iental Protection Development April 1,993
hington, DC 20460
ntinci WatGrbornG
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EPA's Office of Research and Development
The Office of Research and Development (ORD)
conducts an integrated program of scientific research
and development on the sources, transport and fate
processes, monitoring, control, and assessment of risk
and effects of environmental pollutants. These activi-
ties are implemented through its headquarters offices
and National Research Laboratories and Centers. The
research focuses on key scientific and technical is-
sues to generate knowledge supporting sound deci-
sions today, and to anticipate the complex challenges
of tomorrow. With a strong, forward-looking re-
search program, less expensive more effective solu-
tions can be pursued and irreversible damage to the
environment can be prevented.
Front cover photo by Lang Photography.
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"The reported case total for the epidemic nears
three-quarters of a million. Since the beginning
of the epidemic in January 1991, the total
number of reported cases is 746,968 with 6,448
deaths."
(Cholera Epidemic in the Americas, CDC Update, February 11, 1993)
Although the above-listed statis-
tics are alarming, the risk that exten-
sive outbreaks of waterbome cholera
will occur in the United States is
minimal. Effective treatment of
drinking water and sewage, coupled
with adequate personal hygiene
habits, has contributed to a success-
ful line of defense against the spread
of cholera in the U.S. Still, the ease
of international travel has guaran-
teed the import of a wide variety of
diseases not generally considered to
be native to North America. Addi-
tionally, although fatalities caused
by waterbome diseases have de-
clined dramatically in the U.S.
during this century, annual reports of
water-related, microorganism-
induced disease continue to number
in the thousands. Just one water-
bome outbreak of crypto sporidio sis
in western Georgia (1987), for
example, affected an estimated
13,000 people. In the "colonias"
(poor settlements along the Texas-
Mexico border), high levels of
disease have been associated with
the lack of public water supplies and
inadequate waste treatment. While
the words "typhoid fever" fade from
our vocabulary, such terms as "Giar-
dia," "Legionella" and "Norwalk
virus" are becoming more familiar.
The United States Environmen-
tal Protection Agency (U.S. EPA),
through its Office of Research and
Development (ORD), is conducting
research to better understand and
prevent water contamination by
harmful microorganisms. From
monitoring our nation's ground
water systems for viral pathogens...to
developing more effective technol-
ogy for large and small systems...to
providing other nations with critical
technical assistance, ORD scientists
and engineers continue their mission
to ensure safe waters. As the focus of
our efforts adjusts to deal with
emerging challenges, past and cur-
rent successes add to our scientific
arsenal against disease.
Researcher isolating
infectious bacteria in
one ofORD's
pathogen
containment suites.
Printed on Recycled Paper
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Microorganisms Associated with Waterborne Disease
The following groups of microorganisms have been linked with the occurrence of waterbome
disease. As each pathogen is isolated and identified as a threat to water quality, ORD researchers
try to discover the most effective combination of barriers and disinfection methods to minimize risk
of human exposure.
Bacteria. Bacteria are the most widely distributed life
forms. Pathogenic bacteria range in length from
approximately 0.4 to 14 |om (a |im or "micrometer"
equals one one-thousandth of a millimeter) and 0.2 to
1.2 |im in width. Key bacterial pathogens responsible
for waterbome disease include Legionella, Salmonella
iyphi, Shigella, and Vibrio cholerae.
Viruses. Viruses are inactive when outside of a living
host cell. Viruses linked to waterbome disease have
protein coats that provide protection from environ-
mental hazards and range in size from 0.02 to 0.09
|j,m. Unlike bacteria and protozoa, they contain only
one type of nucleic acid (RNA or DNA). Key
pathogens include hepatitis A and Norwalk virus.
i /
Protozoa. Protozoa, common in bodies of water, are
much larger than bacteria and viruses. To survive
harsh environmental conditions, some species can
secrete a protective covering and form a resting stage
called a "cyst." Encystment can protect protozoa from
drinking water disinfection efforts and facilitate the
spread of disease. Key protozoa being studied as
agents of waterbome disease include Giardia and
Cryptosporidium.
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Why Can't Waterborne
Pathogens Be Eliminated?
Microorganisms are present
everywhere in our environment.
Invisible to the naked eye, vast
numbers of these microbes can be
found in soil, air, food and water.
Although humans are essentially
free of microorganisms before birth,
constant circumstances of exposure
(e.g., breathing, eating, and drink-
ing) quickly allow the establishment
of harmless microbial flora in our
bodies.
Microbial pathogens (microor-
ganisms capable of causing disease),
however, can and often do harm
those who become infected. More-
over, diseases that healthy individu-
als "weather" well may prove fatal
to individuals with compromised
immune systems. In some cases, an
infection can persist to create a
"carrier state" where a disease-
causing agent is harbored by the
body (and spread) without any
apparent symptoms.
Waterborne diseases are typi-
cally considered to be those diseases
resulting from ingestion of contami-
nated water. Additional pathways of
infection being studied by EPA
include inhalation of water vapors as
well as body contact during bathing
(opportunistic pathogens) in the
hospital environment.
Since voluntary water ingestion
(drinking water) and bathing are
universal practices and accidental
ingestion during recreational activi-
ties (e.g., swimming, water skiing,
wading) is common, inadequate
protection of water integrity could
lead to widespread outbreaks (the
Centers for Disease Control defines
an outbreak to be two or more cases
of illness that can be traced to a
common source). Because symp-
toms can be mild and short-lived, it
is estimated that only a fraction of
waterborne outbreaks is recognized,
reported and investigated. Of these,
the pathogenic agent is identified
only half of the time. Additionally,
experts believe that some food-
related disease outbreaks may origi-
nate with an initial infection (e.g., of
a restaurant worker) caused by
contaminated drinking water.
