United States
Environmental Protection
Agency
Wastewater Technology Fact Sheet
Bacterial Source Tracking
INTRODUCTION
Pathogens are a major pollutant of water bodies
nationwide according to many states' Clean Water Act
303(d) reports. Various sources contribute pathogens
to contaminated waters, including fecal pollution from
humans, wildlife, and livestock. Besides being potential
pathogens, fecal bacteria (such as Escherichia coif)
can indicate the presence of other waterborne
pathogens. Bacteria from human sources may indicate
the presence of human viruses, while bacteria from wild
and domestic animals may indicate the presence of the
parasites Giardia or Cryptosporidia. The presence
of any fecal bacteria in drinking water is considered a
health hazard. Knowing the source(s) of bacteria in a
water body or water supply is of great value in the
remediation and prevention of further bacterial
contamination. However, it can be difficult to address
water quality impairment effectively without a reliable
method to determine the source of contamination.
Bacterial Source Tracking (BST) is new methodology
used to determine the source of fecal pathogen
contamination in environmental samples.
There are many BST methods available and more are
under development. Interest in applying these
techniques stems from EPA's recent implementation of
the Total Maximum Daily Load (TMDL) study, as
BST techniques appear to provide the best method to
determine the origins of fecal contamination in water
bodies. Projects to develop TMDLs for fecal coliforms
and to design and implement best management
practices (BMPs) to reduce fecal loading in water may
benefit from BST technology (Hager, 2001). This fact
sheet discusses BST methods and presents examples
of BST application to TMDL development and
implementation.
DESCRIPTION
Potential sources of fecal bacteria are generally
grouped into three major categories: human, livestock,
or wildlife. In more urban watersheds, a fourth
category of pets or dogs may be added. Each source
produces unique, identifiable strains of fecal bacteria
because the intestinal environments and selective
pressures to which the bacteria are subjected differ
from source to source.
BST may use one of several methods to differentiate
between potential sources of fecal contamination, all of
which follow a common sequence of analysis. First, a
differentiable characteristic, or fingerprint (such as
antibiotic resistance or DNA), must be selected to
identify various strains of bacteria. A representative
library of bacterial strains and their fingerprints must
then be generated from the human and animal sources
that may impact the water body. Indicator bacteria
fingerprints from the polluted water body are compared
to those in the library and assigned to the appropriate
source category based on fingerprint similarity. BST
methods can be grouped as molecular or non-
molecular methods, according to the characteristic
used to identify or fingerprint the bacteria. Table 1
summarizes the classification of various BST methods.
Molecular methods are also referred to as "DNA
fingerprinting" and are based on the unique genetic
makeup of different strains of fecal bacteria. Molecular
methods rely on genetic variation as the fingerprint to
identify the source of fecal contamination. Three
molecular BST methods are commonly used, including
ribotyping (RT), polymerase chain reaction (PCR), and
pulsed-field gel electrophoresis (PFGE). Procedures
for the RT and PFGE methods are relatively similar
among multiple studies, but substantially different
variations are reported when using PCR methods
(Hagedorn, 2001).
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TABLE 1 CLASSIFICATION OF BST
METHODS
Molecular methods (DMA fingerprinting)
Ribotyping (RT)
Polymerase chain reaction (PCR)
Pulsed-field gel electrophoresis (PFGE)
Non-molecular methods
Biochemical methods
Antibiotic resistance analysis (ARA)
Cell wall fatty acid methyl ester
(FAME)
F-specific coliphage typing
Carbon utilization (BIOLOG)
Chemical methods
Caffeine detection
Optical brightener detection
Source: Parsons, 2001.
Non-molecular methods use non-genetic
characteristics as the fingerprint or basis to differentiate
the source of fecal bacteria, and may be further
subdivided between biochemical and chemical
methods. Biochemical methods are based on the
ability of an organism's genes to actively produce a
biochemical substance. The type and quantity of the
substance(s) produced form the bacterial fingerprint.
