. Role of Biotransformation in Evaluation of MNA of Chlorinated Solvents 1
Intended Use of the Report 2
State of Practice and Emerging State of the Science 3
Section 2. Ecology of Microorganisms that Transform Chlorinated Solvents 5
Bacteria that Gain Energy from Reductive Dechlorination (Halorespiring Bacteria) 6
The Place of Dehalorespiring Bacteria in the Diversity of Life 6
Organisms that Oxidize Chlorinated Ethylenes under Anaerobic Conditions 7
Organisms that Co-Metabolize Chlorinated Ethylenes 7
Aerobic Growth on Chlorinated Ethylenes 7
Section 3. Tools to Assay Microorganisms that Completely Transform Chlorinated Solvents ... 11
Polymerase Chain Reaction (PCR) Assays for Genetic Analysis of the Microbial
Communities 11
Detection of Dehalococcoides Species by the PCR Assay 13
Limitations of the PCR Assay for Dehalococcoides DMA 14
Current State of Practice of PCR Tools to Evaluate Biotransformation of
Chlorinated Solvents 14
Section 4. Dehalococcoides DMA and Rates of Natural Attenuation 17
A Definition of "Generally Useful" Rates of Biological Reductive Dechlorination 17
Site Selection 17
Groundwater Sampling 19
Chemical Analysis 19
Detection of Dehalococcoides by Polymerase Chain Reaction Analysis 20
Calculation of Dechlorination Rates from Monitoring Data 21
Calculation of Dechlorination Rates in Conventional Plumes 21
Relationship Between Dehalococcoides DMA and Dechlorination Rates at
Conventional Plumes 23
Dehalococcoides DMA and Dechlorination Rates over Time in Particular Wells 28
Rates of Natural Attenuation and Density of PCR Products from
Dehalococcoides DMA 31
Biotransformation and Dominant Terminal Electron Accepting Processes 34
Example of Calibration of BIOCHLOR with Distance along a Flow Path 35
Example of Calibration of BIOCHLOR with Time in a Single Monitoring Well 38
Conclusions 39
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Section 5. Geochemical Parameters and Occurrence of Dehalococcoides 41
Synopsis 41
Calibration of Computer Models to Evaluate MNA 42
Sampling Sites 42
Ground Water Sampling, Assay for Dehalococcoides DMA, and Chemical Analyses ....44
Detection of Dehalococcoides DMA 44
Biogeochemistry of Ground Water with Detectable Dehalococcoides DMA 44
Comparison of Geochemistry where Dehalococcoides DMA is Present or Absent 50
A Predictive Model for the Presence of Dehalococcoides DNA 51
Summary and Conclusions 52
Section 6. Presence of Dehalococcoides DMA and the Extent of Biodegradation 55
Enrichment Culture Preparation 55
Sampling and Analysis of Enrichment Cultures 56
Biodegradation of Chlorinated Ethylenes in the Enrichment Cultures 56
Association of Dechlorination in Enrichment Cultures with Dehalococcoides DMA 57
Section 7. Recommendations to Evaluate Biotransformation of Chlorinated Solvents 61
Recommendations for Interpreting Data on Density of DMA in Ground Water 61
Recommendations for Interpreting Geochemistry of Ground Water 63
Recommendations for Selecting Wells for Sampling 63
Recommendations for Sampling and Shipping of Samples 64
Section 8. Data Quality 67
Analysis of Chemical Concentrations 67
Analysis of DMA Concentrations 69
Section 9. References 95
VI
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Figures
Figure 2.1. A phylogenetic tree based on comparisons of sequences in the 16s Ribosomal RNA 8
Figure 3.1. Separation of DMA by Denaturing Gradient Gel Electrophoresis (DGEE) 12
Figure 4.1. The frequency distribution of the maximum concentration of chlorinated solvents and their
transformation products at Department of Defense sites in the United States (from
McNab et al., 2000) 18
Figure 4.2. Location of sites used to survey the relationship between Dehalococcoides DMA in ground
water and the rate of natural attenuation of chlorinated ethylenes in field scale plumes 18
Figure 4.3. Location of monitoring wells and distribution of c/s-DCE at the Western Processing Site in
Kent, Washington, fourth quarter 1994 24
Figure 4.4. Location of monitoring wells and distribution of c/s-DCE in the Upper Saturated Zone Aquifer
at the Landfill 3 Site, Tinker AFB, OK, in 2000 26
Figure 4.5. Comparison of the locations of monitoring wells at the North Beach Site at the U.S. Coast Guard
Support Center in Elizabeth City, North Carolina, to the distribution of PCE in ground water 26
Figure 4.6. Location of monitoring wells and distribution of c/s-DCE in the Intermediate Ground -Water
Zone at Spill Site-4, the former England AFB, Louisiana, March 2002 28
Figure 4.7. Relationship between the density of Dehalococcoides cells as determined by quantitative
PCR and the first order rate of attenuation of c/s-DCE in ground water 32
Figure 4.8. Comparison of the density of Dehalococcoides cells in ground water as determined by
quantitative PCR (real time PCR) to the density of Dehalococcoides DNA as determined
by the semi-quantitative technique that uses the density of the band produced by Gel
Electrophoresis as an estimate of the concentration of amplified DNA 33
Figure 4.9. Input screen to BIOCHLOR with calibration parameters for the North Beach Site 34
Figure 4.10. Correspondence between the measured values for PCE and TCE at the North Beach Site in
2002 and the concentrations that were predicted by calibrating BIOCHLOR using three
different values for the first order rate constant for biotransformation 36
Figure 4.11. Correspondence between the measured values for c/s-DCE and vinyl chloride at the North
Beach Site in 2002 and the concentrations that were predicted by calibrating BIOCHLOR
using three different values for the first order rate constant for biotransformation 37
Figure 4.12. Input screen to BIOCHLOR with calibration parameters for the well A39L010PZ at Area 2500
at England Air Force Base, Alexandria, Louisiana 38
Figure 4.13. Correspondence between predicted and actual values for the chlorinated ethylenes performed
by calibrating BIOCHLOR to field data sampled in different events at the well 39
Figure 5.1. Location of contaminated sites used to compare presence or absence of Dehalococcoides
DNA to the geochemistry of the ground water 42
Figure 6.1. Relationship between the production of ethylene in the enrichment cultures and the
concentration of dissolved oxygen in the corresponding ground water used for the inoculums
of the enrichment cultures 59
VII
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Tables
Table 2.1. Diversity of Bacteria that can Reductively Dechlorinate Ethylenes 7
Table 3.1 Comparison of Advantages and Disadvantages of PCR Tools to Evaluate Biotransformation
of Chlorinated Solvents in Ground Water 15
Table 3.2 Applications of PCR Tools to Evaluate Biotransformation of Chlorinated Solvents in
Ground Water 15
Table 4.1. Location of Specific Sites in the Survey and the Number of Wells Sampled at Each of the
Sites 19
Table 4.2. Calibration Parameters for BIOCHLOR 22
Table 4.3. Relationship between the Concentrations of Chlorinated Ethylenes in Ground Water and
Their Apparent Rates of Dechlorination along an Inferred Flow Path in the Aquifer 23
Table 4.4. Distribution of Chlorinated Ethylenes and Dehalococcoides DMA at Sites that Form
Conventional Plumes 25
Table 4.5. Relationship between the Apparent Rates of Dechlorination along a Flow Path in the Aquifer
and the Detection of Bacterial DMA and Dehalococcoides DMA in Water Samples from a
Monitoring Well in the Plume 27
Table 4.6. Distribution of Chlorinated Ethylenes and Dehalococcoides DMA at Sites at England AFB,
which do not Form Conventional Plumes 29
Table 4.7. Relationship between the Concentrations of Chlorinated Ethylenes in Ground Water and
Their Apparent Rates of Dechlorination over Time in Water Samples from Monitoring Wells
at England AFB, Louisiana 29
Table 4.8. Relationship between the Apparent Rates of Dechlorination over Time and the Detection of
Bacterial DMA and Dehalococcoides DMA in Water Samples from Monitoring Wells at
England AFB, Louisiana 30
Table 4.9. Comparison of Rates on Attenuation to the Overall Geochemical Environment of the Sites
in the Survey 34
Table 5.1. Location of Sites Included in the Survey Comparing the Presence or Absence of
Dehalococcoides DMA to the Geochemistry of the Ground Water 43
Table 5.2 The Intensity Scores of Dehalococcoides DMA and the Concentrations of Chlorinated
Ethylenes and Ethylene in the Wells where Dehalococcoides DMA was Detected 45
Table 5.3. The Concentrations of Nitrate plus Nitrite Nitrogen, Methane, and the ORP Meter Reading
in the Wells where Dehalococcoides DNA was Detected 46
Table 5.4. The Concentrations of Oxygen, Ferrous Iron, and Sulfate in the Wells where Dehalococcoides
DNA was Detected 48
Table 5.5. The Concentration of Dissolved Molecular Hydrogen and Total Organic Carbon, and the
Oxidation/Reduction Potential in the Wells where Dehalococcoides DNA was Detected 49
Table 5.6. The Probability (p) that the Distribution of the Measured Values for Selected Geochemical
Parameters between Ground Water where Dehalococcoides DNA was Present and Ground
Water where Dehalococcoides DNA was not Present is not Statistically Different 51
Table 5.7. Comparisons between the Observed Presence or Absence of Dehalococcoides DNA and the
Concentrations of Nitrate plus Nitrite Nitrogen, Methane, and the ORP Meter Reading in the
Wells 53
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Table 5.8. Comparisons between the Observed Presence or Absence of Dehalococcoides DMA and the
Predicted Probabilities for the Presence of Dehalococcoides DMA 54
Table 6.1. Composition of the Basal Medium (pH 7) 55
Table 6.2. Comparisons of Biotransformation of Chlorinated Ethylenes in Enrichment Cultures to the
Corresponding Presence or Absence of Amplifiable Dehalococcoides DMA in the Ground
Water Sample Used to Inoculate the Enrichment Cultures 58
Table 6.3. Comparison of Biotransformation of Chlorinated Ethylenes in Enrichment Cultures to the
Corresponding Geochemistry of the Ground Water used for Inoculation of the Enrichment
Cultures 60
Table 7.1. Recommendations for Use of PCR Assays to Evaluate Biotransformation of Chlorinated
Solvents 62
Table 8.1. Typical Quality Performance Data for Analysis of TCE in Water 70
Table 8.2. Typical Quality Performance Data for Analysis of c/s-DCE in Water 74
Table 8.3. Typical Quality Performance Data for Analysis of Vinyl Chloride in Water 78
Table 8.4. Typical Quality Performance Data for Analysis of Ethylene in Water or in Gas 82
Table 8.5. Typical Quality Performance Data for Analysis of Methane in Water or in Gas 86
Table 8.6. Typical Quality Performance Data for Analysis of Hydrogen in Gas 90
Table 8.7. Typical Quality Performance Data for Analysis of Nitrate Plus Nitrite Nitrogen in Water 93
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Acknowledgments
Formal peer reviews were provided by Dr. Carolyn Acheson with the U.S.
EPA Office of Research and Development, National Risk Management
Research Laboratory; Dr. Ned Black with U.S. EPA Region 9; Dr. Mitch
Lasat with U.S. EPA Office of Research and Development, Office of
Science Policy; Dr. Donna Fennell at Rutgers University, New Brunswick,
NJ; Dr. James Gossett at Cornell University, Ithaca, NY; Dr. David Ellis with
DuPont Company, Wilmington, DE; Dr. Guy Sewell at East Central State
University, Ada, OK; and Dr. Andrea Leeson, a SERDP/ESTCP Cleanup
Program Manager, Arlington, VA. Courtesy technical reviews were provided
by Mr. Philip Dennis, SIREM Operations Manager, GeoSyntec Consultants,
Guelph, Ontario, Canada, and by Dr. Margaret Findlay and Dr. Sam Fogel
of Bioremediation Consulting, Inc., Watertown, MA.
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Abstract
At most hazardous waste sites where monitored natural attenuation (MNA) of chlorinated solvents in
ground water is successful as a remedy, the chlorinated solvents are biologically degraded to harmless
end products such as ethylene or ethane. Many organisms can degrade chlorinated solvents such as
tetrachloroethylene or trichloroethylene, to dichloroethylene and vinyl chloride. This contributes little to
risk reduction because vinyl chloride is more toxic and more carcinogenic than tetrachloroethylene or
trichloroethylene. The only organisms known to degrade dichloroethylenes and vinyl chloride to ethylene
or ethane are members of the Dehalococcoides group. As a result, these organisms have a critical role
in the evaluation of MNA at chlorinated solvent sites. In recent years, biochemical assays for the pres-
ence of DNA from the organisms have become commercially available. These assays are based on the
polymerase chain reaction (PCR) for the amplification of DNA extracted from ground water. They are
very sensitive and can be very specific.
This report is designed for technical staff in the EPA Regions and in state agencies that require information
on the contribution of Dehalococcoides bacteria to MNA of chlorinated solvents, and information on the
proper application and interpretation of the assays in an evaluation of MNA. This report includes sections
on the role of biotransformation in evaluation of MNA of chlorinated solvents, the ecology of microorganisms
that transform chlorinated solvents, tools to assay microorganisms that transform chlorinated solvents,
the relationship between Dehalococcoides DNA in ground water and rates of natural attenuation at field
scale, the relationship between geochemical parameters and the occurrence of Dehalococcoides DNA
in ground water, and the relationship Dehalococcoides DNA in ground water and behavior of chlorinated
solvents in laboratory treatability studies or microcosm studies done with water from the plume.
XIII
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Section 1.
Role of Biotransformation in Evaluation of MNA of Chlorinated Solvents
Perchloroethylene (PCE) and trichloroethylene (TCE)
are among the most pervasive chlorinated solvents in
ground water at hazardous waste sites. PCE and TCE
are subject to biological and chemical processes that
may further transform them to c/s-dichloroethylene
(c/s-DCE), frans-dichloroethylene (trans-DCE), 1,1-
dichloroethylene (1,1-DCE), vinyl chloride, ethylene,
and ethane. Vinyl chloride is the most hazardous of
the transformation products. Ethylene and ethane
are essentially harmless. A wide variety of microor-
ganisms can transform PCE or TCE to produce the
dichloroethylenes. To date, only microorganisms
from a specific and relatively uncommon group (the
Dehalococcoides group) have been shown to have the
capability to transform c/s-DCE to vinyl chloride, and
then transform vinyl chloride to ethylene. Complete
transformation of PCE or TCE to ethylene can be
an important process contributing to the monitored
natural attenuation of PCE and TCE at hazardous
waste sites.
In the past ten years, very sensitive and specific
biochemical tools have been developed that can
recognize DNAfrom Dehalococcoides organisms. To
carry out an assay for Dehalococcoides organisms,
DNA is isolated from ground water or sediment. Then
short pieces of DNA that are complementary to gene
sequences that are unique to Dehalococcoides or-
ganisms are added to the extract. These short pieces
bind to the Dehalococcoides DNA, and allow the DNA
to be copied by an enzyme called DNA polymerase.
The short pieces that bind to the Dehalococcoides
DNA are often referred to as primers for the DNA
polymerase reaction. The process is repeated for
a number of cycles. Each time the DNA that was
synthesized in the previous reaction becomes the
template for the subsequent reaction. In each cycle
of the chain reaction, the concentration of Dehalococ-
coides DNA is doubled. Finally, the concentration of
Dehalococcoides DNA is high enough that it can be
identified and analyzed by other molecular biology
procedures. The entire process is referred to as the
polymerase chain reaction (PCR).
A recurrent feature in the journal, Remediation, is
the Monitored Natural Attenuation Forum: A Panel
Discussion. In a recent issue (Borden et al., 2003),
a panel of three experts responded to the question:
Recently, there has been a lot of discussion
about the need for Dehalococcoides etheno-
genes to completely break down chlorinated
solvents such as TCE in groundwater. Should
natural attenuation studies automatically in-
clude a test for the microbe? Does the pres-
ence of c/s-DCE and vinyl chloride preclude
the need for microbe testing? Is suitable
groundwater geochemistry or the detection of
this bacterium in groundwater more valuable
in natural attenuation studies?
Their responses are a good summary of the state of
knowledge with respect to the use of PCR tools to
evaluate the contribution of Dehalococcoides bacte-
ria to natural attenuation of chlorinated ethylenes in
ground water (Borden et al., 2003).
One panel member noted that due to the complexity
of most field sites, we rely on multiple lines of evi-
dence to evaluate the behavior of plumes. The panel
member observed that:
"While geochemical data are usually the best
indicators of the potential for complete dechlo-
rination in an aquifer, definitive information
on the presence or absence of Dehalococ-
coides can provide very useful information in
determining whether MNA is an appropriate
approach for a specific site. At present, we do
not understand enough about the distribution
and activity of the currently available assays
to use these results as a primary indicator for
the presence/absence of complete reductive
dechlorination at a site."
A second panel member noted the assay as cur-
rently practiced has a high rate of false positives and
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false negatives. Based on the cost of the PCR assay
compared to the cost for conventional geochemical
parameters, the panel member concluded,
"But clearly the cost benefit analysis of DSA
[Dehalococcoides specific PCR assays] makes
it comparatively less attractive, and due to limi-
tation in sensitivity and interpretation, it should
not be a required analysis at this time."
The third panel member noted that the significance
of PRC data depended on the specific procedure that
was used. She also noted that:
"Another reason that limits the usefulness of
microbial characterization at field sites has
to do with the difficulty associated with using
these data for assessing the efficacy of natural
attenuation. Understanding the microbial pop-
ulation, its activity, and its diversity provides
insight into the biodegradation processes and
pathways but does not yield data that can be
used to estimate concentration declines, gen-
eration of by-products, of clean-up times."
Intended Use of the Report
This report is intended to facilitate the use of DNA
analysis to document the role of Dehalococcoides
organisms in the natural attenuation of PCE, TCE, and
their transformation products in ground water. The
report contains seven sections. This section describes
several innovative genetic tools for evaluating microbial
communities that degrade chlorinated hydrocarbons.
It also describes the relationship between the new
genetic tools and previous technical recommendations
published by EPA/ORD in the Technical Protocol for
Evaluating Natural Attenuation of Chlorinated Solvents
in Ground Water (Wiedemeier et al., 1998).
A short review of the nutritional ecology and physi-
ological diversity of organisms that can degrade
chlorinated solvents in ground water is provided in
Section 2. Ecology of Microorganisms that Transform
Chlorinated Solvents. The new techniques in genetic
analysis that are available to identify and enumerate
specific microorganisms in ground water or aquifer
sediment are described in Section 3. Tools to Assay
Microorganisms that Transform Chlorinated Solvents.
These sections provide background and context for the
detailed discussion of Dehalococcoides organisms in
the remainder of the report. A reader who is familiar
with these topics can skip Sections 2 and 3.
An evaluation of Monitored Natural Attenuation (MNA)
as defined by U.S. EPA in the OSWER Directive (U.S.
EPA, 1999) requires a quantitative understanding of
the behavior of the plume of contamination over time
and space. The critical parameter is the rate of attenu-
ation of concentration of the contaminant over time
and with distance away from the source. To contribute
to an evaluation of MNA, an assay for the presence
or activity of microorganisms must be associated with
the field scale behavior of the plume containing the
microorganisms. Section 4. Dehalococcoides DNA
and Rates of Natural Attenuation at Field Scale com-
pares the achieved rates of natural attenuation of PCE,
TCE, c/s-DCE, and vinyl chloride at several chlorinated
solvent plumes to estimates of the concentration of
Dehalococcoides DNA in ground water provided by the
polymerase chain reaction (PCR) assay. In general,
concentrations of Dehalococcoides DNA that were
high enough to be detected by a commercially avail-
able assay were associated with rates of attenuation
that are useful for MNA.
At most field scale plumes where MNA has been pro-
posed as a remedy for chlorinated solvents in ground
water, biotransformation of the solvents to harmless
end products is an important part of the remedy.
The expected contributions of other processes (such
as sorption or dilution and dispersion) are usually
not adequate to be protective of human health and
the environment. As a consequence, the Technical
Protocol for Evaluating Natural Attenuation of Chlori-
nated Solvents in Ground Water (Wiedemeier et al.,
1998) put a heavy emphasis on biotransformation as
a process to achieve natural attenuation.
The Technical Protocol (1998) used a number of geo-
chemical parameters in a scoring system to predict
whether ground water contained microorganisms that
could biologically transform chlorinated solvents. The
scoring system was criticized by the Committee on
Intrinsic Remediation of the National Research Coun-
cil (NRC, 2000). The Committee recommended that
the scoring system should not be used to evaluate
prospects for MNA.
In Table 2.3 of the Technical Protocol (Wiedemeier,
1998), scores are assigned based in part on analyses
for oxygen, nitrate, iron II, sulfate, methane, molecular
hydrogen, oxidation reduction potential (ORP), pH,
total organic carbon, chloride, BTEX compounds,
temperature, and alkalinity. An assay for the presence
of Dehalococcoides DNA provides direct evidence of
an organism that can completely transform chlorinated
ethylenes. The presence or absence of Dehalococ-
coides DNA can be used to evaluate the information
provided by the geochemical parameters.
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In Section 5. Geochemical Parameters and Occur-
rence of Dehalococcoides DNA, statistics are used
to identify the conventional geochemical parameters
that are associated with the presence of Dehalococ-
coides DNA in ground water. The distribution of the
values for ORR and the concentrations of nitrate and
methane were significantly different between samples
of ground water where Dehalococcoides DNA was
detected and samples where Dehalococcoides DNA
was not detected. As for the other parameters, there
were no significant differences. In Section 5, a statisti-
cal technique is used to derive a formula for the prob-
ability that Dehalococcoides is present in ground water
knowing the concentrations of nitrate and methane,
and the oxidation/reduction potential. The formula is
offered as a replacement for the scoring system in the
Technical Protocol (Wiedemeier et al., 1998).
The process of evaluating MNA as an option at chlo-
rinated solvent sites often involves a laboratory study
to determine whether the contaminated aquifer har-
bors microorganisms that can entirely transform the
contaminants. These studies are often referred to as
engineering treatability studies or microcosm studies.
Laboratory enrichment studies were conducted with
ground water from plumes where the long-term moni-
toring data clearly indicated that biotransformation
processes were responsible for the observed natural
attenuation of the plume. In Section 6. Dehalococcoi-
des DNA and Laboratory Studies of Biodegradation,
the results of the laboratory studies were compared
to the distribution of Dehalococcoides DNA in the
contaminated aquifer.
If the presence of Dehalococcoides DNA indicates
the presence of organisms that will dechlorinate con-
taminants to ethylene, then all the cultures that were
established with ground water containing amplifiable
Dehalococcoides DNA would be expected to de-
chlorinate PCE or TCE to ethylene. If false positives
for the assay are defined as water samples where
Dehalococcoides DNA was detected, but ethylene
was not detected in the enrichment culture, then the
proportion of false positive predictions as evaluated
against the results of all the enrichment cultures that
were constructed was 55%.
An absence of Dehalococcoides DNA would suggest
that these organisms were absent from the ground
water. Because the only organisms known to dechlo-
rinate c/s-DCE and vinyl chloride belong to the Deha-
lococcoides group, the absence of Dehalococcoides
DNA would indicate that dechlorination would not
proceed to vinyl chloride or ethylene. If false negatives
for the assay are defined as water samples where
Dehalococcoides DNA was not detected, but vinyl
chloride or ethylene was detected in the enrichment
culture, then the proportion of false negative predic-
tions as evaluated against the results of all the enrich-
ment cultures that were constructed was 15%. The
proportion of false negative predictions as evaluated
against the results of only those enrichment cultures
that showed dechlorinating activity was 43%.
Although the number of false determinations was
high, the assay can be useful, particularly when the
overall evaluation of natural attenuation is based on
a variety of tests and conditions. Not all strains of
Dehalococcoides can dechlorinate c/s-DCE to vinyl
chloride or ethylene (Duhamel et al., 2004). However,
the unequivocal presence of Dehalococcoides DNA in
a ground water sample strongly suggests, although it
does not prove, that chlorinated ethylenes are being
dechlorinated to ethylene in the aquifer. The deter-
mination is much stronger if it is supported by other
information that would be consistent with dechlorina-
tion to ethylene. A failure to detect Dehalococcoides
DNA in a sample of ground water should not be taken
to mean that dechlorination in the aquifer will stop at
the level of dichloroethylene, and that c/s-DCE and
vinyl chloride will not be degraded.
Section 7. Recommendations to Evaluate Biotransfor-
mation of Chlorinated Solvents provides recommenda-
tions on the interpretation of data on the concentration
of DNA in ground water samples, and on the sampling
protocol.
State of Practice and Emerging State of the
Science
Laboratory research and collection of field data to
prepare this report on the Evaluation of the Role of
Dehalococcoides Organisms in the Natural Attenua-
tion of Chlorinated Ethylenes in Ground Water began
in December 2002. At that time, the literature sug-
gested that organisms in the Dehalococcoides group
were primarily responsible for complete dechlorination
of chlorinated solvents to ethylene, both in the field
and in laboratory cultures. This report is intended to
facilitate the use of DNA analysis to document the role
of Dehalococcoides organisms in the natural attenu-
ation of PCE, TCE, and their transformation products
in ground water at hazardous waste sites.
Future evaluations at U.S. EPA enforcement actions
will be carried out by contractors and consultants to
the responsible parties using commercially available
services for the assay for Dehalococcoides DNA. To
make our research findings consistent with results that
would be obtained at other field sites, we obtained the
-------
assay for Dehalococcoides DNA from a commercial
vendor, instead of doing the assay in-house with EPA
research staff. To generate data that met our data
quality objectives for comparability, all the assays for
Dehalococcoides DNA reported in this study were
conducted by the same commercial vendor using the
same protocol. In December 2002, we were aware
of only one commercial laboratory in the world that
could assay ground water for the presence of Dehalo-
coccoides. Since that time, a number of laboratories
have entered the commercial market.
The assay provided by our vendor was based on
the polymerase chain reaction, and the assay used
primers for the 16S-rRNA gene. This gene codes for
a structural component of the ribosome. As a con-
sequence the nucleotide sequences in the gene tend
to be conserved as the organisms evolve over time.
This makes it possible to recognize organisms that are
currently widely distributed, but which had a common
ancestor. However, the 16S-rRNA gene is not directly
related to metabolism of chlorinated ethylenes, and
it is possible that the assay detected organisms that
belonged to the Dehalococcoides group, but were
not able to metabolize c/s-DCE and vinyl chloride.
Genes have been identified in the Dehalococcoides
group for enzymes that dechlorinate c/s-DCE and vinyl
chloride (Krajmalnik-Brown et al., 2004; Magnuson
et al., 1998; Muller et al., 2004), and PCR assays
for genes encoding for a Vinyl Chloride Reductase
enzyme are now commercially available. There may
be many Vinyl Chloride Reductase genes, and an
assay for only one of the Vinyl Chloride Reductase
genes might fail to identify the capacity to reduce vinyl
chloride to ethylene in the mixed microbial community
in a contaminated plume. However, if a PCR assay
detects DNA for a Vinyl Chloride Reductase, it is very
likely that the microbial community has the capacity
to degrade vinyl chloride.
The U.S. Federal Government has provided substan-
tial funding for research to development new tools
for genetic analysis of organisms that are capable of
degrading chlorinated organic contaminants in ground
water. Many talented people work in this field, their
research is bearing fruit, and new applications are
coming into the market place. This report is based on
the state of commercial practice in 2004. It is already
out of date as far as new applications or potential
applications of genetic analysis. However, use of the
polymerase chain reaction to assay for the 16S-rRNA
gene of Dehalococcoides is still widespread, and there
is need of a report that documents the performance of
the assay to evaluate natural attenuation of chlorinated
solvents in ground water.
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Section 2.
Ecology of Microorganisms that Transform Chlorinated Solvents
The ecology of organisms that biologically transform
chlorinated ethylenes has recently been reviewed
(Bradley, 2003). This Section is a short summary of
the available literature. It is intended to provide the
necessary background for subsequent sections of this
report. Bradley (2003) is recommended to any reader
who is interested in more information on the ecology
of organisms that degrade chlorinated ethylenes.
Chlorinated solvents such as PCE and TCE are
xenobiotic compounds. They have low molecular
weight and are lipophilic, volatile, and nonflammable.
This makes them effective solvents for oil and grease.
The commercial use and production of PCE and TCE
began in the 1920s and peaked in the 1970s. Use
declined thereafter because they were suspected to
be carcinogens.
The evolutionary origin of microbes capable of
transforming chlorinated solvents is uncertain. Many
chlorinated hydrocarbons occur naturally in the en-
vironment, including TCE and PCE (Gribble, 1994)
and vinyl chloride (Keppler et al., 2002). As a result,
microbial enzymes may have evolved that are special-
ized for degrading organochlorine compounds (Lee et
al., 1998). However, the concentrations of chlorinated
ethylenes in plumes of contaminated ground water
are much higher than their natural concentrations.
Chlorinated solvents such as PCE and TCE have been
in the environment at high concentrations for only a
short time. The dechlorinating bacteria may have
evolved to degrade other substrates and by chance
have the capability to degrade chlorinated solvents.
Alternatively, in only a few decades, enzymes that
were originally evolved to degrade other substrates
may have adapted to degrade chlorinated solvents.
The mechanisms involved in microbial metabolism of
chlorinated solvents can be broadly classified into two
categories: oxidation reactions and reductive dechlo-
rination. Oxidation is a process where the chlorinated
solvents are oxidized to carbon dioxide or other benign
compounds. Reductive dechlorination is a process
where a chlorine atom is removed and replaced with
a hydrogen atom. Typically, reductive dechlorination
occurs under anaerobic conditions. An electron donor
is required to carry out reductive dechlorination.
PCE and TCE are highly oxidized compounds and
therefore are most susceptible to reductive dechlorina-
tion. In general, reductive dechlorination of PCE or
TCE occurs by sequential dechlorination from PCE to
TCE to DCE isomers to VC to ethylene. Depending
upon environmental conditions, this sequence may be
interrupted and not go all the way to ethylene (Wiede-
meier et al., 1998). Reductive dechlorination may be
performed by bacteria that couple their growth with
the dechlorination of the chloroethylene or by bacte-
ria that do not benefit from the dechlorination. In the
former case, the process is known as halorespiration
or dehalorespiration, and the bacteria are referred
to as halorespiring bacteria. In the latter case, the
process is a co-metabolic reaction where the growth
of the bacteria is supported by metabolism of other
compounds.
During reductive dechlorination, all three isomers of
DCE can theoretically be produced. However, Bouwer
(1994) reported that under the influence of biotrans-
formation, c/s-DCE is a more common intermediate
than trans-DCE and 1,1-DCE. Compared with PCE
and TCE, the dichloroethylene isomers and vinyl
chloride are not as highly oxidized. Conventional
wisdom holds that they are not as readily reduced in
chemical reactions. As a consequence, the biological
reductive dechlorination of dichloroethylene and vinyl
chloride should be slower and less extensive than is
the case for PCE and TCE. This perspective was
based on a comparison of the Gibbs free energy for
complete dechlorination of each chlorinated ethylene;
however, the chlorinated ethylenes are dechlorinated
in a step-wise fashion. A better way to evaluate the
energy yield is to compare the yield of each separate
dechlorination reaction. The Gibbs free energy for
reductive dechlorination of PCE to TCE, of TCE to
DCE, of DCE to vinyl chloride, and vinyl chloride to
ethylene is -171.8, -166.1, -144.8, and -154.5 kilo
Joules per mole of chlorinated ethylene, respectively
(Dolfing, 2000). The energy yields for each succes-
sive dechlorination are essential equivalent. The rates
-------
of reductive dechlorination of PCE, TCE, c/s-DCE
and vinyl chloride should be equivalent. Cupples et
al. (2004b) compared dechlorination of c/s-DCE and
vinyl chloride by Dehalococcoides strain VS. The
maximum growth rates were equivalent, the maximum
rate of utilization of vinyl chloride was within 75% of
the maximum rate of utilization of c/s-DCE, and half
saturation constants were equivalent.
The energy yield for reductive dechlorination is high
relative to other anaerobic processes. The Gibbs free
energy for nitrate reduction, sulfate reduction and
methanogenesis is -112, -38, and -33 kilo Joules per
mole of substrate consumed (Dolfing, 2000).
The DCE isomers and vinyl chloride also have the
potential to undergo oxidation to carbon dioxide
or acetate (Bradley and Chapelle, 1998). Aerobic
growth on PCE and TCE as an electron donor has
never been reported. However, Ryoo et al. (2000)
reported that PCE can be degraded co-metabolically
under aerobic conditions by oxygenase enzymes,
and the co-metabolic oxidation of TCE has been well
demonstrated (Wilson and Wilson, 1985; Nelson et
al., 1988; Ensley, 1991; Chang and Alvarez-Cohen,
1994). In ground water, different organisms with dif-
ferent metabolic pathways may share the responsibility
for natural attenuation of chlorinated solvents. The
following sections describe the microorganisms that
may be involved in biotransformation of chlorinated
ethylenes.
Bacteria that Gain Energy from Reductive
Dechlorination (Halorespiring Bacteria)
The halorespiring bacteria couple reductive dechlo-
rination to growth. They were first described by Hol-
liger and his co-workers (1993) who obtained a highly
purified enrichment culture that was able to grow by
the reduction of PCE to c/s-DCE using hydrogen as
the electron donor. The active organism was named
Dehalobacter restrictus. Soon after, many different
genera of bacteria capable of respiring chlorinated
ethylenes were isolated. Table 2.1 lists many of the
organisms that have been reported in the literature.
Some of the halorespiring organisms can only grow
on a very limited range of substrates. They use only
hydrogen as the electron donor and couple growth
only to the reduction of chlorinated compounds.
Examples are Dehalobacter restrictus, Strain TEA,
Dehalococcoides ethenogenes strain 195, and De-
halococcoides sp. strain BAV1. Other halorespiring
organisms are less restricted. They are able to use
a number of different electron donors and acceptors
for growth. Examples are Dehalospirillum multivorans
(Sulfospirillum) and various Desulfitobacterium slrams
including Desulfitobacterium strain PCE1, Desul-
fitobacterium strain PCE-S, and Desulfitobacterium
frapp/er/TCE1.
The Place of Dehalorespiring Bacteria in the
Diversity of Life
To understand the diversity of microbial communities
and the evolutionary relationships between various
kinds of microbes, microbiologists have compared the
DNA sequences of genes that encode for enzymes
that carry out the metabolic processes of interest
and the DNA sequences of genes that encode for
important structural components of the microbial cell.
