Preventing Waterbo

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 activities are implemented through its headquarters offices

and National Research Laboratories and Centers. The

research focuses on key scientific and technical issues to generate knowledge supporting sound decisions today, and to anticipate the complex challenges

of tomorrow. With a strong, forward-looking research program, less expensive more effective solutions can be pursued and irreversible damage to the

environment can be prevented.

Front cover photo by Lang Photography.
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 statistics are alarming, the risk that extensive outbreaks of waterborne 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 successful line of defense against the spread

of cholera in the U.S. Still, the ease

of international travel has guaranteed the import of a wide variety of

diseases not generally considered to

be native to North America. Additionally, although fatalities caused

by waterborne diseases have declined dramatically in the U.S.

during this century, annual reports of

water-related, microorganisminduced disease continue to number

in the thousands. Just one waterborne outbreak of cryptosporidiosis

in western Georgia (1987), for

example, affected an estimated

13,000 people. In the “colonias”

(poor settlements along the TexasMexico 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 “Giardia,” “Legionella,” and “Norwalk

virus” are becoming more familiar.

The United States Environmental 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 technology 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 current successes add to our scientific

arsenal against disease.

Re

infe

one 

pat

con

Printed 
The following groups of microorganisms have been linked with the occurrence of wat

disease. As each pathogen is isolated and identified as a threat to water quality, ORD 

try to discover the most effective combination of barriers and disinfection methods to 

of human exposure.

Bacteria. Bacteria are the most widely d

forms. Pathogenic bacteria range in leng

approximately 0.4 to 14 μm (a μm or “m

equals one one-thousandth of a millimet

1.2 μm in width. Key bacterial pathogen

for waterborne disease include Legionell

typhi, Shigella, and Vibrio cholerae.

Viruses. Viruses are inactive when outsi

host cell. Viruses linked to waterborne d

protein coats that provide protection from 

mental hazards and range in size from 0.

μm. Unlike bacteria and protozoa, they c

one type of nucleic acid (RNA or DNA)

pathogens include hepatitis A and Norw

Protozoa. Protozoa, common in bodies o

much larger than bacteria and viruses. T

harsh environmental conditions, some sp

secrete a protective covering and form a 

called a “cyst.” Encystment can protect p

drinking water disinfection efforts and fa

spread of disease. Key protozoa being st

agents of waterborne disease include Gia

Cryptosporidium.

2
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 drinking) quickly allow the establishment

of harmless microbial flora in our

bodies.

Microbial pathogens (microorganisms capable of causing disease),

however, can and often do harm

those who become infected. Moreover, diseases that healthy individuals “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 diseasecausing agent is harbored by the

body (and spread) without any

apparent symptoms.

Waterborne diseases are typically considered to be those diseases

resulting from ingestion of contaminated 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 activities (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 symptoms can be mild and short-lived, it

is estimated that only a fraction of

reported and investigated. Of these,

the pathogenic agent is identified

only half of the time. Additionally,

experts believe that some foodrelated disease outbreaks may originate with an initial infection (e.g., of

a restaurant worker) caused by

contaminated drinking water.

Bacteria, viruses and protozoa

are the microorganism groups containing pathogens of primary concern

in the study of waterborne diseases.

To eliminate these pathogens from

our water, especially from our drinking 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 unworkable. 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 anthropogenic (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 disinfectants.

To overcome these obstacles to

disinfection, successful treatment of

drinking and waste water generally

includes a series of steps. The flowcharts 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

tha

an

ing

1.7 

wa

To 

drin

con

as 

pro

viru

ade

tim

dis

(ch

this 

mu

Ads

clu

par

inh

tion 

Cl 

3
Water

To Distribution

System

Screening

Coagulation/

Rapid Mixing

Flocculation

Sedimentation

Filtration

Disinfection

Clear

Holding

Tank

a significant health 

waters heavily load

causing microorgan

the effectiveness of 

drinking water treat

Such advances as ul

disinfection system

tigated as a wastewa

option several years 

ently becoming mor

cepted and reliable 

design enhancemen

ogy has been demon

capable of meeting 

tion criteria without 

dangerous disinfect

What Progress 

Made?

Early in this cen

borne diseases of ch

the U.S. were typho

amebiasis. Of the 1,

associated with wat

breaks between 192

were attributed to ty

while 102 were cau

Overall, 83% of the 

prior to 1936 and le

occurred after 1970

the number of outbr

nity water systems s

about half as great a

documented during 

this century. The re

ties and number of o

cates that progress h

the prevention of ce

diseases. Much of th

been the result of in

mentation of import

practices (e.g., filtra

tion, sewage treatm

progress has been si

nation’s water conti

source of disease. It 

ously monitored for 

fecal contamination

Figure 1. Simplified

flowchart of drinking

water treatment

processes.

tant must be maintained throughout

the distribution system to guard

against potential problems (e.g.,

microorganisms entering through

breaks in distribution lines or regrowth).