Bacteria, viruses and protozoa
are the microorganism groups con-
taining pathogens of primary concern
in the study of waterborne diseases.
To eliminate these pathogens from
our water, especially from our drink-
ing water, seems theoretically
straightforward. Simply mix in a
disinfectant, allow adequate contact
time to assure inactivation (rendering
the microbes unable to produce
disease), and pump the water into the
distribution lines.
In reality, many conditions
render the above scenario unwork-
able. The physical characteristics of
the water, primarily represented by
dissolved and suspended solids
content, can affect the disinfection
process. The chemical content, both
naturally occurring and anthropo-
genic (i.e., generated by humans),
can also interfere with the chemical
reactions desired during treatment
and disinfection. Finally, pathogens
associated (i.e., imbedded in or
clumped) with higher organisms
(e.g., algae, rotifers, worms) may be
protected from the action of disinfec-
tants.
To overcome these obstacles to
disinfection, successful treatment of
drinking and waste water generally
includes a series of steps. The flow-
charts in Figures 1 and 2 depict the
steps involved in typical drinking
and waste water treatment processes.
In the case of drinking water
disinfection, once the impurities have
been removed, enough disinfectant is
added to inactivate pathogens. Addi-
It is estimated
that swimmers
and waders may
ingest from 0.3 to
1.7 ounces of
water per outing.
To kill or inactivate
drinking water
contaminants such
as bacteria (B),
protozoa (P), and
viruses (V),
adequate contact
time with the
disinfectant
(chlorine or Cl in
this representation)
must be allowed.
Adsorption to and
clumping of solid
particles (S) can
inhibit the disinfec-
tion process.
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Raw
Water
Screening
Coagulation/
Rapid Mixing
Flocculation
Filtration
Disinfection
To Distribution
System
Figure 1. Simplified
flowchart of drinking
water treatment
processes.
tionally, a residual level of disinfec-
tant must be maintained throughout
the distribution system to guard
against potential problems (e.g.,
microorganisms entering through
breaks in distribution lines or re-
growth).
Proper distribution system opera-
tion and maintenance practices are
essential deterrents of pathogen
entry, recovery and survival. These
practices (according to Geldreich et
al, 1992) include:
• Systematic flushing of the entire
distribution system "to get more
movement of the chlorine residual
into all parts of the pipe
network...to remove static water
from slow-flow sections, deadends
and stratified water in storage tanks
on a periodic basis;"
• Effecting repairs and replacement
of distribution line components
(e.g., broken mains and service
meters) in a sanitary manner (i.e.,
soil-free replacement parts,
disinfection and flushing of
repaired lines, valves and fittings);
• Preventing pathogens from being
drawn into the distribution system
by maintaining continuous positive
pressure and preserving barriers be-
tween public water supplies and
sewage or storm water drainage;
• Varying the sample sites during
routine monitoring to produce data
more representative of the entire
system.
While the importance of source
water treatment to ensure safe drink-
ing water seems obvious, the need to
devote equal effort to pathogen
reduction in wastewater is not al-
ways recognized. The release of
untreated or inadequately treated
wastewater into source waters drawn
upon by other communities presents
a significant health risk. Source
waters heavily loaded with disease-
causing microorganisms can reduce
the effectiveness of "downstream"
drinking water treatment processes.
Such advances as ultraviolet light
disinfection systems, initially inves-
tigated as a wastewater disinfection
option several years ago, are pres-
ently becoming more widely ac-
cepted and reliable with recent
design enhancements. This technol-
ogy has been demonstrated to be
capable of meeting existing disinfec-
tion criteria without the release of
dangerous disinfection by-products.
What Progress Has Been
Made?
Early in this century, the water-
borne diseases of chief concern in
the U.S. were typhoid fever and
amebiasis. Of the 1,087 deaths
associated with waterborne out-
breaks between 1920 and 1991, 943
were attributed to typhoid fever
while 102 were caused by amebiasis.
Overall, 83% of the deaths occurred
prior to 1936 and less than 1%
occurred after 1970. Additionally,
the number of outbreaks in commu-
nity water systems since 1945 is
about half as great as the number
documented during the first half of
this century. The reduction in fatali-
ties and number of outbreaks indi-
cates that progress has been made in
the prevention of certain waterborne
diseases. Much of the progress has
been the result of increased imple-
mentation of important treatment
practices (e.g., filtration, disinfec-
tion, sewage treatment). Although
progress has been significant, the
nation's water continues to be a
source of disease. It must be rigor-
ously monitored for indicators of
fecal contamination.
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Some Waterborne Diseases of
Concern in the United States
Disease
Microbial
Agent
General Symptoms
Amebiasis
Protozoan
(Entamoeba histolytica)
Abdominal discomfort, fatigue,
diarrhea, flatulence, weight loss
Campylo-
bacteriosis
Bacterium
(Campylobacter jejuni)
Fever, abdominal pain, diarrhea
Cholera
Bacterium
(Vibrio cholerae)
Watery diarrhea, vomiting, occasional
muscle cramps
Cryptospor-
idiosis
Protozoan
(Cryptosporidium parvum)
Diarrhea, abdominal discomfort
Giardiasis
Protozoan
(Giardia lamblia)
Diarrhea, abdominal discomfort
Hepatitis
Virus
(hepatitis A)
Fever, chills, abdominal discomfort,
jaundice, dark urine
Shigellosis
Bacterium
(Shigella species)
Fever, diarrhea, bloody stool
Typhoid fever
Bacterium
(Salmonella typhi)
Fever, headache, constipation, appetite
loss, nausea, diarrhea, vomiting,
appearance of an abdominal rash
Viral
Gastroenteritis
Viruses
(Norwalk, rotavirus and
other types)
Fever, headache, gastrointestinal
discomfort, vomiting, diarrhea
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Raw
Wastewater
Grit
Primary
Sludge
Secondary
Sludge
To further
treatment or
discharge
Figure 2.