The gene sequence that encodes for 16S rRNA is
commonly used to compare evolutionary relationships
between bacteria. All living cells on earth contain an
organelle called a ribosome that assembles proteins
from amino acids. Because 16S rRNA is an important
component of ribosomes, portions of the 16S rRNA
sequence are highly conserved due to the pivotal role
of protein synthesis in cell metabolism. Based on the
similarity of 16S rRNA sequences, a phylogenetic tree
of microbial groups has been constructed (Figure 2.1).
All life on earth is divided into three great domains; the
organisms with a cell nucleus (eukarya), the bacteria,
and the archaea. The archaea are single-celled and of
similar size as the bacteria but are more closely related
to the eukarya in their genetics and biochemistry. The
archaea include the methanogens and other members
that live in extreme environments. Most common
soil and ground water bacteria that degrade organic
contaminants are bacteria and fall into the division
Proteobacteria, the Bacteroides/Cytophaga group,
and the Gram positive bacteria. The cell wall of the
Gram positive bacteria has a characteristic structure
that can be recognized using a specific staining tech-
nique, the Gram stain. The Gram positive bacteria are
divided into a high C+G group and a low C+G group
based on the relative proportion of the bases guanine
and cytosine in their DNA.
Many halorespirers (Dehalobacter, Desulfitobacte-
rium) fall within the group of low C+G Gram positives;
however, some halorespirers are found within very
distantly related phylogenetic groups. This distribu-
tion indicates that the ability to dechlorinate is not
restricted to one phylogenetic cluster and may have
evolved separately. On the other hand, no halorespir-
ing organisms have yet been isolated that belong to
the archaeal domain (Middeldorp et al., 1999).
Of the organisms described to date, only Dehalococ-
coides species, which are phylogenetically located
within the green non-sulfur bacteria, are capable of
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Table 2.1. Diversity of Bacteria that can Reductively Dechlorinate Ethylenes*
Isolate or Culture
Closest Phylogenetic
Affiliation
Dechlorination
Steps Performed
Reference
Dehalobacter restrictus
Dehalospirillum
multivorans, renamed
Sulfospirillum
Desulfitobacterium strain
PCE1
Desulfuromonas
chloroethenica
strain MS-1
strain TEA
Desulfitobacterium sp.
strain PCE-S
Dehalococcoides
ethenogenes strain 195
Desulfitobacterium
frapp/er/TCE1
Clostridium bifermentans
strain DPH-1
Dehalococcoides sp.
strain CBDB1
Desulfitobacterium sp.
strain Y51
Desulfitobacterium
metallireducens
Desulfuromonas
michiganenis
Dehalococcoides sp.
strain BAV1
Low G+C Gram positive
bacteria
Proteobacteria, e subdivision
Desulfitobacterium
a Gram positive bacterium
Geobacter
Enterobacteriaceae
Gram positive bacteria
Low G+C
Desulfitobacterium
a Gram positive bacterium
Green, nonsulfur bacteria
Desulfitobacterium
a Gram positive bacterium
Clostridium
Dehalococcoides
ethenogenes
Desulfitobacterium
a Gram positive bacterium
Desulfitobacterium
a Gram positive bacterium
Geobacter
Dehalococcoides
etheneogenes
PCE to c/s-DCE Holliger et al., 1993
PCE to c/s-DCE
PCE to TCE
Scholz-Muramatsu et
al., 1995
Gerritseet al., 1996
PCE to c/s-DCE Krumholz et al., 1996
DOC t~ „,-„ noc Sharma and McCarty
PCE to c/s-DCE -iqq«
PCE to c/s-DCE Wild et al., 1996
PCE to c/s-DCE Miller et al., 1997
PCE to ethylene Maymo-Gatell et al.,
PCE to c/s-DCE Gerritse et al., 1999
PCE to c/s-DCE
PCE to trans-
DCE
PCE to c/s-DCE
PCE to c/s-DCE
PCE to c/s-DCE
c/s-DCE to
ethylene
Chang et al., 2000
Adrian et al., 2000
Suyamaetal., 2002
Finneran et al., 2003
Sung et al., 2003
Heetal., 2003a,b
* Extracted from Major et al. (2003).
dechlorinating lower chlorinated ethylenes (i.e., dichlo-
roethylene and vinyl chloride) and coupling growth
with the dechlorination. For instance, Dehalococ-
coide ethenogenes strain 195 obtains energy from all
dechlorination steps except the final step from vinyl
chloride to ethylene (Maymo-Gatell et al., 1999,2001).
Dehalococcoides sp. strain BAV1 grows on vinyl
chloride and all the dichloroethylene isomers (He et
al., 2003a, 2003b). All laboratory mixed cultures that
dechlorinate PCE or TCE beyond c/s-DCE have been
found to contain organisms in the Dehalococcoides
phylogenetic group (Adamson and Parkin, 2000; Ellis
et al., 2000; Fennell et al., 2001; Duhamel et al., 2002;
Richardson et al., 2002; Cupples et al., 2003; Dennis
et al., 2003). Certain stains of Dehalococcoides can
also partially degrade polychlorobiphenyls (PCBs),
chlorobenzenes and dioxins (Fennell et al., 2004)
and dichloroethane and dibromoethane (Thomson
and Vidumsky, 2003). There is a group of Chloroflexi
bacteria that is closely related to Dehalococcoides
(but is not the same genus) that also dechlorinates
PCBs, chlorobenzenes, and PCE (Miller et al., 2005;
Watts et al., 2005; Wu et al., 2002a , 2002b).
If natural attenuation is to be a remedy for ground wa-
ter contamination with chlorinated ethylenes, the chlo-
-------
rinated ethylenes must be completely dechlorinated
to harmless products. Because the Dehalococcoides
group is the only known group of organisms that can
grow by carrying out the reductive dechlorination of
dichloroethylene or vinyl chloride, it has a critical role
in any evaluation of monitored natural attenuation in
anaerobic ground water.
Section 3 of this report discusses the use of tech-
niques for genetic analysis to recognize members of
the Dehalococcoides group in ground water and aqui-
fer sediment. The Dehalococcoides group is unusual
in that its sequences of ribosomal RNA group with
sequences shared by the green non-sulfur bacteria
(i.e., the Chloroflexi ). The green non-sulfur bacteria
are green because they contain a form of chlorophyll.
They are phototrophic, meaning that they gain energy
from light and grow in the presence of light, but they
do not produce oxygen during photosynthesis.
Cytophagales
Bacteroides-Cytophaga-Flexibacteria
Cyanobacteria
blue-green algae
Proteobacteria
Many gram negative bacteria including
Geobacter, Desulfobacter, Pseudomonas,
and Enterobacteriaceae such as
Escherichia
Low G+C gram positive
bacteria
Dehalobacter and
Desulfitobacterium
Green non-sulfur
bacteria
Actinobacteria
Dehalococcoides
e.g. Actinomycetes
and Nocardia
0.10 change per nucleotide
in the 16s RNA gene
Archeae
Figure 2.1. A phylogenetic tree based on comparisons of sequences in the 16s Ribosomal RNA. The length of the
thin lines is roughly proportional to the evolutionary distance between groups of organisms. The thick
lines represent the range within a particular group. Redrawn and simplified from Figure 1 in Hugenholtz
etal., 1998.
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Organisms that Oxidize Chlorinated
Ethylenes under Anaerobic Conditions
Oxidation usually occurs in the presence of molecu-
lar oxygen; however, oxidation of c/s-DCE and vinyl
chloride may also occur under some anaerobic con-
ditions (Bradley and Chapelle, 1998, 2000a, 2000b;
reviewed in Bradley, 2003). Bradley and Chapelle
(2000b) showed that vinyl chloride can be oxidized to
acetate by an interesting class of anaerobic bacteria
called acetogens. They oxidize the organic compound
solely as part of their energy metabolism. They do
not use the chlorinated ethylene as a substrate to
build microbial cell constituents. Although their role
in natural attenuation of chlorinated ethylenes in an-
aerobic aquifers may be as significant as that of the
Dehalococcoides group, very little is known about
them, and there are no techniques that are currently
commercially available to estimate their contribution.
Organisms that Co-Metabolize Chlorinated
Ethylenes
Under some circumstances, dechlorination of chlo-
rinated compounds is not coupled to growth. The
reaction is catalyzed by an enzyme or cofactor that
is fortuitously produced by the microbes for other
purposes. When this occurs, the process is called
co-metabolism. Chlorinated ethylenes can be co-
metabolized under aerobic conditions and under
anaerobic conditions.
PCE and TCE can be co-metabolically dechlorinated
by many types of anaerobic organisms, including
certain species of methanogens such as Methanosar-
cina maze/ (Fathepure and Boyd, 1988) and certain
acetogens such as Acetobacterium woodii (Egli et
al., 1988) and Sporomusa ovata (Terzenbach and
Blaut, 1994); and sulfate-reducing bacteria (Bagley
and Gossett, 1989). Only a small fraction of the total
reducing equivalents derived from the oxidation of
electron donors is used to reduce the chlorinated
compounds.
Some bacteria contain certain enzymes that can for-
tuitously catalyze the oxidation of partially chlorinated
compounds. These monoxygenase or dioxygenase
enzymes require molecular oxygen as a substrate.
Oxygen and a primary substrate are required for
growth of the organisms. When these bacteria grow on
primary substrates like methane, propane, propene,
methanol, toluene, or phenol, they can co-metaboli-
cally oxidize partially chlorinated ethylenes such as
TCE, c/s-DCE, or vinyl chloride (Wilson and Wilson
1985; McCarty and Semprini, 1994; Reij et al., 1995;
Fitch et al., 1996). The only plausible co-substrates
in a contaminated aquifer are methane or aromatic
hydrocarbons such as toluene. If oxygen is available,
bacteria can grow rapidly on the primary substrates,
and exhaust supplies of either the primary substrate or
oxygen. Ground water in a contaminated aquifer can
be expected to have the primary substrate or oxygen,
but not both. Aerobic co-oxidation might occur at the
fringe of an anaerobic plume where contaminated
ground water containing methane mixes with ground
water containing oxygen. However, the process is
unlikely to make a substantial contribution to natural
attenuation of chlorinated ethylenes.
Aerobic Growth on Chlorinated Ethylenes
There is no report on microbial growth supported by
the oxidation of PCE or TCE. However, DCE and vinyl
chloride can be directly oxidized by some bacteria. For
instance, aerobic bacteria such as Actinomycetalessp.
(Phelps et al., 1991), Mycobacterium sp. (Hartmans
and de Bont, 1992), Rhodococcus sp. (Malachowsky
et al., 1994), Pseudomonas sp. (Verce et al., 2000),
and Nocardioides sp. (Coleman et al.; 2002a, 2002b)
can grow on vinyl chloride as the sole carbon source.
Similarly, c/s-DCE has been shown to be utilized as the
sole carbon source by some microorganisms (Bradley
and Chapelle, 2000a, 2000b; Coleman et al., 2002a,
2002b; Olaniran et al., 2004).
Coleman et al. (2002b) determined the distribution of
aerobic bacteria that could metabolize vinyl chloride
in ground water from monitoring wells at chlorinated
solvent spill sites. They were able to isolate vinyl chlo-
ride oxidizing strains from 15 of 24 samples of ground
water or aquifer sediment. Aerobic organisms that
degrade vinyl chloride are widely distributed. Reduc-
ing conditions are required to produce vinyl chloride
or c/s-DCE from TCE or PCE in ground water, yet
oxygen is required for the further aerobic metabolism
of c/s-DCE or vinyl chloride. Degradation of c/s-DCE
and vinyl chloride by aerobic microorganisms can only
be important at the fringe of a plume where DCE and
vinyl chloride, produced in the plume by anaerobic
microbial processes, are mixed with uncontaminated
ground water containing oxygen. Aerobic biodegrada-
tion is probably not important in ground water plumes,
unless the plume is very long. However, conditions
conducive to aerobic biodegradation occur in the
bed sediments of streams and lakes where plumes
of contaminated ground water discharge to aerobic
surface water.
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Section 3.
Tools to Assay Microorganisms that
Completely Transform Chlorinated Solvents
The only organisms that are known to completely
transform chlorinated ethylenes to harmless products
are members of the Dehalococcoides group of bac-
teria. This section discusses an analytical procedure
called the polymerase chain reaction (PCR) that is
used to amplify DNA from the Dehalococcoides group
of bacteria when they are present in samples of ground
water or aquifer sediment, and procedures used to
detect and measure the amplified DNA produced by
PCR. This section discusses the application of PCR
technology to evaluate the presence and distribution
of Dehalococcoides bacteria in ground water or aquifer
sediments, and discusses limitation of the PCR assays
for detecting Dehalococcoides bacteria.
The identification and enumeration of the members of a
microbial community have traditionally been achieved
by cultivation techniques such as plate counting. How-
ever, it has been estimated that the portion of microbes
obtained by traditional cultivation techniques amounts
to only 0.1 to 1 % of the total diversity (Amann et al.,
1995). As a result, non-plating techniques, including
molecular genetic methods, have been developed
that can detect and identify microbes in their natural
environment based on variations in the sequences of
base pairs in their DNA and RNA (e.g. Akkermans et
al., 1995). Most of these techniques are based on
amplification of DNA that encodes the gene for 16S
rRNA, using the polymerase chain reaction (PCR).
The amplified DNA is detected or characterized by a
variety of techniques including denaturing gradient gel
electrophoresis (DGGE) (Muyzer et al., 1993) and real
time PCR (Lee et al., 1993; Livak et al., 1995).
Polymerase Chain Reaction (PCR) Assays
for Genetic Analysis of the Microbial
Communities
The development of PCR is a major step forward in the
study of microorganisms in the environment (Erlich,
1989). To prepare for the assay, a target organism
is identified or selected. It is necessary to know the
DNA sequences from the target organism. Based on
that knowledge, short sequences of DNA are syn-
thesized ("primers") that are complementary to the
gene sequences in the target organism. To carry out
a PCR assay using the primers, DNA is isolated from
ground water or sediment samples. Then, the primers
are combined in a reaction mixture and bind to the
target DNA, if it is present, and allow the DNA to be
copied by an enzyme called DNA polymerase. The
process is repeated for a number of cycles, usually
30 to 40. Each time the DNA that was synthesized
in the previous reaction becomes the substrate for
the subsequent reaction. In each cycle of the chain
reaction, the concentration of target DNA is doubled.
Finally, the concentration of target DNA (the amplicon)
is high enough that it can be identified and analyzed
by optical or chemical procedures. The entire process
is referred to as a polymerase chain reaction (PCR).
Electrophoresis is a technique to separate large
molecules such as DNA based in part on their ionic
charge. If a direct current is imposed across a gel
containing the large molecules in a solution, the
molecules will migrate to one electrode or the other
depending on their charge.
The migration of the large molecules through the
electric field is controlled by the strength of the field,
the ionic charge of the large molecules, and the size
and shape of the molecules. Because DNA has a
net negative charge, DNA will migrate through the
gel toward the positive electrode.
In cells, DNA molecules occur as a duplex or double
helix of two separate strands of DNA. The two strands
of the double helix are held together by hydrogen
bonds between the two strands. The DNA duplex
or double helix is folded into a three-dimensional
structure that is held together by hydrogen bonding
and interactions between hydrophobic regions in the
DNA molecule. The two strands of DNA in the double
helix can be separated from each other by chemicals
and heat.
DNA molecules are commonly separated by a varia-
tion of electrophoresis called denaturing gradient gel
electrophoresis (DGGE). The gel contains a gradient
of chemicals that cause the DNA double helix to unfold
into single strands. As a DNA molecule moves through
11
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the gradient in the gel, the two strands of DNA start to
separate or unfold. The unfolded DNA assumes a dif-
ferent shape, which produces more drag and reduces
the mobility of the DNA molecule through the gel.
Eventually, the partially unfolded DNA molecule will
become so entangled in the gel matrix that it will stop
moving. Small differences in the genetic sequence
have a strong effect on the position in the gradient
where the DNA molecule unfolds. As a result, small
differences can cause the DNA molecule to take up
different positions along the gradient in the gel. The
fragments of DNA separate from each other and take
up unique positions along the electrical gradient in
the gel. When the DNA in the electrophoresis gel is
stained, it shows up as bands (See Figure 3.1).
To identify the organism that supplied the DNA that
was extracted from an environmental sample or an
enrichment culture, the position of the band of un-
known DNA is compared to the positions of the bands
of DNA from known organisms. Because the DNA
from a particular organism has been amplified by PCR
before analysis by DGGE, the combination of the two
techniques can detect very sparse populations of
individual organisms in mixed microbial communities.
The DNA from bands can be excised, reamplified by
PCR, and sequenced, and the source of the DNA
can be identified by comparing the sequences of the
DNA isolated from the environmental sample to the
sequence in known organisms.
Wells for Extracts
Negative Electrode
Electrophoresis Gel
\
Positive Electrode
Figure 3.1. Separation of DNA by Denaturing Gradient Gel Electrophoresis (DGGE). Wells are cut into a rectan-
gular piece of gel. A different extract containing DNA is placed in each well. The DNA dissolves from
the extract into the gel. An electrical current is imposed across the gel, causing the DNA molecules to
migrate. Based on their ionic charge, size, and three-dimensional shapes, different pieces of DNA move
different distances, causing the pieces of DNA to separate.
12
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Detection of Dehalococcoides Species by
the PCR Assay
Although many microbial groups are involved in
reductive dechlorination of chlorinated ethylenes,
Dehalococcoides is the only group known to completely
dechlorinate PCE/TCE to ethylene (Maymo-Gatell et
al., 1997; Major et al., 2003). A recent paper reports
that 17 putative reductive dehalogenases and five
hydrogenase complexes are encoded in the genes
of Dehalococcoides ethenogenes, indicating this
organism is highly evolved to utilize halogenated
organic compounds and hydrogen (Seshadri et al.,
2005). Hence, detection of Dehalococcoides species
may be a useful tool for assessing the efficiency of
natural attenuation or engineered bioremediation at
chlorinated solvent contaminated sites.
Currently (as of 2005), a polymerase chain reaction
(PCR) assay based on primers for genes encoding
for 16S-rRNA is the major tool for the detection of
Dehalococcoides species (Loffler et al., 2000; Fen-
nell et al., 2001; Hendrickson et al., 2002; He et al.,
2003a, 2003b).
Loffler et al. (2000) were the first to publish an assay
for the density of Dehalococcoides bacteria based on
PCR. In their study, they used a two-step nested primer
PCR approach using universal bacterial primers
followed by a second PCR with the Dehalococcoides-
targeted primers. The Dehalococcoides-iargeied
primers were designed based on the 16S rRNA
sequence of Dehalococcoides sp. strain FL2, which
was closely related to Dehalococcoides ethenogenes
(96.9% sequence similarity). Since Dehalococcoides
sp. strain FL2 was not available in pure culture, the
sensitivity of the Dehalococcoides-iargeied primers
was evaluated using serially diluted plasmid DNA
containing the 16S rRNA gene of strain FL2. One
to 10 copies of the 16S rRNA gene of strain FL2
were found to be sufficient to yield the expected
PCR product. This nested PCR approach was used
to detect Dehalococcoides populations in river and
aquifer sediments, and the results were confirmed by
microcosm studies.
Fennell and her co-workers designed a Dehalococ-
coides primer set based on the 16S rRNA gene of
Dehalococcoides ethenogenes strain 195 and used
it for the detection of Dehalococcoides at a TCE-
contaminated site (Fennell et al., 2001). In their
study, separate PCR reactions were performed with
universal primers and with Dehalococcoides primers.
If no product was obtained directly with the specific
primers, a nested approach was performed using the
products from the universal PCR as the template for
PCR with the specific primers. The detection limits
were found to be approximately 103 cells per 0.5 g soil
for the direct PCR and 5 to 10 cells per 0.5 g soil for
the nested approach.The results of PCR analysis were
supported by field data and microcosm studies.
Hendrickson and his co-workers developed a PCR
assay to conduct an extensive survey for the pres-
ence of Dehalococcoides at multiple chloroethylene-
contaminated sites (Hendrickson et al., 2002). In
their study, seven Dehalococcoides primer sets were
designed based on the 16S rRNA gene sequence
of Dehalococcoides ethenogenes strain 195, which
was originally isolated at Cornell University, and De-
halococcoides group sequences found in enrichment
cultures originally isolated from sites at Victoria, Texas,
and Pinellas, Florida. The sensitivities of the primer
sets ranged from 10 to 1,000 copies of the gene per
reaction mixture. The developed PCR assay was ap-
plied to ground water samples and soil samples col-
lected from 24 sites. Positive results were obtained at
21 sites where full dechlorination of chloroethylenes
to ethylene occurred. Phylogenetic analysis of the am-
plicons confirmed that Dehalococcoides sequences
formed a unique 16S rRNA group, which could be
divided into three subgroups (Pinellas, Victoria, and
Cornell) based on specific base substitution patterns
in variable regions 2 and 6 of the Dehalococcoides
16S rRNA gene sequence.
In general, the direct PCR technique using gel elec-
trophoresis to detect the amplified gene product is a
qualitative or semi-quantitative method. More recently,
real time PCR (RT-PCR) has been developed to pre-
cisely quantify the density of the Dehalococcoides
population (Lendvay et al., 2003; He et al., 2003a,
2003b). RT-PCR does not involve the use of gel
electrophoresis to detect the amplified DNA, but it
still makes use of the same principles of amplifica-
tion as standard PCR. In RT-PCR amplification, a
fluorescently labelled probe targeting 16S rRNA gene
sequences of Dehalococcoides was included in the
reaction. The fluorescence of the dye attached to the
amplified DNA is measured during the "extension"
phase of the polymerase chain reaction, allowing "real-
time" monitoring of the accumulation of the replicated
DNA by the instrument during each cycle of the poly-
merase chain reaction. The number of cycles that are
required to accumulate a certain amount or so-called
"threshold" concentration of copied DNA is used to
quantitatively determine the starting concentration of
DNA. This technique has been applied to measure
the abundance of a Dehalococcoides population in
a chloroethylene-contaminated aquifer undergoing
active remediation (Lendvay et al., 2003).
13
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Limitations of the PCR Assay for
Dehalococcoides DMA
Although Dehalococcoides organisms are closely re-
lated phylogenetically, their capacity to transform chlo-
rinated ethylenes can be quite different (Duhamel et
al., 2004; He et al., 2003a, 2003b). Dehalococcoides
strain CBDB1 can dechlorinate chlorobenzenes, but
cannot grow on PCE or TCE (Adrian et al., 2000).
Dehalococcoides ethenogenes strain CBDB1 and
Dehalococcoides sp. strain 195 have 98% identity over
1,422 nucleotides of the 16S rRNA gene sequence.
However, strain CBDB1 can only convert PCE to trans-
DCE, while strain 195 converts PCE to vinyl chloride
and ethylene (Fennell et al. 2004). Dehalococcoides
ethenogenes 195 and Dehalococcoides strain FL2
both grow on TCE or dichloroethylenes, but cannot
grow on vinyl chloride, while Dehalococcoides sp.
strain BAV1 can grow on vinyl chloride (He et al.,
2003b). Although these strains are closely related
and are capable of dechlorinating some of the same
substrates, they did not share the capacity to grow
using vinyl chloride, which is critical to complete
transformation of chlorinated ethylenes to harmless
products.
The PCR primers used in commercially available
assays are designed to detect as many Dehalococ-
coides strains as possible. They are not designed
to distinguish between the different strains. It would
be nearly impossible to design primers for 16S rRNA
genes that could distinguish strains that dechlorinate
dichloroethylenes and vinyl chloride from strains that
do not. Even if the PCR products were cloned and
sequenced from strains that did or did not dechlori-
nate dichloroethylenes and vinyl chloride, it is unlikely
that the relatively short amplicons that are typically
obtained would contain enough information to dis-
tinguish between strains (personal communication,
Donna Fennell, Rutgers University).
One alternative to searching for a structural gene
associated with the Dehalococcoides group is to
create primers for the genes for the dehalogenase
enzymes that actually carry out the transformation
of TCE or vinyl chloride to ethylene. Some strains of
Dehalococcoides are known to express an enzyme
that can dechlorinate TCE to ethylene (Magnuson et
al., 1998, Fennell etal., 2004) or an enzyme that can
dechlorinate vinyl chloride to ethylene (Muller et al.,
2004, Krajmalnik-Brown etal., 2004). Genes for these
enzymes have been sequenced, and a quantitative
PCR assay for these gene sequences is commercially
available. This is an area of active research, and new
dehalogenase primers are being developed. Until a
comprehensive catalogue of primers for dehalogenase
genes can be developed, there is a strong chance
of a false negative in interpreting the absence of the
dehalogenase gene. Although a particular gene may
be absent, other dehalogenase genes that were not
recognized by the primer may be expressed in the mi-
crobial population. However, the interpretation of the
detection of PCR product from dehalogenase genes
is straightforward. If the product is amplified by the
primer, the gene is present in the population.
Current State of Practice of PCR Tools
to Evaluate Biotransformation of
Chlorinated Solvents
In 2005, the Strategic Environmental Research and
Development Program (SERDP) and the Environ-
mental Security Technology Certification Program
(ESTCP) of the U.S. Department of Defense organized
a workshop to evaluate the application of molecular
biological tools to environmental remediation. The
report of the workshop is a useful summary of the
state of practice and needs for technology develop-
ment (Alleman et al., 2005). Table 3.1 presents the
consensus opinion of the experts that participated
in the workshop concerning the advantages and
disadvantages of PCR tools that might be used to
evaluate biotransformation of chlorinated solvents in
ground water, and Table 3.2 presents the consensus
opinion on the applications of the PCR tools and the
frequency at which they are used.
14
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Table 3.1 Comparison of Advantages and Disadvantages of PCR Tools to Evaluate Biotransformation of Chlori-
nated Solvents in Ground Water. Summarized from Table 2 of Alleman et al. (2005)
Tool
Perceived Advantage
Perceived Disadvantage
Direct PCR
Easy to perform
False negatives
Nested PCR
Unsurpassed sensitivity
Requires two PCR steps
Quantitative PCR
for 16s rRNA
gene
Provides information on presence/ absence/
abundance of organisms of interest; nearly
reaches the sensitivity of nested PCR; com-
mercially available for few key organisms
(e.g. Dehalococcoides spp.); estimates of
total bacterial numbers are possible
Does not provide confirmation of
activity; sampling, handling, and
analysis are not standardized
Quantitative PCR
for functional
genes
Provides information on presence/ absence/
abundance of functional gene of interest;
commercially available for few key genes
(e.g. reductase dehalogenase genes)
For DNA, does not provide confor-
mation of activity; sampling, han-
dling, and analysis are not stan-
dardized
Quantitative PCR
for messenger
RNA
Provides information on gene expression
(i.e. activity); quantitative approaches under
development
Relative instability of RNA presents
sampling and preservation chal-
lenges; not commercially available
to a significant extent; sampling,
handling, and analysis are not stan-
dardized
Table 3.2 Applications of PCR Tools to Evaluate Biotransformation of Chlorinated Solvents in Ground Water. Sum-
marized from Table 2 of Alleman et al. (2005)
Tool
Current Applications
Current
Relative
Frequency
of Use
Comments
Direct PCR
Screening tool for presence/ab-
sence [of DNA for putative active
organisms]
Moderate
Replaced by quantitative PCR
Nested PCR
Screening tool for presence/ab-
sence [of DNA for putative active
organisms]
Moderate
Replaced by quantitative PCR
Quantitative
PCR for 16s
rRNA gene
Screening tool for presence/ab-
sence of desired or indicator or-
ganisms; monitoring of growth and
distribution of individual organisms
High
[Would benefit from] expansion to
wider range of organisms; stan-
dardized procedures; availability
of standards
Quantitative
PCR for func-
tional genes
Screening tool for presence/ab-
sence of target functional genes;
monitoring of distribution and pro-
liferation of individual genes
Low
Needs wider range of functional
genes; extension to mRNA; stan-
dardized procedures; availability
of standards
Quantitative
PCR for messen-
ger RNA
A few experimental applications
for confirming expression of func-
tional genes.
Low
Needs wider range of genes of
interest; standardization of ap-
proach; clarification of how mRNA
abundance relates to activity
15
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Section 4.
Dehalococcoides DNA and Rate of Natural Attenuation
Many evaluations of chlorinated solvent contamination
in ground water use mathematical models to project
the future behavior of the plume. The models are very
sensitive to the rate constants for degradation (Newell
et al., 2002). Biological reductive dechlorination can
be an important mechanism for the removal of chlo-
rinated solvents from many anoxic aquifers; however,
there appears to be a significant variation in the rates
and extent of dechlorination from one plume to the
next (Suarez and Rifai, 1999).
Hendrickson et al. (2002) reported that there was
a strong association between the presence of
Dehalococcoides DNA and complete dechlorination to
ethylene. Lendvay et al. (2003) further demonstrated
a quantitative relationship between Dehalococcoides
DNA and ethylene production. To determine if there
is a valid association between Dehalococcoides
DNA in ground water and the observed rates of
dechlorination at field scale, a survey was conducted
at selected field sites. Rate constants for attenuation
of chlorinated solvents at field scale were extracted
from the monitoring data, and then the rate constants
were compared to the presence or density of
Dehalococcoides DNA in water from monitoring wells
at the sites.
As is discussed under the subsection titled, Rates of
Natural Attenuation and Density of PCR Products
from Dehalococcoides DNA, the monitoring wells did
not efficiently sample the Dehalococcoides organisms
in the aquifer, and the number of Dehalococcoides
cells recovered in a liter of well water was a small
fraction of the number of cells that were exposed to a
liter of ground water in the aquifer. Most of the Deha-
lococcoides cells were probably attached to sediment
particles. As a result, there was not a quantitative
relationship between the rates of natural attenuation
at field scale and the density of Dehalococcoides cells
in ground water from monitoring wells.
A Definition of "Generally Useful" Rates of
Biological Reductive Dechlorination
There is no legally mandated time frame for monitored
natural attenuation of contaminants in ground water
(U.S. Environmental Protection Agency, 1999). For
purposes of discussion, a time frame for remediation of
30 years will be assumed. The Maximum Contaminant
Levels (MCLs) for PCE and TCE in drinking water are
5 ug/L. McNab et al. (2000) evaluated the distribution
of contaminants in more than 200 chlorinated solvent
plumes. Figure 4.1 shows the cumulative frequency
distribution of the maximum concentrations of each
separate chlorinated hydrocarbon in each of the
plumes and the first order rates of degradation that
are necessary to reduce the maximum concentrations
to meet a MCL of 5 ug/L in 30 years.
Fifty percent of the plumes had maximum concentrations
of chlorinated hydrocarbons greater than 8,000 ug/L.
The rate of natural biodegradation necessary to re-
duce concentrations from 8,000 ug/L to 5 ug/L in 30
years would be 0.23 per year (calculated by a first-
order kinetic model). Similarly, the rates of degradation
necessary to reduce 10% and 90% of the plumes in
the survey of McNab et al. (2000) from their maximum
concentrations to their MCLs within 30 years would be
0.043 per year and 0.32 per year, respectively. For
purposes of evaluating the data at our study sites, a
rate of 0.3 per year can be considered a "generally
useful" rate constant for monitored natural attenuation
of chlorinated ethylenes in ground water. This "gener-
ally useful" rate should not be applied at other sites
without due consideration of site specific conditions.
If the initial concentrations of chlorinated ethylenes are
low, or if the time allowed to reach MCLs is long, a
slower rate might be acceptable at a particular site.
Site Selection
Eight sites at six locations were selected for the
survey because they had good records of long-term
monitoring. This data made it possible to extract rate
constants for attenuation of the chlorinated solvents.
These sites are the Western Processing Site at Kent,
Washington; Landfill Number 3 (LF3) and Fire Train-
ing Area Number 2 (FTA2) at Tinker Air Force Base,
Oklahoma; the North Beach Site at the U.S. Coast
Guard Support Center in Elizabeth City, North Caro-
lina; Spill Site Number 17 (SS-17) at Altus Air Force
Base, Oklahoma; the Target Area 1 Site at Dover Air
Force Base, Delaware; and Area 800 and Area 2500
at the former England Air Force Base, Louisiana. See
Figure 4.2 for a map showing the locations of the sites
within the United States.
17
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10000000
1000000 -
100000 -
o
10000
1000
I
o 100
o
10
0 10 20 30 40 50 60 70 80 90 100
Frequency (Percent of Plumes)
Figure 4.1. The frequency distribution of the maximum concentration of chlorinated solvents and their transformation
products at Department of Defense Sites in the United States (from McNab et al., 2000). The arrows
identify the first order rates of biotransformation that are required to reach a Maximum Contaminant
Level of 5 ug/L within thirty years at 10% of sites, at 50% of sites, and at 90% of sites.
Western Processing,
Kent, WA
Target Area I,
Dover AFB, DE
North Beach, USGC
Support Center
SS-17,AltusAFB,OK
LF-3, Tinker AFB, OK
FTA-2, Tinker AFB, OK
Figure 4.2. Location of sites used to survey the relationship between Dehalococcoides DNA in ground water and
the rate of natural attenuation of chlorinated ethylenes in field scale plumes.
18
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The plume at the Western Processing Site was ex-
tracted with a pump and treat system from September
1988 through April 2000. The plume at site LF3 has
been extracted by a pump and treat system since early
1999. The plume at site SS-17 has been extracted
with a two-phase vacuum extraction system since
September 1996. At the North Beach Landfill Site,
the source was removed in 1999. There are no engi-
neered remediation actions at the other sites. To avoid
errors in fitting rate constants for natural attenuation,
the rate constants were fit to data that were collected
prior to initiation of any engineered remedies.
Ground Water Sampling
At least two monitoring wells were sampled in the
plume at each site. One well was located at the "hot
spot" and one down gradient. See Table 4.1 for the
number of wells sampled at each site. In most cases,
the water samples were collected with a peristaltic
pump at the well head using a polyethylene plastic
tube inserted into the well. Occasionally, the water
samples were collected using the dedicated submers-
ible pump in the wells. Each well was purged for ap-
proximately one-half hour; at least two casing volumes
were purged before samples were collected.
The dissolved hydrogen in ground water was sampled
using a Microseeps Cell following the bubble strip-
ping method (Mclnnes and Kampbell, 2000). After
sampling for dissolved hydrogen, the effluent from
the pump was directed to pass through an over-flow
cell for measuring pH, temperature, conductivity, and
oxidation reduction potential (ORP) against a Ag/AgCI
reference electrode. Then, the effluent from the pump
was collected to determine the concentrations of
dissolved oxygen and ferrous iron using colorimetric
field test kits. Finally, the effluent from the pump was
directed to fully fill various sample containers for analy-
sis of Dehalococcoides DNA, chlorinated solvents,
dissolved gases (methane and ethylene), inorganic
ions (nitrate plus nitrite, sulfate and chloride), and total
organic carbon (TOC) in the laboratory.