Antibiotic resistance analysis (ARA) is the most
commonly used biochemical BST method. Other
biochemical methods, such as cell wall analysis of fatty
acid methyl ester (FAME), F-specific coliphage typing,
and carbon source utilization (BIOLOG system), are
under development. Chemical methods do not detect
the presence of fecal bacteria, but rely on the
identification of compounds that co-occur with fecal
bacteria in human wastewater to differentiate the
source of fecal pollution. Thus, chemical methods can
only determine whether or not the source of fecal
pathogens is human (Hagedorn, 2001). Examples of
compounds used in chemical BST include caffeine and
optical brighteners commonly used in laundry
detergents.
APPLICABILITY
BST is intended to aid in identifying sources (e.g.,
human, livestock, or wildlife) of fecal contamination in
water bodies. Several states have started to use DNA
fingerprinting to target water quality problems and
formulate a mitigation strategy (Pelley, 1998;
Blankenship, 1996). These techniques can also be
used to direct implementation of effective BMPs to
remove or reduce fecal contamination. For example,
two New Hampshire communities are performing BST
surveys (using the RT method) to determine the
contribution of bacterial contamination from several
specific sources so that BMPs may be put in place to
help rehabilitate water quality. The following is a
summary of one representative survey.
Hampton Harbor, New Hampshire
Hampton Harbor is a tidally dominated, shallow
estuary located at the extreme southeast corner of New
Hampshire. The Hampton Harbor clamflats are closed
for clam harvesting during September and October due
to elevated fecal coliform levels. The flats are open
from November through May but close temporarily if
the rainfall exceeds 0.25 inches. These clamflats are
popular, productive, and accessible to the public.
Despite the construction of a new wastewater
treatment facility in the Town of Seabrook, the bacteria
levels often exceed the limits set by the New
Hampshire Shellfish Program, resulting in flat closures
and frustrated clam diggers. The potential sources of
bacterial contamination include birds (cormorants,
starlings, gulls), domestic animals (cats, dogs, goats,
horses), sanitary wastewater from wastewater
treatment plant failures, and wildlife. The intent of the
survey was to provide information to support
implementation of specific source controls and to
reduce the bacterial contamination to a level that
increases the number of days that the clamflats are
open for recreational harvesting.
Source classification provided by BST is often used in
the development and implementation of TMDL
projects. The information can be used to assign load
reduction allocations to sources in a watershed. For
example, BST techniques have been very useful to
regulatory officials in Virginia, where the ARA method
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has been used in seven TMDL watershed projects to
date.
Virginia Department of Conservation and
Recreation TMDLs
Over 300 stream segments were listed on the
Commonwealth of Virginia's 303(d) list for fecal
coliform bacteria violations. The uncertainty inherent in
identifying specific sources of fecal coliform bacteria in
the streams has hindered development of the TMDLs.
BST studies were applied to three stream segments
(Accotink Creek, Blacks Run, and Christians Creek)
to provide more accurate waste-load allocations and
enhance the development and defensibility of the
TMDLs. In each, the RT method was used to
determine the dominant sources of fecal coliform in the
impaired stream segments. The source-tracking
distribution determined in each segment were used to
modify and strengthen the waste-load allocations in the
TMDL watershed model. In addition, DNA testing is
underway in the Muddy Creek, Lower Dry River, Mill
Creek, and Pleasant Valley watersheds as part of their
TMDL implementation plans.
Cedar Creek, Hall Creek, Byers Creek, Hutton
Creek, and Lower Blackwater River were also placed
on the Commonwealth of Virginia's 303(d) list because
of violations of the fecal coliform bacteria water quality
standard. In fulfilling the state requirement to develop
a TMDL Implementation Plan, a framework was
established to reduce fecal coliform levels and achieve
the water quality goals for which TMDL allocations
were developed. BST analysis using the ARA method
was performed as part of the TMDL implementation.
