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United States Office of Research and EPA/640/K-93/001
Environmental Protection Development April 1993
Agency Washington, DC 20460
Preventing Waterborne
Disease
A Focus on EPA’s
Research
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.
“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- prevent water contamination by
tics are alarming, the risk that exten- harmful microorganisms. From
sive outbreaks of waterborne cholera monitoring our nation’s ground
will occur in the United States is water systems for viral pathogens...to
minimal. Effective treatment of developing more effective technol-
drinking water and sewage, coupled ogy for large and small systems...to
with adequate personal hygiene providing other nations with critical
habits, has contributed to a success- technical assistance, ORD scientists
ful line of defense against the spread and engineers continue their mission
of cholera in the U.S. Still, the ease to ensure safe waters. As the focus of Researcher isolating
of international travel has guaran- our efforts adjusts to deal with infectious bacteria in
teed the import of a wide variety of emerging challenges, past and cur- one of ORD’s
diseases not generally considered to rent successes add to our scientific pathogen
be native to North America. Addi- arsenal against disease. containment suites.
tionally, although fatalities caused
by waterborne 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-
borne outbreak of cryptosporidiosis
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
Printed on Recycled Paper
Microorganisms Associated with Waterborne Disease
The following groups of microorganisms have been linked with the occurrence of waterborne
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 µm (a µm or “micrometer”
equals one one-thousandth of a millimeter) and 0.2 to
1.2 µm in width. Key bacterial pathogens responsible
for waterborne disease include Legionella, Salmonella
typhi, Shigella, and Vibrio cholerae.
Viruses. Viruses are inactive when outside of a living
host cell. Viruses linked to waterborne disease have
protein coats that provide protection from environ-
mental hazards and range in size from 0.02 to 0.09
µm. Unlike bacteria and protozoa, they contain only
one type of nucleic acid (RNA or DNA). Key
pathogens include hepatitis A and Norwalk virus.
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 waterborne disease include Giardia and
Cryptosporidium.
2
Why Can’t Waterborne waterborne outbreaks is recognized,
Pathogens Be Eliminated? reported and investigated. Of these, It is estimated
the pathogenic agent is identified that swimmers
Microorganisms are present and waders may
everywhere in our environment. only half of the time. Additionally,
ingest from 0.3 to
Invisible to the naked eye, vast experts believe that some food-
1.7 ounces of
numbers of these microbes can be related disease outbreaks may origi-
water per outing.
found in soil, air, food and water. nate with an initial infection (e.g., of
Although humans are essentially a restaurant worker) caused by
free of microorganisms before birth, contaminated drinking water.
constant circumstances of exposure Bacteria, viruses and protozoa
(e.g., breathing, eating, and drink- are the microorganism groups con-
ing) quickly allow the establishment taining pathogens of primary concern
of harmless microbial flora in our in the study of waterborne diseases.
bodies. To eliminate these pathogens from
Microbial pathogens (microor- our water, especially from our drink-
ganisms capable of causing disease), ing water, seems theoretically
however, can and often do harm straightforward. Simply mix in a
those who become infected. More- disinfectant, allow adequate contact
over, diseases that healthy individu- time to assure inactivation (rendering
als “weather” well may prove fatal the microbes unable to produce
to individuals with compromised disease), and pump the water into the Cl Cl
immune systems. In some cases, an distribution lines.
infection can persist to create a In reality, many conditions
“carrier state” where a disease- render the above scenario unwork-
causing agent is harbored by the able. The physical characteristics of
the water, primarily represented by B P
body (and spread) without any
apparent symptoms. dissolved and suspended solids
B
content, can affect the disinfection V S
Waterborne diseases are typi-
cally considered to be those diseases process. The chemical content, both
resulting from ingestion of contami- naturally occurring and anthropo-
nated water. Additional pathways of genic (i.e., generated by humans),
infection being studied by EPA can also interfere with the chemical
reactions desired during treatment Cl
include inhalation of water vapors as
well as body contact during bathing and disinfection. Finally, pathogens
(opportunistic pathogens) in the associated (i.e., imbedded in or
To kill or inactivate
hospital environment. clumped) with higher organisms
drinking water
Since voluntary water ingestion (e.g., algae, rotifers, worms) may be contaminants such
(drinking water) and bathing are protected from the action of disinfec- as bacteria (B),
universal practices and accidental tants. protozoa (P), and
ingestion during recreational activi- To overcome these obstacles to viruses (V),
ties (e.g., swimming, water skiing, disinfection, successful treatment of adequate contact
drinking and waste water generally time with the
wading) is common, inadequate
includes a series of steps. The flow- disinfectant
protection of water integrity could (chlorine or Cl in
lead to widespread outbreaks (the charts in Figures 1 and 2 depict the this representation)
Centers for Disease Control defines steps involved in typical drinking must be allowed.
an outbreak to be two or more cases and waste water treatment processes. Adsorption to and
of illness that can be traced to a In the case of drinking water clumping of solid
common source). Because symp- disinfection, once the impurities have particles (S) can
toms can be mild and short-lived, it been removed, enough disinfectant is inhibit the disinfec-
is estimated that only a fraction of added to inactivate pathogens. Addi- tion process.