Proper distribution system operation 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 between 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 drinking water seems obvious, the need to

devote equal effort to pathogen

reduction in wastewater is not always recognized. The release of

untreated or inadequately treated

wastewater into source waters drawn

4
Some Waterborne Diseases of

Concern in the United States

Protozoan

(Entamoeba histolytica)

Bacterium

(Campylobacter jejuni)

Bacterium

(Vibrio cholerae)

Protozoan

(Cryptosporidium parvum)

Protozoan

(Giardia lamblia)

Virus

(hepatitis A)

Bacterium

(Shigella species)

Bacterium

(Salmonella typhi)

Viruses

(Norwalk, rotavirus and

other types)

Abdominal discomfort, f

diarrhea, flatulence, wei

Fever, abdominal pain, d

Watery diarrhea, vomitin

muscle cramps

Diarrhea, abdominal disc

Diarrhea, abdominal disc

Fever, chills, abdominal 

jaundice, dark urine

Fever, diarrhea, bloody s

Fever, headache, constip

loss, nausea, diarrhea, vo

appearance of an abdom

Fever, headache, gastroi

discomfort, vomiting, di

Amebiasis

Campylobacteriosis

Cholera

Cryptosporidiosis

Giardiasis

Hepatitis

Shigellosis

Typhoid fever

Viral

Gastroenteritis

Microbial

Agent

Disease General Symptom

5
Secondary Treatment

Wastewater

Primary

Sludge

Grit

Secondary

Sludge

To further

treatment or

discharge

Primary Treatment

Figure 2.

Simplified flowchart

of typical

wastewater

treatment

processes.

Pretreatment

Sedimentation

Biological

Treatment

Sedimentation

through monitoring, 

public notification o

problems.

Relating to preve

borne disease, the SD

EPA to:

1) set numerical 

referred to as Ma

taminant Levels 

highest allowable 

concentrations in 

or treatment tech

ments for contam

water supplies;

2) issue regulatio

monitoring of all 

certain unregulat

nants, depending 

of people served 

the source of the 

and the contamin

found;

3) set criteria und

tems are obligate

from surface wat

must also develo

states to determin

tems have to filte

4) develop disinf

all public water s

5) require all stat

Wellhead Protec

designed to prote

of contamination 

wells that supply 

water systems.

Through the Sur

Treatment Rule (SW

set treatment require

microbiological cont

public water systems 

water sources (and g

sources under the dir

In 1974, Congress passed the

Safe Drinking Water Act (SDWA)

setting up a regulatory program

among local, state, and federal agencies 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

6
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, including turbidity (suspended particulate matter) removal and disinfection requirements, is adequate

for filtered systems; and

4) all systems must be operated

by qualified operators as determined 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 microorganisms in drinking water distribution systems can lead to the deterioration of water quality and even noncompliance of a supply. Regrowth

has largely been associated with

heterotrophic bacteria (i.e., those

bacteria – including pathogens – that

require preformed organic compounds 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.

es

the

10

vio

SD

Ne

the

MC

Of 

ma

mi

vio

sm

Sin

me

pro

en

in 

wa

co

wh

an

are 

(AOC) is the portion of the total

organic carbon (TOC) dissolved in

water that is easily used by microorganisms 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 established 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 occurring 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, enhance7
ments, and calibratin

hydraulic models. It 

insight into how cha

source utilization, pu

storage levels, use of 

ment, and targeted p

replacement would a

water quality.

In support of sm

and non-community 

people) drinking wat

systems, ORD resear

signing, modifying a

“Hybrid Drinking W

Package Plants.” The

plants are factory-bu

mounted, and ready 

the field with minim

tion. They exhibit lo

than custom designe

onsite and can incorp

drinking water treatm

Promising technolog

sidered for incorpora

membranes, advance

filters, and photocata

By merging, modify

ing conventional trea

with innovative treat

gies, a broader varie

nants (including path

removed and SDWA 

be facilitated.

Concern has rece

over the ability of ce

protozoan (Cryptosp

to survive treatment 

enter the distribution 

in its project entitled 

Particulate Removal 

designing and testing 

tive filtration techniq

cally remove the cys

are slow sand (see F

aceous earth, and co

sand filtration proces

this research will bui

work with filtration 

lamblia.