The samples for chlorinated solvents and the samples
for dissolved gases were preserved with 1 % trisodium
phosphate. The samples for nitrate plus nitrite were
preserved with acid (five drops of 50% sulfuric acid to
50 ml of water sample). The samples for Dehalococ-
coides DNA were packed in ice and stored in coolers
prior to shipment for analysis.
Chemical Analysis
Ground water samples for the analysis of chlorinated
solvents were prepared in an automatic static head-
space sampler (U.S. EPA, 1988). The samples were
analyzed by gas chromatography with a mass spec-
trometer detector. The reporting limits were 1.0 ug/L
for all the analytes. The concentrations of ethylene and
methane in ground water samples were determined
using a headspace equilibration technique (Kampbell
and Vandegrift, 1998). The gaseous components in
the headspace were separated by gas chromatogra-
phy and then measured with a thermal conductivity
detector. The reporting limits were 1 ug/L in the origi-
nal aqueous phase for both gases. The concentration
of dissolved H2 was measured on a RGA3 Reduction
Gas Analyzer equipped with a 60/80 molecular sieve
5A column and a reduction gas detector. The report-
ing limit was 1.0 nM in the water originally sampled.
Nitrate plus nitrite were analyzed using Lachat Flow
Injection Analyses. The reporting limit was 0.1 mg/L
as nitrogen. Sulfate and chloride were analyzed
using Waters Capillary Electrophoresis. The report-
ing limits were 0.5 mg/L for sulfate and 1.0 mg/L for
chloride. Total Organic Carbon was determined by
a Dohrman DC-80 Carbon Analyzer with a reporting
Table 4.1. Location of Specific Sites in the Survey and the Number of Wells Sampled at Each of the Sites
Location
Altus Air Force Base, Oklahoma
Dover Air Force Base, Delaware
U.S. Coast Guard Support Center,
Elizabeth City, North Carolina
England Air Force Base, Louisiana
Tinker Air Force Base, Oklahoma
Kent, Washington
Site
Spill Site 17 (SS-17)
Target Area 1
The North Beach Landfill Site
Area 800
Area 2500
Landfill Number 3 (LF-3)
Fire Training Area Number 2 (FTA-2)
The Western Processing NPL Site
No. Wells Sampled
6
8
6
4
6
5
2
6
19
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limit of 0.5 mg/L. The dissolved oxygen, temperature,
pH, ORP, and conductivity were measured by corre-
sponding electrodes and meters. The concentration
of ferrous iron, hydrogen sulfide, and alkalinity were
determined in the field using a Chemetrics Kit Model
K-6010D for ferrous iron, a Hach Kit Model HS-C for
sulfide, and a Hach Kit Model ALAPMG-L for alkalin-
ity. The reporting limits for ferrous iron, sulfide, and
alkalinity were 0.1, 0.5, and 20 mg/L, respectively.
Detection of Dehalococcoides by Poly-
merase Chain Reaction Analysis
Dehalococcoides DNA in ground water samples was
analyzed by SiREM (Guelph, Ontario) using their
Gene-Trac Test. The test employs the polymerase
chain reaction using primer sets specific to DNA se-
quences in the 16S rRNA gene of the Dehalococcoi-
des group (see Section 3 for a discussion). Each test
used four primer sets, three of which were designed
to target various sequences that were specific to the
Dehalococcoides 16S rRNA gene. A fourth primer
set amplified sequences that were shared by most
members of the True Bacteria (see Figure 2.1) and
was used as a control to confirm that DNA extracted
from the sampled ground water could be amplified
successfully by the polymerase chain reaction.
Bacteria were filtered from 1.0 liter of the ground
water sample using a 0.45 um sterile nylon filter, and
DNA was extracted from the bacteria. The DNA was
extracted and prepared in 55 uL of water that was free
of DNAse and pyrogens. Then, 1 uL of the DNA ex-
tract was used in the PCR reaction mixture. The PCR
product was separated by gel electrophoresis and then
stained to visualize the PCR products produced from
the amplification specific for Dehalococcoides 16S
rRNA gene and from the amplification for the general
(universal) bacterial 16S rRNA gene.
The presence of Dehalococcoides DNA in the samples
was assessed as either "Detected" or "Not Detected"
based on interpretation of an electronic image of the
stained band of DNA in the electrophoresis gel (see
Figure 3.1). Detects (gel bands) were quantified
using densitometry software and assigned a "band
intensity percentage" using the relative intensity of
the strongest bands obtained to the intensity of the
positive control containing 105gene copies. Workers
with experience with PCR are generally reluctant to
make a quantitative association between the quantity
of PCR product and the number of gene copies in the
original sample. As a consequence, SiREM assigned
a "test intensity score" as follows: if the value was 0%
of positive control, a score of (-) was assigned; if the
value was smaller than 3% of positive control, a score
of (+/-) was assigned; if the value was in the range
of 4% to 33% of positive control, a score of (+) was
assigned; if the value was in the range of 34% to 66%
of positive control, a score of (++) was assigned; if
the value was in the range of 67% to 100% of positive
control, a score of (+++) was assigned; and if the value
was larger than 100% of positive control, a score of
(++++) was assigned.
SiREM has compared the semi-quantitative "intensity
score" with the results of quantitative PCR analyses
done on the same samples. When the score was (+),
the approximate range of gene copies was 103 to 105
per liter; when the score was (++), the approximate
range of gene copies was 104 to 106 per liter; when
the score was (+++), the approximate range of gene
copies was 105 to 106 per liter; and when the score
was (++++), the approximate range of gene copies
was 106 to 10s per liter. The effective detection limit
of the PCR reaction for Dehalococcoides DNA using
gel electrophoresis was 500 to 5,000 gene copies
of Dehalococcoides DNA per liter of ground water
extracted.
The density of Dehalococcoides cells in the water
samples was determined using Quantitative Real-time
PCR of 16S rRNA genes. The assays were performed
in the laboratory of Elizabeth Edwards at the University
of Toronto, Ontario, Canada. The quantitation was
performed using primers identical to those used in
a commercial test (Quantitative Gene-Trac, SiREM,
Guelph, Ontario). The primers are specific for variable
regions of the Dehalococcoides 16S rRNA gene and
produce an amplicon of 512 base pairs in length and
are similar to those described by Hendrickson et al.
(2002) and protected under US patent US6894156B2
(Hendrickson and Ebersole). Real-time quantitative
PCR (q-PCR) reactions (50 ul) were performed in
duplicate using 25 ul of 2X DyNAmo SYBR Green
qPCR Master Mix (MJ Research Inc., MA), 1.0 ul of
water containing 25 pmol of each primer and 19 ul
of DNase and RNase-free water (Sigma) and 4 ul of
template DNA which were gently mixed at room tem-
perature and transferred into a 96 well plate (Opticon™
Systems) and sealed with 8-strip Ultraclear caps (MJ
research Inc., Waltham, MA).
Real-time PCR was performed with a DNA Engine
Opticon 2 System (MJ Research Inc., MA) with initial
denaturation at 94 °C for 10 min; 45 cycles of 94 °C for
45 seconds, annealing at 60 °C for 45 seconds, and
extension of 72 °C for 50 seconds. Standard curves
of Ct versus log1016S rRNA gene copy number were
produced using known quantities of cloned Dehalo-
coccoides 16S rRNA genes. The standard curves
were used to estimate the number of 16S rRNA gene
copies in the ground water samples. Verification of the
20
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specificity and identity of the PCR products was deter-
mined by melting curve analysis performed between
72 °C and 95 °C using the Opticon Monitor Software.
The detection limit is near 2,000 gene copies per liter
of ground water extracted.
As mentioned above, SiREM uses primers patented by
DuPont. Hendrickson et al. (2002) sequenced ampli-
cons of the primers to determine the specificity of the
primers for Dehalococcoides. Occasionally SiREM will
sequence the amplicons at the request of their clients.
In every case, the amplicons have been highly similar
(percent similarity >90%) to known Dehalococcoides
organisms (personal communication, Philip Dennis,
SiREM, Guelph, Ontario, Canada). Amplicons from
samples provided in this study were not sequenced.
Purified sterile water was processed with every ten
samples to serve as negative controls or "DNA blanks."
If the controls produced a visible band, the test results
were repeated or invalidated. In some cases where
the bands from the controls were very weak, that fact
was noted in the case narrative, and the test results
were reported.
If no Dehalococcoides DNA was recovered, PCR
was conducted with a universal primer to determine
if amplifiable concentrations of bacterial DNA were
present in the sample. Genomic DNA from E. co//was
used as the positive control for the assays with the
universal primer. The sensitivity of the assay with the
universal primer has not been explicitly determined,
but it should be the same or slightly lower than the
assay with Dehalococcoides primers based on the
fact that fewer cycles of PCR are performed and that
the amplification products, for the most part, are lon-
ger (personal communication, Philip Dennis, SiREM,
Guelph, Ontario, Canada). The amplification of the
bacterial primer was scored as "detected," "trace," or
"not detected."
Calculation of Dechlorination Rates from
Monitoring Data
The BIOCHLOR decision support system was used
to calculate the rates of reductive dechlorination of
chlorinated ethylenes (available at http://www.epa.
gov/ada/csmos.html) (Aziz et al., 2000). The BIO-
CHLOR software simulates remediation of dissolved
solvents by natural attenuation at chlorinated solvent
release sites. It is based on the Domenico analytical
solutions to the solute transport equation and has
the ability to simulate one-dimensional advection,
three-dimensional dispersion, linear adsorption, and
sequential biotransformation of chlorinated ethylenes
by reductive dechlorination. It assumes biotransforma-
tion follows a pseudo-first order rate law. Parameters
used to calibrate BIOCHLOR to the plumes are listed
in Table 4.2. A detailed example of one of the calibra-
tions is provided at the end of this section.
At six sites in five locations (see Table 4.3), the con-
tamination in ground water formed a conventional
plume. Each plume had a region of high contamination
associated with the source and a region with lower
concentrations extending away from the source in
the direction of ground water flow. BIOCHLOR was
calibrated to field data on contaminant concentrations
from single sampling events. Site-specific information
was collected from reports or papers on the study
sites (Acree and Ross, 2003; Altus Air Force Base,
2002; Dover Air Force Base, 2003; Landau Associ-
ates, 1995, 2002; Tinker Air Force Base, 1999, 2002;
Wilson et al., 1997). To avoid errors in fitting rate
constants for natural attenuation, the rate constants
were fit to data that were collected prior to initiation
of any engineered remedies.
At two sites (Area 800 and Area 2500) at England AFB,
Louisiana, there was no discernable overall direction
of ground water flow in the plumes. The sites were
overlaid by a bayou that communicated with the con-
taminated aquifer. Ground water in the aquifer flowed
toward the bayou or away from the bayou depending
on the seasons and recent precipitation events. As
a result, concentration isopleths of TCE, c/s-DCE,
and vinyl chloride were arranged in concentric circles
that were centered about the source area. Therefore,
time series data on contaminant concentrations and
dummy variables for hydrologic properties (seepage
velocity 100 ft/yr, longitudinal dispersivity 10 ft and
retardation factor 1) were inserted into BIOCHLOR to
extract the rate constants of dechlorination over time
in particular wells.
Calculation of Dechlorination Rates in
Conventional Plumes
Table 4.3 shows the relationship between the concen-
trations of chlorinated ethylenes in the ground water
plumes and their apparent rates of dechlorination
along a flow path in the aquifer. A wide variety of con-
centrations were represented in the survey; however a
relatively narrow range of rate constants was deduced.
There did not appear to be any consistent relationship
between the concentration of the chlorinated ethylenes
and their first order rate of biotransformation.
At the Western Processing Site, field data before
implementation of a pump and treat system were
used for calibrating BIOCHLOR. The rate constants
compared very favorably with the rate constants de-
21
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Table 4.2.
Calibration Parameters for BIOCHLOR
Parameter
Seepage
Velocity
(m/yr)
Coefficient
Dispersion
(m)
Ratio Lateral
to Longitudinal
Dispersion
Ratio Vertical
to Longitudinal
Dispersion
Retardation
Factor
Simulation Time
(years)
Modeled Area
Width
(meters)
Modeled Area
Length
(meters)
Source
Thickness
(meters)
Source Width
(meters)
Kent, WA
Western
Processing
1988
20
2.4
0.2
0.2
1.1
30
120
240
9
60
Tinker AFB, OK
Landfill 3
Sept.
1997
29
7.6
0.1
none
3
60
150
760
6
76
Nov.
2002
29
7.6
0.1
none
3
60
150
760
6
76
FTA2
Aug.
1997
29
45
0.1
none
3
30
150
460
6
76
Altus AFB,
OK
SS17
March
2003
8.3
45
0.1
none
1.3
30
180
460
6
60
USCG Support
Center, Eliza-
beth City, NC
North Beach
1997
8.5
0.6
0.2
0.2
1.1
60
120
60
9
60
Oct.
2002
0.8
0.2
0.2
1.1
60
120
76
9
60
Dover
AFB, DE
Target
Area 1
July
1997
37
7.6
0.2
0.2
1.0
60
300
760
15
150
rived from that site by other methods (Lehmicke et
al., 2000, data cited in Table 4.3).
At the other sites, when possible, field data from
independent sampling events were used to calibrate
BIOCHLOR. This was done to evaluate the variability
over time of the rate constants that were extracted
from the field data. There was agreement in the rate
constants extracted for the LF3 plume from data col-
lected in 1997 before the operation of an extraction
system and in 2002 after the operation of the extrac-
tion system. The rate constant we extracted from data
on the FTA2 plume was faster than a rate constant
extracted at the same site in 1999 by a contractor for
the U.S. Air Force (Parsons, Inc., cited in Tinker Air
Force Base, 1999). There was agreement in the rate
constants extracted from the North Beach Site using
data collected in 1997 before source removal efforts
and data collected in 2002 after source removal.
The lower plume in the Target Area 1 is currently at-
tenuating more rapidly near the source areas than
in the far field. As a result, the current profile of
concentrations with distance from the source is in-
verted. Therefore, we chose to calibrate BIOCHLOR
to historical data collected in 1997. There was good
22
-------
Table 4.3. Relationship between the Concentrations of Chlorinated Ethylenes in Ground Water and Their Apparent
Rates of Dechlorination along an Inferred Flow Path in the Aquifer
Facility/ Location
Western Processing
Kent, WA
LF3 (landfill)
Tinker AFB, OK
North Beach, USCG Support
Center
Elizabeth City, NC
FTA2 (fire training)
Tinker AFB, OK
SS17Site
Altus AFB, OK
Target Area 1
(Lower Plume)
Dover AFB, DE
Date
1988a
1999
4/2003
9/1 997 c
11/2002
1997d
1 0/2002
8/1 997
11/2002
3/2003
7/1 997
1997f
PCE
TCE
c/s-
DCE
VC
Concentration near source
(ug/L)
5.3
8
2000
561
6.1
7.5
38.5
680
97
28
105
52
9440
9330
8160
2400
10000
26
0.34
38000
28400
74
25
1200
977
264
560
460
44
1.59
23000
20400
30.8
<1
1.7
2.9
4.28
<1
Transect TA to TB
Transect TB to TC
Wells TA to TB
Wells TB to TC
PCE
TCE
c/s-
DCE
VC
Pseudo-first order
rate constant for
dechlorination
(per year)
0.1
0.1
0.1
0.26
0.18
0.14
0.48
1
1
0.1 e
0.3
0.01
0.1
0.23
0.07
0.06
0.22
0.6
0.6 b
1
1
0.3
0.3
0.1
0.31
0.27
0.08
0.28
3
1 b
3
3
1
1
0.3
0.3
0.2
data collected before the operation of pump and treat system;b data calculated from the half-lives for cis-DCE (1,1 years) and VC
(0.55 year) obtained by Lehmicke et al. (2000); ° data collected before the operation of extraction system, except that data near
source were from the earliest date available (August 2001);d data collected in May or December of 1997 before source removal;e
rate estimate obtained by Parsons (Tinker Air Force Base, 1999); ' data obtained by Ei et al. (2002).
agreement between the rate constants extracted using
BIOCHLOR and rate constants previously extracted
by Ei et al. (2002) using a transect approach or well-
to-well comparisons (Table 4.3).
Relationship between Dehalococcoides
DNA and Dechlorination Rates at
Conventional Plumes
Dehalococcoides DNA was measured and determined
to be present in 24 contaminated wells at six sites
where the contamination in ground water formed con-
ventional plumes. In nine wells at three of the sites,
Dehalococcoides DNA was unequivocally detected.
Table 4.4 compares the concentration of Dehalococ-
coides DNA as determined by the semi-quantitative
PCR test using gel electrophoresis (Gene-TracTest),
and the concentration determined by quantitative
real time PCR, to the concentrations of chlorinated
ethylenes and ethylene in individual wells at the three
sites.
Figure 4.3 compares the locations of the monitoring
wells at the Western Processing Site in Kent, Wash-
ington, to the distribution of cis-DCE in the plume
in 1994. At the Western Processing Site, reductive
dechlorination was essentially complete when the
wells were sampled for Dehalococcoides DNA in
2003 (Table 4.4). Wells at the original source area
of the plume and a well down gradient in the plume
had detectable concentrations of ethylene (Table 4.4).
One of the source area wells and the down gradient
well had detectable concentrations of vinyl chloride.
23
-------
100 meters
o
15M45B
O
Well Sampled for
Dehalococcoides
Well to Contour
Concentrations
Figure 4.3. Location of monitoring wells and distribution of cis-DCE at the Western Processing Site in Kent, Wash-
ington, fourth quarter 1994.
The wells in the former source area and the down
gradient well had intermediate concentrations of De-
halococcoides DNA based on the PCR assay with
gel electrophoresis, and concentrations of Dehalococ-
coides cells ranging from 105 to 107 per liter based on
quantitative real time PCR. Ethylene, vinyl chloride,
and Dehalococcoides DNA were not detected in the
three background wells that had never experienced
the contaminants.
Figure 4.4 compares the location of the monitoring
wells at the Landfill 3 Site at Tinker AFB, Oklahoma,
to the distribution of cis-DCE in 2000. Four of the
wells were originally considered to be in the plume
(wells 2-259B, 83BR, 2-299B, and 2-292B), and well
2-304B was originally considered to be a background
well. Dechlorination was less extensive than at the
Western Processing Site, but the concentrations of
chlorinated ethylenes were reduced to a major extent
along the flow path (Table 4.4). When sampled as
part of this survey, the background well 2-304B had
low concentrations of cis-DCE and vinyl chloride.
Dehalococcoides DNA was detected in all the wells,
including the background well. The estimated cell
density varied from 10s per liter in the most contami-
nated well at the source to 106 per liter in the down
gradient wells (Table 4.4).
Figure 4.5 compares the distribution of wells at the
North Beach Site at the U.S. Coast Guard Support
Center at Elizabeth City, North Carolina, to the distri-
bution of PCE in 1997, before excavation of a portion
of the source. In 1996 and 1997, the concentrations
of PCE in the most contaminated well (GW 3-30 in
Figure 4.5) varied from 2,000 ug/liter to 2,900 ug/liter
(data not shown). When the plume was sampled in
2002 for Dehalococcoides DNA, the concentration of
PCE in well GW 3-30 was 561 ug/liter (Table 4.4).
Dechlorination at the North Beach Site was less ex-
tensive than at the Western Processing Site or the
Landfill 3 Site (Table 4.4). Vinyl chloride was only
detected in one well, and ethylene was not detected
in any of the wells. The well where vinyl chloride
was detected (MW1) was down gradient of the well
in the source area. Dehalococcoides DNA was only
detected in the down gradient well where vinyl chloride
was detected.
24
-------
Table 4.4. Distribution of Chlorinated Ethylenes and Dehalococcoides DNA at Sites that Form Conventional
Plumes
Well
Position
PCE
TCE
c/s-
DCE
Vinyl
Chloride
Ethylene
M9/L
DMA Score*
Cell
Density
cells/L
Western Processing Site 4/29/2003
15T2B
6M6B
15T4B
15M17B
15M39B
15M45B
Source
Source
Down
Gradient
Back-ground
Back-ground
Back-ground
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
100% of positive control =(++++),
Table 4.5 shows the relationship between the appar-
ent rates of dechlorination along the flow path and the
detection of bacterial DNA and Dehalococcoides DNA
in water samples from a monitoring well in the plume.
At sites where Dehalococcoides DNA was detected
in at least one monitoring well (Western Processing,
LF3, and North Beach), the dechlorination rates of
c/s-DCE and vinyl chloride were equal to 0.3 per year,
or were greater than 0.3 per year.
"Generally useful" rates of dechlorination also oc-
curred in a plume where Dehalococcoides DNA was
not detected. In the lower plume at Target Area 1,
the extracted rate of attenuation of vinyl chloride
was 0.3 per year, but Dehalococcoides DNA was not
detected (Table 4.5). Bacterial DNA was detected in
water from this plume even though Dehalcoccoides
DNA was not detected.
The SS-17 Site was oxic, and reductive dechlorina-
tion was not expected. This site was included in the
survey as a control. The extracted rate constant was
very low, 0.01 per year. As would be expected, De-
halococcoides DNA was not detected.
25
-------
200 meters
Sludge Pits
Well Sampled for
Dehalococcoides
Well to Contour
Concentrations
Figure 4.4. Location of monitoring wells and distribution of cis-DCE in the Upper Saturated Zone Aquifer at the
Landfill 3 Site, Tinker AFB, OK, in 2000.
Pasquotank River
Canal
Direction of Ground Water Flow
Monitoring Wells used in the
calculation of rate constants
Monitoring Wells analyzed for
Dehalococcoides DMA and used
in the calculation of rate constants
Monitoring Wells not used in the
calculation of rate constants
© Monitoring Wells analyzed for
Dehalococcoides DMA but not used
in the calculation of rate constants
Figure 4.5. Comparison of the locations of monitoring wells at the North Beach Site at the U.S. Coast Guard Sup-
port Center in Elizabeth City, North Carolina, to the distribution of PCE in ground water.
26
-------
Table 4.5. Relationship between the Apparent Rates of Dechlorination along a Flow Path in the Aquifer and the
Detection of Bacterial DMA and Dehalococcoides DMA in Water Samples from a Monitoring Well in the
Plume
Facility/
Location
Western Processing
Kent, WA
LF3 (landfill)
Tinker AFB, OK
North Beach, USCG
Support Center
Elizabeth City, NC
FTA2 (fire training)
Tinker AFB, OK
SS17Site
Altus AFB, OK
Target Area 1
(Lower Plume)
Dover AFB, DE
Date
1988a
4/2000
4/2003
9/1 997 c
11/2002
1997d
1 0/2002
8/1 997
11/2002
3/2003
7/1 997
PCE
TCE
c/s-
DCE
vc
Rate of Dechlorination
(per year)
0.3
0.1
0.1
1
1
0.1 e
0.3
0.01
0.1
0.6
1
1
1
0.3
0.1
3
3
3
3
1
0.3
Bacterial
DMA
Primer
Not Used
Not Used
Not Used
Not Used
Detected
Trace
Detected
Dehalococcoides
DMA
Primers
Detected
3 of 3
3 of 3
2 of 3
0
0
0
Maximum
Score*
+++ b
++
++++
++++
' maximum score detected by any of the Dehalococcoides primers, 0% of positive control = (-), <3% = (+/-), 4% to 33% = (+), 34%
to 66% = (++), 67% to 100% = (+++), >100% of positive control =(++++).
a data collected before the operation of pump and treat system; b data obtained by Hendrickson et al. (2002, personal communica-
tion); c data collected before the operation of extraction system, except that data near source were from the earliest date available
(August 2001);d data collected in May or December of 1997 before source removal; e rate estimate obtained by Parsons (Tinker
Air Force Base, 1999); ' data obtained by Ei et al. (2002),
27
-------
Dehalococcoides DNA and Dechlorination
Rates over Time in Particular Wells
At the two sites at England Air Force Base, there
was no detectable net direction of ground water flow.
The site is in the floodplain of the Red River, and
the direction of flow varies widely with the seasons.
The direction of flow also varies with the elevation of
water in a body of surface water (Le Tig Bayou) that
lies above the contaminated ground water. The dis-
tribution of contaminant concentrations forms a "bull's
eye" pattern around the two sources of contamination.
Figure 4.6 compares the location of water wells in
two separate plumes to the distribution of c/s-DCE in
March, 2002. Wells at one site (Area 2500) showed
extensive dechlorination, and wells at the other site
(Area 800) showed very limited dechlorination (Table
4.6). However, Dehalococcoides DNA was detected
in both plumes.
Table 4.7 compares the rates of attenuation over time
in individual wells to the concentrations of chlorinated
ethylenes. Two values are entered under "Date Col-
lected" in Table 4.7. The first date is the earliest date
in the data set used to extract rate constants. The
second date is the last date used to extract rate con-
stants and the date that the samples were collected
to assay for Dehalococcoides DNA. In many wells, it
was not possible to calibrate BIOCHLOR and extract
rate constant because the chlorinated ethylene was
absent, or because concentrations increased over
time. When it was possible to extract rate constants,
there was not a consistent relationship between the
concentrations of chlorinated ethylenes in ground wa-
ter and the rates of dechlorination over time that were
extracted by calibrating BIOCHLOR to the monitoring
data (Table 4.7).
Seven wells were sampled for analysis of Dehalococ-
coides DNA (Tables 4.7 and 4.8). There was also
no consistent relationship between the presence of
Dehalococcoides DNA and the trend in concentrations
of chlorinated ethylenes over time (Table 4.8). Well
A39L010PZ at Area 2500 behaved like the wells in
the conventional plumes discussed above. Dehalo-
coccoides DNA was present, and the dechlorination
rates of c/s-DCE and vinyl chloride were both 0.3 per
year. In contrast, Well A39L011PZ at Area 2500
contained Dehalococcoides DNA, but the concentra-
tion of vinyl chloride increased over time rather than
decreased. Well A39L009PZ at Area 800 contained
Dehalococcoides DNA, but the rate of degradation
of TCE and c/s-DCE was very slow, and Well #23 at
Area 2500 contained Dehalococcoides DNA, but the
concentrations of both c/s-DCE and vinyl chloride were
increasing over time.
AREA 800
Well#19 Q
#17
v
Q •
A39L009PZ O
> 50 ng/L
>10ng/L
> 5 ng/L
> 1 ng/L
A39L015PZ
O
500 meters
O
•
Well Sampled for
Dehalococcoides
Well to Contour
Concentrations
A39L011PZ
• AREA 2500
Figure 4.6. Location of monitoring wells and distribution ofcis-DCEin the Intermediate Ground-Water Zone at Spill
Site-4, the former England AFB, Louisiana, March 2002.
28
-------
Table 4.6. Distribution of Chlorinated Ethylenes and Dehalococcoides DMA at Sites at England AFB, which do not
Form Conventional Plumes
Well
PCE
TCE
c/s-DCE
Vinyl
Chloride
Ethylene
M9/L
DMA
Score*
Cell
Density
cells/L
Area 800
SS45L001 MW
A39L009PZ
WELL #17
WELL #19
<1
<1
<1
<1
249
20.8
20.5
<1
126
52.6
30.1
<1
4.46
5.21
3.88
3.85
0.02
<1
<1
<1
-
+ (10%)
-
-
6.7 x105
Area 2500
WELL # 005
A39L011PZ
A39L010PZ
WELL #23
WELL #3
A39L015PZ
<1
<1
<1
<1
1.3
<1
<1
<1
<1
<1
<1
<1
0.58
<1
<1
92
25.6
<1
166
102
95.5
60.6
39.1
0.395
0.014
0.02
0.895
<1
0.007
<1
-
+++ (88%)
++ (37%)
+ (30%)
-
-
4.0 x106
1 .2 x 1 06
4.8 x105
' maximum score detected by any of the Dehalococcoides primers, 0% of positive control = (-), <3% = (+/-), 4% to 33% = (+), 34%
to 66% = (++), 67% to 100% = (+++), >100% of positive control =(++++).
Table 4.7. Relationship between the Concentrations of Chlorinated Ethylenes in Ground Water and Their Apparent
Rates of Dechlorination over Time in Water Samples from Monitoring Wells at England AFB, Louisi-
ana
Location
Area 2500
Area 2500
Area 2500
Area 800
Area 800
Area 800
Area 800
Well
A39L01 1 PZ
A39L010PZ
Well #23
A39L009PZ
Well #17
Well #19
Date
Collected
9/1997
4/2003
6/1997
4/2003
5/2000
4/2003
6/1997
4/2003
6/1997
4/2003
3/1999
4/2003
3/1999
4/2003
TCE
c/s-DCE
VC
Concentration
(M9/L)
<1
<1
<1
<1
<1
<1
35.8
20.8
451
249
9.8
20.5
<1
<1
189
<1
9.5
<1
<1
92
80.3
52.6
4.3
126
<1
30.1
<1
<1
59.2
102
549
95.5
<1
60.6
<1
5.2
0.5
4.5
<1
3.9
<1
3.8
TCE
c/s-DCE
VC
Rate of Dechlorination
(per year)
No
TCE
0.1
0.1
TCEt
No
TCE
1
0.3
c/s-DCE t
0.1
1
c/s-DCE t
No c/s-
DCE
vet
0.3
vet
1
3
vet
vet
29
-------
Table 4.8. Relationship between the Apparent Rates of Dechlorination over Time and the Detection of Bacterial
DMA and Dehalococcoides DMA in Water Samples from Monitoring Wells at England AFB, Louisiana
Plume
Area
2500
Area
2500
Area
2500
Well
A39L01 1 PZ
A39L010PZ
Well #23
Date
Collected
9/1 997
4/2003
6/1 997
4/2003
5/2000
4/2003
TCE
c/s-DCE
VC
Rate of Dechlorination
(per year)
No TCE
1
0.3
c/s-DCE
t
vet
0.3
vet
Bacterial
DMA
Primer
Not Used
Not Used
Not Used
Dehalococcoides
DMA
+++ 3
of 3
++ 3 of 3
+ 2 of 3
Cell
Density
cells/L
4.0 x 106
1.2 x106
4.8 x 105
Area 800
Area 800
Area 800
Area 800
A39L009PZ
Well #17
Well #19
6/1 997
4/2003
6/1 997
4/2003
3/1 999
4/2003
3/1 999
4/2003
0.1
0.1
TCE t
No TCE
0.1
1
c/s-DCE
t
No c/s-
DCE
1
3
vet
vet
Not Used
Detected
Detected
Detected
+ 1 of 3
0
0
0
6.7 x 105
' maximum score detected by any of the Dehalococcoides primers, 0% of positive control = (-), <3% = (+/-), 4% to 33% = (+), 34%
to 66% = (++), 67% to 100% = (+++), >100% of positive control =(++++).
Again, "generally useful" rates of dechlorination also
occurred in some wells where Dehalococcoides DNA
was not detected. In Well SS45L001MW at Area 800,
the dechlorination rates of c/s-DCE and vinyl chloride
were rapid (1 and 3 per year, respectively), but no
Dehalococcoides DNA was detected even though
bacterial DNA was detected. This can be considered
a false negative for the PCR assay. The assay may
have failed to detect Dehalococcoides bacteria that
were present in the aquifer, or the transformation was
carried out by a strain of Dehalococcoides bacteria
that was not recognized by the PCR primer, or the
transformation may have been carried out by other
bacteria all together.
As would be expected, Dehalococcoides DNA was
absent in oxygenated ground water where chlorinated
ethylene concentrations were for the most part below
detection limits (Well #19 at area 800).
The rate of attenuation of concentrations of chlorinated
ethylenes over time in monitoring wells is strongly
influenced by the rate of physical and chemical weath-
ering of the residual contamination in the source area
of the plume (Newell et al., 2002). This residual can
be TCE present as a non-aqueous phase liquid, or
TCE that is sorbed to aquifer material. Apparently, the
concentration of chlorinated ethylenes in the wells at
England AFB was controlled by the rate of dissolu-
tion or desorption of the chlorinated ethylenes from
the source material, and not by biotransformation of
the chlorinated ethylenes dissolved in ground water.
When the concentrations of chlorinated ethylenes are
controlled by dissolution and sorption, a PCR assay
for Dehalococcoides DNA should not be expected to
predict the rate of attenuation in concentration over
time. The PCR assay is not useful to predict the rate
of natural attenuation in the source areas of plumes.
30
-------
Rates of Natural Attenuation and Density of
PCR Products from Dehalococcoides
DNA
This section compares the rates of natural attenu-
ation at field scale to the density of PCR products
from Dehalococcoides DNA in monitoring wells. The
comparison indicates that samples of ground water
from wells do not adequately represent the density of
Dehalococcoides bacteria in aquifers.
For a number of reasons, any association between
Dehalococcoides DNA and rates of natural attenuation
at field scale must be purely empirical. As discussed
in the sections above, Dehalococcoides cells may be
present in the aquifer, but not sampled in the ground
water produced by a monitoring well. Dehalococ-
coides cells may be present in the aquifer, but they
may be dead or inactive. Dehalococcoides DNA for
the 16S rRNA gene may be extracted from stains of
Dehalococcoides that are not capable of biological
transformation of c/s-DCE or vinyl chloride.
To evaluate the effects of any sampling bias caused
by sampling monitoring wells as opposed to sampling
the aquifer sediment, the density of Dehalococcoides
DNA in monitoring wells and rates of attenuation at
field scale were compared to rates in a field study
of active anaerobic bioremediation (Lendvay et al.,
2003) and the rates in a laboratory culture of Deha-
lococcoides (Cupples et al., 2004b). Lendvay et al.
(2003) compared the density of Dehalococcoides
DNA in sediment as measured by the quantitative real
time polymerase chain reaction (q-PCR) to rates of
removal of chlorinated ethylenes during in-situ biore-
mediation of a PCE plume. In a demonstration plot
that was inoculated with a Dehalococcoides-conlaim-
ing culture, the initial concentration of c/s-DCE was
near 970 ug/L. This concentration is higher than the
half saturation constant of 320 ug/L determined for
the Victoria culture of Dehalococcoides (Cupples et
al., 2004a). However, the removal of cis-DCE in the
data presented by Lendvay is roughly first order on
concentration. The plume of PCE was converted to
a plume that was almost entirely c/s-DCE in 8 days,
and then 36% of the c/s-DCE plume was converted to
ethylene in an additional 12 days, 70% was converted
to ethylene in 28 days, and 92% was converted to
ethylene in 35 days. This corresponds to pseudo-
first order rate constants for degradation of c/s-DCE
of 13.6, 15.7, and 26.3 per year, respectively. The
average rate was near 19 per year.