3
tionally, a residual level of disinfec- upon by other communities presents
Raw tant must be maintained throughout a significant health risk. Source
Water the distribution system to guard waters heavily loaded with disease-
against potential problems (e.g., causing microorganisms can reduce
microorganisms entering through the effectiveness of “downstream”
breaks in distribution lines or re- drinking water treatment processes.
Screening growth). Such advances as ultraviolet light
Proper distribution system opera- disinfection systems, initially inves-
tion and maintenance practices are tigated as a wastewater disinfection
essential deterrents of pathogen option several years ago, are pres-
Coagulation/ entry, recovery and survival. These ently becoming more widely ac-
Rapid Mixing
practices (according to Geldreich et cepted and reliable with recent
al., 1992) include: design enhancements. This technol-
ogy has been demonstrated to be
• Systematic flushing of the entire capable of meeting existing disinfec-
Flocculation
distribution system “to get more tion criteria without the release of
movement of the chlorine residual dangerous disinfection by-products.
into all parts of the pipe
network...to remove static water
What Progress Has Been
from slow-flow sections, deadends
Sediment- Made?
and stratified water in storage tanks
ation on a periodic basis;” Early in this century, the water-
borne diseases of chief concern in
• Effecting repairs and replacement the U.S. were typhoid fever and
of distribution line components amebiasis. Of the 1,087 deaths
(e.g., broken mains and service associated with waterborne out-
Filtration meters) in a sanitary manner (i.e., breaks between 1920 and 1991, 943
soil-free replacement parts, were attributed to typhoid fever
disinfection and flushing of while 102 were caused by amebiasis.
repaired lines, valves and fittings); Overall, 83% of the deaths occurred
Disinfection
prior to 1936 and less than 1%
• Preventing pathogens from being
occurred after 1970. Additionally,
drawn into the distribution system
the number of outbreaks in commu-
by maintaining continuous positive
nity water systems since 1945 is
Clear pressure and preserving barriers be-
about half as great as the number
Holding tween public water supplies and
documented during the first half of
Tank sewage or storm water drainage;
this century. The reduction in fatali-
• Varying the sample sites during ties and number of outbreaks indi-
routine monitoring to produce data cates that progress has been made in
more representative of the entire the prevention of certain waterborne
To Distribution system. diseases. Much of the progress has
System been the result of increased imple-
While the importance of source mentation of important treatment
water treatment to ensure safe drink- practices (e.g., filtration, disinfec-
ing water seems obvious, the need to tion, sewage treatment). Although
devote equal effort to pathogen progress has been significant, the
Figure 1. Simplified reduction in wastewater is not al- nation’s water continues to be a
flowchart of drinking ways recognized. The release of source of disease. It must be rigor-
water treatment untreated or inadequately treated ously monitored for indicators of
processes. wastewater into source waters drawn fecal contamination.
4
Some Waterborne Diseases of
Concern in the United States
Disease Microbial General Symptoms
Agent
Amebiasis Protozoan Abdominal discomfort, fatigue,
(Entamoeba histolytica) diarrhea, flatulence, weight loss
Campylo- Bacterium Fever, abdominal pain, diarrhea
bacteriosis (Campylobacter jejuni)
Cholera Bacterium Watery diarrhea, vomiting, occasional
(Vibrio cholerae) muscle cramps
Cryptospor- Protozoan Diarrhea, abdominal discomfort
idiosis (Cryptosporidium parvum)
Giardiasis Protozoan Diarrhea, abdominal discomfort
(Giardia lamblia)
Hepatitis Virus Fever, chills, abdominal discomfort,
(hepatitis A) jaundice, dark urine
Shigellosis Bacterium Fever, diarrhea, bloody stool
(Shigella species)
Typhoid fever Bacterium Fever, headache, constipation, appetite
(Salmonella typhi) loss, nausea, diarrhea, vomiting,
appearance of an abdominal rash
Viral Viruses Fever, headache, gastrointestinal
Gastroenteritis (Norwalk, rotavirus and discomfort, vomiting, diarrhea
other types)
5
proper drinking water quality
Raw
through monitoring, and provide
Wastewater
public notification of contamination
problems.
