EPA/640/K-93/001April 1993

United StatesEnvironmental ProtectionAgency

Office of Research andDevelopmentWashington, DC 20460

Preventing WaterborneDisease

A Focus on EPA’sResearch

EPA’s Office of Research and Development

The Office of Research and Development (ORD)conducts an integrated program of scientific researchand development on the sources, transport and fateprocesses, monitoring, control, and assessment of riskand effects of environmental pollutants. These activi-ties are implemented through its headquarters officesand National Research Laboratories and Centers. Theresearch focuses on key scientific and technical is-sues to generate knowledge supporting sound deci-sions today, and to anticipate the complex challengesof tomorrow. With a strong, forward-looking re-search program, less expensive more effective solu-tions can be pursued and irreversible damage to theenvironment can be prevented.

Front cover photo by Lang Photography.

“The reported case total for the epidemic nearsthree-quarters of a million. Since the beginningof the epidemic in January 1991, the totalnumber of reported cases is 746,968 with 6,448deaths.”

(Cholera Epidemic in the Americas, CDC Update, February 11, 1993)

Although the above-listed statis-tics are alarming, the risk that exten-sive outbreaks of waterborne cholerawill occur in the United States isminimal. Effective treatment ofdrinking water and sewage, coupledwith adequate personal hygienehabits, has contributed to a success-ful line of defense against the spreadof cholera in the U.S. Still, the easeof international travel has guaran-teed the import of a wide variety ofdiseases not generally considered tobe native to North America. Addi-tionally, although fatalities causedby waterborne diseases have de-clined dramatically in the U.S.during this century, annual reports ofwater-related, microorganism-induced disease continue to numberin the thousands. Just one water-borne outbreak of cryptosporidiosisin western Georgia (1987), forexample, affected an estimated13,000 people. In the “colonias”(poor settlements along the Texas-Mexico border), high levels ofdisease have been associated withthe lack of public water supplies andinadequate waste treatment. Whilethe words “typhoid fever” fade fromour vocabulary, such terms as “Giar-dia,” “ Legionella,” and “Norwalkvirus” are becoming more familiar.

The United States Environmen-tal Protection Agency (U.S. EPA),through its Office of Research andDevelopment (ORD), is conductingresearch to better understand and

prevent water contamination byharmful microorganisms. Frommonitoring our nation’s groundwater systems for viral pathogens...todeveloping more effective technol-ogy for large and small systems...toproviding other nations with criticaltechnical assistance, ORD scientistsand engineers continue their missionto ensure safe waters. As the focus ofour efforts adjusts to deal withemerging challenges, past and cur-rent successes add to our scientificarsenal against disease.

Researcher isolatinginfectious bacteria inone of ORD’spathogencontainment suites.

Printed on Recycled Paper

Microorganisms Associated with Waterborne Disease

The following groups of microorganisms have been linked with the occurrence of waterbornedisease. As each pathogen is isolated and identified as a threat to water quality, ORD researcherstry to discover the most effective combination of barriers and disinfection methods to minimize riskof human exposure.

Bacteria. Bacteria are the most widely distributed lifeforms. Pathogenic bacteria range in length fromapproximately 0.4 to 14 µm (a µm or “micrometer”equals one one-thousandth of a millimeter) and 0.2 to1.2 µm in width. Key bacterial pathogens responsiblefor waterborne disease include Legionella, Salmonellatyphi, Shigella, and Vibrio cholerae.

Viruses. Viruses are inactive when outside of a livinghost cell. Viruses linked to waterborne disease haveprotein coats that provide protection from environ-mental hazards and range in size from 0.02 to 0.09µm. Unlike bacteria and protozoa, they contain onlyone type of nucleic acid (RNA or DNA). Keypathogens include hepatitis A and Norwalk virus.

Protozoa. Protozoa, common in bodies of water, aremuch larger than bacteria and viruses. To surviveharsh environmental conditions, some species cansecrete a protective covering and form a resting stagecalled a “cyst.” Encystment can protect protozoa fromdrinking water disinfection efforts and facilitate thespread of disease. Key protozoa being studied asagents of waterborne disease include Giardia andCryptosporidium.

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Why Can’t WaterbornePathogens Be Eliminated?

Microorganisms are presenteverywhere in our environment.Invisible to the naked eye, vastnumbers of these microbes can befound in soil, air, food and water.Although humans are essentiallyfree of microorganisms before birth,constant circumstances of exposure(e.g., breathing, eating, and drink-ing) quickly allow the establishmentof harmless microbial flora in ourbodies.

Microbial pathogens (microor-ganisms capable of causing disease),however, can and often do harmthose who become infected. More-over, diseases that healthy individu-als “weather” well may prove fatalto individuals with compromisedimmune systems. In some cases, aninfection can persist to create a“carrier state” where a disease-causing agent is harbored by thebody (and spread) without anyapparent symptoms.

Waterborne diseases are typi-cally considered to be those diseasesresulting from ingestion of contami-nated water. Additional pathways ofinfection being studied by EPAinclude inhalation of water vapors aswell as body contact during bathing(opportunistic pathogens) in thehospital environment.

Since voluntary water ingestion(drinking water) and bathing areuniversal practices and accidentalingestion during recreational activi-ties (e.g., swimming, water skiing,wading) is common, inadequateprotection of water integrity couldlead to widespread outbreaks (theCenters for Disease Control definesan outbreak to be two or more casesof illness that can be traced to acommon source). Because symp-toms can be mild and short-lived, itis estimated that only a fraction of

waterborne outbreaks is recognized,reported and investigated. Of these,the pathogenic agent is identifiedonly half of the time. Additionally,experts believe that some food-related disease outbreaks may origi-nate with an initial infection (e.g., ofa restaurant worker) caused bycontaminated drinking water.