Lendvay et al. (2003) acquired sediment samples
from three depths in the center of demonstration plot
(the injection zone) and from three depths near the
extraction wells at the periphery of the demonstration
plot. Averaged across the six samples, the aquifer
sediment in the plot that was inoculated contained 8 x
105 Dehalococcoides cells per gram wet sediment. If
the sediment contained 0.11 ml of pore water per gram
wet weight (water filled porosity 25%), the density of
Dehalococcoides DNA in the sediment corresponds
to 7 x 109 cells in or exposed to a liter of pore water.
Substrate was supplied to a control plot, but the ground
water was not inoculated with Dehalococcoides organ-
isms. However, the aquifer contained native strains of
Dehalococcoides, and c/s-DCE was degraded to eth-
ylene. The initial concentration of c/s-DCE was near
1,500 ug/L. Transformation of cis-DCE began after a
period of acclimation lasting 87 days. There was 27%
conversion of c/s-DCE to ethylene in the subsequent
27 days and 76% conversion in the subsequent 34
days, corresponding to first order rates of 8.2 and 15.3
per year. The average rate was near 12 per year. The
aquifer sediment in the plot that was not inoculated
contained on average 3.6 x 104 Dehalococcoides cells
per gram which corresponds to 3.2 x 10s cells in or
exposed to a liter of pore water.
The first order rates of attenuation of c/s-DCE are
compared to the density of Dehalococcoides cells in
Figure 4.7. There was a little more than one-order
of magnitude difference in the density of Dehalo-
coccoides cells that achieved a rate of degradation
near 20 per year in the in-situ bioremediation study
of Lendvay et al. (2003). This is reasonably good
agreement between two independent field-scale esti-
mates of the effect of the density of Dehalococcoides
cells on the rate of transformation of c/s-DCE in the
aquifer. This estimate will be compared to the rate of
natural biotransformation at the natural attenuation
sites. It is important to note that the estimate is for
only two pilot-scale plots at one site. We could not
find another study in the literature that compared the
rates of biodegradation of c/s-DCE to the density of
Dehalococcoides cells in the sediment. Until other
studies are reported, there is no benchmark to de-
termine whether the rates achieved by Lendvay et al.
(2003) are typical.
If the rate of biotransformation of c/s-DCE by an indi-
vidual cell of Dehalococcoides is pseudo-first order
on concentration, then the overall rate of transforma-
tion should be proportional to the number of active
cells. Two cells would degrade c/s-DCE twice as fast
as one cell, and so on. The grey shape in Figure 4.7
bounds the area where the rate of biotransformation
is proportional to the cell density in the plot that was
bioaugmented with an active culture of Dehalococ-
31
-------
coides, and the plot that relied on the native strains
of Dehalococcoides. The density of Dehalococcoides
cells in two of the monitoring wells at the MNA sites
fell within the grey shape, indicating that the perfor-
mance of the native Dehalococcoides stains in the
plumes undergoing natural attenuation was roughly
equivalent to the performance of the Dehalococcoides
stains at the active in-situ bioremediation plots. Based
on the concentration of cells in the water from the
monitoring wells, the performance in the other wells
was substantially better than in the bioremediation
demonstration plots. The rates were up to one order
of magnitude faster than would be expected from the
density of Dehalococcoides cells.
The solid square in Figure 4.7 is the first order rate of
transformation of c/s-DCE by a laboratory culture of
Dehalococcoides strain VS (Cupples et al., 2004b).
The culture was growing at 20° C with optimal con-
centrations of molecular hydrogen. The first order rate
of c/s-DCE transformation was calculated by dividing
the maximum rate of transformation of c/s-DCE by the
half saturation constant for c/s-DCE transformation.
The dotted line in Figure 4.7 extrapolates the perfor-
mance of the laboratory culture to cell densities that
were determined by quantitative PCR in monitoring
wells at the study sites. Based on the concentration
of Dehalococcoides cells in ground water from moni-
toring wells, the Dehalococcoides organisms in the
aquifer at the field sites performed as well or better
than the culture growing under optimal conditions in
the laboratory.
It is possible, but unlikely, that the native Dehalococ-
coides organisms at our survey sites were more ef-
ficient than the organisms reported by Lendvay et al.
(2003) and Cupples et al. (2004b). It is possible, but
unlikely, that other organisms were primarily respon-
sible for natural attenuation of chlorinated ethylenes
at our study sites. The most likely explanation is that
the monitoring wells did not efficiently sample the
Dehalococcoides organisms in the aquifer, and that
the number of Dehalococcoides cells recovered in a
liter of well water was a small fraction of the number
of cells that were exposed to a liter of ground water in
the aquifer. Most of the Dehalococcoides cells were
probably attached to sediment particles.
If the relationship between the density of Dehalococ-
coides cells and the rates of c/s-DCE degradation in
10000
1000
100
10
I
e
0.01
• Laboratory Culture Strain VS
• In Situ Bioremediation with Bioaugmentation
A In Situ Bioremediation with Native Organisms
D England AFB
O Western Processing
ATinkerAFBLF-3
O North Beach
1.E+05 1.E+06 1.E+07 1.E+08 1.E+09
Density Dehalococcoides Cells (Cells per Liter)
1.E+10
Figure 4.7. Relationship between the density of Dehalococcoides cells as determined by quantitative PCR and the
first order rate of attenuation of c/s-DCE in ground water. The data points with an open symbol are from
ground water samples collected at natural attenuation sites as presented in Tables 4.3 and 4.6. The
data points with a solid diamond symbol or a solid triangle symbol are from sediment samples from a
site where biological reductive dechlorination was used to clean up a PCE spill (Lendvay et al., 2003).
The data point with a solid square symbol is from a laboratory study ofcis-DCE transformation by De-
halococcoides strain VS growing under optimum conditions (Cupples et al., 2004b).
32
-------
the bioremediation study of Lendvey et al. (2003) is ex-
trapolated to a "generally useful" rate of bioremediation
for natural attenuation of 0.3 per year, then a density
of near or greater than 1 x 107 Dehalococcoides cells
per liter is necessary to produce a useful rate of natu-
ral attenuation (compare Figure 4.7). Dennis (2005)
reported that ethylene as a transformation product
was detected in 78% of ground water samples where
the density of Dehalococcoides cells was greater than
1 x 104 cells per liter but less than 9.9 x 105 cells per
liter. When the density of Dehalococcoides cells var-
ied between 1 x 106 cells per liter and 9.9 x 107 cells
per liter, ethylene was detected in 83% of the wells.
The detection limit for the quantitative real time PCR
assay for Dehalococcoides is near 2 x 103 cells per
liter. The assay can easily detect Dehalococcoides
cells if they are present at densities that cause the
accumulation of ethylene, or that can produce "gener-
ally useful" rates of natural attenuation. At its present
level of development, the PCR assay has adequate
sensitivity. False negative reports from the assay
are most likely caused by a failure of a ground water
sample to adequately represent the true density of
Dehalococcoides cells in the aquifer.
In addition to problems associated with sampling bias
with water from monitoring wells, the interpretation of
the semi-quantitative PCR assay based on gel elec-
trophoresis is further complicated by variability in the
estimate of the amplified DNA. Figure 4.8 compares
the actual density of Dehalococcoides gene copies to
the intensity score reported by the semi-quantitative
test in Tables 4.4 and 4.6. For a given intensity score,
the number of gene copies varied by as much as two
orders of magnitude. When the intensity scores were
greater than (++), there was no observed increase
in the number of Dehalococcoides gene copies with
an increase in the intensity score. When an intensity
score was assigned, the density of Dehalococcoides
gene copies was uniformly greater than 1 x 105 cells
per liter. However, there was no quantitative relation-
ship between the different intensity scores.
1.E+09
1.E+08
8 1.E+07
£ 1.E+06
O
CL
-------
Biotransformation and Dominant Terminal
Electron Accepting Processes
Table 4.9 compares the geochemistry of the ground
water at the studied sites. Based on the concen-
tration of soluble electron acceptors and reduced
metabolic products, the ground water at each site was
classified and assigned to categories based on the
inferred dominant electron accepting process. Most
of the water samples fell into more than one category.
This may reflect spatial heterogeneity in the aquifer
with water from different geochemical environments
contributing to the water sampled from the well. It
may also reflect concurrent geochemical processes
occurring in the aquifer.
The SS-17 site at Altus AFB and the Area 800 site at
the former England AFB were included in the survey
as controls where reductive dechlorination and the
presence of Dehalococcoides DNA were not expected.
With the exception of well A39L009PZ at the Area 800
site, the water at these two sites is oxygenated, and
the concentration of c/s-DCE and vinyl chloride are a
Table 4.9. Comparison of Rates on Attenuation to the Overall Geochemical Environment of the Sites in the
Survey
Site
Well
SS45L001 MW
at Area 800
Well 2-259B at
LF3
Well T4 at West-
ern Processing
Well GM3-30 at
North Beach
Well MW-5 at
North Beach
Well 2-62B
at FTA2 at Tin-
ker AFB
Well MW212D
at Target Area 1
Well DM353D at
Target Area 1
Well
A39L009PZ at
Area 800
Well WL080 at
SS17site,
Altus AFB
Well #23 at
Area 2500
Rate
per year
c/s-DCE
1
c/s-DCE
1
c/s-DCE
0.6
c/s-DCE
0.3
c/s-DCE
0.3
TCE
0.3
c/s-DCE
0.1
c/s-DCE
0.1
c/s-DCE
0.1
TCE
0.01
NONA
Geochemistry3
oxic, iron
reducing
iron reducing
iron reducing and
methanogenic
sulfate reducing
sulfate reducing
nitrate reducing,
sulfate reducing,
methanogenic
nitrate reducing,
methanogenic
no dominant
process
iron reducing,
sulfate reducing
oxic or nitrate
reducing
iron reducing,
methanogenic
02
NO3+NO2-
Fe(ll)
so4-2
CH4
mg/L
3.8
0.2
0.2
0.2
0.3
0.1
0.4
0.7
0.1
0.7
0.1
0.22
0.03
0.04
<0.1
<0.1
5.32
2.17
0.53
0.08
1.58
0.08
2.5
9
7.5
0.5
0.2
-
<0.1
<0.1
7
<0.1
12
35.7
<0.1
<0.1
59.7
5.72
138
0.71
0.36
18.6
1670
<0.1
0.505
3.1
20.7
0.060
0.055
0.938
3.26
0.247
0.19
0.003
3.11
H2
nM
-
-
2.38
<0.4
7.97
7.69
1.19
10.47
6.54
<0.4
2.27
1 oxic: dissolved oxygen was greater than 0.5 mg/L; iron-reducing: iron II was greater than 0.5 mg/L; methanogenic: methane was near
or greater than 1 mg/L; nitrate-reducing: nitrate plus nitrite -N was greater than 0.5 mg/L, and oxygen was not available; sulfate
reducing: oxygen was not available, and the concentration of sulfate was greater than 20 mg/L.
34
-------
small fraction of the concentration of TCE. Water in the
lower plume at Target Area 1 is reducing in wells near
the source (MW212D in Table 4.9), but oxygenated in
a down gradient well (DM353D in Table 4.9). Water in
the other plumes is iron-reducing, sulfate-reducing, or
methanogenic. The rate of attenuation of chlorinated
ethylenes in the ground water plumes in this survey
could not be correlated with a unique dominant ter-
minal electron accepting process (Table 4.9).
Example of Calibration of BIOCHLOR with
Distance along a Flow Path
The plume of PCE at the North Beach Landfill Site at
the U.S. Coast Guard Support Center in Elizabeth City,
North Carolina, will be used to illustrate the process
of extracting rate constants for natural attenuation
along a flow path in the aquifer. Figure 4.5 is a map
showing the location of monitoring wells in the plume.
The plume of contamination is contained in a shallow
confined aquifer extending from depths of six to ten
meters below land surface. The plume is crossed by
a shallow agricultural drainage ditch that does not
seem to communicate with the plume.
Monitoring wells (GM3-30, GP23D, GM6-30, MW1,
MW6, MW10, and MW7) along the centerline of the
plume were used to extract the rate constants (some
of the wells including GM3-30, GP23D, MW1, and
MW6 were sampled for analysis of Dehalococcoides
DNA). To calculate the rate constants, site-specific
estimates of the hydrological parameters of the aquifer
(Wilson et al., 1997) and field data on contaminant
concentrations in 2000 were entered into the BIO-
CHLOR software. Figure 4.9 shows the BIOCHLOR
input screen.
The first order rate constants were estimated by cali-
brating BIOCHLOR to field data following a forward,
trial-and-error process until all the predicted concen-
trations of all the chlorinated ethylenes best matched
their field data.
The output of BIOCHLOR compares the measured
concentrations of chlorinated ethylenes along a flow
path to the expected concentrations along the center-
line of the plume. At a real field site, the distribution
of contaminants in monitoring wells may not match
BIOCHLOR Natural Attenuation Decision Support System
Verstantl
TYPE OF CHLORINATED SOLVENT:
1.ADVECTION
Seepage Velocity'
o
Hydraulic Conductivity
Hydraulic Gradient
Effective Porosity
2. DISPERSION
Alpha x Calc Method
(Alpha y)/ (Alpha x)
(Alpha i) I (Alpha x)
3. ADSORPTION
Retardation Factor"
or
Soil Bulk Density, rho
FractJOnOrganicCarbon. foe
Partition Coefficient
Elnenes
Ethanes
5. GENERAL
Simulation Time*
Modeled Area Width1
Vs
(ity K
j
R
X)
2 (ft)
27.8
t-
2.8E.Q2
0.0007
02
(fi/yr) Modeled A
Zone 1 Le
(cm/Bee) Zone 2 Le
(Hit)
(.) 6. SOURC
Change Alpha x
| SOU*
Elizabeth City North Beach
I historical
Data Input Instructions:
115 1. Enter value directly....or
*• °r 2. Calculate by filling in gray
0.02 cells Press Enter. Ihen
(To restore formulas, hrt "Restore Formulas" button )
• Data used directly tn model.
Test if
Btotransfonmallon
is OccurhrHj
TYPE: Single Planar
, Vertical Plane Source: Determine Source Well
Location and Input Solvent Concentrations
02
(-)
2E-01 l(-)
Calc Method
Source Thickness In Sat. Zone'
(kg
PCE TCE
TCE —> OCE
DCE —> VC
VC ETH_
Zone 2 CHLJ—H^
PCE -» TCE
TCE —» QCE
DCE -» VC
VC —* ETH
ETH —* Ethane
-let Order Dcciiy Corf
M'V?
Wldlh" (ft)
Cone. (mg/Lf
PCE
TCE
DCE
VC
ETH
Common R(
1
1
125
30
1
jaed in mo
(L/kg)
(L/kg)
(L/kg)
(L/kg)
(Ukg)
»l}'-
1.1
1.1
7.8
2.6
1.1
' 1.1 '
(•)
(-)
(-)
("I
(-)
PCE
TCE
DCE
VC
ETH
2.0
1
07
03
0
7. FIELD DATA FOR COMPARISON
PCE Cone- (mg/L)
TCE Cone. (mgrL)
OCE Cone (mart)
VC Cone. {mg/L)
ETH Cone. (rogfL)
Dist- from Source (ft)
View of Plume Looking Down
Observed Cenlerline Cone at Monitoring Walls
t 0.22 O.S11 0167 0.83 0.459
0.105 0.067 0.076 0,015 0.21 0114
0.074 0.086 0.1 0.0128 0.24 0.1 64
0,0308 0.018 0.016 0.003 0.0*2 0.034
I ! I I ! I !
0 126 1752 1782 192 240
6. CHOOSE TYPE OF OUTPUT TO SEE:
Figure 4.9. Input screen to BIOCHLOR with calibration parameters for the North Beach Site.
35
-------
the assumptions of the mathematical model. The
available wells often do not lie along the centerline.
The screened intervals of the monitoring wells may
not match the vertical distribution of the contaminant
in the aquifer. The flow direction of ground water can
vary over time, moving a well into or out of a flow path
from the source. The concentrations of contaminants
in different regions of the source area are usually vari-
able, and different monitoring wells may sample water
that originated from different regions in the source
area. The actual rates of biotransformation probably
vary from one location to the next in an aquifer.
As would be expected, there was scatter in the field
data when concentrations were plotted against dis-
tance along the inferred flow path (Figure 4.10 and
Figure 4.11). A sensitivity analysis revealed that if the
rates of transformation varied by a factor of three, it
was possible to clearly identify a rate that was the best
fit to the data in the calibrations. Calibrations were
examined at rate constants of 0.01 per day, 0.03 per
day, 0.1 per day, 0.3 per day, 1 per day, 3 per day,
and 10 per day. The rate constant producing the best
match to the field data for each chlorinated ethylene
was selected as the best estimate of the rate.
10.000
E 1.000-
o
-£ 0.100-
-------
1.000
0.3 per year
0.001
50
100
150
200
250
300
Distance (feet)
1.000
50 100 150 200
250 300
Distance (feet)
Figure 4.11. Correspondence between the measured values forcis-DCEand vinyl chloride at the North Beach Site in
2002 and the concentrations that were predicted by calibrating BIOCHLOR using three different values
for the first order rate constant for biotransformation. The dotted line was considered the best calibration
to the field data.
37
-------
Calibration started with PCE, then proceeded toTCE,
then to c/s-DCE, and then to vinyl chloride. Then each
calibration was checked again to see if calibration of
the down stream transformation products had affected
the calibration of the parent chlorinated ethylenes.
Figure 4.10 and Figure 4.11 display the correspon-
dence between the measured concentrations and
concentrations predicted by the BIOCHLOR software.
The obtained rate constants for reductive dechlorina-
tion of PCE, TCE, c/s-DCE, and vinyl chloride were
0.1, 1, 0.3, and 1 per year, respectively.
Example of Calibration of BIOCHLOR with
Time in a Single Monitoring Well
To extract rate constants for natural attenuation over
time in a particular well, data from a series of sam-
pling events were used. Data from the first sampling
event were used as the source data. Data from other
sampling events were arranged in an order accord-
ing to their time interval to the first sampling date,
similar to the way data were arranged according to
their distance to the source along a flow path in a
plume. Dummy variables for hydrologic properties
were inserted into BIOCHLOR to extract the rate
constants of dechlorination over time in the particular
well, as opposed to over distance along a flow path.
Well A39L01OPZ at Area 2500 at the former England
AFB in Alexandria, Louisiana was used to illustrate
the process. Figure 4.12 shows the BIOCHLOR input
screen for this well.
The rate constants were estimated by calibrating BIO-
CHLOR to field data from a series of sampling events
following a forward, trial-and-error process until all the
predicted concentrations best matched their field data.
Figure 4.13 displays the correspondence between
the predicted and actual values performed by the
BIOCHLOR software. The obtained rate constants for
reductive dechlorination of c/s-DCE and vinyl chloride
were both about 0.3 per year.
BIOCHLOR Natural Attenuation Decision Support System
England AFB
A39LQ10PZ
TYPE OF CHLORINATED SOLVENT: EttWnMi *
Ethanes
I.ADVECTtON
Seepage Velocity' Vs
or
Hydraulic Conductivity K
Hydraulic Gradient 1
Effective Porosity n
2. DISPERSION
Aloha x- 10 (in
10D.O
1*
2.5E-02
0.0018
0.38
Calt.
Alpha x
(Alpha z) /(Alpha i)- 1 E-9 (.)
3. ADSORPTION
or
Soil Bulk Density, rno I 1.59 1 (kn/L)
FractionOrgmicCarbon. foe 1 2 SE^t I <-)
Partition Coefficient Koc "*
PCE 426 lUfcg)
TCE 130 (LJVq)
DCE 125 lUkg)
VC 30 (LJkal
ETH 302 lUtal
Common R (used in model}' =
.45
14
.13
03
.32
a CO '
("Pi
(cmlaec)
(rW)
(-)
,-,
4. BIOTRANSFORMATION .1« On)« CUuy CoetncUnf
S. SENERAL
Simulation Time"
Modeled Area Width'
Modeled Area Length'
Zone 1 Length'
Zone 2 Length'
«. SOURCE DATA
Source Cpllo
Source Thldinc
VWrJIfl' (ft)
Cone. (mfj/L)-
PCE
TCE
DCE
VC
ETH
is
ss rn Sal. Zone*
Y1
50
C1
0
9.46
54EJ.O
0
30 (yt) ; <•
100 (It) IV ^^>-J>
GOO tit) 1
600 (It)
0 (H) ^am *"
TYPE: ConHnuoua /
Sincjte Planar /
1 '0 I"" ttt-
0 ^//
0 /
0 / :
y/
7. FIELD DATA FOR COMPARISON /_ /
PCE Cone. (mg^L>
TCE Cone. (mcJL)
OCE Cone. (mryL
.0 .0 .i 1.0
9.46 4.52 6.7 2.8
Data Input Instructions:
us 1. Enter value tfcreefly..,.or
* * 2. Calculate by liiling in gray
O.fj2 cells. Press Enter, then
(To restore formulae, hil "Restore Formula&" bunco }
Data used directly in modal
Tesl if
Biolransrormatiom
ts Occurring
Natural Attenuation
Screening Protocol
Vertical Plane Source: Determine Source Well
Locator sraJ Inpwl Solvent Concentrations
View of Plume Looking Down
Observed Cenlerllne Cone, al Moniloring Wells
dryrl
ivCConc. (mgi-L]
IfiTHCcnc. (msrl)
iTime from First Samptinfl (day)
JDatfl Data Callaetad
.0
9.46
54S.O
0
6/1JS7
.0
4.52
605.0
25
.5
6.7
619.0
50
1.0
2.8
4200
175
ana? town 3/1/97
10.0
3.0
380.0
294247
flVMO
1.0
12.0
470.0
338
1.0
1.01
330.0
379
10
1.0
74.0
4BO
.0
55.5
8950
5B6
KKUIOO 3/1«1 3121X2 V3O3
Figure 4.12. Input screen to BIOCHLOR with calibration parameters for the well A39L01OPZat Area 2500 at England
Air Force Base, Alexandria, Louisiana.
38
-------
1000
100
I
8 1
-DCE Prediction
VC Prediction
DCE Field Data
x VC Field Data
0.01
0 200 400 600
Time From First Sampling Date (day)
Figure 4.13. Correspondence between predicted and actual values for the chlorinated ethylenes performed by cali-
brating BIOCHLOR to field data sampled in different events at the well. A39L010PZat England Air Force
Base, Alexandria, Louisiana.
Conclusions
The commercially available assay for Dehalococcoides
DNA had adequate sensitivity to detect concentrations
of Dehalococcoides DNA that could sustain "generally
useful" rates on natural attenuation of c/s-DCE and
vinyl chloride.
The assay is not appropriate in the source area of
plumes where the concentrations of chlorinated sol-
vents are controlled by dissolution and desorption of
chlorinated solvents from residual contamination. The
assay is more appropriate for those areas of a plume
outside the source area where the concentrations of
chlorinated solvents are controlled by biotransforma-
tion of chlorinated solvents already in solution.
The field scale rates of natural attenuation were
faster than would be expected from the density of
Dehalococcoides DNA in ground water samples from
monitoring wells, suggesting that monitoring wells
failed to adequately sample the Dehalococcoides
cells in the aquifer. If assays for Dehalococcoides
DNA are to be used to make quantitative predictions
of the rates of natural attenuation, it will generally be
necessary to acquire and analyze samples of the
aquifer sediment.
39
-------
40
-------
Section 5.
Geochemistry of Ground Water and Occurrence of Dehalococcoides
Synopsis
The Technical Protocol for Evaluating Natural Attenu-
ation of Chlorinated Solvents in Ground Water (Wi-
edemeier et al., 1998) used a number of geochemical
parameters in a scoring system to predict whether
ground water could reasonably be expected to contain
microorganisms that could cause biotransformation of
chlorinated solvents. The scoring system was based
on professional judgment. It was not validated against
any data set that compared selected geochemical
parameters against the presence or absence of mi-
croorganisms that might be capable of transforma-
tion of chlorinated solvents. The scoring system was
criticized by the Committee on Intrinsic Remediation
of the National Research Council (NRC, 2000). The
Committee recommended that the scoring system
should not be used to evaluate prospects for MNA.
Because the Dehalococcoides group is the only known
group of organisms that can grow by carrying out
the reductive dechlorination of DCE or vinyl chloride,
it plays a critical role in the evaluation of monitored
natural attenuation of chlorinated ethylenes in an-
aerobic ground water. To determine the association
between the presence of Dehalococcoides and the
critical biogeochemical parameters that define the
habitat of these organisms, various contaminated sites
across the United States were sampled. This Section
compares the distribution of selected geochemical
parameters to the distribution of Dehalococcoides
DNA in ground water at the sites. A commercially
available polymerase chain reaction (PCR) assay
was used to detect Dehalococcoides organisms in
the water samples. The presence or absence of De-
halococcoides DNA was compared to the values for
selected geochemical parameters.
Not every strain of Dehalococcoides can dechlorinate
every chlorinated ethylene. As discussed in Section
3, many strains cannot dechlorinate the dichloroethyl-
enes or vinyl chloride to ethylene. However, every or-
ganism that has been identified that can dechlorinate
the dichloroethylenes and vinyl chloride to ethylene is
a member of the Dehalococcoides group. An assay
for the presence of Dehalococcoides DNA provides
direct evidence for the presence of a strain of bacteria
that might be capable of completely dechlorinating
chlorinated ethylenes.
Knowledge of the growth of Dehalococcoides species
is derived primarily from studies in the laboratory.
Dehalococcoides are strict anaerobes. To date, they
are known to use only chlorinated organic compounds
as electron acceptors and molecular hydrogen (H2)
as an electron donor. They do not use nitrate, nitrite,
fumarate, ferric iron, sulfate, sulfite, thiosulfate, sulfur,
or oxygen as electron acceptors (Maymo-Gatell et al.,
1997; Heetal., 2003a; Adrian etal., 2000). In a con-
taminated aquifer, multiple terminal electron accepting
processes may occur at the same time. In contami-
nated aquifers, Dehalococcoides organisms may have
to compete with other H2-consuming bacteria for the
shared electron donors (Lee et al., 1998). Obviously,
the growth of Dehalococcoides can be influenced by
the geochemical environment.
Concentrations of nitrate, methane, and the oxidation/
reduction potential were associated with the presence
or absence of Dehalococcoides DNA with a statistical
confidence of 95%. The association of the other geo-
chemical parameters with the presence or absence
of Dehalococcoides DNA was not significant at 95%
confidence. Logistical regression was applied to the
data set to develop a formula (equation 5.1) that uses
the concentrations of nitrate, methane, and the oxida-
tion/reduction potential to calculate the probability that
Dehalococcoides is present in ground water.
The formula has several potential applications. A
calculated probability would be useful to screen wells
at a site in order to decide which wells should be
sampled for a PCR assay. As discussed below, it can
be used to improve the calibration of computer models
of natural attenuation in ground water.
The formula provides a simple and rapid way to
calculate the probability that a specific dechlorinating
organism (Dehalococcoides) is present at the site, as
opposed to the scoring system in the Technical Protocol
(Wiedemeier et al., 1998). However, a calculated
41
-------
probability that an organism exists in ground water is
not equivalent to a PCR assay for its presence, and
probabilities calculated from geochemical parameters
should not be used to replace PCR assays.
Calibration of Computer Models to Evaluate
MNA
Often the computer models that are used to evalu-
ate monitored natural attenuation are distributed
parameter models. In these models, different rates
of biotransformation can be assigned to each cell
of the model, based on the conditions pertaining to
that cell. BIOCHLOR (Aziz et al., 2000), a simple
screening model for monitored natural attenuation of
chlorinated solvents, allows the user to assign two
rates of natural biotransformation, depending on the
local conditions. To properly calibrate these computer
models, it is necessary to know the distribution of the
capacity to transform chlorinated ethylenes through-
out the aquifer. Data from the PCR assay may not
be available from every well at a site. The calculated
probability of Dehalococcoides organisms based on
the geochemistry of ground water in a well could be
used to assign rate constants to cells in a model. If
Dehalococcoides organisms are expected, a rate
constant characteristic of reductive dechlorination
at that site would be assigned. If Dehalococcoides
organisms are not expected, the rate constant for
reductive dechlorination would be set to zero in that
particular region of the aquifer.
Sampling Sites
Samples were collected from fifteen plumes at ten
locations across the United States as shown in Fig-
ure 5.1 and listed in Table 5.1. The contamination
occurred in a variety of geological matrices including
consolidated sandstone and siltstone; unconsolidated
sandy clay; silty to fine sand; fine to course sand;
and sands and gravels. The contamination sources
included industrial landfills, airbase fire training areas,
Western Processing, Kent, WA
FF-87, Newark AFB, OH
Site 35, Vandenberg AFB, CA
^
SS-17,AltusAFB, O
OU-1,AltusAFB, OK
Site 41, Rickenback AFB, OH
Target Area 1,
Dover AFB, DE
Building 79,
USCG Support
Center,
Elizabeth City, NC
\North Beach Landfill,
\JSCG Support
Center,
Elizabeth City, NC
Area 6, Carswell AFB, TX /
LF-3, Tinker AFB, OK
Area 2500, England AFB, LA
Area 800, England AFB, LA
OFTA, Tinker AFB, OK
FTA-2, Tinker AFB, OK
Figure 5.1. Location of contaminated sites used to compare presence or absence of Dehalococcoides DNA to the
geochemistry of the ground water. The dashed lines identify sites under monitored natural attenuation.
The solid lines identify sites under active remediation.
42
-------
Table 5.1. Location of Sites Included in the Survey Comparing the Presence or Absence of Dehalococcoides DMA
to the Geochemistry of the Ground Water
Location
Altus AFB,
Oklahoma
Carswell AFB,
TX
Dover AFB,
DE
Vandenberg
AFB, CA
USCG
Elizabeth City,
NC
England AFB,
LA
Rickenbacker
AFB, OH
Newark AFB,
OH
Tinker AFB,
OK
Kent, WA
Site
SS-17
OU-1
Area 6
Target Area
1
Site 35
Building 79
North
Beach Site
Area 800
Area 2500
Site 41
FF-87
LF-3
FTA-2
OFTA
Western
Processing
Geochemistry3
Treatment
Area
I,S,M
I,S,M
I,S,M
I,S,M
I,S,M
I,S,M
i,s
O, N
I, M
S, M
I,S,M
O,N,S,M
N,S,M
O,N
I,M
Background
Geochemistry
O,N
O,N
O,N
O,N
O,N
O,N
O,N
O,N
O
S,M
I
O
O,N
O,N
I,M
Remedial Technology
Soybean oil, surfactant,
yeast extract and
lactate
Plant mulch permeable
reactive barrier
Iron permeable reactive
barrier
Soybean oil injection
Molasses injection
Iron permeable reactive
barrier
MNA
MNA
MNA
MNA
Soybean oil injection
MNA
MNA
Chemical oxidation
MNA attenuation
No.
Wells
Sampled
6
5
5
8
3
11
6
4
6
4
3
5
2
6
6
O=oxic: dissolved oxygen was greater than 0.5 mg/L; N=nitrate-reducing: nitrate plus nitrite -N was greater than 0.5 mg/L, and
oxygen was not available; l=iron-reducing: iron II was greater than 0.5 mg/L; S=sulfate reducing: oxygen was not available, and the
concentration of sulfate was greater than 20 mg/L; M=methanogenic: methane was near or greater than 1 mg/L
surface impoundments for industrial wastes, and ac-
cidental spills of solvents. At most sites, the major
contaminants wereTCE, c/s-DCE, and vinyl chloride.
In three sites (Target Area 1, FF-87, and North Beach),
PCE was one of the major contaminants.
A variety of remediation technologies were applied at
these sites. An emulsion of soybean oil, surfactant,
yeast extract, and lactate was injected into a barrier
in the source zone at one site. A permeable reactive
barrier composed of shredded tree mulch, cotton gin
compost, and sand was installed across a plume
of TCE in ground water at another site. Permeable
reactive barriers composed of zero-valent iron and
sand were constructed across the plumes at two sites.
Soybean oils were injected into the plume at two sites.
Molasses was injected into a barrier in the source
zone at one site. An oxidizing reagent (a mixture of
potassium permanganate and Fenton's reagent) was
injected into the plume at one site. Seven other sites
were managed through monitored natural attenuation.
At the LF-3 site at Tinker AFB, Oklahoma, ground
water has been extracted from the down gradient
portion of the plume in recent years.
43
-------
Ground Water Sampling, Assay for
Dehalococcoides DNA, and Chemical
Analyses
These procedures and analyses were conducted as
described in Section 4.
Detection of Dehalococcoides DMA
A total of 81 monitoring wells were sampled from the
15 sites. Most of these wells were contaminated with
chlorinated ethylenes at concentrations above their
maximum contaminant levels (MCLs) in drinking water.
In 26 wells, Dehalococcoides DNA was unequivocally
detected. These 26 wells were distributed over 12
sites as shown in Table 5.2.
Table 5.2 compares the concentrations of chlorinated
ethylenes and ethylene to the intensity score of De-
halococcoides DNA in the wells where Dehalococ-
coides DNA was detected. The extent of reductive
dechlorination was generally associated with the
intensity score of Dehalococcoides DNA. That is, in
10 of the 12 wells that had higher intensity scores
of Dehalococcoides DNA (scores of ++ or more),
complete dechlorination to ethylene was observed.
In contrast, in 9 of the 14 wells that had lower inten-
sity scores of Dehalococcoides DNA (scores of +),
dechlorination only proceeded to c/s-DCE or vinyl
chloride. However, it should be noted that complete
dechlorination to ethylene was also observed in four
wells that had lower intensity scores of Dehalococ-
coides DNA (scores of +).
A monitoring well is not an ideal instrument to sample
bacteria in aquifers. In many aquifers, the bacteria
are primarily associated with surfaces; they are not
planktonic. If the water produced by a well has slight
turbidity from silt or clay-sized particles, bacteria as-
sociated with these particles will be sampled with the
ground water. If the screen of the well has a sand
pack and if the well has been properly developed,
turbidity will be low, and bacteria in the aquifer may
not be sampled effectively. Detectable concentra-
tions of bacterial DNA were recovered from 86% of
the wells sampled, and detectable concentrations of
Dehalococcoides DNA were recovered from 32% of
the wells sampled.
In 53 samples, Dehalococcoides DNA was not de-
tected unequivocally and in two ground water samples,
the assay for Dehalococcoides DNA was inconclusive
(intensity of the band in the electrophoresis gel was
less than 3% of the positive control, intensity scores
of +/-). For the 53 negative samples, the control using
a universal bacterial PCR primer was performed to
determine whether bacterial DNA of any type could
be extracted from the water sample. The absence
of bacterial DNA may indicate that sampling of the
biomass from the aquifer was ineffective, as opposed
to indicating that Dehalococcoides was not present in
the aquifer. Most of the samples contained bacterial
DNA; only 11 of the 53 samples of ground water did
not contain enough bacterial DNA to allow the ampli-
fied DNA to be detected in the electrophoresis gel.