Results indicated contributions of fecal coliform from
livestock, human, and wildlife sources. The wildlife
contribution alone was enough to push fecal coliform
levels beyond the standard at five sampling sites, while
human sources alone were high enough to violate the
standard at five sampling sites. Livestock sources were
sufficient to violate the standard at eight of eleven
sampling sites. In the Cedar and Hutton Creek
watersheds, livestock appeared to be an issue
throughout the watershed, while in the Hall/Byers
Creek watershed, livestock problems appeared limited
to smaller tributaries (e.g. Indian Run and Tattle
Branch). Human sources seemed most significant in
the Hall/Byers and Hutton Creek watersheds. The
quantity of control measures required during
implementation was determined and progress toward
end goals will be assessed during implementation
through tracking control measure installations and
continued water quality monitoring. Water quality
monitoring will include fecal coliform enumeration and
BST analysis. BST will provide an indication of the
effectiveness of specific groups of control measures,
specifically agricultural and urban. Implementation was
scheduled to begin in July 2001, with the final goal
being the delisting of the impaired segments from the
Commonwealth of Virginia's 303(d) List of Impaired
Waters by 2011.
ADVANTAGES AND DISADVANTAGES
In general, molecular BST methods may offer the most
precise identification of specific types of sources, but
are limited by high per-isolate costs and detailed, time-
consuming procedures. They are also not yet suitable
for assaying large numbers of samples in a reasonable
time frame. Biochemical BST methods are simpler,
faster, less expensive, and allow large numbers of
samples to be assayed in a short period of time.
BST development is so new that little research
comparing individual methods is complete. Results of
initial studies should become available over the next
few years.
The United States Department of Agriculture recently
funded a two-year study to compare three BST
methods using E. coll and Enterococcus: ARA,
PFGE, and RT. The merits of these methods will be
compared by a) accuracy, cost, and processing time;
b) determining the geographic range of the libraries;
and c) assessing the utility of each method in field
experiments. This comparison and development of
BST methodology is intended to refine BMP
implementation and focus resources on pollution
sources for water quality impairments.
The United States Geologic Survey is developing a
program to identify sources of fecal bacteria in the
waters of Berkeley County, West Virginia. At least
five methods will be tested for their ability to determine
animal sources of fecal bacteria in water samples (RT,
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PFGE, ARA, PCR, and BIOLOG carbon-utilization).
The three objectives of this project include building
source libraries for the five methods, comparing
methods to see which is best to determine sources, and
using the best method to identify sources of bacteria in
water resources of Berkeley County. This study will
provide source libraries for five promising methods to
identify bacteria sources, quantitative information on
which method(s) works best, determination of bacteria
sources for ten domestic wells that contain bacteria,
and determination of bacteria sources for five large
public-supply springs. The libraries and methods will
be applicable to both surface and ground water in
Berkeley County and surrounding areas.
PERFORMANCE
Many BST techniques are undergoing intensive
research that leads to rapid change in existing methods
and the creation of new methods. BST technologies
are quickly becoming proven and should be used by
federal and state regulatory agencies to address
sources of fecal bacterial pollution in water. Although
they are still experimental, BST methods represent the
best tools available to determine pathogen TMDL load
allocations and TMDL implementation plan
development. The following are examples of BST
technique performance in specific watershed studies.
Antibiotic Resistance Analysis (ARA) Method
Holmans Creek, Virginia
Holmans Creek watershed was listed on the
Commonwealth of Virginia's 1998 303(d) TMDL
Priority List of Impaired Waters based on violations of
the fecal coliform bacteria water quality standard.
There are several potential fecal coliform sources in this
watershed, including the non-point sources of wildlife,
livestock, individual residential sewerage systems, and
land application of manure and litter. Beef cattle,
poultry and dairy are the major livestock operations in
the Holmans Creek watershed. Residential sewerage
in the watershed consists of direct discharges from
straight pipes (homes without facilities to treat their
waste discharge), privies, and failing septic systems.
BST analysis using the ARA method was used to
classify sources of the fecal bacteria found in the
polluted water. Results of the BST analysis suggest
that the primary source of fecal pollution is human,
constituting just under half of the total fecal coliform
deposited into the waters of Holmans Creek. Wildlife
and cattle sources each contribute approximately one-
fourth of the total fecal coliform loads in the watershed.
Poultry were determined to be a minor contributor to
fecal coliform pollution in Holmans Creek, contributing
one-tenth of the total fecal load.