Relating to prevention of water-
borne disease, the SDWA required
Grit Pretreatment
Primary Treatment
EPA to:
1) set numerical standards,
referred to as Maximum Con-
taminant Levels (MCLs — the
Primary Sediment-
highest allowable contaminant
Sludge ation concentrations in drinking water)
or treatment technique require-
ments for contaminants in public
water supplies;
2) issue regulations requiring
monitoring of all regulated and
Biological
Secondary Treatment
certain unregulated contami-
Treatment
nants, depending on the number
of people served by the system,
the source of the water supply,
and the contaminants likely to be
found;
Secondary Sediment-
Sludge ation
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;
To further
treatment or 4) develop disinfection rules for
discharge
all public water supplies; and
Figure 2. In 1974, Congress passed the 5) require all states to develop
Simplified flowchart Safe Drinking Water Act (SDWA) Wellhead Protection Programs
of typical setting up a regulatory program designed to protect from sources
wastewater among local, state, and federal agen- of contamination areas around
treatment wells that supply public drinking
cies to help ensure the provision of
processes.
safe drinking water in the U.S. The water systems.
states are expected to administer and
enforce these regulations for public Through the Surface Water
water systems (systems that either Treatment Rule (SWTR), EPA has
have 15 or more service connections set treatment requirements to control
or regularly serve an average of 25 or microbiological contaminants in
more people daily for at least 60 days public water systems using surface
each year). Public water systems water sources (and ground-water
must provide water treatment, ensure sources under the direct influence of
6
surface water). These requirements Assimilable organic carbon
include the following: (AOC) is the portion of the total Currently, it is
organic carbon (TOC) dissolved in estimated that
1) treatment must remove or water that is easily used by microor- there will be over
inactivate at least 99.9% of ganisms as a carbon source (i.e., 100,000
Giardia lamblia cysts and nutrients). ORD researchers are violations of the
99.99% of viruses; currently investigating treatment SDWA annually.
processes to control AOC. One Nearly half of
2) all systems must disinfect, and these will be for
promising process is biologically
are required to filter if certain MCL violations.
active filtration wherein bacterial
source water quality criteria and Of these, the
communities are intentionally estab-
site-specific criteria are not met; majority will be
lished in the filters to use up, or
biodegrade, the AOC as it passes microbiological
3) the regulations set criteria for violations by
determining if treatment, includ- through. This treatment process must
be employed before final disinfection small systems.
ing turbidity (suspended particu-
late matter) removal and disin- so that bacteria escaping from the
fection requirements, is adequate filter can be properly controlled. As
for filtered systems; and described in Figure 1, most water
utilities do not disinfect with chlorine
4) all systems must be operated until late in the treatment train. This
by qualified operators as deter- limits the formation of disinfection
mined by the states. by-products (i.e., those compounds
like chloroform produced when
chlorine reacts with naturally occur-
Current EPA Research – ring organic carbon).
Barriers to Contamination To accomplish disinfection
earlier in treatment, some water Single step
Although water treatment and
utilities employ ozonation. While membrane filter
disinfection techniques are quite procedure for
effective at microbe reduction, ozone is a very strong disinfectant, it
enumerating E. coli
finished drinking water is not sterile. also converts a portion of the TOC
in recreational
Survival and regrowth of microor- into AOC. ORD researchers are waters. The yellow
ganisms in drinking water distribu- examining the advantages (e.g., colonies are E. coli
tion systems can lead to the deterio- disinfection of bacteria, viruses and while the blue, red
ration of water quality and even non- protozoan cysts, control of color, and purple colonies
compliance of a supply. Regrowth control of taste and odor, enhance- are other coliforms.
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.
7
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
In situ cytotoxicity test for heterotrophic bacteria found in “Hybrid Drinking Water Treatment
drinking water. Heterotrophs recovered from drinking water Package Plants.” These package
form individual yellow colonies (left) that can be transferred to a plants are factory-built, skid-
tissue cell culture (right). Formation of plaques (i.e., clear areas mounted, and ready to be operated in
caused by destruction of infected cells) in the tissue culture can the field with minimal site prepara-
indicate virulence and signal the need for further action. tion. They exhibit lower capital cost
than custom designed facilities built
ment of coagulation, and partial onsite and can incorporate any
oxidation of the naturally occurring drinking water treatment process.
organic carbon that reacts with Promising technologies being con-
chlorine) and disadvantages of ozone sidered for incorporation include
(e.g., enhancement of AOC, conver- membranes, advanced oxidation, bag
sion of bromide to bromate, and filters, and photocatalytic oxidation.