Simplified flowchart
of typical
wastewater
treatment
processes.
In 1974, Congress passed the
Safe Drinking Water Act (SDWA)
setting up a regulatory program
among local, state, and federal agen-
cies to help ensure the provision of
safe drinking water in the U.S. The
states are expected to administer and
enforce these regulations for public
water systems (systems that either
have 15 or more service connections
or regularly serve an average of 25 or
more people daily for at least 60 days
each year). Public water systems
must provide water treatment, ensure
proper drinking water quality
through monitoring, and provide
public notification of contamination
problems.
Relating to prevention of water-
borne disease, the SDWA required
EPA to:
1) set numerical standards,
referred to as Maximum Con-
taminant Levels (MCLs — the
highest allowable contaminant
concentrations in drinking water)
or treatment technique require-
ments for contaminants in public
water supplies;
2) issue regulations requiring
monitoring of all regulated and
certain unregulated contami-
nants, depending on the number
of people served by the system,
the source of the water supply,
and the contaminants likely to be
found;
3) set criteria under which sys-
tems are obligated to filter water
from surface water sources; it
must also develop procedures for
states to determine which sys-
tems have to filter;
4) develop disinfection rules for
all public water supplies; and
5) require all states to develop
Wellhead Protection Programs
designed to protect from sources
of contamination areas around
wells that supply public drinking
water systems.
Through the Surface Water
Treatment Rule (SWTR), EPA has
set treatment requirements to control
microbiological contaminants in
public water systems using surface
water sources (and ground-water
sources under the direct influence of
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surface water). These requirements
include the following:
1) treatment must remove or
inactivate at least 99.9% of
Giardia lamblia cysts and
99.99% of viruses;
2) all systems must disinfect, and
are required to filter if certain
source water quality criteria and
site-specific criteria are not met;
3) the regulations set criteria for
determining if treatment, includ-
ing turbidity (suspended particu-
late matter) removal and disin-
fection requirements, is adequate
for filtered systems; and
4) all systems must be operated
by qualified operators as deter-
mined by the states.
Current EPA Research -
Barriers to Contamination
Although water treatment and
disinfection techniques are quite
effective at microbe reduction,
finished drinking water is not sterile.
Survival and regrowth of microor-
ganisms in drinking water distribu-
tion systems can lead to the deterio-
ration of water quality and even non-
compliance of a supply. Regrowth
has largely been associated with
heterotrophic bacteria (i.e., those
bacteria - including pathogens - that
require preformed organic com-
pounds as carbon and energy
sources). Bacterial growth occurs on
the walls of the distribution system
(referred to as "biofilms") and in the
water either as free living cells or
cells attached to suspended solids. A
multi-faceted phenomenon, bacterial
regrowth is influenced primarily by
temperature, residence time in mains
and storage units, the efficacy of
disinfection, and nutrients.
Assimilable organic carbon
(AOC) is the portion of the total
organic carbon (TOC) dissolved in
water that is easily used by microor-
ganisms as a carbon source (i.e.,
nutrients). ORD researchers are
currently investigating treatment
processes to control AOC. One
promising process is biologically
active filtration wherein bacterial
communities are intentionally estab-
lished in the filters to use up, or
biodegrade, the AOC as it passes
through. This treatment process must
be employed before final disinfection
so that bacteria escaping from the
filter can be properly controlled. As
described in Figure 1, most water
utilities do not disinfect with chlorine
until late in the treatment train. This
limits the formation of disinfection
by-products (i.e., those compounds
like chloroform produced when
chlorine reacts with naturally occur-
ring organic carbon).
To accomplish disinfection
earlier in treatment, some water
utilities employ ozonation. While
ozone is a very strong disinfectant, it
also converts a portion of the TOC
into AOC. ORD researchers are
examining the advantages (e.g.,
disinfection of bacteria, viruses and
protozoan cysts, control of color,
control of taste and odor, enhance-
Currently, it is
estimated that
there will be over
100,000
violations of the
SDWA annually.
Nearly half of
these will be for
MCL violations.
Of these, the
majority will be
microbiological
violations by
small systems.
Single step
membrane filter
procedure for
enumerating E. coli
in recreational
waters. The yellow
colonies are E. coli
while the blue, red
and purple colonies
are other conforms.
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In situ cytotoxicity test for heterotrophic bacteria found in
drinking water. Heterotrophs recovered from drinking water
form individual yellow colonies (left) that can be transferred to a
tissue cell culture (right). Formation of plaques (i.e., clear areas
caused by destruction of infected cells) in the tissue culture can
indicate virulence and signal the need for further action.
In the lesser
developed
countries,
waterborne
disease is still a
major problem.
The World
Health
Organization has
estimated that
more people die
annually of
water-related
diarrheal
illnesses than of
cancer or AIDs.
ment of coagulation, and partial
oxidation of the naturally occurring
organic carbon that reacts with
chlorine) and disadvantages of ozone
(e.g., enhancement of AOC, conver-
sion of bromide to bromate, and
formation of its own disinfection by-
products like formaldehyde).
The project entitled "EPANET"
involves the development and testing
of a water quality model for drinking
water distribution systems. The
EPANET model is a computer
program that performs extended
period simulation of hydraulic and
water quality behavior within water
distribution networks. It tracks the
flow of water in each pipe, the pres-
sure at each pipe junction, the height
of water in each tank, and the con-
centration of a contaminant through-
out the network during a multiple
time period simulation. Water age
and source tracing can also be simu-
lated.
EPANET can be useful for
analyzing the loss of disinfectant
residual, designing water quality
sampling programs, performing
drinking water exposure risk assess-
ments, and calibrating network
hydraulic models. It can provide
insight into how changes in water
source utilization, pumping water
storage levels, use of satellite treat-
ment, and targeted pipe cleaning and
replacement would affect drinking
water quality.