Stevenson Creek, Florida
The Stevenson Creek basin encompasses
approximately 6,000 acres in central Pinellas County,
Florida. In keeping with the objectives of the
Stevenson Creek Watershed Management Plan, a
BST study was initiated to identify the dominant
source(s) of fecal contamination to Stevenson Creek in
Clearwater, Florida. The ARA method was chosen
because it can assess the source of indicator organisms
based on a much larger subset of the bacterial
population than molecular methods can. The dominant
sources of fecal coliform over the course of the study
were wild animal, dog, and human, with the overall
trend indicating that wild animal isolates comprised the
majority of fecal coliforms obtained when colony
forming units (CFU) counts exceeded the acceptable
limit of 200 CFU per 100 mL. While human input was
not the major cause of elevated fecal coliform levels for
most of the samples analyzed for this study, the
domination of some small populations by human
isolates suggests that human sources contribute to
low-level background contamination. This occurs
when fecal coliform populations are low, near the
transition to dry season, and perhaps few isolates are
washed into surface waters from draining storm water.
Lowering water tables may also draw wastewater from
small, otherwise innocuous leaks. Overall, there was
little evidence of acute human fecal contamination on a
large scale; however, human sources may influence two
sampling sites, detectable despite the presence of fecal
coliforms from other sources. The human input alone
for these two sites in one month was high enough to
violate water quality standards.
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Pulsed-Field Gel Electrophoresis (PFGE)
Method
Eastern Shore, Virginia
DNA fingerprinting using PFGE proved helpful when
an oyster farmer on Virginia's Eastern Shore was faced
with the closure of his shellfish beds due to elevated
levels of E. coli. Failing septic tanks were assumed to
be the primary source of the fecal pollution, but a
survey of septic systems in the sparsely populated
watershed indicated that they were not the cause, and
it became necessary to identify other potential sources.
The highest levels of coliform bacteria were measured
in the small tidal inlets and rivulets of the wetlands
located upstream of local houses, shifting suspected
sources from human to other sources. Researchers
collected fecal samples from raccoon, waterfowl, otter,
muskrat, deer, and humans in the area and used DNA
fingerprinting to confirm the suspicion that the source
was not anthropogenic in nature. Comparing E. coli
from the shellfish beds against the fingerprints of known
strains in the DNA library, the researchers linked the
in-stream E. coli to deer and raccoon (mostly
raccoon). Several hundred animals, including 180
raccoon, were removed from areas adjacent to the
wetlands. E. coli levels subsequently declined by 1 to
2 orders of magnitude throughout the watershed,
allowing threatened areas of the tidal creeks to be
reopened to shellfishing.
Four Mile Run, Virginia
Four Mile Run is listed on the Commonwealth of
Virginia's 303(d) listing for elevated levels of fecal
coliform bacteria. The Northern Virginia Regional
Commission is currently developing a TMDL for the
Four Mile Run watershed, with the final draft to be
submitted to Virginia Department of Environmental
Quality by March 1, 2002. Four Mile Run is an urban
stream with no agricultural runoff. The watershed is
home to 183,000 people, just over 9,000 per square
mile. The dominant land use in the watershed is
medium to high density residential housing. Seven
central business districts exist within this 20 square mile
watershed, and two high-capacity interstates pass
through the watershed along with numerous primary
and secondary roadways. The watershed is
approximately 40 percent impervious. A large pet
population accompanies the dense human population in
the watershed. As to potential fecal sources, there is
little manufacturing industry to generate point source
discharges and there are no combined sewers in the
majority of the watershed. Sanitary sewers serve more
than 99.9 percent of the watershed population. The
number of septic systems in the watershed is believed
to be less than 50. The PFGE method of BST analysis
was conducted on E. coli DNA from seasonally varied
stream and sediment samples in the watershed. Results
of the analysis show that waterfowl contribute over
one-third (38 percent) of the bacteria, humans and pets
together account for over one-fourth (26 percent), and
raccoons account for 15 percent of the contamination,
with deer (9 percent) and rats (11 percent) also
contributing. The predominant non-human sources
include wildlife species with intimate association with
the waterways.