formation of its own disinfection by- By merging, modifying, and adapt-
products like formaldehyde). ing conventional treatment trains
The project entitled “EPANET” with innovative treatment technolo-
involves the development and testing gies, a broader variety of contami-
of a water quality model for drinking nants (including pathogens) can be
water distribution systems. The removed and SDWA compliance can
In the lesser EPANET model is a computer be facilitated.
developed program that performs extended Concern has recently mounted
countries, period simulation of hydraulic and over the ability of certain pathogenic
waterborne water quality behavior within water protozoan (Cryptosporidium) cysts
disease is still a distribution networks. It tracks the to survive treatment processes and
major problem. flow of water in each pipe, the pres- enter the distribution system. ORD,
The World sure at each pipe junction, the height in its project entitled “Evaluation of
Health of water in each tank, and the con- Particulate Removal Processes,” is
Organization has centration of a contaminant through- designing and testing the most effec-
estimated that out the network during a multiple tive filtration techniques to physi-
more people die time period simulation. Water age cally remove the cysts. Being studied
annually of and source tracing can also be simu- are slow sand (see Figure 3), diatom-
water-related lated. aceous earth, and coagulation/rapid
diarrheal EPANET can be useful for sand filtration processes. Results of
illnesses than of analyzing the loss of disinfectant this research will build upon earlier
cancer or AIDs. residual, designing water quality work with filtration of Giardia
sampling programs, performing lamblia.
8
Flow
Overflow Control
Supernatant
Water
Raw
Ventilation
Water Active Biological
Layer V-Notch Weir
Sand
Filterbed
Underdrains
Filter Effluent
Flow
Measurement
Current EPA Research — parts of the country. Seasonal and Figure 3. Basic
Pathogenic Intestinal geographic differences have been elements of a slow
Protozoa recognized and data on concentra- sand filter.
During the last 20 years, signifi- tions in various waters have been
cant improvements have been made collected.
in the quantitative methods for Work on cross-species infectiv-
detecting pathogenic intestinal ity of animal and human cysts has
protozoa (particularly Giardia and established that beavers and musk-
Cryptosporidium) in water. Addi- rats may at least be secondary reser-
tionally, there has been progress in voirs for giardiasis. Also, while it
standardizing these methods. Current appears that avian cysts are not
methods, however, are still time- infective for mammals, we cannot In Minnesota, 100%
consuming and skill-intensive (re- now distinguish avian and mamma- of the muskrats and
quiring highly trained analysts), and lian cysts in a water sample. The 7% of the beavers
lack the ability to indicate viability goal of an ORD project entitled examined were
(whether the cyst is dead or alive) or “Development of Gene Probes for positive for Giardia.
Speciation of Giardia” is to develop In four
infectivity. The latter item has
and test the application of genetic Northeastern states
clouded the development of quantita-
and molecular probe methods to (Maine, New
tive risk assessments.
allow classification of detected Hampshire, New
The cosmopolitan nature of
Giardia species. York, and
intestinal protozoa, and the certainty Gene sequences have been Vermont), the
that all surface water supplies must mapped in species of Giardia shed corresponding
be contaminated with these organ- by animals (e.g., herons and mice) figures were 94%
isms, has been established through and compared with corresponding for muskrats and
studies in animals (beavers, musk- gene sequences in human-hosted 17% for beavers.
rats, and birds) and by occurrence Giardia. Preliminary results indicate
Erlandsen et al., 1990.
studies in sewage throughout all that through these mapped se-
9
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
Trophozoite to allow testing for viable cysts only.
Practical methods for isolation,
identification and quantification of
waterborne 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 Waterborne
Disease” is to develop innovative
immunologic methods for the detec-
tion, identification and enumeration
of pathogenic microorganisms.
Immunologic methods may provide
Cyst
the sensitivity and specificity needed
for detection since low numbers of
Two-stage life cycle quences, once labelled with a detect- target organisms may be present in
of Giardia lamblia: able probe, human type Giardia can large volumes of water along with
the active be differentiated from bird and high numbers of the normal flora and
trophozoite stage mouse Giardia. Probes have been fauna.
and the synthesized and experiments show To accomplish this, the patho-
environmentally-
that one reacts with 10 different G. genic agents will be isolated and
resistant, resting
cyst stage. Cellular lamblia human isolates but not with their antigens (proteins that stimulate
components shown G. psittaci (associated with birds) or the body to produce antibodies) will
above include G. muris (associated with mice). It is be used to produce specific antisera
nuclei (blue), hoped that progress in speciation of for immunologic tests (e.g., immun-
axonemes (red), Giardia can be applied to the study ofluorescent assay, enzyme immuno-
and median bodies of Cryptosporidium. assay, radioimmunoassay).