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.

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, conversion of bromide to bromate, and

formation of its own disinfection byproducts 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 pressure at each pipe junction, the height

of water in each tank, and the concentration of a contaminant throughout the network during a multiple

time period simulation. Water age

and source tracing can also be simulated.

EPANET can be useful for

analyzing the loss of disinfectant

residual, designing water quality

sampling programs, performing

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.

8
Erla

Fig

ele

san

Active Biological

Layer

Raw

Water

Flow

Measurement

Ventila

V-Notc

Supernatant

Water

Overflow Flow

Control

In M

of t

7% 

exa

pos

In fo

Nor

(Ma

Ham

Yor

Ver

cor

figu

for 

17% 

parts of the country. Seasonal and

geographic differences have been

recognized and data on concentrations in various waters have been

collected.

Work on cross-species infectivity of animal and human cysts has

established that beavers and muskrats may at least be secondary reservoirs for giardiasis. Also, while it

appears that avian cysts are not

infective for mammals, we cannot

now distinguish avian and mammalian 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 seCurrent EPA Research —

Pathogenic Intestinal

Protozoa

During the last 20 years, significant improvements have been made

in the quantitative methods for

detecting pathogenic intestinal

protozoa (particularly Giardia and

Cryptosporidium) in water. Additionally, there has been progress in

standardizing these methods. Current

methods, however, are still timeconsuming and skill-intensive (requiring highly trained analysts), and

lack the ability to indicate viability

(whether the cyst is dead or alive) or

infectivity. The latter item has

clouded the development of quantitative risk assessments.

The cosmopolitan nature of

intestinal protozoa, and the certainty

that all surface water supplies must

be contaminated with these organisms, has been established through

studies in animals (beavers, muskrats, and birds) and by occurrence

studies in sewage throughout all

Sand

Filterbed

Underdrains

9
cance of positive rep

allow establishment 

standards under the S

objectives of the pro

Probes for the Detec

zoan Parasites” and 

Stress Proteins as a M

Giardia Cyst Viabili

cover, separate and a

genetic sequences (D

associated with viab

If these specific sequ

identified, probes ca

to allow testing for v

Practical method

identification and qu

waterborne pathogen

dia are not yet availa

and identification me

needed before contro

be evaluated and reg

sions can be made re

treatment processes 

goal of ORD’s proje

munological Method

of Etiological Agent

Disease” is to develo

immunologic method

tion, identification an

of pathogenic microo

Immunologic metho

the sensitivity and sp

for detection since lo

target organisms ma

large volumes of wa

high numbers of the 

fauna.

To accomplish th

genic agents will be 

their antigens (protei

the body to produce 

be used to produce s

for immunologic tes

ofluorescent assay, e

assay, radioimmunoa

Because standard

for detecting pathoge

not now exist, confu

interpretation of resu

quences, once labelled with a detectable 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 methods are unable to distinguish viable

from nonviable cysts. A practical

detection method for viable cysts

Two-stage life cycle

of Giardia lamblia:

the active

trophozoite stage

and the

environmentallyresistant, resting

cyst stage. Cellular

components shown

above include

nuclei (blue),

axonemes (red),

and median bodies

(green).

Cyst

Trophozoite

10
goal of the project entitled Stan

dardized Methods for Detecting

Pathogenic Protozoa in Water” is to

develop standardized methods for

detecting Giardia and Cryptosporidium in water. These methods will

also assist EPA in assessing research

findings relevant to regulatory activities 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 unavailability 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 Cryptosporidium infection.

The objective of the project

entitled “Cryptosporidium Infectious

Dose and Immune Response” is to

determine Cryptosporidium infectious dose and the associated immune 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 contaminant level regulations.

In preparation for development

of disinfection and disinfection byproduct rules, information on the

occurrence of Giardia cysts and

Cryptosporidium oöcysts in source

waters and throughout the drinking

water treatment process must be

project entitled Cyst and

Oöcyst Levels in the Ohio

River,” ORD is monitoring monthly one raw

water sample (collected

from the river) plus

samples from five different points in the drinking

water treatment process.

Although current methods are based on microscopic examination of

concentrated samples obtained from

large volumes of water, immunofluorescence membrane assays and gene

probe techniques are being used for

this project. Findings from this

project will also be used in a nationwide 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 investigation program. Since that time, computer hardware and software have

been introduced which may increase

the potential for improved efficiency

of disease reporting. Although

cryptosporidiosis

outbreaks

have been associated

with drinking water, the relative

significance of drinking water in the

transmission of this disease is unknown. The project entitled “Surveillance of Waterborne Disease/

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.