In support of small community
and non-community (less than 3300
people) drinking water treatment
systems, ORD researchers are de-
signing, modifying and testing
"Hybrid Drinking Water Treatment
Package Plants." These package
plants are factory-built, skid-
mounted, and ready to be operated in
the field with minimal site prepara-
tion. They exhibit lower capital cost
than custom designed facilities built
onsite and can incorporate any
drinking water treatment process.
Promising technologies being con-
sidered for incorporation include
membranes, advanced oxidation, bag
filters, and photocatalytic oxidation.
By merging, modifying, and adapt-
ing conventional treatment trains
with innovative treatment technolo-
gies, a broader variety of contami-
nants (including pathogens) can be
removed and SDWA compliance can
be facilitated.
Concern has recently mounted
over the ability of certain pathogenic
protozoan (Cryptosporidium) cysts
to survive treatment processes and
enter the distribution system. ORD,
in its project entitled "Evaluation of
Particulate Removal Processes," is
designing and testing the most effec-
tive filtration techniques to physi-
cally remove the cysts. Being studied
are slow sand (see Figure 3), diatom-
aceous earth, and coagulation/rapid
sand filtration processes. Results of
this research will build upon earlier
work with filtration of Giardia
lamblia.
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Overflow
Raw
Water
Ventilation
V-Notch Weir
Flow
Measurement
^F
Filter Effluent
Current EPA Research —
Pathogenic Intestinal
Protozoa
During the last 20 years, signifi-
cant improvements have been made
in the quantitative methods for
detecting pathogenic intestinal
protozoa (particularly Giardia and
Cryptosporidium) in water. Addi-
tionally, there has been progress in
standardizing these methods. Current
methods, however, are still time-
consuming and skill-intensive (re-
quiring highly trained analysts), and
lack the ability to indicate viability
(whether the cyst is dead or alive) or
infactivity. The latter item has
clouded the development of quantita-
tive risk assessments.
The cosmopolitan nature of
intestinal protozoa, and the certainty
that all surface water supplies must
be contaminated with these organ-
isms, has been established through
studies in animals (beavers, musk-
rats, and birds) and by occurrence
studies in sewage throughout all
parts of the country. Seasonal and
geographic differences have been
recognized and data on concentra-
tions in various waters have been
collected.
Work on cross-species infectiv-
ity of animal and human cysts has
established that beavers and musk-
rats may at least be secondary reser-
voirs for giardiasis. Also, while it
appears that avian cysts are not
infective for mammals, we cannot
now distinguish avian and mamma-
lian cysts in a water sample. The
goal of an ORD project entitled
"Development of Gene Probes for
Speciation of Giardia" is to develop
and test the application of genetic
and molecular probe methods to
allow classification of detected
Giardia species.
Gene sequences have been
mapped in species of Giardia shed
by animals (e.g., herons and mice)
and compared with corresponding
gene sequences in human-hosted
Giardia. Preliminary results indicate
that through these mapped se-
Figure 3. Basic
elements of a slow
sand filter.
In Minnesota, 100%
of the muskrats and
7% of the beavers
examined were
positive forGiardia.
In four
Northeastern states
(Maine, New
Hampshire, New
York, and
Vermont), the
corresponding
figures were 94%
for muskrats and
17% for beavers.
Erlandsenefa/., 1990.
9
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Cyst
Two-stage life cycle
ofGiardia lamblia:
the active
trophozoite stage
and the
environmentally-
resistant, resting
cyst stage. Cellular
components shown
above include
nuclei (blue),
axonemes (red),
and median bodies
(green).
quences, once labelled with a detect-
able probe, human type Giardia can
be differentiated from bird and
mouse Giardia. Probes have been
synthesized and experiments show
that one reacts with 10 different G.
lamblia human isolates but not with
G. psittaci (associated with birds) or
G. muris (associated with mice). It is
hoped that progress in speciation of
Giardia can be applied to the study
of Cryptosporidium.
Current Giardia detection meth-
ods are unable to distinguish viable
from nonviable cysts. A practical
detection method for viable cysts
would allow assessing the signifi-
cance of positive reports and may
allow establishment of numerical
standards under the SDWA. The
objectives of the projects "Molecular
Probes for the Detection of Proto-
zoan Parasites" and "Induction of
Stress Proteins as a Measure of
Giardia Cyst Viability" are to dis-
cover, separate and amplify specific
genetic sequences (DNA or RNA)
associated with viable Giardia cysts.
If these specific sequences can be
identified, probes can be developed
to allow testing for viable cysts only.
Practical methods for isolation,
identification and quantification of
waterbome pathogens such as Giar-
dia are not yet available. Isolation
and identification methods are
needed before control methods can
be evaluated and regulatory deci-
sions can be made regarding required
treatment processes and MCLs. The
goal of ORD's project entitled "Im-
munological Methods for Detection
of Etiological Agents of Waterbome
Disease" is to develop innovative
immunologic methods for the detec-
tion, identification and enumeration
of pathogenic microorganisms.
Immunologic methods may provide
the sensitivity and specificity needed
for detection since low numbers of
target organisms may be present in
large volumes of water along with
high numbers of the normal flora and
fauna.
To accomplish this, the patho-
genic agents will be isolated and
their antigens (proteins that stimulate
the body to produce antibodies) will
be used to produce specific antisera
for immunologic tests (e.g., immun-
ofluorescent assay, enzyme immuno-
assay, radioimmunoassay).
Because standardized procedures
for detecting pathogenic protozoa do
not now exist, confusion in the
interpretation of results obtained by
10
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different laboratories occurs. The
goal of the project entitled "Stan-
dardized Methods for Detecting
Pathogenic Protozoa in Water" is to
develop standardized methods for
detecting Giardia and Cryptospo-
ridium in water. These methods will
also assist EPA in assessing research
findings relevant to regulatory activi-
ties under the SDWA.