(green). Current Giardia detection meth- Because standardized procedures
ods are unable to distinguish viable for detecting pathogenic protozoa do
from nonviable cysts. A practical not now exist, confusion in the
detection method for viable cysts interpretation of results obtained by
10
different laboratories occurs. The collected. Through the
goal of the project entitled “Stan- project entitled “Cyst and
Cell
dardized Methods for Detecting Oöcyst Levels in the Ohio
Pathogenic Protozoa in Water” is to River,” ORD is monitor-
develop standardized methods for ing monthly one raw
Chromosome
detecting Giardia and Cryptospo- water sample (collected
ridium in water. These methods will from the river) plus
also assist EPA in assessing research samples from five differ-
findings relevant to regulatory activi- ent points in the drinking
ties under the SDWA. water treatment process.
Cryptosporidium is the only Although current meth-
microorganism on the Office of ods are based on micro- Gene (Linear stretch of DNA)
Ground Water and Drinking Water’s scopic examination of
list of contaminants to be addressed concentrated samples obtained from
in the next round of regulations. large volumes of water, immunofluo-
Quantitative risk assessment for this rescence membrane assays and gene
organism is hampered by the un- probe techniques are being used for
availability of any human infectious this project. Findings from this
dose data and by the scarcity of project will also be used in a nation-
animal dose data. Additionally, very wide survey for occurrence of these
little is known of the longevity and organisms in water supplies.
protective ability of the body’s In the early 1980s, a waterborne
immune response to Cryptospo- disease study in Washington State
ridium infection. suggested that certain elements were
The objective of the project required for a good waterborne
entitled “Cryptosporidium Infectious disease surveillance and investiga-
Dose and Immune Response” is to tion program. Since that time, com-
determine Cryptosporidium infec- puter hardware and software have
tious dose and the associated im- been introduced which may increase
mune response in human volunteers. the potential for improved efficiency
Organisms known to of disease reporting. Although
be infectious for cryptosporidiosis
humans will be out-
obtained from
infected calves From 1986 through 1990,
and administered 20 waterborne outbreaks due to
in drinking water
intestinal protozoa were reported in the U.S.;
to the volunteers.
these outbreaks occurred in 10 states and
Conclusions drawn
affected more than 15,000 people.
from this project
could help shape
future maximum contami-
nant level regulations.
In preparation for development breaks
of disinfection and disinfection by- have been associated
product rules, information on the with drinking water, the relative
occurrence of Giardia cysts and significance of drinking water in the
Cryptosporidium oöcysts in source transmission of this disease is un-
waters and throughout the drinking known. The project entitled “Surveil-
water treatment process must be lance of Waterborne Disease/
11
Cryptosporidiosis Epidemiological more information on the nature and
An epidemiological Feasibility Study” is underway to: 1) extent of viral contamination in our
investigation systematically evaluate waterborne nation’s waters. The objective of
involves the study disease strategies, computer software ORD’s project entitled “Practical
and occurrence of and educational programs in local Methods for Monitoring Viral Patho-
disease within a and state health departments, and 2) gens in Surface and Groundwater
population. In the design an epidemiological study to Source Waters to Define Level of
study of address the significance of drinking Treatment” is to develop improved
waterborne water in the transmission of methods for detection of waterborne
disease, cryptosporidiosis. Products from viruses. In addition to supporting
epidemiological these efforts could shed light on the EPA’s risk assessment efforts relat-
data can indicate a understanding and tracking of water- ing to water quality, these methods
need for additional borne disease outbreaks throughout will provide the means to support the
drinking water the world. establishment of new virological
treatment (e.g., standards and to permit the formation
filtration). of effective options for regulatory
Current EPA Research —
decisions.
Viruses
This project will focus on the
Traditionally, methods for detect- development of biotechnology meth-
ing and identifying viruses have ods based on recognition of viral-
relied on slow cell culture methods. specific nucleic acids within infected
Existing methods may underestimate cell systems. The use of a biotechnol-
the quantity of viruses present or ogy approach that employs DNA
alternatively produce false negatives probes to detect the presence of
when viruses are actually present in viruses is faster, less expensive and
sampled water. Some viruses (e.g., easier to perform than traditional
hepatitis A and Norwalk) simply plaque assay methods.
cannot be detected by the commonly The Science Advisory Board's
used cell culture/plaque assay meth- (SAB) Reducing Risk report to EPA
ods. Given the health risks presented describes pollutants in drinking water
by viruses, it is essential to develop 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
25-49
0-24
Source: 1990 State Section 305(b) Water Quality Reports
12
human health. With over 50% of the Once virus particles
U.S. population relying on ground infect cells in a
water as their primary source of single layer tissue
drinking water, the need for ground- culture, cellular
damage (clear areas
water resource protection, including
or “plaque-forming
protection from pathogens, is clear.