11

Gene (Linear str

Chromosome

Cell
extent of viral contam

nation’s waters. The 

ORD’s project entitl

Methods for Monito

gens in Surface and 

Source Waters to De

Treatment” is to dev

methods for detectio

viruses. In addition t

EPA’s risk assessme

ing to water quality, 

will provide the mea

establishment of new 

standards and to perm

of effective options f

decisions.

This project will 

development of biote

ods based on recogn

specific nucleic acid

cell systems. The use 

ogy approach that em

probes to detect the p

viruses is faster, less 

easier to perform tha

plaque assay method

The Science Adv

(SAB) Reducing Ris

describes pollutants 

as one of the four hig

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 waterborne disease outbreaks throughout

the world.

Current EPA Research —

Viruses

Traditionally, methods for detecting 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 methods. Given the health risks presented

by viruses, it is essential to develop

Source: 1990 State Section 305(b) Water Quality Reports

Percentage of State Populations Served by Ground Water for Dom

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).

12

LEGE

Percent of P

25-49

50-74

75-10

0-24
U.S. population relying on ground

water as their primary source of

drinking water, the need for groundwater resource protection, including

protection from pathogens, is clear.

“Monitoring of Ground Water

for Human Enteric Viruses” is a

current project to address the mandate of the SDWA that EPA establish treatment requirements and

criteria for ground water systems. A

virus occurrence survey of vulnerable ground water systems is being

performed to support requirements

for minimum levels of virus inactivation and ultimately a ground water

disinfection rule. A number of public

ground water systems will be identified and ranked according to vulnerability 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 contaminated 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 gastroenteritis usually is not determined.

Since the viral density in environmental 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 particle 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 determine the virus concentration. The

project entitled “Develop a DoseLabelled

genetic

probe

Target DNA sequence

sin

cul

dam

or 

uni

aga

bec

The 

use

ide

cou

pur

viru

Hy

blu

targ

seq

ma

by 

col

13
often eaten raw or in

cooked, subjecting s

human wastes consti

health risk.

Because there ar

waterborne virus typ

cause clinical outbre

monitoring efforts ar

ensure the virologica

waters and particular

of water and wastew

practices. This inform

be provided through 

toring and assessmen

each major pathway 

deposition of human 

into the nation’s wat

ruses are responsible 

illnesses ranging fro

myocarditis to centra

tem disorders to acut

The general recomm

been that drinking w

free of human enteri

recreational water vi

The goal of the proje

“National Monitorin

ment Program: Statu

Term Trends in Hum

Pollutants in the Aqu

tems” is to establish 

survey program focu

following five factor

monitoring sites base

likely to have broad 

importance; 2) field 

will result in the coll

equate and represent

volumes to safeguard 

negatives; 3) concen

dures to increase the 

viruses so that they c

assayed; 4) standard 

viral detection using 

and classical plaque 

niques; and 5) parall

chemical analysis th

determine the quality 

source.

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 established; and 4) a human volunteer

study will be initiated to determine

the amount of virus particles required 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 Methods and Exposure Response Assessment/Viruses” is being conducted to

develop methods for detecting and

enumerating contamination of shellfish and shellfish growing waters by

human enteric viruses. Shellfish

growing in polluted waters are

known to concentrate these viruses

Preparing stock

tissue cell cultures

for isolation and

identification of

viruses in water

samples.

14
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 detection of total coliforms and E. coli in

a broth medium in 24 hours; however, there is no equivalent method

for use with membrane filters. Development of such a method will

allow those who prefer to obtain

counts of these organisms in their

distribution systems to use a membrane filter method and to have

results within the 24-hour time

frame. Through the project “Development of a Membrane Filter Medium 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. Logistical concerns in sample handling and

holding require evaluation of conditions for optimizing sample stability

and longevity. No current regulations

exist for handling samples for analysis 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 coliform 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

pre

col

in d

is u

ind

pos

mic

con

Wh

col

det

col

ana

pe

E. coli is new. Data from available

methods for detecting chlorinedamaged 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 evaluate and compare the abilities of a

commercial method (Colilert) and a

standard coliform method (ECMUG) to recover low numbers of

chlorine-stressed E. coli from potable water. Pure cultures of E. 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 multiple

tubes by

the three

methods.

These

experiments

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 cholerae. The

epidemic in Latin America has

prompted a renewed interest in

control measures for this disease.