Biogeochemistry of Ground Water with
Detectable Dehalococcoides DNA
The geochemistry of the water in the 26 wells where
Dehalococcoides DNA was detected was evaluated
to determine the geochemical environment that was
associated with the presence of Dehalococcoides
DNA. It is important to remember that a monitoring
well can produce ground waters from different por-
tions of an aquifer and may mix and blend ground
waters that have different geochemical properties.
The presence or absence of a particular biogeo-
chemical parameter, such as molecular oxygen, in
well water containing Dehalococcoides DNA does not
necessarily mean that the Dehalococcoides cells in
the aquifer occupied ground water with the average
biogeochemical characteristics of the water produced
from the monitoring well.
Table 5.3 presents data on concentrations of nitrate
plus nitrite, concentrations of methane, and the meter
reading for oxidation/reduction potential for the 26
wells. As will be discussed later in this section, these
were the parameters with the strongest association
with the presence of Dehalococcoides DNA. In gen-
eral, the concentrations of nitrate plus nitrite were low;
most were below the detection limit. Concentrations
of methane were high, usually above 2 mg/L, and the
oxidation/reduction electrode potential was low, usu-
ally less than -50 mV against the Ag/AgCI reference
electrode.
The associations are reasonable based on the current
understanding of biological reductive dechlorination.
Nitrate can serve as a direct inhibitor of reductive
dechlorination (Nelson et al., 2002) and may prevent
the colonization of the aquifer by Dehalococcoides
organisms. More importantly, nitrate may also serve
as a competing terminal electron acceptor, and de-
plete concentrations of H2 to concentrations that are
below the concentration that is necessary to sustain
the growth of Dehalococcoides bacteria (Fennell and
Gossett. 1998; Yang and McCarty, 1998). The primary
cause for the association of the absence of nitrate
with the presence of Dehalococcoides DNA may be
44
-------
Table 5.2 The Intensity Scores of Dehalococcoides DMA and the Concentrations of Chlorinated Ethylenes and
Ethylene in the Wells where Dehalococcoides DMA was Detected
Site
SS-17, Altus
AFB, OK
OU-1, Altus
AFB, OK
Area 6, Carswell
AFB, TX
Target Area 1 ,
Dover AFB, DE
Site 35,
Vandenberg
AFB, CA
Building 79,
USCG Support
Center, Elizabeth
City, NC
Area 800,
England AFB,
LA
Area 2500,
England AFB,
LA
Site 41,
Rickenbacker
AFB, OH
LF-3, Tinker
AFB, OK
Western
Processing,
Kent, WA
North Beach,
USCG Support
Center, Elizabeth
City, NC
Wells
TSMW-5
WL410
PESMP09
WHGLTA071
WHGLTA072
MW236S
MW20
MW13
ML22.5-0
ML22.5-8
A39L009PZ
A39L011PZ
A39L010PZ
Well#23
MW103
MW104
MW105
83BR
2-259B
2-299B
2-292B
2-304B
6M6B
T2
T4
MW1
Dhc DMA
Score
++++
+
+
++++
+
+
++
+
++
+
+
+++
++
+
++
+
+
++++
++
+
+
+
++
++
+
++++
Concentration (ug/L)
PCE
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
1.3
1.2
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
0.5
8.0
0.4
0.4
<0.3
<0.3
<0.3
<0.3
380
TCE
64
41
25
2.4
2.4
<0.4
210
250
1300
36
21
<0.4
<0.4
<0.4
19
180
<0.4
270
28
230
220
0.5
<0.4
<0.4
<0.4
81
c/s-DCE
250
15
227
1.7
17
<0.2
140
13
22
41
53
<0.2
<0.2
92
5400
1700
3.0
13
28000
0.8
1.0
0.5
<0.2
<0.2
0.3
79
vc
1300
<1.0
1.7
1.0
1.2
11
170
<0.2
5.8
20
5.2
100
96
61
940
250
10
4.0
20000
<0.2
0.7
<0.2
<0.2
<0.2
1.6
12
Ethylene
91
<1.5
<1.5
58
36
<1.5
2.9
<1.5
9.0
na
<1.5
20
90
<1.5
41000
1500
1700
<1.5
5.3
<1.5
<1.5
<1.5
3.0
8.0
6.0
<1.5
na: not analyzed
" maximum score detected by any of the Dehalococcoides primers. 0% of positive control = (-), <3% = (+/-), 4% to 33% = (+), 34%
to 66% = (++), 67% to 100% = (+++), >100% of positive control =(++++).
45
-------
Table 5.3. The Concentrations of Nitrate plus Nitrite Nitrogen, Methane, and the ORP Meter Reading in the Wells
where Dehalococcoides DNA was Detected
Site/Location
SS-17, Altus AFB, OK
OU-1, AltusAFB, OK
Area 6, Carswell AFB, TX
Target Area 1 , Dover AFB, DE
Site 35, Vandenberg AFB, CA
Building 79, USCG Support
Center, Elizabeth City, NC
Area 800, England AFB, LA
Area 2500,
England AFB, LA
Site 41 , Rickenbacker AFB,
OH
LF-3, Tinker AFB, OK
Western Processing, Kent, WA
North Beach, USCG Support
Center, Elizabeth City, NC
Well
TSMW-5
WL410
PESMP 09
WHGLTA071
WHGLTA072
MW236S
MW20
MW13
ML22.5-0
ML22.5-8
A39L009PZ
A39L01 1 PZ
A39L010PZ
WELL #23
MW103
MW104
MW105
83BR
2-259B
2-299B
2-292B
2-304B
6M6B
T2
T4
MW1
Dhc DNA
Score*
++++
+
+
++++
+
+
++
+
++
+
+
+++
++
+
+
+
+
++++
++
+
+
+
++
++
+
++++
NO3+NO2-N
(mg/L)
<0.1
<0.1
<0.1
<0.1
<0.1
1.3
0.4
8.6
0.1
<0.1
<0.1
0.1
<0.1
<0.1
<0.1
<0.1
<0.1
0.2
<0.1
2.6
2.9
0.2
<0.1
<0.1
<0.1
0.1
CH4
(mg/L)
1.7
0.7
4.3
4.5
3.6
0.001
4.5
8.3
1.7
na
0.2
2.0
3.8
3.1
1.2
0.019
0.97
0.03
3.1
<0.001
<0.001
<0.001
13
19
21
0.1
Meter ORP
(mV)
-210
-82
-52
-180
-200
270
-150
-61
-19
-64
-77
-94
-140
-110
na
na
na
150
-160
330
210
280
180
-98
-100
na
na: not analyzed
" maximum score detected by any of the
to 66% = (++), 67% to 100% = (+++),
Dehalococcoides primers. 0% of positive control = (-),
>100% of positive control = (++++).
<3% = (+/-), 4% to 33% = (+), 34%
46
-------
an indirect association with dissolved oxygen. The
absence of nitrate or nitrite is generally associated
with the absence of dissolved oxygen. The presence
of Dehalococcoides DNA in oxygenated ground water
is not expected based on laboratory studies that show
that Dehalococcoides organisms are strict anaerobes
(Maymo-Gatell et al., 1997; He et al., 2003a, He et
al., 2003b).
The accumulation of methane would be expected
in anoxic ground water with high concentrations of
dissolved hydrogen. The accumulation of methane
would suggest that supplies of dissolved hydrogen
are also adequate to allow proliferation of Dehalococ-
coides organisms. The low electrode potentials (ORP)
reflect reducing conditions that would favor growth of
strict anaerobic bacteria such as Dehalococcoides
organisms.
Data on the association of Dehalococcoides DNA
and concentrations of oxygen, iron (II), and sulfate
are presented in Table 5.4. As mentioned above, the
presence of Dehalococcoides DNA is not expected
in oxic conditions. In general, ground water that con-
tained Dehalococcoides DNA had low concentrations
of dissolved oxygen. The four oxic wells that contained
Dehalococcoides DNA (83BR, 2-299B, 2-292B, and
2-304B) were all located in site LF-3 at Tinker AFB.
For several years, this plume has been managed by
extracting ground water from the down gradient portion
of the plume. Monitoring wells 83BR, 2-299B, 2-292B,
and 2-304B were located adjacent to the pumped
wells. The source area of the plume (represented by
well 2-259B) was strongly anaerobic and had a high
intensity score of Dehalococcoides DNA. The influ-
ence of the pumped wells may have mixed oxygenated
ground water with the anaerobic plume that contained
Dehalococcoides DNA. The PCR assay can detect
DNA from non-viable Dehalococcoides cells.
At three sites, Dehalococcoides DNA was not detected
in any of the wells that were sampled (site FF-87 in
Newark AFB, Ohio, and sites FTA-2 and OFTA in
Tinker AFB, Oklahoma). At these three sites, the
dissolved oxygen level was generally high (data not
shown) with the median concentrations ranging from
0.8 mg/L to 7.7 mg/L.
In most cases, the presence of Dehalococcoides
DNA was associated with detectable concentrations
of iron (II) (Table 5.4). The scoring system presented
in the Technical Protocol for Evaluating Natural At-
tenuation of Chlorinated Solvents in Ground Water
(Wiedemeier et al., 1998) suggested that reductive
dechlorination should not be expected in ground water
with sulfate concentrations above 20 mg/L. In this
survey, Dehalococcoides DNA was found in several
ground waters with concentrations of sulfate above
100 mg/L. In this survey, there is no evidence that
sulfate inhibits growth of Dehalococcoides organisms
or prevents reductive dechlorination in contaminated
aquifers (Table 5.4).
Biological reductive dechlorination requires a source
of reducing power either as dissolved hydrogen or as
an organic substrate. Table 5.5 compares the pres-
ence of Dehalococcoides DNA to the concentrations
of dissolved hydrogen and total organic carbon (TOC).
Yang and McCarty (1998) found that a mixed culture
growing on benzoate with c/s-DCE available as an
electron acceptor (at 28° C) poised the hydrogen
concentration at 2 nanomolar (nM). They interpreted
this concentration as the minimum concentration of
hydrogen that would support utilization by organisms
carrying out reductive dechlorination. Fennell and
Gossett (1998) reported that the lowest hydrogen con-
centration that would support dechlorination (at 35° C)
was as low as 1.5 nM. Following their approach, the
Technical Protocol for Evaluating Natural Attenuation
of Chlorinated Solvents in Ground Water (Wiedemeier
et al., 1998) predicts that reductive dechlorination of
solvents will occur if the concentration of hydrogen ex-
ceeds 1 nM. The concentration of dissolved hydrogen
was measured in 18 of the 26 wells that contained
detectable concentrations of Dehalococcoides DNA.
Of the 18 wells, 14 had dissolved hydrogen equal to
or greater than 1.0 nM (Table 5.5).
The Technical Protocol for Evaluating Natural
Attenuation of Chlorinated Solvents in Ground
Water (Wiedemeier et al., 1998) suggests that
biological reductive dechlorination is favored
when concentrations of TOC are greater than
20 mg/L. Detectable concentrations of Dehalococcoi-
des DNA were found in ground water with much lower
concentrations of TOC, in the range of 1 to 2 mg/L.
In Table 5.5, wells were assigned to redox conditions
based on the concentrations of methane, sulfate,
ferrous iron, and oxygen as follows: conditions were
considered to be methanogenic when methane was
greater than 1.0 mg/L; sulfate and iron reducing when
methane was less than 1.0 mg/L, sulfate was greater
than 20 mg/L, and ferrous iron was detected; and oxic
when dissolved oxygen was greater than 1.0 mg/L,
and ferrous iron and methane were not detected.
This assignment was done to facilitate comparisons
between general redox conditions and the presence
or absence of Dehalococcoides DNA. Of the 26 wells
where Dehalococcoides DNA was detected, 17 had
47
-------
Table 5.4. The Concentrations of Oxygen, Ferrous Iron, and Sulfate in the Wells where Dehalococcoides DMA was
Detected
Site/Location
SS-17, AltusAFB, OK
OU-1, AltusAFB, OK
Area 6, Carswell AFB, TX
Target Area 1 , Dover AFB, DE
Site 35, Vandenberg AFB, CA
Building 79, USCG Support
Center, Elizabeth City, NC
Area 800, England AFB, LA
Area 2500,
England AFB, LA
Site 41 , Rickenbacker AFB,
OH
LF-3, Tinker AFB, OK
Western Processing, Kent, WA
North Beach, USCG Support
Center, Elizabeth City, NC
Well
TSMW-5
WL410
PESMP 09
WHGLTA071
WHGLTA072
MW236S
MW20
MW13
ML22.5-0
ML22.5-8
A39L009PZ
A39L011PZ
A39L010PZ
WELL #23
MW103
MW 104
MW105
83BR
2-259B
2-299B
2-292B
2-304B
6M6B
T2
T4
MW1
Dhc DMA
Score*
++++
+
+
++++
+
+
++
+
++
+
+
+++
++
+
+
+
+
++++
++
+
+
+
++
++
+
++++
(mg/L)
0.2
0.2
0.3
0.1
0.1
2.3
<0.1
0.1
0.2
0.1
0.1
0.5
0.2
0.1
na
na
na
1.5
0.2
2.9
4.9
3.8
0.5
0.1
0.2
0.1
Fe(ll)
(mg/L)
3.0
0.2
0.7
1.5
0.4
0.8
30
0.3
3.0
<0.1
7.0
14
16
12
na
na
na
<0.1
9.0
<0.1
<0.1
<0.1
0.5
2.3
7.5
0.9
SO 2
(mg/L)
880
520
1100
<5
<5
56
23
740
280
21
19
<5
<5
<5
470
110
260
42
<5
64
52
21
<5
<5
<5
77
na: not analyzed
" maximum score detected by any of the
to 66% = (++), 67% to 100% = (+++),
Dehalococcoides primers. 0% of positive control = (-),
>100% of positive control = (++++).
<3% = (+/-), 4%to33% = (+), 34%
48
-------
Table 5.5. The Concentration of Dissolved Molecular Hydrogen and Total Organic Carbon, and the Oxidation/Re-
duction Potential in the Wells where Dehalococcoides DMA was Detected
Site/Location
SS-17, Altus AFB, OK
OU-1, AltusAFB, OK
Area 6, Carswell AFB, TX
Target Area 1 , Dover AFB,
DE
Site 35, Vandenberg AFB,
CA
Building 79, USCG Sup-
port Center, Elizabeth City,
NC
Area 800,
England AFB, LA
Area 2500,
England AFB, LA
Site 41 , Rickenbacker
AFB, OH
LF-3, Tinker AFB, OK
Western Processing, Kent,
WA
North Beach, USCG Sup-
port Center, Elizabeth City,
NC
Well
TSMW-5
WL410
PESMP 09
WHGLTA071
WHGLTA072
MW236S
MW20
MW13
ML22.5-0
ML22.5-8
A39L009PZ
A39L011PZ
A39L010PZ
WELL #23
MW 103
MW 104
MW105
83BR
2-259B
2-299B
2-292B
2-304B
6M6B
T2
T4
MW1
Dhc DMA
Score*
++++
+
+
++++
+
+
++
+
++
+
+
+++
++
+
+
+
+
++++
++
+
+
+
++
++
+
++++
Redox Condition
Methanogenic
Sulfate and iron
reducing
Methanogenic
Methanogenic
Methanogenic
Oxic
Methanogenic
Methanogenic
Methanogenic
Sulfate and iron
reducing
Methanogenic
Methanogenic
Methanogenic
Methanogenic
Methanogenic
Methanogenic
Sulfate and iron
reducing
Methanogenic
Oxic
Oxic
Oxic
Methanogenic
Methanogenic
Methanogenic
Sulfate and iron
reducing
\
(nM)
4.6
1.0
1.0
>10
0.4
6.7
>10
>10
3.7
0.9
6.5
>10
0.8
2.3
na
na
na
na
na
na
na
na
6.4
2.0
2.4
0.2
TOC
(mg/L)
8.7
9.9
15
0.8
9.6
4.6
910
4.8
2.1
5.6
1.1
1.9
2.1
1.4
na
na
na
8.8
92
<0.5
<0.5
<0.5
13
21
16
na
na: not analyzed
" maximum score detected by any of the Dehalococcoides primers. 0% of positive control = (-), <3% = (+/-), 4% to 33% = (+), 34%
to 66% = (++), 67% to 100% = (+++), >100% of positive control =(++++),
49
-------
methanogenic conditions, 4 had sulfate and iron re-
ducing conditions, and 4 had oxic conditions. There
was a strong association between a high intensity
score for Dehalococcoides DNA and low redox po-
tential. With two exceptions, all the wells with higher
intensity scores of Dehalococcoides DNA (scores of
++ or more) had methanogenic conditions.
In general, the geochemical habitat of Dehalococ-
coides, as defined by the parameters measured, was
wide. In the 26 wells where Dehalococcoides DNA
was detected, the pH ranged from 4.7 to 7.4, the
temperature ranged from 13.4° C to 33.4° C, the con-
centration of BTEX ranged from not detected to more
than 800 mg/L, the alkalinity ranged from 60 mg/L to
680 mg/L, the electrical conductivity was in the range
of 200 us/cm to 5430 us/cm, and the concentration of
chloride was in the range of 5 to 1270 mg/L.
Comparison of Geochemistry where
Dehalococcoides DNA is Present or
Absent
The geochemical parameters discovered as significant
by the multiple testing were used as the explanatory
variables in a statistical model using logistic regres-
sion. The prediction accuracy of the model was
evaluated by comparing the observed response and
the predicted probability for the presence of Dehalo-
coccoides DNA.
To determine whether a particular biogeochemical
parameter could predict the presence or absence
of Dehalococcoides DNA in ground water, the dis-
tribution of values for that parameter in water where
Dehalococcoides DNA was detected was compared
to the distribution in water where Dehalococcoides
DNA was not detected. The samples that did not have
enough DNA to be amplified by the PCR assay and
the samples with inconclusive test results for Dehalo-
coccoides DNA were excluded from the comparison.
A two-sample Kolmogorov-Smirnov test was used to
compare the data distributions for each geochemical
parameter.
Fourteen geochemical parameters were compared.
To deal with the problem of multiple comparisons, the
False Discovery Rate (FDR) developed by Benjamin!
and Hochberg (1995) was used to control the propor-
tion of significant results that were in fact type I errors
(false positive). The two-sample Kolmogorov-Smirnov
test was performed for each biogeochemical param-
eter to generate a probability (p) that there was no
difference between the distribution of the parameter
in wells where Dehalococcoides DNA was detected
and the distribution in wells where Dehalococcoides
DNA was not detected. This is the probability of a false
positive, the probability that the distributions appear to
be different when in fact they are not different.
A False Discovery Rate 0 of 0.05 (that is, in every
20 significant results for different parameters, the ex-
pected number of false positives is one) was used in
this study. The fourteen separate p-values were sorted
in ascending order p(1), p(2), ...p(14). All geochemical
parameters with p-values p< p(j) were rejected where
j was the largest index for which
0.05*7
(J) ~ 14
Table 5.6 shows the probability (p) that the distribu-
tions of each biogeochemical parameter are not differ-
ent between water with and water without Dehalococ-
coides DNA as predicted by the Kolmogorov-Smirnov
test. Table 5.6 also shows the FDR threshold for the
values of p. To be protected from false positives, the
value of p must be less than the FDR threshold. When
the FDR threshold was set at a probability 0 of 0.05,
significant test results were obtained for three of the
biogeochemical parameters presented in Table 5.5.
The distribution of the values of the concentration of
NO3"1 plus NO2"1-N, of ORP, and of the concentration
of CH4was significantly different between the samples
of ground water where Dehalococcoides DNA was
detected and samples where Dehalococcoides DNA
was not detected. As for the other parameters, (O ,
H2, Fe (II), SO;2, TOC, CM, BTEX compounds, alkalin-
ity, electrical conductivity, pH and temperature) there
were no significant differences in the distribution of
the values of the parameters at the specified FDR
controlling level (Table 5.6).
A series of Chi-square tests were performed on each
of these three parameters. It was found that the
proportion of NO3~1 plus NO2~1-N below 0.5 mg/L, the
proportion of ORP below 0 mV, and the proportion of
CH4 over 0.5 mg/L were significantly different between
the water samples where Dehalococcoides DNA was
either present or absent at 95% confidence level.
The two-sample Kolmogorov-Smirnov test was per-
formed using SPSS software. The Chi-square test was
performed using the Data Analysis Tool in Microsoft
Excel. The logistic regression was performed using
SAS software.
50
-------
Table 5.6. The Probability (p) that the Distribution of the Measured Values for Selected Geochemical Parameters
between Ground Water where Dehalococcoides DNA was Present and Ground Water where Dehalo-
coccoides DNA was not Present is not Statistically Different. To be Accepted as Statistically Significant,
the Value of ( p) for a Parameter must be Less than the Value for the Threshold for the False Discover
Rate (FDR)
Parameter
Sample Size
(No. Wells where
Dehalococcoides DNA)
Detected
Not
Detected
Z value a
p-value
(2-tailed)
Index
Number j
Threshold
for FDR b
Parameters found to be statistically significant at 90% confidence or greater
NCV+NCV1 -N
ORP
CHA
26
20
25
39
30
40
1.874
1.848
1.687
0.002
0.002
0.007
1
2
3
0.004
0.007
0.011
Parameters not found to be statistically significant at 90% confidence
Fe(ll)
TOC
SO/2
0,
Alkalinity
Temperature.
BTEX
PH
Electrical Conductivity
CM
23
22
26
23
15
17
19
23
17
18
39
32
42
40
18
24
26
37
24
32
1.544
1.457
1.196
1.080
1.017
0.943
0.919
0.761
0.704
0.636
0.017
0.029
0.114
0.194
0.252
0.336
0.367
0.608
0.705
0.813
4
5
6
7
8
9
10
11
12
13
0.014
0.018
0.021
0.025
0.029
0.032
0.036
0.039
0.043
0.046
J Z value in the Kolmogorov-Smirnov test,
' Threshold for False Discovery Rate, defined as ~^, where j is the index number of each parameter, and n is the total number of
parameters compared. In this study, the 0 was 0,05, and n was 14,
A Predictive Model for the Presence of
Dehalococcoides DMA
It would be useful to have a predictive model for the
presence of Dehalococcoides DNA based on the
three simple geochemical parameters (methane,
nitrate plus nitrite nitrogen, and oxidation reduction
potential) that were found to be statistically correlated
with the presence or absence of Dehalococcoides
DNA. For this purpose, a logistic regression with
a binary response for the presence or absence of
Dehalococcoides DNA was used to model the prob-
ability of the presence of Dehalococcoides DNA as a
function of the biogeochemical parameters expressed
as continuous variables. In the model, the response
variable (Y) had two values: Y=1 for the presence of
Dehalococcoides DNA and Y=0 for the absence of
Dehalococcoides DNA. The three biogeochemical
parameters (ORP, CH4 and NO3'1 plus NO2'1-N) were
the explanatory variables. The model was as follows
(Equation 5.1):
logit (p) = log
l-p
-0.1370 - 0.0050 x ORP + 0.1328 x CHt - 0.0468 x NO;1 plus NO;1 - N
where p is the probability of the presence of Dehalo-
coccoides DNA, ORP\s the value of oxidation reduc-
tion potential against a silver/silver chloride reference
electrode in millivolts, CH4 is the concentration of
methane in mg/L, and NO?1 plus NO21-N is the con-
centration of nitrate plus nitrite nitrogen in mg/L.
A total of 45 cases (wells) from ten sites were used
to develop the model. Of the 45 cases, 20 had re-
sponse value of 1, and 25 had response value of 0.
The generalized coefficient of determination (R2) of
the model was 0.2497, and the Max-rescaled R2 was
0.3343. The parameter estimates indicated that the
probability for the presence of Dehalococcoides DNA
increased with the increase of CH4 or the decrease of
ORP or NO -1 plus NO ~1-N. Table 5.7 compares the
51
-------
geochemical data from the wells used to develop the
model to the presence or absence of Dehalococcoides
DNA. Table 5.8 compares the presence or absence of
Dehalococcoides DNA to the predicted probability for
the presence of Dehalococcoides DNA and the lower
and upper 95% confidence limits for the predicted
probability. Wells are listed in Table 5.8 from the well
with the highest probability that Dehalococcoides DNA
is present to the well with the lowest probability that
Dehalococcoides DNA is present.
The model accurately predicted the observed pres-
ence or absence of Dehalococcoides DNA in most
of the wells. In 15 of the 20 wells that had observed
response of 1, the predicted probability for the pres-
ence of Dehalococcoides DNA was greater than 0.50.
In 23 of the 25 wells that had observed response of
0, the predicted probability for the presence of Deha-
lococcoides DNA was less than 0.50.
There were seven "outlier" wells where the predicted
probabilities for the presence of Dehalococcoides DNA
were greatly different from the observed responses.
They were well PESMP07 at site OU-1 on Altus AFB,
OK; well MW236S at the Target Area 1 site at Dover
AFB, DE; well 15M45B at the Western Processing
site in Kent, WA; and wells 83BR, 2-292B, 2-299B,
and 2-304B at the LF-3 site at Tinker AFB, OK. The
behavior of the wells at the LF-3 site at Tinker AFB,
OK, may be a result of the ground water extraction
system, which may have blended anaerobic water
from the plume bearing the Dehalococcoides DNA
with clean aerobic ground water. The discrepancy of
the other sites cannot be explained.
It is possible that the high concentration of methane,
low concentrations of nitrate, and low values for redox
potential are better predictors of biological reductive
dechlorination than the presence or absence of De-
halococcoides DNA. However, we did not attempt a
statistical comparison of the achieved rates of attenu-
ation at field scale to the values of the geochemical
parameters. Fitted rates of attenuation of c/s-DCE
and vinyl chloride were available from only three
sites (Table 4.5). These are too few data for a robust
comparison.
Summary and Conclusions
For the evaluation of natural attenuation of chlorinated
solvents, particularly in an anoxic aquifer, it's important
to know if Dehalococcoides organisms are present.
The presence of Dehalococcoides DNA may be used
to directly demonstrate the occurrence of biotransfor-
mation at the site. The predictive model established
in this report provides a simple and rapid way to es-
timate the presence of Dehalococcoides organisms
based on geochemical condition. This is very useful
considering the cost associated with the analysis of
Dehalococcoides DNA.
The predictive model can be used to screen samples
to be submitted for the biochemical assay. Fennell and
her coworkers discovered that Dehalococcoides or-
ganisms are heterogeneously distributed in the aquifer
(Fennell etal., 2001). To obtain useful DNA data from
the field, it is a good practice to calculate the prob-
ability that Dehalococcoides is present based on the
more easily available geochemical information. The
water samples that most likely have Dehalococcoides
can be submitted to the biochemical assay.
It should be noted that the predictive model is not
intended to replace the biochemical assay. A calcu-
lated probability that Dehalococcoides exists in ground
water is not equivalent to a direct biochemical assay
for its presence. However, under conditions where
data from a direct biochemical assay are not avail-
able, a calculated probability could be used to properly
calibrate computer models of natural attenuation. In
particular, a rate constant for biotransformation of
lower chlorinated ethylenes such as c/s-DCE and vinyl
chloride is not appropriate to be applied to regions of
the aquifer where the geochemical parameters sug-
gest that Dehalococcoides organisms should not be
expected.
Any well that blends ground water from different geo-
chemical environments will produce confusing data.
The predictions based on Equation 5.1 will work best
from monitoring wells with relatively short screens.
The short screens minimize the blending of ground
water from different geochemical environments. The
predictions of Equation 5.1 should not be applied to
pumped treatment wells if the wells produce water that
is obviously not in geochemical equilibrium.
52
-------
Table 5.7. Comparisons between the Observed Presence or Absence of Dehalococcoides DMA and the Concen-
trations of Nitrate plus Nitrite Nitrogen, Methane, and the ORP Meter Reading in the Wells
Site/Location
Western Processing, Kent, WA
Western Processing, Kent, WA
Western Processing, Kent, WA
OU-1, AltusAFB, OK
Area 6, Carswell AFB, TX
Area 6, Carswell AFB, TX
Site 35, Vandenberg AFB, CA
SS-17, AltusAFB, OK
LF-3, Tinker AFB, OK
Area 2500, England AFB, LA
Site 35, Vandenberg AFB, CA
Area 2500, England AFB, LA
OU-1, AltusAFB, OK
Western Processing, Kent, WA
Area 2500, England AFB, LA
SS-17, AltusAFB, OK
Area 800, England AFB, LA
Area 800, England AFB, LA
Western Processing, Kent, WA
Target Area 1 , Dover AFB, DE
Area 6, Carswell AFB, TX
Area 800, England AFB, LA
Western Processing, Kent, WA
Area 6, Carswell AFB, TX
Area 800, England AFB, LA
FTA-2, Tinker AFB, OK
OU-1, AltusAFB, OK
OU-1, AltusAFB, OK
LF-3, Tinker AFB, OK
FTA-2, Tinker AFB, OK
OFTA, Tinker AFB, OK
OFTA, Tinker AFB, OK
OFTA, Tinker AFB, OK
LF-3, Tinker AFB, OK
SS-17, AltusAFB, OK
SS-17, AltusAFB, OK
Target Area 1 , Dover AFB, DE
SS-17, AltusAFB, OK
Target Area 1 , Dover AFB, DE
LF-3, Tinker AFB, OK
OFTA, Tinker AFB, OK
Target Area 1 , Dover AFB, DE
OFTA, Tinker AFB, OK
SS-17, AltusAFB, OK
LF-3, Tinker AFB, OK
Well
T4
T2
15M45B
PESMP 07
WHGLTA071
WHGLTA072
MW20
TSMW-5
2-259B
A39L010PZ
MW13
WELL #23
PESMP 09
6M6B
A39L011PZ
WL410
A39L009PZ
WELL #19
15M39B
MW236D
WHGLFE002
WELL #17
15M17B
LF04-4E
SS45L001 MW
2-62B
WL019
WL250
83BR
2-393B
2-440B
2-1 44B
2-394B
2-292B
WL082
WL094
DM353D
WL090
MW236S
2-304B
2-1 43B
MW101S
2-329B
WL080
2-299B
Observed
Response
present
present
absent
absent
present
present
present
present
present
present
present
present
present
present
present
present
present
absent
absent
absent
absent
absent
absent
absent
absent
absent
absent
absent
present
absent
absent
absent
absent
present
absent
absent
absent
absent
present
present
absent
absent
absent
absent
present
Nitrate+ Nitrite
N (mg/L)
0.04
0.04
0.04
0.07
0.04
0.01
0.35
0.07
0.03
0.07
8.6
0.08
0.04
0.04
0.12
0.05
0.08
0.8
0.03
0.04
1.2
0.1
0.04
0.23
0.22
5.3
1.6
0.7
0.2
0.5
3.10
0.61
2.0
2.9
2.0
2.6
0.5
3.7
1.3
0.2
3.4
1.1
7.3
1.6
2.6
CH4
(mg/L)
21
19
12
4.6
4.5
3.6
4.5
1.7
3.1
3.8
8.3
3.1
4.3
13
2.0
0.7
0.2
0.002
1.8
0.2
0.3
0.02
2.1
2.4
0.5
0.9
0.09
0.005
0.03
<0.001
<0.001
0.9
0.06
0
0
0.001
0.3
<0.001
0.001
<0.001
<0.001
0.003
0.007
0.003
<0.001
ORP
(mV)
-100
-98
-93
-200
-180
-196
-150
-210
-160
-140
-61
-110
-52
180
-94
-82
-77
-24
73
51
54
82
170
180
150
110
130
140
150
170
150
220
210
210
230
230
270
240
270
280
260
290
240
330
330
' LCL: lower 95% confidence limit for the predicted probability;b UCL: upper 95% confidence limit for the predicted probability.
53
-------
Table 5.8. Comparisons between the Observed Presence or Absence of Dehalococcoides DMA and the Predicted
Probabilities for the Presence of Dehalococcoides DMA
Site/Location
Western Processing, Kent, WA
Western Processing, Kent, WA
Western Processing, Kent, WA
OU-1, AltusAFB, OK
Area 6, Carswell AFB, TX
Area 6, Carswell AFB, TX
Site 35, Vandenberg AFB, CA
SS-17, AltusAFB, OK
LF-3, Tinker AFB, OK
Area 2500, England AFB, LA
Site 35, Vandenberg AFB, CA
Area 2500, England AFB, LA
OU-1, AltusAFB, OK
Western Processing, Kent, WA
Area 2500, England AFB, LA
SS-17, AltusAFB, OK
Area 800, England AFB, LA
Area 800, England AFB, LA
Western Processing, Kent, WA
Target Area 1 , Dover AFB, DE
Area 6, Carswell AFB, TX
Area 800, England AFB, LA
Western Processing, Kent, WA
Area 6, Carswell AFB, TX
Area 800, England AFB, LA
FTA-2, Tinker AFB, OK
OU-1, AltusAFB, OK
OU-1, AltusAFB, OK
LF-3, Tinker AFB, OK
FTA-2, Tinker AFB, OK
OFTA, Tinker AFB, OK
OFTA, Tinker AFB, OK
OFTA, Tinker AFB, OK
LF-3, Tinker AFB, OK
SS-17, AltusAFB, OK
SS-17, AltusAFB, OK
Target Area 1 , Dover AFB, DE
SS-17, AltusAFB, OK
Target Area 1 , Dover AFB, DE
LF-3, Tinker AFB, OK
OFTA, Tinker AFB, OK
Target Area 1 , Dover AFB, DE
OFTA, Tinker AFB, OK
SS-17, AltusAFB, OK
LF-3, Tinker AFB, OK
Well
T4
T2
15M45B
PESMP 07
WHGLTA071
WHGLTA072
MW20
TSMW-5
2-259B
A39L010PZ
MW13
WELL #23
PESMP 09
6M6B
A39L011PZ
WL410
A39L009PZ
WELL #19
15M39B
MW236D
WHGLFE002
WELL #17
15M17B
LF04-4E
SS45L001MW
2-62B
WL019
WL250
83BR
2-393B
2-440B
2-1 44B
2-394B
2-292B
WL082
WL094
DM353D
WL090
MW236S
2-304B
2-1 43B
MW101S
2-329B
WL080
2-299B
Observed
Response
present
present
absent
absent
present
present
present
present
present
present
present
present
present
present
present
present
present
absent
absent
absent
absent
absent
absent
absent
absent
absent
absent
absent
present
absent
absent
absent
absent
present
absent
absent
absent
absent
present
present
absent
absent
absent
absent
present
Predicted
Probability
0.958
0.949
0.867
0.811
0.799
0.789
0.765
0.756
0.746
0.743
0.705
0.692
0.668
0.651
0.643
0.591
0.567
0.487
0.435
0.407
0.394
0.366
0.329
0.327
0.306
0.306
0.301
0.296
0.287
0.268
0.260
0.246
0.220
0.207
0.203
0.194
0.183
0.182
0.174
0.174
0.171
0.162
0.154
0.132
0.128
LCLa
0.315
0.334
0.449
0.526
0.519
0.491
0.500
0.407
0.461
0.477
0.087
0.440
0.436
0.108
0.387
0.320
0.292
0.259
0.254
0.219
0.226
0.191
0.150
0.149
0.151
0.075
0.162
0.152
0.135
0.126
0.109
0.105
0.099
0.083
0.086
0.077
0.062
0.059
0.063
0.053
0.057
0.054
0.017
0.037
0.037
UCLb
0.999
0.999
0.981
0.943
0.936
0.936
0.914
0.934
0.909
0.901
0.984
0.865
0.84
0.966
0.837
0.815
0.807
0.721
0.634
0.628
0.592
0.584
0.576
0.574
0.522
0.707
0.491
0.496
0.508
0.482
0.501
0.475
0.422
0.432
0.408
0.410
0.434
0.441
0.399
0.442
0.416
0.398
0.655
0.372
0.363
' LCL: lower 95% confidence limit for the predicted probability;b UCL: upper 95% confidence limit for the predicted probability
54
-------
Section 6.