Cryptosporidium is the only
microorganism on the Office of
Ground Water and Drinking Water's
list of contaminants to be addressed
in the next round of regulations.
Quantitative risk assessment for this
organism is hampered by the un-
availability of any human infectious
dose data and by the scarcity of
animal dose data. Additionally, very
little is known of the longevity and
protective ability of the body's
immune response to Cryptospo-
ridium infection.
The objective of the project
entitled "Cryptosporidium Infectious
Dose and Immune Response" is to
determine Cryptosporidium infec-
tious dose and the associated im-
mune response in human volunteers.
Organisms known to
be infectious for
humans will be
obtained from
infected calves
and administered
in drinking water
to the volunteers.
Conclusions drawn
from this project
could help shape
future maximum contami-
nant level regulations.
In preparation for development
of disinfection and disinfection by-
product rules, information on the
occurrence of Giardia cysts and
Cryptosporidium oocysts in source
waters and throughout the drinking
water treatment process must be
Cell
Chromosome
collected. Through the
project entitled "Cyst and
Oocyst Levels in the Ohio
River," ORD is monitor-
ing monthly one raw
water sample (collected
from the river) plus
samples from five differ-
ent points in the drinking
water treatment process.
Although current meth-
ods are based on micro-
scopic examination of
concentrated samples obtained from
large volumes of water, immunofluo-
rescence membrane assays and gene
probe techniques are being used for
this project. Findings from this
project will also be used in a nation-
wide survey for occurrence of these
organisms in water supplies.
In the early 1980s, a waterborne
disease study in Washington State
suggested that certain elements were
required for a good waterborne
disease surveillance and investiga-
tion program. Since that time, com-
puter hardware and software have
been introduced which may increase
the potential for improved efficiency
of disease reporting. Although
cryptosporidiosis
out-
From 1986 through 1990,
20 waterborne outbreaks due to
intestinal protozoa were reported in the U.S.;
these outbreaks occurred in 10 states and
affected more than 15,000 people.
breaks
have been associated1
with drinking water, the relative
significance of drinking water in the
transmission of this disease is un-
known. The project entitled "Surveil-
lance of Waterborne Disease/
Gene (Linear stretch of DMA)
11
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An epidemiological
investigation
involves the study
and occurrence of
disease within a
population. In the
study of
waterborne
disease,
epidemiological
data can indicate a
need for additional
drinking water
treatment (e.g.,
filtration).
Cryptosporidiosis Epidemiological
Feasibility Study" is underway to: 1)
systematically evaluate waterborne
disease strategies, computer software
and educational programs in local
and state health departments, and 2)
design an epidemiological study to
address the significance of drinking
water in the transmission of
Cryptosporidiosis. Products from
these efforts could shed light on the
understanding and tracking of water-
borne disease outbreaks throughout
the world.
Current EPA Research —
Viruses
Traditionally, methods for detect-
ing and identifying viruses have
relied on slow cell culture methods.
Existing methods may underestimate
the quantity of viruses present or
alternatively produce false negatives
when viruses are actually present in
sampled water. Some viruses (e.g.,
hepatitis A and Norwalk) simply
cannot be detected by the commonly
used cell culture/plaque assay meth-
ods. Given the health risks presented
by viruses, it is essential to develop
more information on the nature and
extent of viral contamination in our
nation's waters. The objective of
ORD's project entitled "Practical
Methods for Monitoring Viral Patho-
gens in Surface and Groundwater
Source Waters to Define Level of
Treatment" is to develop improved
methods for detection of waterborne
viruses. In addition to supporting
EPA's risk assessment efforts relat-
ing to water quality, these methods
will provide the means to support the
establishment of new virological
standards and to permit the formation
of effective options for regulatory
decisions.
This project will focus on the
development of biotechnology meth-
ods based on recognition of viral-
specific nucleic acids within infected
cell systems. The use of a biotechnol-
ogy approach that employs DNA
probes to detect the presence of
viruses is faster, less expensive and
easier to perform than traditional
plaque assay methods.
The Science Advisory Board's
(SAB) Reducing Risk report to EPA
describes pollutants in drinking water
as one of the four highest risks to
Percentage of State Populations Served by Ground Water for Domestic Supply
LEGEND
Percent of Population
• 75-100
• 50-74
CH 25-49
CH 0-24
Source: 1990 State Section 305(b) Water Quality Reports
12
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human health. With over 50% of the
U.S. population relying on ground
water as their primary source of
drinking water, the need for ground-
water resource protection, including
protection from pathogens, is clear.
"Monitoring of Ground Water
for Human Enteric Viruses" is a
current project to address the man-
date of the SDWA that EPA estab-
lish treatment requirements and
criteria for ground water systems. A
virus occurrence survey of vulner-
able ground water systems is being
performed to support requirements
for minimum levels of virus inactiva-
tion and ultimately a ground water
disinfection rule. A number of public
ground water systems will be identi-
fied and ranked according to vulner-
ability to fecal contamination. Of
these, 25 systems will be monitored
for the presence of viruses through
tissue culture methods and gene
probe techniques.
Norwalk and Norwalk-like
viruses cause viral gastroenteritis
(the second leading cause of illness
in the U.S.) in consumers of con-
taminated water and food. Since
these viruses cannot be grown and
identified in tissue cultures, they
cannot be detected in water samples
by current monitoring techniques. A
small amount of Norwalk virus is
available for studies. This virus
preparation has been isolated from
stool samples of infected individuals
and used in an enzyme immunoassay
for the detection of Norwalk virus.
Because immunologic methods
require a high virus concentration,
the etiologic agent responsible for
most waterborne outbreaks of gastro-
enteritis usually is not determined.