Through the project entitled “Inactivation of Vibrio cholerae Biotype El

Tor and Biotype Classical by Chlorination,” it has been determined that

the strain responsible for the epidemic in Peru is capable of reverting

to a variant which is more resistant

The 

she

col

ind

pre

col

pos

sam

sup

con

15
drinking water. Cert

amoebae (protozoa) 

multiplication of L. p

drinking water syste

bae may also be resp

enhancing the virule

a microorganism to c

the Legionellae and 

them from adverse e

factors such as high 

chlorine disinfection

entitled “Multiplicat

Legionellae in Amoe

ment of Virulence” w

the effect of intracell

Legionella in amoeb

and as protection aga

and high temperature

this, a method will b

study the ability of v

amoebae to provide 

niche for the multipl

Legionellae under ad

mental conditions. C

Legionella isolates a

amoebae that result i

Legionella after intra

will be used to study 

intracellular growth 

Preliminary studies o

amoebae to supply ir

Legionellae growing 

showed no obvious a

between growth and 

tion.

EPA is required 

establish appropriate 

regulations for potab

project entitled “Dev

for Identifying Poten

Pathogens in Drinkin

develop a data base o

health hazards (i.e., p

associated with bacte

found in water distri

To accomplish this, 

species will be comp

nitrous oxides or imm

agents, and the anim

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 community- and hospital-acquired pneumoThis one step

method

(developed by

ORD) allows

enterococci (blue

colonies)

enumeration in just

24 hours.

A rugose (roughsurfaced) variant

(above left) of Vibrio

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).

16
lenged via

the gastrointestinal

route.

Although

virulence is

usually

measured in

vivo (animal

research), the

need for

extensive

animal

testing can be

significantly reduced by the development 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 determine the potential pathogenicity of

bacteria found in potable water

distribution systems. Additionally,

gene probe and other assays to

identify known opportunistic pathogens will be developed and evaluated.

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 sloughing 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 drinking) water treatment in small communities 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 appropriate 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-ofentry, whole house filters. To accomplish 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.

(a) (b) 

Tw

me

for 

ent

rec

(a) 

per

col

pre

(c) 

col

hal

as 

17
meeting the above criteria has been

found, a volunteer population of

appropriate size will be selected

from among the water system customers. 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 disease and the use of GAC filters is

observed, health advisory guidelines

will be established or processes that

will eliminate the causative organisms will be developed.

fecal pollution throu

waters or ingestion o

shellfish causes grea

the pollution is of hu

animal origin. Befor

risks of human versu

pollution can be asse

sary to develop a mi

method for distingui

from animal pollutio

methods detect fecal 

not reveal the source

of the project entitled 

Distinguish Non-Hu

Pollution from Hum

tion” is to develop a 

specific for E. coli th

human intestine for u

tor of the presence o

contamination in wa

will be field tested a

which fecal pollution 

from human sources

from animal sources 

sources.

Shigella species 

most common and si

gens associated with 

EPA researcher

using the

transmission

electron microscope

to detect pathogens

unable to be

detected by other

methods.

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.

18
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 conventional methods because of their

biochemical similarities to E. coli.

The use of current gene probe technology in the project entitled “Detection 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

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 disinfectionresistant 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.

Hu

bac

sub

ana

19
EPA Publications

The EPA publications listed below may provide more detailed information 

subjects discussed in this document. These references and additional copies 

chure can be requested at no charge (while supplies are available) from EPA’

Environmental Research Information (CERI). Once the CERI inventory is exha

clients will be directed to the National Technical Information Service (NTIS) w

ments can be purchased.

Environmental Pollution Control Alternatives: Drinking Water Treatment for Sm

nities, EPA/625/5-90/025.

Methods for the Investigation and Prevention of Waterborne Disease Outbrea

600/1-90/005a.

Microbiological Methods for Monitoring the Environment — Water and Wastes

8-78/017.

Seminar Publication: Control of Biofilm Growth in Drinking Water Distribution 

EPA/625/R-92/001.

Test Methods for Escherichia coli and Enterococci in Water by the Membrane 

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 Epidemiolog

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. Pr

Giardia spp. in beaver and muskrat populations in northeastern states and Minnesota. A

Micro., 56: 31-36.

Geldreich, E.E., K.R. Fox, J.A. Goodrich, E.W. Rice, R.M. Clark, and D.L. Swerdlow. 19

for a water supply connection in the Cabool, Missouri disease outbreak of E. coli 0157:H

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, Robe

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 Reasone

and Eugene Rice of the National Risk Management Research

Laboratory. Thanks to Al Lang and Jim O’Dell for photographic

support.