Presence of Dehalococcoides DNA and the Extent of Biodegradation
Presence of Dehalococcoides DNA in ground water
may not indicate that the bacteria are active and
degrading the chlorinated solvents. On the other
hand, the failure to detect Dehalococcoides using a
particular PCR primer does not prove that the aqui-
fer does not contain Dehalococcoides organisms or
other bacteria that can degrade chlorinated solvents
to ethylene. Laboratory microcosm studies conducted
with sediment or enrichment cultures conducted with
ground water from a contaminated site play a key
role in determining if the bacteria at a site have the
potential for complete dechlorination (David Ellis,
DuPont Company, Wilmington, Delaware, Personal
Communication, September 16, 2005). To determine
if dechlorination of chlorinated ethylenes could be car-
ried out by indigenous microorganisms in the samples
of ground water, ground water was used to establish
enrichment cultures. The results of laboratory studies
were compared to the distribution of Dehalococcoi-
des DNA in the contaminated ground water at field
scale. These experiments were intended to establish
a connection between the extent of observed activity
and the presence of Dehalococcoides as detected
by PCR.
Enrichment Culture Preparation
Ground Water samples for inoculation were collected
from PCE and TCE contaminated wells at the SS-17
Site at Altus AFB, OK; the OU-1 Site at Altus AFB, OK;
Area 6 at Carswell AFB, TX; Target Area 1 at Dover
AFB, DE; Site 35 at Vandenberg AFB, CA; Building
79 at the USCG Support Center, Elizabeth City, NC;
the North Beach Site at the USCG Support Center,
Elizabeth City, NC; Area 800 at England AFB, LA;
Area 2500 at England AFB, LA; the LF-3 Site at Tinker
AFB, OK; the OFTA Site at Tinker AFB, OK; and the
Western Processing Site at Kent, WA (described in
Table 5.1). A total of 76 ground water samples were
collected for constructing the enrichment cultures.
Ground water samples were collected in sterile 40 ml
VOA vials with no preservatives. The samples were
collected with no headspace in the vials.
Enrichment cultures were prepared in 160 ml serum
bottles in an anaerobic glove box. For each ground
water sample, two enrichment cultures were con-
structed in the same manner except that propionate
(5 mM) was added as an additional elector donor in
one enrichment culture but not in the other. Each
enrichment culture contained 100 ml autoclaved basal
medium and 25 ml ground water (as an inoculum) and
was amended with 10 ml of a PCE stock solution in
water or 1.5 ml of a TCE stock solution in water to
produce a nominal concentration of 100 uM in the
enrichment culture (nominal concentration corrects
for headspace-liquid partitioning equilibrium within a
bottle). The enrichment culture was sealed with a Tef-
lon-lined butyl rubber septum and an aluminum crimp
cap. The composition of the basal medium is shown
in Table 6.1. The enrichments contained 0.1 g/L yeast
extract, which is adequate to completely dechlorinate
100uMof PCE.
Five rounds of enrichment cultures were constructed
depending on the date the ground water samples were
collected. In each round of enrichment cultures, two
controls (with and without propionate) were prepared
in the same manner except that an additional 25 ml
of basal medium were added to each vial instead of
ground water. The controls were preserved with 1 %
trisodium phosphate and autoclaved for 45 minutes
at 121 °C. All the enrichment cultures were incubated
statically on their sides in an anaerobic glove box at
room temperature (roughly 20°C).
The enrichment cultures are similar to the microcosm
studies described in the RABITT protocol (Battelle Me-
morial Institute etal., 2002; Morse etal., 1998), and to
microcosm studies conducted commercially (Findlay
and Fogel, 2000). The concentrations of chlorinated
solvents, of yeast extract, and of primary substrate
are similar. An important exception is the absence
of aquifer sediment in the enrichment cultures in this
study. The RABITT protocol suggests 50 g sediment
and 50 ml of ground water. At many hazardous waste
55
-------
sites, the site owner cannot afford to collect sediment
for the laboratory studies, and the studies are often
done with ground water from established monitoring
wells (Margaret Findlay, Bioremediation Consulting
Incorporated, Watertown, MA, Personal Communica-
tion, November 3, 2005).
Table 6.1.
Composition of the Basal Medium (pH 7)
Composition
NH4CI
MgS04-7H20
CaCI2-2H20
yeast extract
resazurin
KH2P04
Na2HPO4
EDTA
FeSO4-7H2O
CaCI2-6H2O
MnCI2-4H2O
NiCI2-6H20
ZnSO4-7H2O
H2Se03
H3B03
CuCI2-2H20
NaMoO4-2H2O
CoCI2-6H2O
p-aminobenzoic
acid
folic acid
lipoic acid
nicotinic acid
riboflavine
thiamine
panthotenic acid
pyridoxamine
vitamin B12
biotine
Concentration
1.0
0.1
0.05
0.1
1.0
0.0066
0.013
1.0
2.0
0.2
0.03
0.02
0.1
0.02
0.3
0.01
0.033
0.2
100
50
100
200
100
200
100
500
100
20
Unit
g/L
g/L
g/L
g/L
mg/L
Molar
Molar
ug/L
ug/L
ug/L
ug/L
ug/L
ug/L
ug/L
ug/L
ug/L
ug/L
ug/L
ug/L
ug/L
ug/L
ug/L
ug/L
ug/L
ug/L
ug/L
ug/L
ug/L
Sampling and Analysis of Enrichment
Cultures
Resazurin was used as a visual indicator of redox
condition in the enrichment cultures (purple indicat-
ing an oxidizing condition, and the absence of color
indicating a reduced condition). Enrichment cultures
were sampled periodically (nondestructively) using
small samples of liquid or headspace. The samples
were analyzed for chlorinated ethylenes (PCE, TCE,
DCE isomers, and vinyl chloride), dissolved gases
(methane, ethylene, ethane, and hydrogen), and or-
ganic acids (propionate and acetate).
For analysis of chlorinated ethylenes, as well as for
analysis of fatty acids, 1 ml of liquid was taken from
each enrichment culture using a 1 ml glass syringe
and injected to the bottom of a 40 ml VOA vial contain-
ing 40 ml RO (reverse osmosis) water and 2 drops
of concentrated hydrochloric acid. The vial was im-
mediately sealed after sample collection. Analysis
of chlorinated ethylenes was performed using auto-
mated purge and trap gas chromatography. Organic
acids were analyzed using high performance liquid
chromatography.
For analysis of methane, ethylene, and ethane, 0.2 ml
of gas was taken from the headspace of each enrich-
ment culture into a 2.0 ml gas tight syringe; the sample
was diluted to 2.0 ml with nitrogen and then injected
into a GC for analysis. The high purity nitrogen used
to dilute the sample was free of detectable concen-
trations of methane, ethylene, and ethane. Henry's
Law was used to calculate the original concentrations
of methane, ethylene, and ethane in the water in the
enrichment cultures.
For analysis of hydrogen, a gas sample of 0.2 ml was
taken from the headspace of each enrichment culture
into a 2.0 ml gas tight syringe; the sample was diluted
to 2.0 ml with nitrogen and then injected into a RGA3
Reduction Gas Analyzer. The high purity nitrogen
used to dilute the sample was free of detectable
concentrations of molecular hydrogen. The original
concentrations of dissolved molecular hydrogen in
the water in the enrichment cultures were calculated
using Henry's Law.
Biodegradation of Chlorinated Ethylenes in
the Enrichment Cultures
All the enrichment cultures were incubated for at
least 20 months. Some enrichment cultures were
incubated for more than 30 months. Out of 152
enrichment cultures from ground water, ethylene as
56
-------
a dechlorination end product was observed in 28
enrichments. These 28 enrichments originated from
17 ground water samples (15 had been spiked with
propionate and 13 had not). Of the 28 enrichments,
ethylene as the only end product (100% recovery of
the total chlorinated ethylenes on a molar basis) was
observed in eight cases; ethylene as the major end
product (68%-99% recovery of the total chlorinated
ethylenes) was observed in four cases; and a smaller
amount of ethylene (1%-16% recovery of the total
chlorinated ethylenes) was observed in 16 cases.
Vinyl chloride as a dechlorination end product was
observed in nine enrichments.These nine enrichments
originated from six ground water samples (2 had been
spiked with propionate and 7 had not). The observed
molar recovery of total chlorinated ethylenes as vinyl
chloride ranged from 1% to 24%.
Dichloroethylenes (mainly c/s-DCE) as a dechlorina-
tion end product, were observed in 28 enrichments.
These 28 enrichments originated from 15 ground
water samples (14 had been spiked with propionate
and 14 had not). The observed molar recovery of
total chlorinated ethylenes as total DCE (sum of three
isomers) ranged from 1 % to 74%.
In three enrichment cultures amended with PCE,
a trace of TCE was detected as a transformation
product. No transformation of the added PCE or
TCE was observed in the other 84 ground water
amended enrichment cultures. No transformation
of the added PCE or TCE was observed in the ten
sterile controls.
For most ground water samples, the spike of propio-
nate as an additional electron donor did not seem to
greatly increase the rate and extent of dechlorination
in the enrichments. Complete dechlorination to ethyl-
ene or partial dechlorination to DCE or vinyl chloride
frequently occurred in the enrichments without the
spike of propionate. This observation was not sur-
prising because a significant amount of yeast extract
(100 mg/L) was contained in the basal medium in the
enrichment cultures. Acetate in the range of 0.02 to
3.7 mM (1.2 to 220 mg/L) was detected in the enrich-
ments without the spike of propionate, probably due
to fermentation of yeast extract in the basal medium
and organic compounds in the ground water.
The measurement of dissolved molecular hydrogen
also confirmed the availability of molecular hydrogen
as an electron donor. In nearly all of the enrichment
cultures (96%), the dissolved hydrogen concentra-
tions were greater than 1 nM, a condition favorable
for reductive dechlorination. The concentration of
molecular hydrogen was greater than 9 nM in 75%
of the enrichments, greater than 130 nM in 50% of
the enrichments, and greater than 3000 nM in 25% of
the enrichments. There was no statistically significant
difference in the distribution of dissolved hydrogen
concentration between the enrichment cultures spiked
with propionate and the enrichment cultures without
the spike of propionate (significance level a=0.05).
Association of Dechlorination in Enrichment
Cultures with Dehalococcoides DNA
Prior to constructing the enrichment cultures, ground
water samples collected from the same wells were
analyzed for the presence or absence of Dehalococ-
coides DNA. Table 6.2 compares the extent of bio-
transformation of the chlorinated ethylenes obtained
in the enrichment cultures to the presence or absence
of Dehalococcoides DNA in the field samples used to
establish the enrichments.
Dehalococcoides DNA was detected in water samples
from 22 wells out of a total of 72 sampled. The water
was used to establish 44 enrichment cultures (one
with propionate, one without for each of the 22 wells).
Of the 44 enrichment cultures, 20 showed complete
biotransformation to ethylene. The 20 microcosms
corresponded to 11 of the 22 ground water samples
where Dehalococcoides DNA was detected. In two
ground water samples where Dehalococcoides DNA
was detected, dechlorination proceeded only as far as
DCE in the four corresponding enrichment cultures.
Vinyl chloride as an end product was not detected in
enrichment cultures that were inoculated with ground
water that contained amplifiable Dehalococcoides
DNA. If dechlorination proceeded past dichloroeth-
ylene, it went all the way to ethylene.
Dehalococcoides DNA was not detected in water
samples from 50 wells out of the 72 wells that were
used to establish the enrichment cultures. Complete
dechlorination to ethylene was detected in only 7 of the
100 enrichment cultures that were constructed from
the ground water samples that did not have amplifi-
able Dehalococcoides DNA. Dechlorination to vinyl
chloride was detected in 8 enrichment cultures and
dechlorination to DCE was detected in 20 of the 100
enrichment cultures. If Dehalococcoides DNA was
not detected, the majority of the cultures that showed
activity stopped at the production of DCE.
The capacity to transform PCE or TCE to c/s-DCE is
common in anaerobic bacteria. The PCR assay for
Dehalococcoides DNA is conventionally interpreted as
an assay for organisms that might have the capabil-
57
-------
Table 6.2. Comparisons of Biotransformation of Chlorinated Ethylenes in Enrichment Cultures to the Correspond-
ing Presence or Absence of Amplifiable Dehalococcoides DMA in the Ground Water Sample Used to
Inoculate the Enrichment Cultures
Biotransformation
Product Detected on the
Last Sampling Date
No Product
Dichloroethylene
Vinyl Chloride
Ethylene
Number of Enrichment
Cultures where the Presence
of Dehalococcoides DMA
was detected in the Field
20 Of 44
4 of 44
Oof 44
20 Of 44
Number of Enrichment
Cultures where the Presence
of Dehalococcoides DMA
was not detected in the Field
65 of 1 00
20 of 1 00
8 of 100
7 of 100
The test results for the presence of Dehalococcoides DMA in two ground water samples were inconclusive (score of+/-); therefore, the
enrichment cultures constructed from these two samples were not included in the comparison. Twelve enrichment cultures prepared
using material from the North Beach Site were amended with PCE as the source contaminant. Of the 12 enrichment cultures, three
had TCE as the biotransformation end product on the last sampling date (not shown in Table 6,2),
ity to completely transform chlorinated ethylenes to
ethylene. Out of 44 enrichment cultures established
with ground water with detectable concentrations of
Dehalococcoides DNA, 24 cultures failed to produce
ethylene. If false positives for the assay are defined
as water samples where Dehalococcoides DNA was
detected, but ethylene was not detected in the enrich-
ment culture, then 55% of the cultures were false posi-
tives. As will be discussed later, it is possible that the
community of dechlorinating organisms was damaged
during collection, transport, or storage of the sample
prior to construction of the enrichment cultures. It is
also possible that conditions in the enrichment cultures
failed to support growth of organisms that could and
did grow in the aquifer, and could dechlorinate PCE or
TCE to ethylene. If the proportion of false positives is
calculated on the basis of cultures that produced any
transformation product, then 4 out of 24 enrichment
cultures failed to produce ethylene. On this basis, the
proportion of false positives is 17%.
It is also possible that the Dehalococcoides DNA de-
tected by the PCR assay belonged to strains that could
not transform chlorinated ethylenes to ethylene.
Although the number of false positives in our survey
is high, the PCR assay can still be useful to evaluate
monitored natural attenuation. This is particularly true
when the overall determination is based on a variety
of tests and conditions. The interpretation of the PCR
assay is more straightforward if it is supported by other
information that would be consistent with on-going
dechlorination to ethylene in the aquifer. Additional
information might include geochemical conditions that
are conducive to the growth of Dehalococcoides spe-
cies, a reduction in the concentration of vinyl chloride
in ground water over time, and the accumulation of
significant concentrations of ethylene.
An absence of Dehalococcoides DNA would suggest
that Dehalococcoides organisms were absent from
the ground water and that dechlorination would not
proceed to vinyl chloride or ethylene. If false negatives
for the assay are defined as water samples where
Dehalococcoides DNA was not detected, but vinyl
chloride or ethylene was detected in the enrichment
culture, then the proportion of false negatives is 15
out of 100 enrichments, or 15%. When Dehalococcoi-
des DNA was not detected in the ground water used
to establish the cultures, 35 out of 100 enrichments
showed production of some transformation product
(dichloroethylene, vinyl chloride, or ethylene). The
cultures transformed PCE or TCE to ethylene in 7 out
of the 35 enrichments, to vinyl chloride in 8 of the 35
enrichments, and stopped at the level of DCE in 20
of the 35 enrichments. The proportion of false nega-
tive predictions, as evaluated against the number of
enrichment cultures that showed production of at least
one of the transformation products, was 15 out of 35
cultures, or 43%. A failure to detect Dehalococcoides
DNA in a sample of ground water using the PCR as-
say should not be taken to mean that dechlorination
in the aquifer will stop at the level of DCE.
Several interactions might account for the complete
lack of dechlorination activity in roughly half of the
microcosms. First, the activity of dechlorinating bac-
teria might be suppressed by competition from other
hydrogen utilizing bacteria such as the methanogens.
Yang and McCarty (1998) reported that dechlorinators
competed best against methanogens and acetogens
when the hydrogen level was maintained between 2
58
-------
and 11 nM. The basal medium in the microcosms is
a much richer nutritional environment than the con-
taminated ground water at the sites. In the enrichment
cultures, the hydrogen level was greater than 11 nM
in 72% of the enrichment cultures. This may have
posed a competitive advantage to other bacteria over
dechlorinating bacteria.
Another possibility is that the living dechlorinating
bacteria in the ground water samples that were re-
turned to the laboratory for the enrichment study were
killed by oxygen before the enrichment cultures were
constructed. Many of the water samples had detect-
able concentrations of dissolved oxygen. The oxygen
may have entered the well water in the monitoring well
when oxygenated ground water in uncontaminated
portions of the aquifer was mixed with the contami-
nant plume. The oxygen may also have entered the
well water from the atmosphere when the well was
sampled.
A plot of the relationship between the production of
ethylene in the enrichment cultures against the dis-
solved oxygen concentration measured in the field in
the corresponding ground water used to inoculate the
culture indicated that the dechlorination activity was
strongly influenced by the dissolved oxygen concen-
tration (Figure 6.1). The production of ethylene was
expressed as the molar ratio of the final concentration
of ethylene to the initial concentration of PCE orTCE
supplied to the enrichments. In all 20 enrichment
cultures where Dehalococcoides DNA was detected
in the ground water used to establish the enrichment
cultures, and the enrichment cultures had accumu-
lated ethylene, the corresponding dissolved oxygen
concentration in the well water sample used to estab-
lish the enrichment culture was below 0.5 mg/L. This
observation agrees with reports that Dehalococcoides
organisms are strict anaerobes. The same relation-
ship held for the enrichments where Dehalococcoides
DNA was not detected in the ground water used to
establish the enrichment culture. With two exceptions,
no ethylene was produced in any enrichment that was
inoculated with ground water that had dissolved oxy-
gen concentrations higher than 0.5 mg/L. In the two
exceptional enrichments, only trace concentrations
of ethylene were detected (the molar ratio of the final
concentration of ethylene to the initial concentration
of TCE was less than 0.01).
1.6
S 1.4 H
jo
o
S1.2H
_
a.
a.
LU
O
CD
0)
f
LLJ
O
O
0
o
n
a
0.8-
0.6 -
2 0.4 -
0.2 -
o Dehalococcoides DNA detected
n Dehalococcoides DNA not detected
i — D n ^ 1 3
1 1 i |i~
-ee-
012345678
Dissolved Oxygen in Well Water (mg/L)
Figure 6.1. Relationship between the production of ethylene in the enrichment cultures and the concentration of
dissolved oxygen in the corresponding ground water used for the inoculums of the enrichment cultures.
Diamond symbols are ground water samples where Dehalococcoides DNA was detected, and square
symbols are ground water samples where Dehalococcoides DNA was not detected.
59
-------
Table 6.3 compares the extent of dechlorination in the
enrichment cultures with the geochemical character of
the ground water used to inoculate the culture. (See
Section 5 for the definition of the geochemical cat-
egories.) Dechlorination of PCE and TCE to ethylene
or vinyl chloride occurred most frequently and most
extensively in the enrichment cultures inoculated with
methanogenic ground water. Dechlorination to vinyl
chloride or ethylene was limited in cultures inoculated
with water that was sulfate-reducing, iron-reducing, or
oxic. However, dechlorination to DCE was frequent
and extensive when the cultures were inoculated
with ground water that was sulfate-reducing or iron-
reducing.
Table 6.3. Comparison of Biotransformation of Chlorinated Ethylenes in Enrichment Cultures to the Corresponding
Geochemistry of the Ground Water used for Inoculation of the Enrichment Cultures
Geochemistry
Methanogenic
Sulfate and/or iron reducing
Oxic
Methanogenic
Sulfate and/or iron reducing
Oxic
Methanogenic
Sulfate and/or iron reducing
Oxic
Number of Enrichment Cultures in Category
Mole Percent of Final Ethylene to Initial PCE or TCE
No Ethylene
29
52
44
<1%
5
0
2
1 %-50%
9
0
0
>50%
9
2
0
Mole Percent of Final VC to Initial PCE or TCE
NoVC
39
51
44
<1%
1
3
1
1-50%
8
0
1
50%
4
0
0
Mole Percent of Final DCE to Initial PCE or TCE
No DCE
25
29
38
<1%
18
18
7
1-50%
9
7
1
50%
0
0
0
60
-------
Section 7.
Recommendations to Evaluate Biotransformation of Chlorinated Solvents
Based on the performance of PCR assays at the
sites in this study that were undergoing natural at-
tenuation of chlorinated solvents, the authors offer a
number of recommendations for collecting samples
for PCR assays and interpreting the data provided.
The recommendations are summarized in Table 7.1
and are discussed in detail in the remainder of this
section.
Recommendations for Interpreting Data on
Density of DMA in Ground Water
The OSWER Directive on MNA (U.S. EPA 1999) iden-
tifies three lines of evidence that can be used to sup-
port the selection of MNA as a remedy. The first line
of evidence is historical monitoring data that provide a
clear and meaningful trend of decreasing concentra-
tions or contaminant mass overtime. The second line
of evidence is hydrogeologic and geochemical data
that can be used to demonstrate indirectly the types
of natural attenuation processes active at the site,
and the rate at which such processes will reduce con-
taminant concentrations to required levels. The third
line of evidence is data from the field or microcosm
studies which directly demonstrate the occurrence
of a particular natural attenuation process at the site
and its ability to degrade the contaminant of concern.
The presence of Dehalococcoides DNA in an aquifer
can contribute to the third line of evidence.
As specified in the OSWER Directive (U.S. EPA 1999),
unless EPA or the overseeing regulatory authority de-
termines that historical data (the first line of evidence)
are of sufficient quality and duration to support a deci-
sion to use MNA, data characterizing the nature and
rates of natural attenuation processes at the site (the
second line of evidence) should be provided. Where
the latter are inadequate or inconclusive, data from
microcosm studies (or genetic analysis, the third line
of evidence) may also be necessary.
Data provided from analysis of DNA in water samples
from wells are a semi-quantitative lower boundary
on the density of organisms in the aquifer. The
microorganisms may be attached to aquifer solids,
and as a consequence, not adequately sampled by
ground water from a monitoring well. As a practical
matter, many evaluations of the distribution of De-
halococcoides DNA will be done with ground water
samples from permanent wells. These evaluations
done with samples of ground water will be subject
to false negatives.
As a consequence, the density of Dehalococcoi-
des cells in ground water does not provide direct
evidence for a particular rate of biotransformation of
chlorinated solvents. An assay for Dehalococcoides
DNA in ground water does not provide the second
line of evidence for natural attenuation. However, an
assay for Dehalococcoides DNA in ground water can
readily provide the third line of evidence for natural
attenuation.
Although the PCR assay for Dehalococcoides DNA
in ground water can only provide the third line evi-
dence, the assay has two desirable features. The
assays are relatively inexpensive, and they can be
preformed in a short period of time. The PCR assay
can reduce uncertainty in the role and contribution
of biological reductive dechlorination to monitored
natural attenuation in plumes of chlorinated ethylenes
in ground water.
The strong possibility of false negatives for the pres-
ence of Dehalococcoides DNA makes it important
that no interpretation be put on a failure to recover
Dehalococcoides DNA from a water sample. The
fact that Dehalococcoides DNA is not detected in a
sample of well water does not mean that Dehalococ-
coides organisms are absent in the aquifer. This is
particularly true if DNA corresponding to a universal
bacterial primer is absent from the water sample. If
the assay fails to detect Dehalococcoides DNA, it sim-
ply fails to contribute to the third line of evidence.
If the third line of evidence is critical to accepting
MNA as a remedy, then other means to provide the
third line of evidence are necessary, or MNA should
be rejected. If the third line of evidence is not critical,
61
-------
Table 7.1. Recommendations for Use of PCR Assays to Evaluate Biotransformation of Chlorinated Solvents
Concern
Recommendation
Does PCR for Dehalococcoides
DNA provide the Second Line of
Evidence for MNA?
PCR should not be expected to provide the Second Line of Evidence
when applied to water samples from monitoring wells.
Does PCR for Dehalococcoides
DNA provide the Third Line of
Evidence for MNA?
The presence of DNA from Dehalococcoides provides the Third Line
of Evidence. However, the absence of DNA from Dehalococcoides
should not be interpreted as the absence of biological natural attenu-
ation.
Can PCR for Dehalococcoides
DNA provide the Second Line of
Evidence in the future?
It will be necessary to extract DNA from sediment samples, and
compare data on density of Dehalococcoides cells to rates attained
in benchmark field studies.
How should sites be "scored" to
determine whether site charac-
terization of biological process-
es is justified?
The scoring system in the EPA Technical Protocol to Evaluate Natu-
ral Attenuation at Chlorinated Solvents in Ground Water should be
replaced with Equation 5.1 in Section 5 of this report.
How can limited PCR data from
a few wells be extrapolated to
other wells in a contaminated
aquifer?
Rather than assume that PCR data from a few wells apply to an
entire aquifer, use Equation 5.1 to estimate whether it is likely that
Dehalococcoides is present in ground water from a particular well.
Which wells should have the
highest priority for a PCR assay
for Dehalococcoides?
Sample wells screened in material with high hydraulic conductivity
compared to the rest of the aquifer, and wells with high concentra-
tions of transformation products. Sample an equal number of wells in
the source area, in the region with intermediate concentrations, and
at the toe of the plume.
What precautions are
needed to sample monitoring
wells for a PCR assay for
Dehalococcoides?
Avoid cross contamination. Use dedicated sampling tubing. The EPA
low flow procedure is not optimal for sampling for Dehalococcoides.
Pump as rapidly as possible.
What precautions are needed to
collect water samples for a PCR
assay for Dehalococcoides?
Collect one liter samples into plastic bottles that have never been
used for another purpose. Collect spare samples from each well.
Store on ice in the field before transportation to the laboratory. Pro-
tect samples from cross contamination during storage and transpor-
tation. Prepare shipping container to keep samples cool during trans-
portation to the laboratory.
What controls are needed to
document data quality?
Prepare field blanks and field duplicates as specified in the Quality
Assurance Plan for the site. Include positive and negative controls
for amplification of DNA in the PCR assay, and include a control for
extraction of bacterial DNA from the samples.
62
-------
then the presence of Dehalococcoides DNA merely
strengthens a decision that was based on the first two
lines of evidence. The absence of Dehalococcoides
DNA fails to strengthen a decision that was made on
the first two lines of evidence.
Whenever possible, aquifer solids should be sampled
and assayed for Dehalococcoides DNA. This will
reduce the chance of false negative results for the
presence of Dehalococcoides DNA as determined
by the qualitative assay using electrophoresis, or
the density of Dehalococcoides cells as determined
by the quantitative real time PCR assay. If aquifer
solids are extracted and analyzed, there is at least
a possibility that at some time in the future it will be
possible to relate the density of active organisms as
revealed by a PCR assay to the achieved rate of re-
ductive dechlorination at field scale. At many sites, it
is possible to recover core samples quickly and at low
cost with push technology (e.g. GeoProbe® tools). At
other sites, the cost of acquiring core samples may
be prohibitively expensive.
Recommendations for Interpreting
Geochemistry of Ground Water
The scoring system in the Technical Protocol for Evalu-
ating Natural Attenuation of Chlorinated Solvents in
Ground Water (Wiedemeier et al., 1998) was offered
as a screening mechanism to identify ground water
where biological reductive dechlorination is likely to oc-
cur; however, the scoring system has been criticized.
Equation 5.2 as discussed in Section 5 provides a
simple and rapid way to calculate the probability that
a specific dechlorinating organism, Dehalococcoides,
is present at the site. The probability that Dehalococ-
coides DNA occurs in the ground water, as calculated
by Equation 5.2, should replace the scoring system.
However, a calculated probability that an organism
exists in ground water is not equivalent to a PCR
assay for its presence, and probabilities calculated
from geochemical parameters should not be used to
replace PCR assays.
Often the computer models that are used to evaluate
monitored natural attenuation are distributed param-
eter models. To properly calibrate these computer
models, it is necessary to know the distribution of the
capacity to transform chlorinated ethylenes through-
out the aquifer. Data from the PCR assay may not
be available from every well at a site. The calculated
probability of Dehalococcoides organisms based on
the geochemistry of ground water in a well could be
used to assign rate constants to cells in a model. If
Dehalococcoides organisms are expected, a rate
constant characteristic of reductive dechlorination
at that site would be assigned. If Dehalococcoides
organisms are not expected, the rate constant for
reductive dechlorination would be set to zero in that
particular region of the aquifer.
Recommendations for Selecting Wells for
Sampling
At many chlorinated solvent sites, computer models
have been used to describe the previous behavior of
the plume and make future projections of its natural at-
tenuation over time. Often these computer projections
are an important part of the conceptual model of a site.
Frequently, the calibration of the computer models will
assume a uniform rate constant for biotransformation
across the entire plume or major portions of the plume.
The calibration of the model frequently assumes that
the rate constant will be sustained into the future. If
biotransformation carried out by Dehalococcoides
organisms is the primary process for natural attenua-
tion, an assay for Dehalococcoides DNA can be used
to test these assumptions about uniform distribution
and sustainability of biotransformation. Dehalococ-
coides organisms should be uniformly present in those
portions of the aquifer where the computer model
projects a rate constant for biotransformation, and the
populations of Dehalococcoides organisms should be
sustained during long-term monitoring.
At most sites, there is little value in sampling every
well for analysis of Dehalococcoides DNA. Wells
with relatively high hydraulic conductivity should be
sampled from the more permeable portions of the
aquifer. These are the portions of the aquifer with
the greatest capacity to transport contaminated water
and may be the portions that provide the most risk
of impacting a receptor. Wells with higher hydraulic
conductivity should be selected for assays for Deha-
lococcoides DNA.
Wells with higher relative concentrations of transfor-
mation products provide circumstantial evidence that
biotransformation has occurred at some point along
the flow path from the source to the well. However,
the high concentrations of transformation products
do not prove that the transformation occurred in the
portion of the aquifer immediately proximate to the
monitoring well. Nonetheless, the wells with higher
relative concentrations of vinyl chloride and ethylene
should be sampled.
Wells should be sampled at various positions along the
flow path from the source to the most down gradient
wells containing detectable concentrations of chlori-
nated solvents. For the specific purpose of evaluating
in situ biotransformation of chlorinated solvents, there
63
-------
is little value in sampling background wells. Equation
5.2 as discussed in Section 5 can be used to identify
wells where the geochemical environment is favorable
for Dehalococcoides species, and to select wells for
a PCR assay for Dehalococcoides species. Sample
an equal number of wells in the source area, in the
region with intermediate concentrations, and in the
distal portion of the plume where concentrations are
within a factor of ten to one hundred of the Maximum
Contaminant Level (MCL) or other relevant clean-up
goals.
There is little value in sampling for Dehalococcoides
DNA in every round of sampling. A better strategy
is to perform a comprehensive baseline assessment
across a plume as part of the selection of MNA as
a remedy, and perform a second comprehensive
assessment before the performance of the remedy
goes for review. The biogeochemical parameters,
particularly concentrations of nitrate, methane, and
ORP should be determined at the same time ground
water is collected for determination of Dehalococ-
coides DNA.
Recommendations for Sampling and
Shipping of Samples
To avoid any cross-contamination, equipment to purge
and sample ground water should not be moved from
one well to another even if an attempt is made to
sterilize or decontaminate the equipment. The PCR
assay can amplify DNA, even though the organisms
have been killed. Water samples should be obtained
using a dedicated pump or pump tubing, freshly
installed pump tubing, a disposable pump that has
not been used on another well, or a new clean bailer
and line. Neither the bailer nor the line should have
been used on another well. If possible, avoid using
bailers. If water is produced with a peristaltic pump at
the well head, the peristaltic pump tubing and effluent
tubing should be replaced immediately before a well
is purged and sampled. A minimum of one casing
volume should be purged before the sample is taken.
It is better to purge the casing volume plus a volume
equal to the porosity of the sand pack around the well
screen, if one is present.
The EPA low-flow sampling protocol is designed to
produce a sample that is free of turbidity and sus-
pended solids. If Dehalococcoides cells are sorbed
to sediment particles in the aquifer, they may not be
sampled efficiently. Sediment and turbidity in the water
sample do not interfere with the extraction of bacterial
DNA. To increase the turbidity and suspended solids
in the water sample, purge and sample the well at the
fastest rate the pump will allow. Set the pump or the
end of the sampling tube at the center of the screened
interval of the monitoring well.
Water samples for analysis of Dehalococcoides DNA
should be collected in a one liter plastic bottle con-
structed of polypropylene (e.g. Nalgene®) or high
density polyethylene. The bottles should be new and
should never have been used for another purpose.
Use the bottles as supplied by the manufacturer. Do
not attempt to clean the bottles before use. If the
original shipping container has been opened, the
empty bottles should be shipped to the field site with
the lid firmly attached to the bottle.
Fill the bottle in the field and screw the lid back on
the bottle to make a tight seal. As far as possible,
avoid exposing the water sample to the atmosphere.
Fill the bottle with the fill tube at the bottom of the
bottle. Do not allow the water sample to flow down
the inside of the bottle.
Label the sample with a permanent marker on a strip
of labeling tape that entirely circles the bottle. Provide,
at a minimum, the complete name of the well being
sampled, the name of the facility or location where
the well is installed, the date and time the sample
was collected, and the name or initials of the person
collecting the samples. If a chain of custody form is
required, fill in the required information at the same
time. Seal the sample bottle in a plastic bag with a
ZipLock® closure or equivalent. Remove excess air
from the bag before the closure is sealed. The pur-
pose of the bag is to prevent cross contamination of
the samples in case one of the sample bottles leaks
during shipment. Place the bottles on water ice im-
mediately after they are collected, labeled, and sealed
in a plastic bag. Keep the bottles on ice until they are
packaged for shipment for analysis.