Since the viral density in environ-
mental samples is normally too low
for direct immunologic detection and
since there is no known cell culture
method for this virus, the genetic
material of the Norwalk virus par-
ticle must currently be amplified
using a biotechnology approach
called polymerase chain reaction
(PCR).
Although known to be highly
infectious, the infectious dose for
Norwalk virus is unknown. The only
safety-tested virus inoculum (a
microorganism-containing specimen
that has been shown to be free of
other pathogens) available cannot be
used for infectious dose studies
because it is not possible to deter-
mine the virus concentration. The
project entitled "Develop a Dose-
Once virus particles
infect cells in a
single layer tissue
culture, cellular
damage (clear areas
or "plaque-forming
units" in the brown
agar depicted at left)
becomes apparent.
The plaque assay is
used for
identification,
counting, and
purification of
Hybridized probe (in
blue) binds with
target genetic
sequence (in red) to
make it detectable
by radiation or
color.
ULUU11UU
Target DMA sequence
13
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Preparing stock
tissue cell cultures
for isolation and
identification of
viruses in water
samples.
Although the rate of
water transport
through their gills
varies greatly,
oysters have been
found to filter as
many as 154
gallons per day. In
waters exposed to
human sewage,
these shellfish can
filter out and
concentrate
pathogens as well
as food.
Response Curve for Norwalk Virus"
is approaching this problem in four
phases: 1) cell cultures capable of
growing the Norwalk virus will be
developed; 2) Norwalk viruses will
be grown in cell culture, purified and
safety-tested for use in volunteer
studies; 3) a measure of the number
of total and infectious virus particles
in the purified sample will be estab-
lished; and 4) a human volunteer
study will be initiated to determine
the amount of virus particles re-
quired to cause disease.
The Clean Water Act stipulates
that the nation's rivers, lakes, and
coastal waters be swimmable and
fishable. Water quality standards
based on established criteria to
achieve this goal must be developed.
The project entitled "Shellfish Meth-
ods and Exposure Response Assess-
ment/Viruses" is being conducted to
develop methods for detecting and
enumerating contamination of shell-
fish and shellfish growing waters by
human enteric viruses. Shellfish
growing in polluted waters are
known to concentrate these viruses
during feeding. Since shellfish are
often eaten raw or insufficiently
cooked, subjecting shellfish waters to
human wastes constitutes a public
health risk.
Because there are more than 100
waterbome virus types that may
cause clinical outbreaks in humans,
monitoring efforts are essential to
ensure the virological safety of
waters and particularly the reliability
of water and wastewater treatment
practices. This information can only
be provided through increased moni-
toring and assessment programs of
each major pathway leading to the
deposition of human enteric viruses
into the nation's waters. These vi-
ruses are responsible for serious
illnesses ranging from hepatitis to
myocarditis to central nervous sys-
tem disorders to acute gastroenteritis.
The general recommendation has
been that drinking water should be
free of human enteric viruses and that
recreational water viral limits be set.
The goal of the project entitled
"National Monitoring and Assess-
ment Program: Status and Long-
Term Trends in Human Enteric Virus
Pollutants in the Aquatic Ecosys-
tems" is to establish a national viral
survey program focusing on the
following five factors: 1) selection of
monitoring sites based on those most
likely to have broad public health
importance; 2) field sampling that
will result in the collection of ad-
equate and representative sample
volumes to safeguard against false
negatives; 3) concentration proce-
dures to increase the density of
viruses so that they can be effectively
assayed; 4) standard protocols for
viral detection using both gene probe
and classical plaque assay tech-
niques; and 5) parallel biological and
chemical analysis that will serve to
determine the quality of the water
source.
14
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Current EPA Research —
Bacteria
The new National Primary
Drinking Water Regulations require
that all drinking water samples
testing positive for total coliforms be
further tested for the presence of
either fecal coliforms or E. coli.
There is a method currently available
that allows the simultaneous detec-
tion of total coliforms and E. coli in
a broth medium in 24 hours; how-
ever, there is no equivalent method
for use with membrane filters. De-
velopment of such a method will
allow those who prefer to obtain
counts of these organisms in their
distribution systems to use a mem-
brane filter method and to have
results within the 24-hour time
frame. Through the project "Devel-
opment of a Membrane Filter Me-
dium for the Simultaneous Detection
of Total Coliforms and E. coli" a
membrane filter medium on which
both total coliforms and E. coli can
be distinguished from noncoliforms
will be developed and patented.
E. coli are fecal organisms that
when present in drinking water are
indicative of fecal pollution. Logisti-
cal concerns in sample handling and
holding require evaluation of condi-
tions for optimizing sample stability
and longevity. No current regulations
exist for handling samples for analy-
sis of E. coli. Through the project
entitled "Optimal Sample Holding
Conditions for Analysis of Fecal E.
coli in Drinking Water," sample
temperature and holding time will be
determined for E. coli or fecal colif-
orm analysis methods (i.e., Colilert
and M-FC agar). Relative recovery
of methods and storage conditions
will be assessed for optimal E. coli
recovery.
The requirement (through the
SDWA amendments) to test all
coliform-positive drinking water
samples for either fecal coliforms or
E. coli is new. Data from available
methods for detecting chlorine-
damaged E. coli in drinking water
are limited. The objective of the
project entitled "Detection of Low
Numbers of Chlorine-Stressed E.
coli in Drinking Water" is to evalu-
ate and compare the abilities of a
commercial method (Colilert) and a
standard coliform method (EC-
MUG) to recover low numbers of
chlorine-stressed E. coli from po-
table water. Pure cultures ofE. coli
will be washed, nutrient-stressed in
finished drinking water, and treated
with chlorine. The chlorine-stressed
E. coli will then be enumerated,
diluted to levels that would be found
in marginally unsafe drinking water
and
assayed
in mul-
tiple
tubes by
the three
methods.