units” in the brown
“Monitoring of Ground Water agar depicted at left)
for Human Enteric Viruses” is a becomes apparent.
current project to address the man- The plaque assay is
date of the SDWA that EPA estab- used for
lish treatment requirements and identification,
criteria for ground water systems. A counting, and
virus occurrence survey of vulner- purification of
able ground water systems is being viruses.
performed to support requirements
for minimum levels of virus inactiva- material of the Norwalk virus par-
tion and ultimately a ground water ticle must currently be amplified
disinfection rule. A number of public using a biotechnology approach
ground water systems will be identi- called polymerase chain reaction
fied and ranked according to vulner- (PCR).
ability to fecal contamination. Of Although known to be highly
these, 25 systems will be monitored infectious, the infectious dose for
for the presence of viruses through Norwalk virus is unknown. The only
tissue culture methods and gene safety-tested virus inoculum (a
probe techniques. microorganism-containing specimen
Hybridized probe (in
Norwalk and Norwalk-like that has been shown to be free of
blue) binds with
viruses cause viral gastroenteritis other pathogens) available cannot be target genetic
(the second leading cause of illness used for infectious dose studies sequence (in red) to
in the U.S.) in consumers of con- because it is not possible to deter- make it detectable
taminated water and food. Since mine the virus concentration. The by radiation or
these viruses cannot be grown and project entitled “Develop a Dose- color.
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
Labelled
stool samples of infected individuals
genetic
and used in an enzyme immunoassay probe
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
Target DNA sequence
for direct immunologic detection and
since there is no known cell culture
method for this virus, the genetic
13
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
waterborne 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-
Preparing stock Response Curve for Norwalk Virus” tem disorders to acute gastroenteritis.
tissue cell cultures is approaching this problem in four The general recommendation has
for isolation and phases: 1) cell cultures capable of been that drinking water should be
identification of growing the Norwalk virus will be free of human enteric viruses and that
viruses in water
developed; 2) Norwalk viruses will recreational water viral limits be set.
samples.
be grown in cell culture, purified and The goal of the project entitled
safety-tested for use in volunteer “National Monitoring and Assess-
studies; 3) a measure of the number ment Program: Status and Long-
of total and infectious virus particles Term Trends in Human Enteric Virus
in the purified sample will be estab- Pollutants in the Aquatic Ecosys-
lished; and 4) a human volunteer tems” is to establish a national viral
study will be initiated to determine survey program focusing on the
the amount of virus particles re- following five factors: 1) selection of
quired to cause disease. monitoring sites based on those most
Although the rate of
The Clean Water Act stipulates likely to have broad public health
water transport
that the nation’s rivers, lakes, and importance; 2) field sampling that
through their gills
coastal waters be swimmable and will result in the collection of ad-
varies greatly,
fishable. Water quality standards equate and representative sample
oysters have been
based on established criteria to volumes to safeguard against false
found to filter as
achieve this goal must be developed. negatives; 3) concentration proce-
many as 154
The project entitled “Shellfish Meth- dures to increase the density of
gallons per day. In
ods and Exposure Response Assess- viruses so that they can be effectively
waters exposed to
ment/Viruses” is being conducted to assayed; 4) standard protocols for
human sewage,
develop methods for detecting and viral detection using both gene probe
these shellfish can
enumerating contamination of shell- and classical plaque assay tech-
filter out and
fish and shellfish growing waters by niques; and 5) parallel biological and
concentrate
human enteric viruses. Shellfish chemical analysis that will serve to
pathogens as well
growing in polluted waters are determine the quality of the water
as food.
known to concentrate these viruses source.
14
Current EPA Research — samples for either fecal coliforms or
Bacteria E. coli is new. Data from available In the U.S., the
methods for detecting chlorine- presence of
The new National Primary coliform bacteria
Drinking Water Regulations require damaged E. coli in drinking water
are limited. The objective of the in drinking water
that all drinking water samples is used as an
testing positive for total coliforms be project entitled “Detection of Low
Numbers of Chlorine-Stressed E. indicator of
further tested for the presence of possible
either fecal coliforms or E. coli. coli in Drinking Water” is to evalu-
ate and compare the abilities of a microbiological
There is a method currently available contamination.