Collect a sample and a spare sample from each well
sampled. Ship both samples for analysis. Identify
which is the sample and which is the spare. The
spare will be available to the laboratory analyst if
there is any problem with preparation or analysis of
the sample. If a field duplicate is desired, collect a
third sample from the well and label the third sample
as the field duplicate.
Prepare water to make a field blank by filtering dis-
tilled water or deionized water through a 0.22 micron
filter into a sterile container. This is best done in the
laboratory before going to the field. To prepare a trip
blank, take the filtered water to the well head of the
well at the site that is most likely to contain Dehalo-
coccoides and pour the previously prepared water
64
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into a one liter plastic bottle just as would be done for
any other sample. Seal the lid, label the bottle, seal
in a plastic bag, store on water ice, and ship to the
laboratory along with the other samples.
Collect and ship the number of trip blanks and field
duplicates that are specified in the quality assurance
project plan for the site.
Package the samples in a plastic cooler with an ad-
equate number of bricks of frozen brine sealed in a
plastic cover (Blue Ice® or equivalent) to keep the
samples at or below 10° C for two days. Do not ship
the samples with water ice. The samples should ar-
rive at the laboratory within five days after they were
originally collected in the field. If the field site is re-
mote from the analytical laboratory, ship the samples
by air freight for overnight delivery. Avoid shipping on
a Friday or the day before a holiday if the receiving
laboratory will not be open for business and not avail-
able to accept and properly store the samples.
The laboratory should provide information in their
report on the procedures used to prepare the water
samples for the polymerase chain assay, on the
number and types of primers used, and the results
with each primer. The laboratory should also pro-
vide information on the number of gene copies that
would be required to provide the minimal density of
Dehalococcoides DNA that can be detected by the
assay. The minimum number of gene copies should
be expressed in gene copies per liter of well water
or gene copies per kilogram sediment, whichever is
appropriate.
Dehalococcoides DNA may be absent in a water
sample because bacteria of any kind were absent in
the sample. To be able to determine if Dehalococcoi-
des organisms are not present in the microbial com-
munity of the aquifer being sampled, it is necessary to
include a primer for a gene that is essentially universal
in bacteria in the polymerase chain reaction assay.
The absence of bacterial DNA suggests that the mi-
crobial community in the aquifer was not effectively
sampled, and that Dehalococcoides organisms may
have been present but were not sampled. Positive
and negative controls should be included in the PCR
assay to ensure that the reaction is working properly
and that contamination of reagents has not occurred.
Amplicons (DNA amplified by the PCR assay using
the Dehalococcoides primers) should be cloned and
sequenced periodically to ensure that the assay is
working as intended.
65
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-------
Section 8.
Data Quality
Analysis of Chemical Concentrations
Laboratory analyses for data presented in Table 4.2,
Table 4.4, Table 4.6, Table 5.2, Table 5.3, Table 5.4,
Table 5.5, Table 5.7, and Table 6.4 were conducted
at the R.S. Kerr Environmental Research Center in
accordance with a Quality Assurance Project Plan
prepared for in-house task 3674 (Monitored Natural At-
tenuation of Chlorinated Solvents). Concentrations of
chlorinated solvents, dissolved gases (methane, eth-
ylene, ethane, and hydrogen), inorganic compounds
(nitrate plus nitrite, sulfate, and chloride), fatty acids
(propionate and acetate), and total organic carbon
(TOC) were determined following in-house Standard
Operating Procedures (SOPs). Chlorinated solvents
were analyzed by automated headspace gas chroma-
tography and mass spectrometry. Methane, ethylene,
and ethane were analyzed by gas chromatography
with a thermal conductivity detector. Hydrogen was
analyzed by RGA3 Reduction Gas Analyzer with a
reduction gas detector. Nitrate plus nitrite was ana-
lyzed by Lachat flow injection analysis. Sulfate and
chloride were analyzed by waters capillary electropho-
resis. Total Organic Carbon (TOC) was analyzed by
Dohrman DC-80 Carbon Analyzer. Propionate and
acetate were analyzed by high performance liquid
chromatography.
Major quality assurance and quality control (QA/QC)
evaluations for the analyses included method blank
(MB), continuing calibration check (CCC), second
source check (QC) using a sample obtained from
the second source as identified by their designated
names, laboratory duplicates (LD), and matrix spike
(MS). Method blank was analyzed in the beginning
and end of sample set. Calibration check standards
were analyzed every ten samples as well as in the
beginning and end of sample set. QC checks were
analyzed every ten samples. Lab duplicates were
analyzed every ten samples. Matrix spikes were
analyzed every twenty samples. The data quality
objectives were as follows: The target analyte in the
method blank would be below method detection limit.
The reported concentration of continuing calibration
check standard, QC check standard, and matrix spike
would agree with the expected concentration plus or
minus 20% of the known concentration (i.e., recovery
of the expected value would be in the range of 80-
120%). Laboratory duplicates would agree with each
other plus or minus 20%. All the samples were held
less than thirty days before analysis.
Table 8.1 summarizes typical data quality forTCE in
the ground water samples and in the water samples
from the enrichment cultures. Two out of 38 of the
calibration check standards did not meet the goal of
± 20% of the nominal values (One calibration check
analyzed on 4/2/03 was reported as 122% of the
nominal value, and one calibration check analyzed on
6/26/03 was reported as 69% of the nominal value.)
One out of 43 of the QC check standards did not
meet the goal of ± 20% of the nominal values (One
QC check analyzed on 4/2/03 was reported as 126%
of the nominal value.) One out of 36 laboratory dupli-
cates did not agree within 20% (The relative percent
difference for the duplicates analyzed on 4/2/03 was
21%.) Four out of 50 method blanks had TCE con-
centration above method detection limit (The values
were 0.99 and 0.675 on 4/2/03, and 1.03 and 0.89 on
4/30/03.) All 25 matrix spikes met the goal of ± 20%
of the expected values.
Several quality controls did not meet the criteria on
date 4/2/03 in the beginning of the analysis. The instru-
ment was recalibrated, and the subsequent checks
met the criteria. All the data for TCE were determined
to be of acceptable quality, and the data were used
in the report.
Table 8.2 summarizes typical data quality for cis-
DCE in the ground water samples and in the water
samples from the enrichment cultures. Five out of 38
of the calibration check standards did not meet the
goal of ± 20% of the nominal values. (Two calibration
checks analyzed on 3/9/03 were reported as 127%
and 129% of the nominal values, one calibration
check on 4/30/03 was reported as 136% the nomi-
nal value, one calibration check analyzed on 6/26/03
was reported as 78% of the nominal value, and one
67
-------
calibration check analyzed on 1/20/04 was 123% of
the nominal value.)
Four out of 43 of the QC check standards did not meet
the goal of ± 20% of the nominal values (One QA
check analyzed on 4/10/03 was reported as 129% of
the nominal value, one QA check analyzed on 4/30/03
was reported as 153% of the nominal value, and two
QA checks analyzed on 2/20/04 were reported as
169% and 145% of the nominal values.) All other
quality controls met the objectives, including all 36
duplicates, which agreed with each other within 20%
difference, all 50 method blanks did not have detect-
able c/s-DCE, and all 25 matrix spikes met the goal
of ± 20% of the expected values.
The chromatographic response for c/s-DCE in the two
QA checks analyzed on 2/20/04 showed evidence
of peak asymmetry suggesting coelution of another
compound. The previous and subsequent recoveries
for c/s-DCE in the continuing calibration checks met
the data quality objectives. On 4/30/03, the reported
values for c/s-DCE might be slightly elevated over
the true values, as shown in the continuing calibra-
tion checks and QC checks. Not all of the calibration
checks met the goal of ± 20% of the nominal values.
When the calibration checks did not meet the goal,
they were still within 36% of the nominal values. An
error of 36% would not change the interpretation
placed on the data. Therefore, all the data for c/s-DCE
were determined to be of acceptable quality, and the
data were used in the report.
Table 8.3 summarizes typical data quality for vinyl
chloride in the ground water samples and in the water
samples from the enrichment cultures. Compared
to TCE and c/s-DCE, vinyl chloride is more volatile
and more easily lost, resulting in more missed data
quality objectives. Three out of 38 of the calibration
check standards did not meet the goal of ± 20% of the
nominal values (One calibration check analyzed on
3/9/03 was reported as 78% of the nominal value, one
calibration check analyzed on 3/30/03 was reported
as 76% of the nominal value, and one calibration
check analyzed on 6/5/03 was reported as 131 % of
the nominal value.) Four out of 43 of the QC check
standards did not meet the goal of ± 20% of the
nominal values (One QC check analyzed on 4/10/03
was reported as 77% of the nominal value, one QC
check analyzed on 6/26/03 was reported as 49% of
the nominal value, and two QC checks analyzed on
10/28/03 were reported as 51 % and 62% of the nomi-
nal values.) Three out of 36 laboratory duplicates did
not agree within 20% (The relative percent differences
were 43.5% for the duplicates analyzed on 3/21/03,
84.1 % for duplicates analyzed on 6/26/03, and 42.9%
for duplicates analyzed on 6/26/03.) One out of 50
method blanks had vinyl chloride concentration above
method detection limit. Five out of 25 matrix spikes
did not meet the goal of ± 20% of the expected values
(See Table 8.3.).
Most of the missed data quality objectives for vinyl
chloride were due to the problem of preparing the
check standards, not the problem of the instrument.
Subsequent samples using the analysis applied a
modified standard preparation technique to minimize
vinyl chloride losses. All the data for vinyl chloride
were determined to be of acceptable quality, and the
data were used in the report.
If there is no data provided for calibration check
controls (CCCs) in Tables 8.1, 8.2, and 8.3, that in-
formation was not included in the report provided by
the analyst.
Table 8.4 summarizes typical data quality for ethylene
in the ground water samples and in the gas phase
of the enrichment cultures. All 98 calibration checks
met the goal of ± 20% of the nominal values. All 24
method blanks did not have detectable ethylene. All
16 laboratory duplicates agreed with each other within
20% difference. All seven matrix spikes met the goal
of ± 20% of the expected values.
All the data for ethylene were determined to be of
acceptable quality, and the data were used in the
report.
Table 8.5 summarizes typical data quality for methane
in the ground water samples and in the gas phase of
the enrichment cultures. All 119 calibration checks
met the goal of ± 20% of the nominal values. All 24
method blanks did not have detectable methane. All
16 laboratory duplicates agreed within 20% except
the duplicates analyzed on 8/25/03 where the relative
percent difference was 23.0%. All seven matrix spikes
met the goal of ± 20% of the expected values.
All the data for methane were determined to be of
acceptable quality, and the data were used in the
report.
Table 8.6 summarizes typical data quality for hydro-
gen in the gas samples stripped from ground water
and in the gas phase of the enrichment cultures. All
106 calibration checks met the goal of ± 20% of the
nominal values. Two out of 22 method blanks had
68
-------
hydrogen concentration above detection limit. Three
out of 12 laboratory duplicates did not agree within
20% (See Table 8.6.).
All the data for hydrogen were determined to be of
acceptable quality, and the data were used in the
report.
Table 8.7 summarizes typical data quality for nitrate
plus nitrite nitrogen in the ground water samples.
All 26 calibration checks met the goal of 20% of the
nominal values. All seven QC checks met the goal
of ± 20% of the nominal values. All ten laboratory
duplicates agreed with each other within 20% differ-
ence. All 16 method blanks had nitrate plus nitrite
nitrogen concentration below quantification limit. All
ten matrix spikes met the goal of ± 20% of the ex-
pected values.
All the data for nitrate plus nitrite nitrogen were deter-
mined to be of acceptable quality, and the data were
used in the report.
Analysis of DMA Concentrations
The analyses of Dehalococcoides DNA were per-
formed by SiREM (Guelph, Ontario) using their
Gene-Trac® Test. The QA/QC was maintained by
implementing clean techniques and control PCR
reactions.
DNA was extracted using a single-use sterile filter unit
and single-use DNA extraction kit. Prior to PCR, all
micropipettes and other equipments used in setting
up reactions were swabbed with DNA AWAY™, and
10% bleach or 70% ethanol to ensure cleanliness and
sterility. PCR reaction mixtures were assembled in a
Forma HEPA flow cabinet to prevent the introduction
of particles and bacteria/DNA that might produce
false positives.
Three types of control reactions were used in the
Gene-Trac procedure: a negative control, a positive
control, and a DNA extraction control. The controls
were conducted and interpreted by SiREM, the
vendor for the PCR assays. The negative control
involved processing sterile water through the same
DNA extraction procedure as the sample. It ensured
that contamination of samples did not occur via the
DNA extraction process, PCR setup, or performance
of the reactions. In the data reported to EPA, a
sample was flagged when DNA was amplified in the
negative control. There was no DNA amplified in the
negative control in the data reported in Tables 4.5,
4.8, or 5.4. The positive control consisted of a PCR
assay containing a cloned Dehalococcoides 16S rRNA
gene. It ensured that all reagents and equipment were
performing properly. If the PCR procedure failed to
amplify the Dehalococcoides 16S rRNA gene, the
data were discarded by SiREM and were not reported
to U.S. EPA. The problem with the PCR procedure
was corrected by SiREM, and the DNA extracted
from the ground water sample was assayed and
reported. The DNA extraction control was performed
when the Dehalococcoides test was negative. In the
DNA extraction control, PCR was performed on the
sample using a universal bacterial PCR primer set.
It was used to determine whether bacterial DNA was
present and extracted from the sample. The results of
the DNA extraction control are provided in Tables 4.5
and 4.8, and are interpreted in Section 4. PCR data
from the Dehalococcoides primers from three wells
sampled at the Area 2500 Site at the former England
AFB, Louisiana, were discarded because the bacterial
DNA primer was not detected.
69
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Table 8.1. Typical Quality Performance Data for Analysis of TCE in Water. All Values are ug/L Unless Otherwise
Indicated
Date Collected
Date Analyzed
CCC Standard Nominal
CCC Standard Measured
Percent of Check Standard
CCC Standard Nominal
CCC Standard Measured
Percent of Check Standard
CCC Standard Nominal
CCC Standard Measured
Percent of Check Standard
QC Standard Nominal
QC Standard Measured
Percent of Check Standard
QC Standard Nominal
QC Standard Measured
Percent of Check Standard
Blank 1
Blank 2
Sample Analysis 1
Laboratory Duplicate 1
Relative Percent Difference
Sample Analysis 2
Laboratory Duplicate 2
Relative Percent Difference
Spike Concentration 1
Sample Concentration 1
Spike Recovery (Percent)
Spike Concentration 2
Sample Concentration 2
Spike Recovery (Percent)
Date Collected
Date Analyzed
CCC Standard Nominal
CCC Standard Measured
Percent of Check Standard
CCC Standard Nominal
CCC Standard Measured
Percent of Check Standard
11/7/02
20
19.3
96.4%
200
189
94.4%
<0.5
<0.5
80.5
75.2
6.8%
200
<0.5
97%
2/24/03
3/9/03
50
44.2
88%
200
168
84%
20
22.1
111%
200
219
1 1 0%
<0.23
<0.23
2180
2130
2.3%
200
28.4
108%
3/6/03
4/2/03
10
12.2
122%
100
113
1 1 3%
1/7/03
10
10.1
1 01 %
100
96
96%
250
235
94%
50
51.0
1 07%
50
48.1
96.2%
<0.39
<0.39
227
225
0.9%
50
<0.39
83.6%
50
232
87.2%
3/11/03
3/21/03
1/24/03
2/11/03
20
21.1
1 06%
200
225
113%
<0.3
<0.3
263
235
1 1 .2%
231
240
3.8%
200
215
1 00%
3/26/03
4/10/03
10
10.6
1 06%
250
259
1 04%
2/4/03
3/6/03
20
21.3
107%
200
194
97%
<0.23
<0.23
<0.23
<0.23
-
200
10.4
105%
4/8/03
4/30/03
10
11.2
1 1 2%
50
45
90%
CCC: Continuing Calibration Check; QC: Second Source Check
70
-------
Table 8.1. Typical Quality Performance Data for Analysis of TCE in Water. All Values are ug/L Unless Otherwise
Indicated continued
CCC Standard Nominal
CCC Standard Measured
Percent of Check Standard
QC Standard Nominal
QC Standard Measured
Percent of Check Standard
QC Standard Nominal
QC Standard Measured
Percent of Check Standard
Blank 1
Blank 2
Sample Analysis 1
Laboratory Duplicate 1
Relative Percent Difference
Sample Analysis 2
Laboratory Duplicate 2
Relative Percent Difference
Spike Concentration 1
Sample Concentration 1
Spike Recovery (Percent)
Spike Concentration 2
Sample Concentration 2
Spike Recovery (Percent)
Date Collected
Date Analyzed
CCC Standard Nominal
CCC Standard Measured
Percent of Check Standard
CCC Standard Nominal
CCC Standard Measured
Percent of Check Standard
CCC Standard Nominal
CCC Standard Measured
Percent of Check Standard
QC Standard Nominal
QC Standard Measured
Percent of Check Standard
QC Standard Nominal
50
42
84%
20
23.4
1 1 7%
200
211
105%
<0.39
<0.39
204
203
0.5%
212
192
9.9%
200
234
100%
4/21/03
5/20/03
10
11.4
114%
50
51.2
102%
100
98
98%
50
52.3
105%
10
8.44
84.4%
25
26.8
107%
50
63.0
126%
0.99
0.675
63.9
51.7
21.1%
100
36.4
81%
4/29/03
5/5/03
20
21.3
106%
200
10
9.7
97%
200
202
1 01 %
<0.07
<0.07
96.7
102
5.3%
100
37.2
95%
5/1 6/03
6/5/03
20
20.3
1 02%
50
250
253
1 01 %
100
111
1 1 1 %
<0.39
<0.39
158
157
0.6%
160
156
2.5%
6/23/03
6/26/03
25
24
96%
100
93.9
93.9%
250
173
69.2%
50
44.8
89.6%
100
108
108%
50
58.1
1 1 6%
1.03
0.89
<0.39
<0.39
-
7/23/03
8/5/03
20
22.3
112%
200
CCC: Continuing Calibration Check; QC: Second Source Check
71
-------
Table 8.1. Typical Quality Performance Data for Analysis of TCE in Water. All Values are ug/L Unless Otherwise
Indicated continued
QC Standard Measured
Percent of Check Standard
Blank 1
Blank 2
Sample Analysis 1
Laboratory Duplicate 1
Relative Percent Difference
Sample Analysis 2
Laboratory Duplicate 2
Relative Percent Difference
Spike Concentration 1
Sample Concentration 1
Spike Recovery (Percent)
Date Collected
Date Analyzed
Spike Concentration 2
Sample Concentration 2
Spike Recovery (Percent)
CCC Standard Nominal
CCC Standard Measured
Percent of Check Standard
CCC Standard Nominal
CCC Standard Measured
Percent of Check Standard
CCC Standard Nominal
CCC Standard Measured
Percent of Check Standard
QC Standard Nominal
QC Standard Measured
Percent of Check Standard
QC Standard Nominal
QC Standard Measured
Percent of Check Standard
Blank 1
Blank 2
Sample Analysis 1
Laboratory Duplicate 1
Relative Percent Difference
Sample Analysis 2
Laboratory Duplicate 2
<0.39
<0.39
344
348
1 .2%
8/7/03
8/12/03
20
19.3
96%
200
214
107%
<0.23
<0.23
127
119
6.5%
106
105
206
103%
<0.23
<0.23
<0.23
<0.23
-
200
<0.23
1 02%
8/20/03
8/22/03
20
20.5
103%
200
210
105%
<0.23
<0.23
1410
1370
2.9%
48.4
97%
<0.07
<0.07
185
182
1 .6%
182
179
1 .7%
200
190
91%
1 0/3/03
1 0/22/03
200
168
84%
500
547
1 09%
20
21
1 05%
200
178
89%
<0.07
<0.07
155
155
0.0%
186
176
<0.39
<0.39
241
231
4.2%
188
202
7.2%
1 0/7/03
11/3/03
100
98.6
98.6%
100
99.6
1 00%
<0.07
<0.07
60.0
57.0
5.1%
218
112%
<0.23
<0.23
<0.23
<0.23
-
100
247
84%
10/24/03
10/28/03
25
24.5
98%
50
49.9
99.8%
100
97.6
97.6%
5
5.48
1 1 0%
50
46.4
92.8%
<0.39
<0.39
147
143
2.8%
150
151
CCC: Continuing Calibration Check; QC: Second Source Check
72
-------
Table 8.1. Typical Quality Performance Data for Analysis of TCE in Water. All Values are ug/L Unless Otherwise
Indicated continued
Relative Percent Difference
Spike Concentration 1
Sample Concentration 1
Spike Recovery (Percent)
Spike Concentration 2
Sample Concentration 2
Spike Recovery (Percent)
Date Collected
Date Analyzed
CCC Standard Nominal
CCC Standard Measured
Percent of Check Standard
CCC Standard Nominal
CCC Standard Measured
Percent of Check Standard
CCC Standard Nominal
CCC Standard Measured
Percent of Check Standard
QC Standard Nominal
QC Standard Measured
Percent of Check Standard
QC Standard Nominal
QC Standard Measured
Percent of Check Standard
Blank 1
Blank 2
Sample Analysis 1
Laboratory Duplicate 1
Relative Percent Difference
Sample Analysis 2
Laboratory Duplicate 2
Relative Percent Difference
Spike Concentration 1
Sample Concentration 1
Spike Recovery (Percent)
Spike Concentration 2
Sample Concentration 2
Spike Recovery (Percent)
0.9%
200
111
102%
200
108
105%
1/14/04
1/20/04
10
8.03
80%
100
92.5
93%
100
92.8
93%
50
46.3
93%
100
96.0
96%
<0.39
<0.39
200
9.3
106%
2/19/04
2/20/04
10
10.8
108%
50
50.4
101%
100
103
103%
10
11.0
1 1 0%
100
99.1
99%
<0.39
<0.39
1.20
1.17
2.5%
91.5
93.2
1.8%
5.5%
100
163
91%
200
3.3
93%
6/4/04
6/1 7/04
20
19.9
1 00%
20
20
1 00%
20
22.7
114%
200
200
1 00%
<0.23
<0.23
80.8
83.1
2.8%
110
108
1 .8%
100
<0.23
1 05%
100
134
99%
100
59.3
94.5%
9/7/04
9/17/04
20
20.6
1 03%
<0.23
<0.23
149
148
0.7%
152
154
1 .3%
100
159
1 05%
100
132
1 03%
0.7%
7/14/05
7/15/05
20
17.8
89%
200
193
96%
20
21
105%
20
21.4
107%
<0.23
<0.23
148
139
6.3%
72.6
63.8
12.9%
100
62.9
95%
100
117
89%
CCC: Continuing Calibration Check; QC: Second Source Check
73
-------
Table 8.2. Typical Quality Performance Data for Analysis of c/s-DCE in Water. All Values are ug/L Unless Otherwise
Indicated
Date Collected
Date Analyzed
CCC Standard Nominal
CCC Standard Measured
Percent of Check Standard
CCC Standard Nominal
CCC Standard Measured
Percent of Check Standard
CCC Standard Nominal
CCC Standard Measured
Percent of Check Standard
QC Standard Nominal
QC Standard Measured
Percent of Check Standard
QC Standard Nominal
QC Standard Measured
Percent of Check Standard
Blank 1
Blank 2
Sample Analysis 1
Laboratory Duplicate 1
Relative Percent Difference
Sample Analysis 2
Laboratory Duplicate 2
Relative Percent Difference
Spike Concentration 1
Sample Concentration 1
Spike Recovery (Percent)
Spike Concentration 2
Sample Concentration 2
Spike Recovery (Percent)
Date Collected
Date Analyzed
CCC Standard Nominal
CCC Standard Measured
Percent of Check Standard
CCC Standard Nominal
CCC Standard Measured
Percent of Check Standard
10/23/02
11/7/02
20
21.1
106%
200
187
93.6%
<0.2
<0.2
79.3
75.7
4.6%
200
<0.2
101%
2/24/03
3/9/03
50
48
95%
200
254
127%
11/25/02
12/19/02
20
21.9
1 1 0%
200
216
108%
<0.2
<0.2
57.1
54.8
4.1%
200
28400
108%
3/6/03
4/2/03
10
9.58
95.8%
100
105
105%
1 2/23/02
1/7/03
10
8.71
87.1%
100
100
1 00%
250
233
93.2%
50
54.8
110%
50
51.1
1 02%
<0.36
<0.36
111
110
0.9%
50
<0.36
95%
50
<0.36
1 06%
3/11/03
3/21/03
1/24/03
2/11/03
20
21.3
1 07%
200
219
110%
<0.2
<0.2
0.48
0.48
0.0%
<0.2
<0.2
-
200
0.48
1 05%
3/26/03
4/1 0/03
10
10.6
1 06%
250
258
1 03%
2/4/03
3/6/03
20
21.6
108%
200
206
103%
<0.2
<0.2
<0.2
<0.2
-
200
72
104%
4/8/03
4/30/03
10
11.0
1 1 0%
50
68.2
136%
CCC: Continuing Calibration Check; QC: Second Source Check
74
-------
Table 8.2. Typical Quality Performance Data for Analysis of c/s-DCE in Water. All Values are ug/L Unless Otherwise
Indicated continued
CCC Standard Nominal
CCC Standard Measured
Percent of Check Standard
QC Standard Nominal
QC Standard Measured
Percent of Check Standard
QC Standard Nominal
QC Standard Measured
Percent of Check Standard
Blank 1
Blank 2
Sample Analysis 1
Laboratory Duplicate 1
Relative Percent Difference
Sample Analysis 2
Laboratory Duplicate 2
Relative Percent Difference
Spike Concentration 1
Sample Concentration 1
Spike Recovery (Percent)
Spike Concentration 2
Sample Concentration 2
Spike Recovery (Percent)
Date Collected
Date Analyzed
CCC Standard Nominal
CCC Standard Measured
Percent of Check Standard
CCC Standard Nominal
CCC Standard Measured
Percent of Check Standard
CCC Standard Nominal
CCC Standard Measured
Percent of Check Standard
QC Standard Nominal
QC Standard Measured
Percent of Check Standard
QC Standard Nominal
QC Standard Measured
50
64
129%
20
22.1
1 1 0%
200
211
105%
<0.36
<0.36
<0.36
<0.36
-
<0.36
<0.36
-
200
<0.36
109%
4/21/03
5/20/03
10
10.3
103%
50
51.1
102%
100
102
102%
50
51.8
104%
100
109
109%
25
24.4
97.6%
50
57.7
115%
<0.14
<0.14
249
<0.14
-
100
40.7
88%
4/29/03
5/5/03
20
18.7
93%
200
195
10
9.4
94%
200
185
92%
<0.06
<0.06
56.7
57.7
1 .7%
100
18.8
95%
5/16/03
6/5/03
20
19.7
99%
50
46.6
250
260
1 04%
100
129
129%
<0.14
<0.14
<0.14
<0.14
-
0.42
0.46
9.1%
6/23/03
6/26/03
25
19.5
78%
100
91.8
91.8%
250
264
1 06%
50
46.6
93.2%
100
117
1 1 7%
50
76.3
153%
<0.14
<0.14
<0.14
<0.14
-
7/23/03
8/5/03
20
21
105%
200
206
CCC: Continuing Calibration Check; QC: Second Source Check
75
-------
Table 8.2. Typical Quality Performance Data for Analysis of c/s-DCE in Water. All Values are ug/L Unless Otherwise
Indicated continued
Percent of Check Standard
Blank 1
Blank 2
Sample Analysis 1
Laboratory Duplicate 1
Relative Percent Difference
Sample Analysis 2
Laboratory Duplicate 2
Relative Percent Difference
Spike Concentration 1
Sample Concentration 1
Spike Recovery (Percent)
Spike Concentration 2
Sample Concentration 2
Spike Recovery (Percent)
Date Collected
Date Analyzed
CCC Standard Nominal
CCC Standard Measured
Percent of Check Standard
CCC Standard Nominal
CCC Standard Measured
Percent of Check Standard
CCC Standard Nominal
CCC Standard Measured
Percent of Check Standard
QC Standard Nominal
QC Standard Measured
Percent of Check Standard
QC Standard Nominal
QC Standard Measured
Percent of Check Standard
Blank 1
Blank 2
Sample Analysis 1
Laboratory Duplicate 1
Relative Percent Difference
Sample Analysis 2
<0.14
<0.14
17.5
19.4
10.3%
8/7/03
8/12/03
20
20.4
102%
200
204
102%
<0.2
<0.2
<0.2
<0.2
-
<0.2
98%
<0.2
<0.2
<0.2
<0.2
-
200
0.34
104%
8/20/03
8/22/03
20
20.8
104%
200
200
100%
<0.2
<0.2
99.7
99.0
0.7%
93%
<0.06
<0.06
0.2
0.2
0.0%
1.2
1.2
0.0%
200
<0.06
94%
200
<0.06
87%
1 0/3/03
1 0/22/03
500
518
1 04%
20
20.2
1 01 %
200
191
95%
<0.06
<0.06
0.18
0.18
0.0%
16.6
<0.14
<0.14
<0.14
<0.14
-
140
105
28.6%
1 0/7/03
11/3/03
100
93.7
93.7%
100
96.2
96%
<0.06
<0.06
127
122
4.0%
103%
<0.2
<0.2
<0.2
<0.2
-
100
12.9
103%
10/24/03
10/28/03
25
21.8
87.2%
50
51
102%
100
95.6
95.6%
5
5.59
1 1 2%
50
46.3
92.6%
<0.14
<0.14
<0.14
<0.14
-
1.62
CCC: Continuing Calibration Check; QC: Second Source Check
76
-------
Table 8.2. Typical Quality Performance Data for Analysis of c/s-DCE in Water. All Values are ug/L Unless Otherwise
Indicated continued
Laboratory Duplicate 2
Relative Percent Difference
Spike Concentration 1
Sample Concentration 1
Spike Recovery (Percent)
Spike Concentration 2
Sample Concentration 2
Spike Recovery (Percent)
Date Collected
Date Analyzed
CCC Standard Nominal
CCC Standard Measured
Percent of Check Standard
CCC Standard Nominal
CCC Standard Measured
Percent of Check Standard
CCC Standard Nominal
CCC Standard Measured
Percent of Check Standard
QC Standard Nominal
QC Standard Measured
Percent of Check Standard
QC Standard Nominal
QC Standard Measured
Percent of Check Standard
Blank 1
Blank 2
Sample Analysis 1
Laboratory Duplicate 1
Relative Percent Difference
Sample Analysis 2
Laboratory Duplicate 2
Relative Percent Difference
Spike Concentration 1
Sample Concentration 1
Spike Recovery (Percent)
Spike Concentration 2
Sample Concentration 2
Spike Recovery (Percent)
<0.2
-
200
<0.2
103%
200
<0.2
100%
1/14/04
1/20/04
10
12.3
123%
100
104
104%
100
113
1 1 3%
50
46.9
94%
100
95.0
95%
<0.14
<0.14
200
250
97%
2/19/04
2/20/04
10
11.5
115%
50
57.7
115%
100
112
112%
10
16.9
169%
100
145
145%
<0.14
<0.14
<0.14
<0.14
-
<0.14
<0.14
-
15.7
5.6%
100
<0.06
94%
200
1.2
91%
6/4/04
6/17/04
20
20.7
1 03%
20
19.8
99%
20
20.8
1 04%
200
217
1 09%
<0.2
<0.2
<0.2
<0.2
-
<0.2
<0.2
-
100
<0.2
1 07%
100
<0.2
1 05%
100
0.2
88%
9/7/04
9/1 7/04
20
19.4
97%
<0.2
<0.2
<0.2
<0.2
-
<0.2
<0.2
-
100
<0.2
1 05%
100
<0.2
1 03%
1.58
2.5%
7/14/05
7/15/05
20
20.2
101%
200
210
105%
20
19.3
97%
20
21.1
105%
200
207
103%
<0.2
<0.2
0.73
0.73
0.0%
<0.2
<0.2
-
100
1.31
94%
100
<0.2
91%
CCC: Continuing Calibration Check; QC: Second Source Check
77
-------
Table 8.3. Typical Quality Performance Data for Analysis of Vinyl Chloride in Water. All Values are ug/L Unless
Otherwise Indicated
Date Collected
Date Analyzed
CCC Standard Nominal
CCC Standard Measured
Percent of Check Standard
CCC Standard Nominal
CCC Standard Measured
Percent of Check Standard
CCC Standard Nominal
CCC Standard Measured
Percent of Check Standard
QC Standard Nominal
QC Standard Measured
Percent of Check Standard
QC Standard Nominal
QC Standard Measured
Percent of Check Standard
Blank 1
Blank 2
Sample Analysis 1
Laboratory Duplicate 1
Relative Percent Difference
Sample Analysis 2
Laboratory Duplicate 2
Relative Percent Difference
Spike Concentration 1
Sample Concentration 1
Spike Recovery (Percent)
Spike Concentration 2
Sample Concentration 2
Spike Recovery (Percent)
Date Collected
Date Analyzed
CCC Standard Nominal
CCC Standard Measured
Percent of Check Standard
CCC Standard Nominal
CCC Standard Measured
Percent of Check Standard
10/23/02
11/7/02
20
20.1
101%
200
180
90.1%
<0.5
<0.5
12
9.93
18.9%
200
<0.5
98%
2/24/03
3/9/03
50
41
82%
200
156
78%
11/25/02
12/19/02
20
21.6
108%
200
208
104%
<0.3
<0.3
0.47
<0.3
-
200
20400
87%
3/6/03
4/2/03
10
10.9
109%
100
113
1 1 3%
12/23/02
1/7/03
10
8.78
88%
100
90.5
91%
250
219
88%
50
50.8
1 02%
50
44.3
88.6%
<0.22
<0.22
78.9
92.0
15.3%
50
<0.22
50%
50
<0.22
56%
3/11/03
3/21/03
1/24/03
2/11/03
20
21.3
1 07%
200
211
1 06%
<0.3
<0.3
<0.3
<0.3
-
<0.3
<0.3
-
200
<0.3
1 01 %
3/26/03
4/1 0/03
10
8.89
89%
250
288
115%
2/4/03
3/6/03
20
20.8
104%
200
196
98%
<0.3
<0.3
<0.3
<0.3
-
200
3.69
103%
4/8/03
4/30/03
10
7.62
76.2%
50
42.3
84.6%
CCC: Continuing Calibration Check; QC: Second Source Check
78
-------
Table 8.3. Typical Quality Performance Data for Analysis of Vinyl Chloride in Water. All Values are ug/L Unless
Otherwise Indicated continued
CCC Standard Nominal
CCC Standard Measured
Percent of Check Standard
QC Standard Nominal
QC Standard Measured
Percent of Check Standard
QC Standard Nominal
QC Standard Measured
Percent of Check Standard
Blank 1
Blank 2
Sample Analysis 1
Laboratory Duplicate 1
Relative Percent Difference
Sample Analysis 2
Laboratory Duplicate 2
Relative Percent Difference
Spike Concentration 1
Sample Concentration 1
Spike Recovery (Percent)
Spike Concentration 2
Sample Concentration 2
Spike Recovery (Percent)
Date Collected
Date Analyzed
CCC Standard Nominal
CCC Standard Measured
Percent of Check Standard
CCC Standard Nominal
CCC Standard Measured
Percent of Check Standard
CCC Standard Nominal
CCC Standard Measured
Percent of Check Standard
QC Standard Nominal
QC Standard Measured
Percent of Check Standard
QC Standard Nominal
50
40
80%
20
23.3
1 1 7%
200
192
96%
<0.22
<0.22
<0.22
<0.22
-
<0.22
<0.22
-
200
<0.22
103%
4/21/03
5/20/03
10
10
100%
50
44.4
88%
100
98.0
98%
50
56.7
1 1 3%
100
113
1 1 3%
25
25.5
102%
50
58.4
1 1 7%
<0.22
<0.22
1290
1290
0.0%
4/29/03
5/5/03
20
22.3
111%
200
10
9.0
90%
200
180
90%
<0.49
<0.49
8.4
5.4
43.5%
100
3.3
125%
100
20.3
81%
5/16/03
6/5/03
20
23.5
118%
50
65.9
1 31 %
250
236
94%
100
76.7
77%
<0.22
<0.22
0.26
0.26
0.0%
<0.22
<0.22
-
6/23/03
6/26/03
25
20.4
81.6%
100
93.5
93.5%
250
252
1 01 %
50
24.4
49%
100
87.9
87.9%
50
55.6
1 1 1 %
<0.22
<0.22
0.41
0.38
7.6%
7/23/03
8/5/03
20
21.2
106%
200
CCC: Continuing Calibration Check; QC: Second Source Check
79
-------
Table 8.3. Typical Quality Performance Data for Analysis of Vinyl Chloride in Water. All Values are ug/L Unless
Otherwise Indicated continued
QC Standard Measured
Percent of Check Standard
Blank 1
Blank 2
Sample Analysis 1
Laboratory Duplicate 1
Relative Percent Difference
Sample Analysis 2
Laboratory Duplicate 2
Relative Percent Difference
Spike Concentration 1
Sample Concentration 1
Spike Recovery (Percent)
Spike Concentration 2
Sample Concentration 2
Spike Recovery (Percent)
Date Collected
Date Analyzed
CCC Standard Nominal
CCC Standard Measured
Percent of Check Standard
CCC Standard Nominal
CCC Standard Measured
Percent of Check Standard
CCC Standard Nominal
CCC Standard Measured
Percent of Check Standard
QC Standard Nominal
QC Standard Measured
Percent of Check Standard
QC Standard Nominal
QC Standard Measured
Percent of Check Standard
Blank 1
Blank 2
Sample Analysis 1
Laboratory Duplicate 1
Relative Percent Difference
Sample Analysis 2
<0.22
1.37
5.28
4.56
14.6%
8/7/03
8/12/03
20
22.1
1 1 0%
200
212
106%
<0.3
<0.3
<0.3
<0.3
-
<0.3
220
1 1 0%
<0.3
<0.3
<0.3
<0.3
-
200
1.59
101%
8/20/03
8/22/03
20
22.9
115%
200
233
1 1 6%
<0.3
<0.3
0.42
0.35
18.2%
<0.49
<0.49
<0.49
<0.49
-
<0.49
<0.49
-
200
<0.49
144%
200
<0.49
141%
1 0/3/03
1 0/22/03
500
470
94%
20
22.7
113%
200
183
92%
<0.49
<0.49
<0.49
<0.49
-
<0.49
<0.22
<0.22
0.22
0.34
42.9%
32.1
78.7
84.1%
1 0/7/03
11/3/03
100
89.4
89.4%
100
117
117%
<0.49
<0.49
97
91.2
6.2%
227
1 1 3%
<0.3
<0.3
<0.3
<0.3
-
100
<0.3
1 1 3%
10/24/03
10/28/03
50
41.7
83.4%
5
2.57
51%
50
30.3
62%
<0.22
<0.22
<0.22
<0.22
-
<0.22
CCC: Continuing Calibration Check; QC: Second Source Check
80
-------
Table 8.3. Typical Quality Performance Data for Analysis of Vinyl Chloride in Water. All Values are ug/L Unless
Otherwise Indicated continued
Laboratory Duplicate 2
Relative Percent Difference
Spike Concentration 1
Sample Concentration 1
Spike Recovery (Percent)
Spike Concentration 2
Sample Concentration 2
Spike Recovery (Percent)
Date Collected
Date Analyzed
CCC Standard Nominal
CCC Standard Measured
Percent of Check Standard
CCC Standard Nominal
CCC Standard Measured
Percent of Check Standard
CCC Standard Nominal
CCC Standard Measured
Percent of Check Standard
QC Standard Nominal
QC Standard Measured
Percent of Check Standard
QC Standard Nominal
QC Standard Measured
Percent of Check Standard
Blank 1
Blank 2
Sample Analysis 1
Laboratory Duplicate 1
Relative Percent Difference
Sample Analysis 2
Laboratory Duplicate 2
Relative Percent Difference
Spike Concentration 1
Sample Concentration 1
Spike Recovery (Percent)
Spike Concentration 2
Sample Concentration 2
Spike Recovery (Percent)
<0.3
-
200
<0.3
1 1 3%
200
<0.3
1 1 6%
1/14/04
1/20/04
10
9.97
100%
100
105
105%
100
108
108%
50
57.2
114%
100
113
1 1 3%
<0.22
<0.22
200
2.62
111%
2/19/04
2/20/04
10
10.7
107%
50
48.9
97.8%
100
103
103%
10
10.9
109%
100
101
101%
<0.22
<0.22
<0.22
<0.22
-
<0.22
<0.22
-
<0.49
-
100
<0.49
93%
200
3.4
87%
6/4/04
6/17/04
20
19.8
99%
20
20.8
1 04%
20
18.0
90%
200
192
96%
<0.3
<0.3
<0.3
<0.3
-
<0.3
<0.3
-
100
<0.3
1 01 %
100
<0.3
91%
100
4.6
89.5%
9/7/04
9/1 7/04
20
20.8
1 04%
<0.3
<0.3
<0.3
<0.3
-
<0.3
<0.3
-
100
<0.3
1 05%
100
<0.3
1 05%
<0.22
-
7/14/05
7/15/05
20
19.9
100%
200
169
84%
20
19.4
97%
20
20.6
103%
200
188
94%
<0.3
<0.3
<0.3
<0.3
-
<0.3
<0.3
-
100
<0.3
100%
100
<0.3
83%
CCC: Continuing Calibration Check; QC: Second Source Check
81
-------
Table 8.4. Typical Quality Performance Data for Analysis of Ethylene in Water or in Gas. The Values for Check
Standard Nominal and Check Standard Measured are ppm (v/v). All Other Values are mg/L Unless
Otherwise Indicated
Date Collected
Date Analyzed
Check Standard Nominal
Check Standard Measured
Percent of Check Standard
Check Standard Nominal
Check Standard Measured
Percent of Check Standard
Check Standard Nominal
Check Standard Measured
Percent of Check Standard
Check Standard Nominal
Check Standard Measured
Percent of Check Standard
Check Standard Nominal
Check Standard Measured
Percent of Check Standard
Blank 1
Blank 2
Sample Analysis 1
Laboratory Duplicate 1
Relative Percent Difference
Sample Analysis 2
Laboratory Duplicate 2
Relative Percent Difference
Spike Concentration
Sample Concentration
Spike Recovery (Percent)
Date Collected
Date Analyzed
Check Standard Nominal
Check Standard Measured
Percent of Check Standard
Check Standard Nominal
Check Standard Measured
Percent of Check Standard
Check Standard Nominal
Check Standard Measured
1 0/23/02
11/13/02
10
11
110%
10
10.7
1 07%
100
104
1 04%
<0.00028
<0.00028
<0.00028
-
<0.00028
<0.00028
-
0.0275
<0.00028
1 04%
2/20/03
2/20/03
10
10.5
1 05%
10
10.2
1 02%
10
10.3
11/25/02
12/4/02
10
10.5
105%
100
108
108%
100
106
106%
10000
10200
102%
<0.00028
<0.00028
<0.00028
-
0.275
<0.00028
1 1 3%
3/6/03
3/11/03
10
10.1
101%
100
107
107%
100
102
12/23/02
12/23/02
10
10.9
109%
10
11.1
111%
10
10.9
109%
10
11.1
111%
100
106
106%
<0.28*
3/11/03
3/19/03
10
10.4
104%
100
106
106%
1/22/03
1/22/03
10
10.1
1 01 %
10
10.4
1 04%
100
109
1 09%
100
107
1 07%
1000
1010
1 01 %
<0.28*
<0.28*
3/25/03
3/25/03
10
10.7
1 07%
10
10.4
1 04%
10
10.5
2/4/03
2/7/03
10
10.9
1 09%
100
106
1 06%
100
108
1 08%
<0.00028
<0.00028
<0.00028
-
2.71
<0.00028
99%
4/8/03
4/14/03
10
10.3
1 03%
100
98
98%
100
106
*The values are ppm (v/v).