Ribotyping (RT) method
Little Soos Creek, Washington
A BST survey was designed to help characterize
sources of fecal coliform bacterial contamination in
Little Soos Creek in southeast King County,
Washington, in response to the impact of existing and
anticipated urban development in the area. Little Soos
Creek has historically been categorized as a Class A
stream, but violates fecal coliform standards for this
classification. The goal of the BST survey was to help
determine the contribution to contamination of the
stream from two potential sources: livestock on hobby
farms and ranches adjacent to the stream and on-site
septic systems close to the stream in highly permeable
soils. Other animal sources were also considered.
Genetic fingerprinting (using ribosomal RNA typing or
RT) was performed on E. coli isolates to effectively
match specific strains of E. coli from a contaminated
site in the stream to its source. The intent was to
provide information to support implementation of
specific source controls. The study identified the
sources of approximately three-fourths of the fecal
coliform contamination, with the primary sources
determined to be cows, dogs, and horses. Although
septage was identified as a contributor to the
contamination problem, it was not indicated as a major
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source. However, even low levels of contribution from
septage suggest the potential for Little Soos Creek to
harbor a number of human viral, bacterial, and parasitic
pathogens associated with human sources. For this
reason, further investigation of the contribution from
septic systems and of human exposure (particularly
children) to the stream may be warranted.
Lower Boise River, Idaho
The Lower Boise River watershed from Lucky Peak
Reservoir to the Snake River near Parma contains
almost one-third of Idaho's population and four major
municipalities, including the city of Boise. An arid
climate (approximately 10 inches of annual rainfall)
makes irrigation a requirement on most farmland. This
irrigation coupled with reuse of pasture water on
irrigated fields results in the contribution of non-point
discharge of fecal coliform bacteria to the Lower Boise
River. In 1994, the Idaho Department of
Environmental Quality (IDEQ) placed the Lower Boise
River on the 303(d) list for impairment of primary and
secondary contact designated uses because fecal
coliform levels exceeded state standards. A draft
TMDL was completed and submitted to the USEPA
on December 1998 and approved on January 2001
with implementation plan due July 2001. The TMDL
indicates that bacteria discharge loads will require more
than 95 percent reductions from non-point source
bacteria loadings to meet the primary contact bacteria
standard. A DNA fingerprinting of coliform bacteria
was conducted to focus bacteria reduction
improvement. E. coll cultures were grown from fecal
samples of cows, sheep, humans, ducks, and geese,
and DNA from these samples was identified. The
maj or bacteria sources in the watershed identified using
the RT method were waterfowl, humans, pets, and
cattle/horses. Waterfowl were clearly the largest
source. The major advantage of using the DNA
fingerprinting tool is the ability to develop accurate
control measures (BMPs) in terms of bacterial sources.
Prior to this study, IDEQ knew there were bacteria
problems, but did not know where to focus control
measures. The results of the BST analysis identify the
major sources, allowing IDEQ to strategically place
BMPs.
University of Georgia/USDA RT comparison
BST methods, including RT, rely on a database of
known source fingerprints to identify environmental
isolates of fecal bacteria. It is not well understood to
what degree these known source fingerprints are
biogeographically variable. This is important because
a fingerprint database developed for one state or region
may or may not be applicable to another. The
objective of a University of Georgia/USDA study
(Hartel et al, 2002) was to use the RT method of BST
analysis to determine the geographic variability of the
fecal bacterium, E. coli, from one location in Idaho and
three locations in Georgia for four animals: cattle,
horse, swine, and poultry. The study identified distance
from the source sample to the watershed as a key
variable for cattle and horses, but not for swine and
poultry. When theE1. coll ribotypes among the animals
were compared at one location, the relative percent
difference between them was 86, 89, 81, and 79 for
each of the four locations, suggesting good ribotype
separation among host animal species at one location.
Achieving a high degree of accuracy in matching
environmental isolates of fecal bacteria to a host origin
database depends on having a large number of isolates
for comparison and using a distance of 175 km or less
(at least for certain host animal species).