that allows the simultaneous detec- commercial method (Colilert) and a
standard coliform method (EC- When total
tion of total coliforms and E. coli in coliforms are
a broth medium in 24 hours; how- MUG) to recover low numbers of
chlorine-stressed E. coli from po- detected, fecal
ever, there is no equivalent method coliform or E. coli
for use with membrane filters. De- table water. Pure cultures of E. coli
will be washed, nutrient-stressed in analysis must be
velopment of such a method will performed.
allow those who prefer to obtain finished drinking water, and treated
counts of these organisms in their with chlorine. The chlorine-stressed
distribution systems to use a mem- E. coli will then be enumerated,
brane filter method and to have diluted to levels that would be found
results within the 24-hour time in marginally unsafe drinking water
frame. Through the project “Devel- and
opment of a Membrane Filter Me- assayed
dium for the Simultaneous Detection in mul-
The golden metallic
of Total Coliforms and E. coli,” a tiple
sheen of the
membrane filter medium on which tubes by colonies at left
both total coliforms and E. coli can the three indicate the
be distinguished from noncoliforms methods. presence of total
will be developed and patented. These coliforms and the
E. coli are fecal organisms that experi- possibility that the
ments sampled water
when present in drinking water are supply is
indicative of fecal pollution. Logisti- will be
repeated contaminated.
cal concerns in sample handling and
holding require evaluation of condi- using
tions for optimizing sample stability naturally
and longevity. No current regulations occurring E. coli from diluted human
exist for handling samples for analy- fecal specimens, contaminated
sis of E. coli. Through the project source waters and effluents.
entitled “Optimal Sample Holding The infectious bacterial agent
Conditions for Analysis of Fecal E. identified from the stools of cholera
coli in Drinking Water,” sample victims is Vibrio cholerae. The
temperature and holding time will be epidemic in Latin America has
determined for E. coli or fecal colif- prompted a renewed interest in
orm analysis methods (i.e., Colilert control measures for this disease.
and M-FC agar). Relative recovery Through the project entitled “Inacti-
of methods and storage conditions vation of Vibrio cholerae Biotype El
will be assessed for optimal E. coli Tor and Biotype Classical by Chlori-
recovery. nation,” it has been determined that
The requirement (through the the strain responsible for the epi-
SDWA amendments) to test all demic in Peru is capable of reverting
coliform-positive drinking water to a variant which is more resistant
15
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
A rugose (rough- to chlorination than the typical this, a method will be established to
surfaced) variant smooth variety of Vibrio cholerae. study the ability of various types of
(above left) of Vibrio Cells of the variant appear to be amoebae to provide a protective
cholerae 01 is able imbedded in a gelatinous mucoid niche for the multiplication of
to form aggregates. Legionellae under adverse environ-
material, facilitating the formation of
ORD studies have
aggregates, which renders them more mental conditions. Combinations of
indicated that this
variant is more resistant to disinfection. Although Legionella isolates and specific
resistant to the variant is more resistant, studies amoebae that result in high yields of
disinfection than the have indicated that all strains are Legionella after intracellular growth
smooth strain readily inactivated through adequate will be used to study the effects of
(above right). chlorination. intracellular growth on virulence.
The Legionella pneumophila Preliminary studies on the ability of
bacterial strains that cause commu- amoebae to supply iron to
nity- and hospital-acquired pneumo- 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
This one step
method project entitled “Develop Methods
(developed by for Identifying Potential Bacterial
ORD) allows Pathogens in Drinking Water” will
enterococci (blue develop a data base on potential
colonies) health hazards (i.e., pathogenicity)
enumeration in just associated with bacteria commonly
24 hours. 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
will be chal-
lenged via
the gastroi-
ntestinal
route.
Although
virulence is
usually
measured in
vivo (animal
research), the
need for
extensive (a) (b) (c)
animal
testing can be
significantly reduced by the develop- ing) water treatment in small com- Two step, 48 hour
ment of a battery of in vitro (cell munities where the treatment system membrane filter test
culture) tests for traits known to be has been overwhelmed by organic for enumerating
virulence-related. This battery can be substances that may be harmful to enterococci in
used to predict the potential an human health. EPA’s Office of recreational waters.
(a) and (b) Two
organism has for causing disease in Ground Water and Drinking Water
perspectives of
exposed populations. Through the (OGWDW), however, does not want colonies (red)
project entitled “Develop In Vitro to recommend the use of these filters present at 24 hours.
Methods for Identifying Potential if the possibility exists that their use (c) At 48 hours,
Bacterial Pathogens in Drinking poses an acute disease risk due to colonies with black
Water,” model systems will be bacteria that grow on the filters. The halos are identified
developed that can be used to deter- health significance of the bacteria as enterococci.
mine the potential pathogenicity of known to adsorb and grow on GAC
bacteria found in potable water filters used in the home will be
distribution systems. Additionally, evaluated. The OGWDW will use
gene probe and other assays to this information to develop appropri-
identify known opportunistic patho- ate controls and regulations for this
gens will be developed and evalu- type of drinking water treatment as
ated. required by the SDWA.