These
experi-
ments
will be
repeated
using
naturally
occurring E. coli from diluted human
fecal specimens, contaminated
source waters and effluents.
The infectious bacterial agent
identified from the stools of cholera
victims is Vibrio choleme. The
epidemic in Latin America has
prompted a renewed interest in
control measures for this disease.
Through the project entitled "Inacti-
vation of Vibrio choleras Biotype El
Tor and Biotype Classical by Chlori-
nation," it has been determined that
the strain responsible for the epi-
demic in Peru is capable of reverting
to a variant which is more resistant
In the U.S., the
presence of
coliform bacteria
in drinking water
is used as an
indicator of
possible
microbiological
contamination.
When total
coliforms are
detected, fecal
coliform orE. coli
analysis must be
performed.
The golden metallic
sheen of the
colonies at left
indicate the
presence of total
coliforms and the
possibility that the
sampled water
supply is
contaminated.
15
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A rugose (rough-
surfaced) variant
(above left) ofVibrio
cholerae 01 is able
to form aggregates.
ORD studies have
indicated that this
variant is more
resistant to
disinfection than the
smooth strain
(above right).
This one step
method
(developed by
ORD) allows
enterococci (blue
colonies)
enumeration in just
24 hours.
to chlorination than the typical
smooth variety of Vibrio cholerae.
Cells of the variant appear to be
imbedded in a gelatinous mucoid
material, facilitating the formation of
aggregates, which renders them more
resistant to disinfection. Although
the variant is more resistant, studies
have indicated that all strains are
readily inactivated through adequate
chlorination.
The Legionella pneumophila
bacterial strains that cause commu-
nity- and hospital-acquired pneumo-
nia are usually spread via finished
drinking water. Certain free living
amoebae (protozoa) support the
multiplication of L. pneumophila in
drinking water systems. These amoe-
bae may also be responsible for
enhancing the virulence (capacity of
a microorganism to cause disease) of
the Legionellae and for protecting
them from adverse environmental
factors such as high temperature and
chlorine disinfection. The project
entitled "Multiplication of
Legionellae in Amoebae and Assess-
ment of Virulence" will determine
the effect of intracellular growth of
Legionella in amoebae on virulence
and as protection against chlorine
and high temperature. To accomplish
this, a method will be established to
study the ability of various types of
amoebae to provide a protective
niche for the multiplication of
Legionellae under adverse environ-
mental conditions. Combinations of
Legionella isolates and specific
amoebae that result in high yields of
Legionella after intracellular growth
will be used to study the effects of
intracellular growth on virulence.
Preliminary studies on the ability of
amoebae to supply iron to
Legionellae growing intracellularly
showed no obvious associations
between growth and iron concentra-
tion.
EPA is required by the SDWA to
establish appropriate controls and
regulations for potable water. ORD's
project entitled "Develop Methods
for Identifying Potential Bacterial
Pathogens in Drinking Water" will
develop a data base on potential
health hazards (i.e., pathogenicity)
associated with bacteria commonly
found in water distribution systems.
To accomplish this, three rodent
species will be compromised using
nitrous oxides or immunosuppressive
agents, and the animals subsequently
16
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will be chal-
lenged via
the gastroi-
ntestinal
route.
Although
virulence is
usually
measured in
vivo (animal
research), the
need for
extensive
animal
testing can be
significantly reduced by the develop-
ment of a battery of in vitro (cell
culture) tests for traits known to be
virulence-related. This battery can be
used to predict the potential an
organism has for causing disease in
exposed populations. Through the
project entitled "Develop In Vitro
Methods for Identifying Potential
Bacterial Pathogens in Drinking
Water," model systems will be
developed that can be used to deter-
mine the potential pathogenicity of
bacteria found in potable water
distribution systems. Additionally,
gene probe and other assays to
identify known opportunistic patho-
gens will be developed and evalu-
ated.
Bacteria common to drinking
water distribution systems colonize
point-of-entry, granular activated
carbon (GAC) filters where they are
able to grow to very high densities.
Subsequent to reaching the high
densities the bacteria begin slough-
ing off the GAC filters. The number
of bacteria in the filter effluent (i.e.,
water flowing out of the filter) is
significantly higher than in the
influent water. This amplification of
bacteria in drinking water is of
concern to EPA because GAC filters
are being considered as a substitute
for central potable (i.e., fit for drink-
(a)
(b)
ing) water treatment in small com-
munities where the treatment system
has been overwhelmed by organic
substances that may be harmful to
human health. EPA's Office of
Ground Water and Drinking Water
(OGWDW), however, does not want
to recommend the use of these filters
if the possibility exists that their use
poses an acute disease risk due to
bacteria that grow on the filters. The
health significance of the bacteria
known to adsorb and grow on GAC
filters used in the home will be
evaluated. The OGWDW will use
this information to develop appropri-
ate controls and regulations for this
type of drinking water treatment as
required by the SDWA.
The objective of ORD's project
entitled "Health Effects Associated
with Point-of-Entry GAC Filters" is
to determine if a significant health
hazard is associated with the use of
granular activated carbon, point-of-
entry, whole house filters. To accom-
plish this, a suitable study site will be
selected based on the following
criteria: 1) the water in the delivery
system must meet EPA and local
drinking water standards; and 2) the
water distribution system should
contain a bacterial population whose
density is as high as possible and still
acceptable under local regulations.
Two step, 48 hour
membrane filter test
for enumerating
enterococci in
recreational waters.
(a) and (b) Two
perspectives of
colonies (red)
present at 24 hours.
(c) At 48 hours,
colonies with black
halos are identified
as enterococci.
17
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Analysis of
potable water or
cooling tower
water for
Legionella
pneumophila
requires
approximately
five to seven
days for growth of
the organisms on
the initial isolation
medium and
another five to
seven days to
confirm the
identity of these
organisms. Gene
probe techniques
could reduce
analysis time to
one day.