82
-------
Table 8.4. Typical Quality Performance Data for Analysis of Ethylene in Water or in Gas. The Values for Check
Standard Nominal and Check Standard Measured are ppm (v/v). All Other Values are mg/L Unless
Otherwise Indicated continued
Percent of Check Standard
Check Standard Nominal
Check Standard Measured
Percent of Check Standard
Check Standard Nominal
Check Standard Measured
Percent of Check Standard
Blank 1
Blank 2
Sample Analysis 1
Laboratory Duplicate 1
Relative Percent Difference
Sample Analysis 2
Laboratory Duplicate 2
Relative Percent Difference
Spike Concentration
Sample Concentration
Spike Recovery (Percent)
Date Collected
Date Analyzed
Check Standard Nominal
Check Standard Measured
Percent of Check Standard
Check Standard Nominal
Check Standard Measured
Percent of Check Standard
Check Standard Nominal
Check Standard Measured
Percent of Check Standard
Check Standard Nominal
Check Standard Measured
Percent of Check Standard
Check Standard Nominal
Check Standard Measured
Percent of Check Standard
Blank 1
Blank 2
1 03%
100
106
1 06%
1000
1080
1 08%
4/21/03
5/1/03
10
11
110%
100
109
1 09%
<0.00057
102%
<0.00057
<0.00057
<0.00057
-
<0.00057
<0.00057
-
0.964
<0.00057
1 1 6%
4/29/03
5/6/03
10
10.2
1 02%
100
108
108%
<0.00057
<0.00028
<0.00028
<0.00028
-
2.68
<0.00028
99%
5/15/03
5/15/03
10
10.3
103%
10
10.4
104%
10
10.2
102%
10
10.5
105%
100
107
107%
1 05%
10
10.4
1 04%
6/20/03
6/20/03
10
10.3
1 03%
10
10.4
1 04%
10
10.6
1 06%
10
10.3
1 03%
10
10.4
1 04%
1 06%
1000
1030
1 03%
10000
10800
1 08%
<0.00057
<0.00057
<0.00057
-
<0.00057
<0.00057
-
1.94
<0.00057
1 00%
7/23/03
7/31/03
10
9.92
99%
100
107
1 07%
<0.00057
<0.00057
*The values are ppm (v/v).
83
-------
Table 8.4. Typical Quality Performance Data for Analysis of Ethylene in Water or in Gas. The Values for Check
Standard Nominal and Check Standard Measured are ppm (v/v). All Other Values are mg/L Unless
Otherwise Indicated continued
Sample Analysis 1
Laboratory Duplicate 1
Relative Percent Difference
Sample Analysis 2
Laboratory Duplicate 2
Relative Percent Difference
Spike Concentration
Sample Concentration
Spike Recovery (Percent)
Date Collected
Date Analyzed
Check Standard Nominal
Check Standard Measured
Percent of Check Standard
Check Standard Nominal
Check Standard Measured
Percent of Check Standard
Check Standard Nominal
Check Standard Measured
Percent of Check Standard
Check Standard Nominal
Check Standard Measured
Percent of Check Standard
Check Standard Nominal
Check Standard Measured
Percent of Check Standard
Blank 1
Blank 2
Sample Analysis 1
Laboratory Duplicate 1
Relative Percent Difference
Sample Analysis 2
Laboratory Duplicate 2
Relative Percent Difference
Spike Concentration
Sample Concentration
Spike Recovery (Percent)
<0.00057
<0.00057
-
1.96
<0.00057
92%
8/6/03
8/6/03
10
10.9
1 09%
10
10.3
1 03%
100
106
1 06%
100
108
1 08%
10000
10900
1 09%
<0.57*
0.003
<0.00057
-
8/20/03
8/25/03
10
9.44
94.4%
100
106
106%
<0.00057
<0.00057
<0.00057
-
<0.00057
<0.00057
-
10/2/03
10/2/03
10
10.4
104%
10
9.9
99%
100
105
105%
100
106
106%
1000
1080
108%
<0.57*
1 0/7/03
1 0/1 6/03
100
101
1 01 %
1000
1030
1 03%
10000
10100
1 01 %
1 00000
97000
97%
<0.0003
<0.0003
<0.0003
-
0.039
0.037
5.3%
<0.00057
<0.00057
-
1 0/22/03
1 0/22/03
10
10.1
1 01 %
100
106
1 06%
1000
1080
1 08%
<0.57*
*The values are ppm (v/v).
84
-------
Table 8.4. Typical Quality Performance Data for Analysis of Ethylene in Water or in Gas. The Values for Check
Standard Nominal and Check Standard Measured are ppm (v/v). All Other Values are mg/L Unless
Otherwise Indicated continued
Date Collected
Date Analyzed
Check Standard Nominal
Check Standard Measured
Percent of Check Standard
Check Standard Nominal
Check Standard Measured
Percent of Check Standard
Check Standard Nominal
Check Standard Measured
Percent of Check Standard
Check Standard Nominal
Check Standard Measured
Percent of Check Standard
Check Standard Nominal
Check Standard Measured
Percent of Check Standard
Blank 1
Blank 2
Sample Analysis 1
Laboratory Duplicate 1
Relative Percent Difference
Sample Analysis 2
Laboratory Duplicate 2
Relative Percent Difference
Spike Concentration
Sample Concentration
Spike Recovery (Percent)
1/14/04
1/14/04
10
9.98
1 00%
100
104
1 04%
100
106
1 06%
1000
1070
1 07%
<0.57*
2/1 8/04
2/1 8/04
10
10.3
103%
10
9.82
98%
10
10.4
104%
10
10.5
105%
100
106
106%
<0.57*
6/1/04
6/1/04
10
10.1
101%
10
10.4
104%
10
10.2
1 02%
100
107
107%
100
108
108%
<0.57*
9/9/04
9/9/04
10
10.3
1 03%
10
10.4
1 04%
100
112
112%
100
110
110%
1000
1100
110%
<0.57*
7/1 3/05
7/1 3/05
10
10.8
1 08%
10
10.1
1 01 %
100
105
1 05%
100
104
1 04%
1000
1020
1 02%
<0.57*
<0.57*
*The values are ppm (v/v).
85
-------
Table 8.5. Typical Quality Performance Data for Analysis of Methane in Water or in Gas. The Values for Check
Standard Nominal and Check Standard Measured are ppm (v/v). All Other Values are mg/L Unless
Otherwise Indicated
Date Collected
Date Analyzed
Check Standard Nominal
Check Standard Measured
Percent of Check Standard
Check Standard Nominal
Check Standard Measured
Percent of Check Standard
Check Standard Nominal
Check Standard Measured
Percent of Check Standard
Check Standard Nominal
Check Standard Measured
Percent of Check Standard
Check Standard Nominal
Check Standard Measured
Percent of Check Standard
Blank 1
Blank 2
Sample Analysis 1
Laboratory Duplicate 1
Relative Percent Difference
Sample Analysis 2
Laboratory Duplicate 2
Relative Percent Difference
Spike Concentration
Sample Concentration
Spike Recovery (Percent)
Date Collected
Date Analyzed
Check Standard Nominal
Check Standard Measured
Percent of Check Standard
Check Standard Nominal
Check Standard Measured
Percent of Check Standard
Check Standard Nominal
Check Standard Measured
10/23/02
11/13/02
100
104
104%
100
106
106%
1000
1056
105%
10000
9340
93%
100000
99800
100%
<0.00003
0.2
0.198
1.0%
0.0551
0.0548
0.5%
0.0098
0.0009
96%
2/20/03
2/20/03
10
10.6
106%
10
9.99
100%
10
10.2
11/25/02
12/4/02
10
10.9
109%
100
106
106%
100
109
109%
10000
10000
100%
<0.0003
<0.0003
<0.0003
-
0.0978
<0.0003
114%
3/6/03
3/11/03
10
10.4
104%
100
109
109%
1000
1040
12/23/02
12/23/02
10
10.7
1 07%
10
10.6
1 06%
10
10.8
1 08%
100
107
1 07%
100
107
1 07%
<0.3*
3/11/03
3/19/03
10
10.5
1 05%
100
109
1 09%
1000
1050
1/22/03
1/22/03
10
10.4
1 04%
10
9.95
1 00%
10
9.88
99%
100
102
1 02%
100
106
1 06%
<0.3*
<0.3*
3/25/03
3/25/03
10
10.8
1 08%
10
9.85
99%
100
105
2/4/03
2/7/03
10
10.6
106%
100
110
1 1 0%
100
106
106%
1000
1060
106%
<0.0003
<0.0003
<0.0003
-
0.971
0.0015
106%
4/8/03
4/14/03
10
9.56
96%
100
97.9
98%
1000
1020
*The values are ppm (v/v).
86
-------
Table 8.5. Typical Quality Performance Data for Analysis of Methane in Water or in Gas. The Values for Check
Standard Nominal and Check Standard Measured are ppm (v/v). All Other Values are mg/L Unless
Otherwise Indicated continued
Percent of Check Standard
Check Standard Nominal
Check Standard Measured
Percent of Check Standard
Check Standard Nominal
Check Standard Measured
Percent of Check Standard
Blank 1
Blank 2
Sample Analysis 1
Laboratory Duplicate 1
Relative Percent Difference
Sample Analysis 2
Laboratory Duplicate 2
Relative Percent Difference
Spike Concentration
Sample Concentration
Spike Recovery (Percent)
Date Collected
Date Analyzed
Check Standard Nominal
Check Standard Measured
Percent of Check Standard
Check Standard Nominal
Check Standard Measured
Percent of Check Standard
Check Standard Nominal
Check Standard Measured
Percent of Check Standard
Check Standard Nominal
Check Standard Measured
Percent of Check Standard
Check Standard Nominal
Check Standard Measured
Percent of Check Standard
Blank 1
Blank 2
102%
10
10.1
101%
100
107
107%
4/21/03
5/1/03
10
11
1 1 0%
100
104
104%
1000
1070
107%
10000
10300
103%
100000
98200
98%
<0. 00042
104%
10000
10100
101%
100000
100000
100%
<0.00042
0.086
0.0854
0.7%
<0.00042
<0.00042
-
0.964
0.007
93%
4/29/03
5/6/03
100
102
102%
10000
10200
102%
100000
97300
97%
<0.00042
1 05%
10000
10300
1 03%
<0.00003
0.007
0.007
0.0%
0.964
<0.00003
1 08%
5/15/03
5/15/03
10
10.1
1 01 %
10
10.5
1 05%
100
102
1 02%
1000
1070
1 07%
10000
10300
1 03%
1 05%
1000
1060
1 06%
10000
9790
98%
6/20/03
6/20/03
10
10.4
1 04%
10
10.3
1 03%
100
100
1 00%
1000
1060
1 06%
10000
10200
1 02%
1 02%
10000
9900
99%
100000
95900
96%
<0. 00042
0.002
0.002
0.0%
0.147
0.145
1 .4%
0.957
0.019
89%
7/23/03
7/31/03
100
101
101%
100
98.8
99%
1000
1030
103%
10000
9850
99%
100000
95300
95%
<0. 00042
<0. 00042
*The values are ppm (v/v).
87
-------
Table 8.5. Typical Quality Performance Data for Analysis of Methane in Water or in Gas. The Values for Check
Standard Nominal and Check Standard Measured are ppm (v/v). All Other Values are mg/L Unless
Otherwise Indicated continued
Sample Analysis 1
Laboratory Duplicate 1
Relative Percent Difference
Sample Analysis 2
Laboratory Duplicate 2
Relative Percent Difference
Spike Concentration
Sample Concentration
Spike Recovery (Percent)
Date Collected
Date Analyzed
Check Standard Nominal
Check Standard Measured
Percent of Check Standard
Check Standard Nominal
Check Standard Measured
Percent of Check Standard
Check Standard Nominal
Check Standard Measured
Percent of Check Standard
Check Standard Nominal
Check Standard Measured
Percent of Check Standard
Check Standard Nominal
Check Standard Measured
Percent of Check Standard
Blank 1
Blank 2
Sample Analysis 1
Laboratory Duplicate 1
Relative Percent Difference
Sample Analysis 2
Laboratory Duplicate 2
Relative Percent Difference
Spike Concentration
Sample Concentration
Spike Recovery (Percent)
0.265
0.229
14.6%
0.964
0.001
97%
8/6/03
8/6/03
100
102
102%
100
99.9
100%
1000
1060
106%
10000
10300
103%
100000
97800
98%
<0.42*
12.3
12.9
4.8%
8/20/03
8/25/03
100
101
101%
100
95.8
96%
1000
1020
102%
10000
9810
98%
100000
94700
95%
<0.00042
1.70
1.35
23.0%
2.44
2.78
13.0%
1 0/2/03
1 0/2/03
10
9.6
96%
100
101
1 01 %
100
102
1 02%
1000
1070
1 07%
10000
10300
1 03%
<0.42*
1 0/7/03
10/16/03
100
107
1 07%
10
10.1
1 01 %
100
103
1 03%
1000
1050
1 05%
<0.0001
2.15
2.15
0.0%
3.85
3.36
13.6%
7.28
7.02
3.6%
10/22/03
10/22/03
10
10.3
103#
100
103
103%
1000
1070
107%
10000
10300
103%
100000
98500
99%
<0.42*
*The values are ppm (v/v).
-------
Table 8.5. Typical Quality Performance Data for Analysis of Methane in Water or in Gas. The Values for Check
Standard Nominal and Check Standard Measured are ppm (v/v). All Other Values are mg/L Unless
Otherwise Indicated continued
Date Collected
Date Analyzed
Check Standard Nominal
Check Standard Measured
Percent of Check Standard
Check Standard Nominal
Check Standard Measured
Percent of Check Standard
Check Standard Nominal
Check Standard Measured
Percent of Check Standard
Check Standard Nominal
Check Standard Measured
Percent of Check Standard
Check Standard Nominal
Check Standard Measured
Percent of Check Standard
Blank 1
Blank 2
Sample Analysis 1
Laboratory Duplicate 1
Relative Percent Difference
Sample Analysis 2
Laboratory Duplicate 2
Relative Percent Difference
Spike Concentration
Sample Concentration
Spike Recovery (Percent)
1/14/04
1/14/04
100
98.4
98%
100
101
101%
1000
1070
107%
10000
10200
102%
100000
97700
98%
<0.42*
2/18/04
2/18/04
10
10.7
107%
100
102
102%
1000
1070
107%
10000
10300
103%
100000
98000
98%
<0.42*
6/1/04
6/1/04
10
10.9
1 09%
100
107
1 07%
1000
1080
1 08%
10000
10500
1 05%
1 00000
99200
99.2%
<0.42*
9/9/04
9/9/04
10
10.7
1 07%
100
105
1 05%
1000
1110
1 1 1 %
10000
11200
112%
1 00000
99800
99.8%
<0.42*
7/1 3/05
7/1 3/05
10
9.88
98.8%
100
101
101%
1000
1040
104%
10000
10300
103%
100000
99800
99.8%
<0.42*
<0.42*
*The values are ppm (v/v).
89
-------
Table 8.6. Typical Quality Performance Data for Analysis of Hydrogen in Gas. All Values are ppm Unless Otherwise
Indicated
Date Collected
Date Analyzed
Check Standard Nominal
Check Standard Measured
Percent of Check Standard
Check Standard Nominal
Check Standard Measured
Percent of Check Standard
Check Standard Nominal
Check Standard Measured
Percent of Check Standard
Check Standard Nominal
Check Standard Measured
Percent of Check Standard
Check Standard Nominal
Check Standard Measured
Percent of Check Standard
Blank 1
Blank 2
Sample Analysis 1
Laboratory Duplicate 1
Relative Percent Difference
Sample Analysis 2
Laboratory Duplicate 2
Relative Percent Difference
Date Collected
Date Analyzed
Check Standard Nominal
Check Standard Measured
Percent of Check Standard
Check Standard Nominal
Check Standard Measured
Percent of Check Standard
Check Standard Nominal
Check Standard Measured
Percent of Check Standard
Check Standard Nominal
Check Standard Measured
10/23/02
11/5/02
1
1.037
103%
5
5.479
1 1 0%
0.5
0.46
96%
1
1.03
103%
5
5.42
108%
<0.19
0.348
0.351
0.9%
2.847
2.042
32.9%
3/25/03
3/25/03
0.5
0.51
102%
1
0.99
99%
1
1
100%
5
4.98
12/23/02
12/23/02
0.5
0.59
1 1 8%
1.19
1.26
92.4%
2.5
2.45
98%
10.1
9.34
98%
10.1
11.0
109%
<0.19
4/8/03
4/14/03
0.5
0.473
95%
0.5
0.534
107%
1
1.03
103%
1
1.05
2/20/03
2/20/03
0.5
0.49
99%
0.5
0.5
1 00%
1
1.07
1 07%
5
5.6
110%
10
9.96
99%
<0.19
4/21/03
5/1/03
1
0.943
94%
1
0.899
90%
5
5.05
1 01 %
10
10.4
3/6/03
3/11/03
0.5
0.54
1 08%
0.5
0.486
97%
1
1.094
1 09%
5
5.489
1 09%
<0.19
0.74
2.67
113.2%
0.33
0.36
8.7%
4/29/03
5/6/03
0.5
0.534
1 07%
1
1.04
1 04%
5
5.06
1 01 %
10
10.5
3/11/03
3/1 9/03
1
0.98
98%
0.5
0.52
104%
1
1.06
106%
5
5.32
106%
<0.19
0.564
0.601
6.4%
5/15/03
5/15/03
0.5
0.533
94%
1
0.939
94%
5
5.01
100%
10
10.6
The values are ppm (v/v).
90
-------
Table 8.6. Typical Quality Performance Data for Analysis of Hydrogen in Gas. All Values are ppm Unless Otherwise
Indicated continued
Percent of Check Standard
Check Standard Nominal
Check Standard Measured
Percent of Check Standard
Blank 1
Blank 2
Sample Analysis 1
Laboratory Duplicate 1
Relative Percent Difference
Sample Analysis 2
Laboratory Duplicate 2
Relative Percent Difference
Date Collected
Date Analyzed
Check Standard Nominal
Check Standard Measured
Percent of Check Standard
Check Standard Nominal
Check Standard Measured
Percent of Check Standard
Check Standard Nominal
Check Standard Measured
Percent of Check Standard
Check Standard Nominal
Check Standard Measured
Percent of Check Standard
Check Standard Nominal
Check Standard Measured
Percent of Check Standard
Blank 1
Blank 2
Sample Analysis 1
Laboratory Duplicate 1
Relative Percent Difference
Sample Analysis 2
Laboratory Duplicate 2
Relative Percent Difference
99%
5
4.9
98%
6/20/03
6/20/03
0.5
0.5
100%
1
1.04
104%
10
10.5
105%
<0.19
105%
5
4.56
91%
<0.19
2.08
2.59
21 .8%
0.669
0.744
10.6%
7/23/03
7/31/03
0.5
0.523
105%
1
1.17
1 1 7%
5
4.4
88%
10
11.1
111%
20
18.8
94%
<0.19
13.9
13.1
5.9%
1 04%
20
21.3
1 06%
<0.19
<0.5
<0.5
-
8/6/03
8/6/03
0.5
0.461
92%
1
0.959
96%
5
5.45
1 09%
10
11
110%
20
18.5
93%
<0.19
1 05%
20
19.5
98%
<0.19
2.5
2.1
1 7.4%
8/20/03
8/25/03
0.5
0.51
1 02%
1
0.96
96%
20
21.6
1 08%
<0.19
0.48
0.42
13.3%
106%
20
20.5
103%
<0.19
10/2/03
10/2/03
0.5
0.585
1 1 7%
1
0.964
96%
5
5.55
1 1 1 %
10
8.89
89%
20
16.9
85%
0.232
10/7/03
10/16/03
1
1.02
1 02%
5
4.6
92%
10
9.52
95%
20
17.6
88%
<0.19
134
136
1 .5%
The values are ppm (v/v).
91
-------
Table 8.6. Typical Quality Performance Data for Analysis of Hydrogen in Gas. All Values are ppm Unless Otherwise
Indicated continued
Date Collected
Date Analyzed
Check Standard Nominal
Check Standard Measured
Percent of Check Standard
Check Standard Nominal
Check Standard Measured
Percent of Check Standard
Check Standard Nominal
Check Standard Measured
Percent of Check Standard
Check Standard Nominal
Check Standard Measured
Percent of Check Standard
Check Standard Nominal
Check Standard Measured
Percent of Check Standard
Blank 1
Blank 2
Sample Analysis 1
Laboratory Duplicate 1
Relative Percent Difference
Sample Analysis 2
Laboratory Duplicate 2
Relative Percent Difference
10/22/03
10/22/03
0.5
0.444
1 1 3%
1
0.991
99%
5
5.23
105%
10
8.98
90%
20
19.5
98%
0.244
1/14/05
1/14/05
1
1.12
112%
5
4.30
1 1 6%
10
9.45
95%
20
18.4
109%
<0.19
2/18/04
2/18/04
1
1.07
1 07%
5
5.13
1 03%
10
9.72
97.2%
10
9.9
99%
20
19
95%
<0.19
6/1/04
6/1/04
0.5
0.586
117%
1
0.931
93.1%
5
4.42
88.4%
10
11.5
115%
20
18.1
90.5%
<0.19
9/9/04
9/9/04
0.5
0.465
93%
1
1.13
1 1 3%
5
5.67
1 1 3%
10
11.1
1 1 1 %
20
18.6
93%
<0.19
7/13/05
7/13/05
0.5
0.459
92%
1
0.937
93.7%
5
4.88
97.6%
10
10.1
101%
20
19.2
96%
<0.19
<0.19
The values are ppm (v/v).
92
-------
Table 8.7. Typical Quality Performance Data for Analysis of Nitrate Plus Nitrite Nitrogen in Water. All Values are
mg/L Unless Otherwise Indicated
Date Collected
Date Analyzed
CCC Standard Nominal
CCC Standard Measured
Percent of Check Standard
CCC Standard Nominal
CCC Standard Measured
Percent of Check Standard
CCC Standard Nominal
CCC Standard Measured
Percent of Check Standard
QC Standard Nominal
QC Standard Measured
Percent of Check Standard
QC Standard Nominal
QC Standard Measured
Percent of Check Standard
Blank 1
Blank 2
Sample Analysis
Laboratory Duplicate
Relative Percent Difference
Spike Concentration
Sample Concentration
Spike Recovery (Percent)
Date Collected
Date Analyzed
CCC Standard Nominal
CCC Standard Measured
Percent of Check Standard
CCC Standard Nominal
CCC Standard Measured
Percent of Check Standard
CCC Standard Nominal
CCC Standard Measured
Percent of Check Standard
QC Standard Nominal
QC Standard Measured
Percent of Check Standard
10/23/02
11/6/02
0.5
0.49
98%
1
0.97
97%
2.5
2.45
98%
7.06
7.06
100%
<0.1
<0.1
<0.1
<0.1
-
10
<0.1
107%
4/21/03
5/1 3/03
1
0.95
95%
5
4.99
100%
1
0.96
96%
2/4/03
2/7/03
2.5
2.36
94%
<0.1
0.62
0.59
5.0%
10
2.04
103%
4/29/03
5/13/03
1
0.95
95%
1
0.96
96%
3/6/03
3/20/03
2.5
2.53
1 01 %
1
0.97
97%
2.99
3.02
1 01 %
<0.1
1.58
1.57
0.6%
10
1.97
110%
7/23/03
8/6/03
2.5
2.39
95.6%
5
4.78
96%
5
4.74
95%
3/11/03
3/26/03
0.5
0.51
1 02%
1
1
1 00%
2.99
3.10
1 04%
<0.1
1.25
1.29
3.1%
10
0.03
1 06%
8/20/03
8/25/03
0.5
0.48
96%
1
0.92
92%
5
4.91
98.2%
10
9.8
98%
4/8/03
4/14/03
0.5
0.45
90%
1
0.93
93%
2.99
3.05
1 02%
<0.1
<0.1
<0.1
-
10
0.06
1 1 6%
10/7/03
10/28/03
0.5
0.5
100%
1
0.93
93%
5
5
100%
10
9.87
98.7%
CCC: Continuing Calibration Check; QC: Second Source Check
93
-------
Table 8.7. Typical Quality Performance Data for Analysis of Nitrate Plus Nitrite Nitrogen in Water. All Values are
mg/L Unless Otherwise Indicated continued
QC Standard Nominal
QC Standard Measured
Percent of Check Standard
Blank 1
Blank 2
Sample Analysis
Laboratory Duplicate
Relative Percent Difference
Spike Concentration
Sample Concentration
Spike Recovery (Percent)
13.3
12.7
95%
<0.1
<0.1
3.39
3.49
2.9%
10
1.26
1 1 6%
13.3
12.7
95%
<0.1
<0.1
<0.1
<0.1
-
10
0.04
109%
21.3
18.9
89%
<0.1
<0.1
11.2
11.1
0.9%
5
<0.004
84.6%
<0.1
<0.1
<0.1
<0.1
-
10
1.86
1 09%
<0.1
<0.1
<0.1
<0.1
-
5
0.04
101%
CCC: Continuing Calibration Check; QC: Second Source Check
94
-------
Section 9.
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