Bacteria common to drinking The objective of ORD’s project
water distribution systems colonize entitled “Health Effects Associated
point-of-entry, granular activated with Point-of-Entry GAC Filters” is
carbon (GAC) filters where they are to determine if a significant health
able to grow to very high densities. hazard is associated with the use of
Subsequent to reaching the high granular activated carbon, point-of-
densities the bacteria begin slough- entry, whole house filters. To accom-
ing off the GAC filters. The number plish this, a suitable study site will be
of bacteria in the filter effluent (i.e., selected based on the following
water flowing out of the filter) is criteria: 1) the water in the delivery
significantly higher than in the system must meet EPA and local
influent water. This amplification of drinking water standards; and 2) the
bacteria in drinking water is of water distribution system should
concern to EPA because GAC filters contain a bacterial population whose
are being considered as a substitute density is as high as possible and still
for central potable (i.e., fit for drink- acceptable under local regulations.
17
After a distribution system Some believe that exposure to
Analysis of
meeting the above criteria has been fecal pollution through recreational
potable water or
found, a volunteer population of waters or ingestion of contaminated
cooling tower
appropriate size will be selected shellfish causes greater health risks if
water for
from among the water system cus- the pollution is of human rather than
Legionella
tomers. The selected population will animal origin. Before the relative
pneumophila
be randomly divided into GAC user risks of human versus animal fecal
requires
and non-user groups. Point-of-entry, pollution can be assessed, it is neces-
approximately
GAC filters will be installed in the sary to develop a microbiological
five to seven
homes of the randomly selected user method for distinguishing human
days for growth of
group. The health status of both from animal pollution. Current
the organisms on
groups will be monitored over a methods detect fecal pollution but do
the initial isolation
predetermined period of time and not reveal the source. The objective
medium and
during this time interval the bacterial of the project entitled “Method to
another five to
population in the water system and Distinguish Non-Human Fecal
seven days to
the filter effluent will be monitored Pollution from Human Fecal Pollu-
confirm the
on a routine basis. In the event of an tion” is to develop a gene probe
identity of these
illness where a bacterial agent is specific for E. coli that inhabit the
organisms. Gene
diagnosed as the cause, the GAC human intestine for use as an indica-
probe techniques
filter will be removed and examined tor of the presence of human fecal
could reduce
for the presence of the organism contamination in water. The probe
analysis time to
determined to be the agent of the will be field tested at several sites in
one day.
disease in that household unit. If an which fecal pollution is exclusively
association between illness or dis- from human sources, exclusively
ease and the use of GAC filters is from animal sources and from mixed
observed, health advisory guidelines sources.
will be established or processes that Shigella species are among the
will eliminate the causative organ- most common and significant patho-
isms will be developed. gens associated with wastewater and
EPA researcher
using the
transmission
electron microscope
to detect pathogens
unable to be
detected by other
methods.
18
sludge. Because of their low infec- mental Protection Agency. The
tive doses, these organisms may be Office of Research and Development
hazardous even if present in low is committed, through the extensive
numbers in wastewaters that are waterborne disease research efforts
recycled for potable use or sludges earlier described, to ensure that the
that are applied to agricultural land. most effective and efficient methods
Shigellae are very difficult to detect are developed to identify, detect, and
in environmental samples by conven- inactivate/remove pathogens that
tional methods because of their may be present in our drinking water
biochemical similarities to E. coli. supplies.
The use of current gene probe tech- Life cycles, mechanisms of
nology in the project entitled “Detec- infection, protective or dormant
tion of Enteroinvasive Shigella in states, emergence of disinfection-
Wastewaters and Sludges” should resistant variants, optimal pathogen
enable us to detect Shigellae in removal techniques, regrowth in
sludges and wastewaters that would distribution lines…all are areas that
appear to be free of these pathogens must be investigated and understood
if analyzed by conventional methods. to afford the water quality safeguards
that are so often taken for granted.
The successes and failures of these
Conclusion research efforts, relayed to the public Human enteric
The protection and enhancement and appropriate federal, state, and bacteria being
of our nation’s water quality remains local agencies, have helped to ensure subcultured in an
a chief concern of the U.S. Environ- safe drinking water. anaerobic hood.
19
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 coli and 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 E. coli 0157:H7. Wat. Res.,
26: 1127-1137.
20
This publication was prepared by Patrick Burke of ORD’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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