EPA researcher
using the
transmission
electron microscope
to detect pathogens
unable to be
detected by other
methods.
After a distribution system
meeting the above criteria has been
found, a volunteer population of
appropriate size will be selected
from among the water system cus-
tomers. The selected population will
be randomly divided into GAC user
and non-user groups. Point-of-entry,
GAC filters will be installed in the
homes of the randomly selected user
group. The health status of both
groups will be monitored over a
predetermined period of time and
during this time interval the bacterial
population in the water system and
the filter effluent will be monitored
on a routine basis. In the event of an
illness where a bacterial agent is
diagnosed as the cause, the GAC
filter will be removed and examined
for the presence of the organism
determined to be the agent of the
disease in that household unit. If an
association between illness or dis-
ease and the use of GAC filters is
observed, health advisory guidelines
will be established or processes that
will eliminate the causative organ-
isms will be developed.
Some believe that exposure to
fecal pollution through recreational
waters or ingestion of contaminated
shellfish causes greater health risks if
the pollution is of human rather than
animal origin. Before the relative
risks of human versus animal fecal
pollution can be assessed, it is neces-
sary to develop a microbiological
method for distinguishing human
from animal pollution. Current
methods detect fecal pollution but do
not reveal the source. The objective
of the project entitled "Method to
Distinguish Non-Human Fecal
Pollution from Human Fecal Pollu-
tion" is to develop a gene probe
specific for E. coli that inhabit the
human intestine for use as an indica-
tor of the presence of human fecal
contamination in water. The probe
will be field tested at several sites in
which fecal pollution is exclusively
from human sources, exclusively
from animal sources and from mixed
sources.
Shigella species are among the
most common and significant patho-
gens associated with wastewater and
18
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sludge. Because of their low infec-
tive doses, these organisms may be
hazardous even if present in low
numbers in wastewaters that are
recycled for potable use or sludges
that are applied to agricultural land.
Shigellae are very difficult to detect
in environmental samples by conven-
tional methods because of their
biochemical similarities to E. coli.
The use of current gene probe tech-
nology in the project entitled "Detec-
tion of Enteroinvasive Shigella in
Wastewaters and Sludges" should
enable us to detect Shigellae in
sludges and wastewaters that would
appear to be free of these pathogens
if analyzed by conventional methods.
Conclusion
The protection and enhancement
of our nation's water quality remains
a chief concern of the U.S. Environ-
mental Protection Agency. The
Office of Research and Development
is committed, through the extensive
waterborne disease research efforts
earlier described, to ensure that the
most effective and efficient methods
are developed to identify, detect, and
inactivate/remove pathogens that
may be present in our drinking water
supplies.
Life cycles, mechanisms of
infection, protective or dormant
states, emergence of disinfection-
resistant variants, optimal pathogen
removal techniques, regrowth in
distribution lines.. .all are areas that
must be investigated and understood
to afford the water quality safeguards
that are so often taken for granted.
The successes and failures of these
research efforts, relayed to the public
and appropriate federal, state, and
local agencies, have helped to ensure
safe drinking water.
Human enteric
bacteria being
subcultured in an
anaerobic hood.
19
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EPA Publications
The EPA publications listed below may provide more detailed information on the
subjects discussed in this document. These references and additional copies of this bro-
chure can be requested at no charge (while supplies are available) from EPA's Center for
Environmental Research Information (CERI). Once the CERI inventory is exhausted,
clients will be directed to the National Technical Information Service (NTIS) where docu-
ments can be purchased.
Environmental Pollution Control Alternatives: Drinking Water Treatment for Small Commu-
nities, EPA/625/5-90/025.
Methods for the Investigation and Prevention of Waterborne Disease Outbreaks, EPA/
600/1-90/005a.
Microbiological Methods for Monitoring the Environment — Water and Wastes, EPA/600/
8-78/017.
Seminar Publication: Control of Biofilm Growth in Drinking Water Distribution Systems,
EPA/625/R-92/001.
Test Methods for Escherichia coliand Enterococci in Water by the Membrane Filter
Procedure, EPA/600/4-85/076.
USEPA Manual of Methods for Virology, EPA/600/4-84/013 and updates.
Waterborne Disease Outbreaks - Selected Reprints of Articles on Epidemiology, Surveil-
lance, Investigation, and Laboratory Analysis, EPA/600/1-90/005b.
Center for Environmental Research Information (CERI)
U.S. Environmental Protection Agency
26 W. Martin Luther King Drive
Cincinnati, OH 45268
Phone: (513) 569-7562 FAX: (513) 569-7566
Cited Literature
Erlandsen, S.L., L.A. Sherlock, W.J. Bemrick, H. Ghobrial and W. Jakubowski. 1990. Prevalence of
Giardia spp. in beaver and muskrat populations in northeastern states and Minnesota. Appl. & Envir.
Micro., 56: 31-36.
Geldreich, E.E., K.R. Fox, J.A. Goodrich, E.W. Rice, R.M. Clark, and D.L. Swerdlow. 1992. Searching
for a water supply connection in the Cabool, Missouri disease outbreak of £. co//0157:1-17. Wat. Res.,
26: 1127-1137.
20
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This publication was prepared by Patrick Burke ofORD's National
Risk Management Research Laboratory, Cincinnati, Ohio. Contribu-
tors and reviewers include Alfred Dufour, Walter Jakubowski, Robert
Safferman, Shay Fout, Gerard Stelma and Terry Covert of the
National Exposure Research Laboratory - Cincinnati, and Robert
Clark, Kim Fox, Edwin Geldreich, Richard Miltner, Donald Reasoner,
and Eugene Rice of the National Risk Management Research
Laboratory. Thanks to Al Lang and Jim O'Dell for photographic
support.
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