COSTS
Given the fact that many BST methods are still in the
research and development phase, there is great
variation for cost per sample (or per isolate) among
different laboratories. Factors that affect cost include
the following:
Analytical method - Molecular BST methods (e.g.
RT) are generally more expensive than non-molecular
methods. In addition, automated techniques are more
expensive but less labor-intensive than manual
techniques for the same method (such as RT).
Size of the database - It is not known what size
database or library of bacterial isolates from known
fecal sources is required for accurate source prediction
in a given watershed. Considerations in the
development of the BST library include the size of the
watershed, the diversity of animal species and human
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sources that may significantly impact water quality, and
the heterogeneity of the population within a given
source species. In many studies, the number of isolates
required to develop the known source database may
make up the majority of total isolates analyzed,
constituting a large fraction of the total cost for the
study.
Number of environmental isolates - The number of
isolates that must be analyzed from the water body of
interest varies among study sites. There may be
multiple isolates from each water sample taken, with
costs generally calculated per isolate.
Level of accuracy - Cost increases in proportion with
accuracy or the percentage of isolates classified
correctly. In some cases 80 percent is considered the
lowest acceptable level of accuracy. More studies are
needed to determine the level of accuracy achievable
by each BST method.
The cost for BST analysis ranges from $25 to $100
per isolate using molecular methods and from $10 to
$30 per isolate for non-molecular methods. These
costs are based on classifying a sample within an
accuracy range of 70 to 90 percent or higher.
However, there is little firm guidance on the required
number of reference fecal samples and isolates
extracted from each sample, causes wide variance in
the total cost for a fecal source tracking project. For
example, the cost for TMDL developments for
Accotink Creek, Blacks Run and Christians Creek in
Virginia by the USGS Richmond office was
approximately $617,000 (total for the three TMDLs),
while the New Hampshire Department of
Environmental Services spent approximately $225,000
to establish the ribotyping laboratory and partially
support the two source tracking surveys. In two
ongoing comparison studies, the cost of the San Juan
Creek Watershed Bacteria Study (California) is
$274,000 (excluding the expenses for laboratory
analysis), while the USD A grant to compare three BST
methods (RT, PFGE and ARA) is $310,000.
REFERENCES
Other Related Fact Sheets
Other EPA Fact Sheets can be found at the following
web address:
http://www.epa.gov/owm/mtbfact.htm
Overview
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ADDITIONAL INFORMATION
Dr. C. Andrew Carson
Department of Veterinary Pathobiology
College of Veterinary Medicine
University of Missouri
201 Connaway Hall
Columbia, MO 65211-5120
Dr. Charles Hagedorn
Department of Crop and Soil Environmental Sciences
Virginia Tech University
Blacksburg, VA 24061-0404
Dr. Valerie J. Harwood
Department of Biology
University of South Florida
4202 East Fowler Ave.
Tampa, FL 33620
Dr. Peter G. Hartel
Department of Crop and Soil Sciences
University of Georgia
3111 Plant Sciences Building
Athens, GA 30602-7274
Dr. Salina Parveen
USDA - Agricultural Research Service
Delaware State University
1200N. DuPontHwy
W.W. Baker Center
Dover, DE 19901
Dr. Michael J. Sadowsky
Department of Soil, Water, and Climate
University of Minnesota
258 Borlaug Hall
1991 Upper Buford Circle
St. Paul, MN 55108
Dr. Mansour Samadpour
Department of Environmental Health
School of Public Health & Community Medicine
University of Washington
Health Sciences Building
Box 357234
Seattle, WA 98195-7234
Dr. Bruce A. Wiggins
Department of Biology
James Madison University
Harrisonburg, VA 22807
The mention of trade names or commercial products
does not constitute endorsement or recommendation
for use by the U. S. Environmental Protection Agency
(EPA).
Office of Water
EPA 832-F-02-010
May 2002
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For more information contact:
Municipal Technology Branch
U.S. EPA
1200 Pennsylvania Avenue, NW
Mail Code 4204M
Washington, D.C. 20460
* 2002 *
THEYEAR'OF
CLEAN WATER
yvrre
Excellence in compliance through optimal tedmicat solutions
MUNICIPAL TECHNOLOGY *
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