Bacteria, viruses and protozoaare the microorganism groups con-taining pathogens of primary concernin the study of waterborne diseases.To eliminate these pathogens fromour water, especially from our drink-ing water, seems theoreticallystraightforward. Simply mix in adisinfectant, allow adequate contacttime to assure inactivation (renderingthe microbes unable to producedisease), and pump the water into thedistribution lines.

In reality, many conditionsrender the above scenario unwork-able. The physical characteristics ofthe water, primarily represented bydissolved and suspended solidscontent, can affect the disinfectionprocess. The chemical content, bothnaturally occurring and anthropo-genic (i.e., generated by humans),can also interfere with the chemicalreactions desired during treatmentand disinfection. Finally, pathogensassociated (i.e., imbedded in orclumped) with higher organisms(e.g., algae, rotifers, worms) may beprotected from the action of disinfec-tants.

To overcome these obstacles todisinfection, successful treatment ofdrinking and waste water generallyincludes a series of steps. The flow-charts in Figures 1 and 2 depict thesteps involved in typical drinkingand waste water treatment processes.

In the case of drinking waterdisinfection, once the impurities havebeen removed, enough disinfectant isadded to inactivate pathogens. Addi-

It is estimatedthat swimmersand waders mayingest from 0.3 to1.7 ounces ofwater per outing.

To kill or inactivatedrinking watercontaminants suchas bacteria (B),protozoa (P), andviruses (V),adequate contacttime with thedisinfectant(chlorine or Cl inthis representation)must be allowed.Adsorption to andclumping of solidparticles (S) caninhibit the disinfec-tion process.

Cl Cl

Cl

B P

VB

S

3

RawWater

To DistributionSystem

Screening

Coagulation/Rapid Mixing

Flocculation

Sediment-ation

Filtration

Disinfection

ClearHolding

Tank

upon by other communities presentsa significant health risk. Sourcewaters heavily loaded with disease-causing microorganisms can reducethe effectiveness of “downstream”drinking water treatment processes.Such advances as ultraviolet lightdisinfection systems, initially inves-tigated as a wastewater disinfectionoption several years ago, are pres-ently becoming more widely ac-cepted and reliable with recentdesign enhancements. This technol-ogy has been demonstrated to becapable of meeting existing disinfec-tion criteria without the release ofdangerous disinfection by-products.

What Progress Has BeenMade?

Early in this century, the water-borne diseases of chief concern inthe U.S. were typhoid fever andamebiasis. Of the 1,087 deathsassociated with waterborne out-breaks between 1920 and 1991, 943were attributed to typhoid feverwhile 102 were caused by amebiasis.Overall, 83% of the deaths occurredprior to 1936 and less than 1%occurred after 1970. Additionally,the number of outbreaks in commu-nity water systems since 1945 isabout half as great as the numberdocumented during the first half ofthis century. The reduction in fatali-ties and number of outbreaks indi-cates that progress has been made inthe prevention of certain waterbornediseases. Much of the progress hasbeen the result of increased imple-mentation of important treatmentpractices (e.g., filtration, disinfec-tion, sewage treatment). Althoughprogress has been significant, thenation’s water continues to be asource of disease. It must be rigor-ously monitored for indicators offecal contamination.

Figure 1. Simplifiedflowchart of drinkingwater treatmentprocesses.

tionally, a residual level of disinfec-tant must be maintained throughoutthe distribution system to guardagainst potential problems (e.g.,microorganisms entering throughbreaks in distribution lines or re-growth).

Proper distribution system opera-tion and maintenance practices areessential deterrents of pathogenentry, recovery and survival. Thesepractices (according to Geldreich etal., 1992) include:

• Systematic flushing of the entiredistribution system “to get moremovement of the chlorine residualinto all parts of the pipenetwork...to remove static waterfrom slow-flow sections, deadendsand stratified water in storage tankson a periodic basis;”

• Effecting repairs and replacementof distribution line components(e.g., broken mains and servicemeters) in a sanitary manner (i.e.,soil-free replacement parts,disinfection and flushing ofrepaired lines, valves and fittings);

• Preventing pathogens from beingdrawn into the distribution systemby maintaining continuous positivepressure and preserving barriers be-tween public water supplies andsewage or storm water drainage;

• Varying the sample sites duringroutine monitoring to produce datamore representative of the entiresystem.

While the importance of sourcewater treatment to ensure safe drink-ing water seems obvious, the need todevote equal effort to pathogenreduction in wastewater is not al-ways recognized. The release ofuntreated or inadequately treatedwastewater into source waters drawn

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Some Waterborne Diseases ofConcern 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 andother types)

Abdominal discomfort, fatigue,diarrhea, flatulence, weight loss

Fever, abdominal pain, diarrhea

Watery diarrhea, vomiting, occasionalmuscle cramps

Diarrhea, abdominal discomfort

Diarrhea, abdominal discomfort

Fever, chills, abdominal discomfort,jaundice, dark urine

Fever, diarrhea, bloody stool

Fever, headache, constipation, appetiteloss, nausea, diarrhea, vomiting,appearance of an abdominal rash

Fever, headache, gastrointestinaldiscomfort, vomiting, diarrhea

Amebiasis

Campylo-bacteriosis

Cholera

Cryptospor-idiosis

Giardiasis

Hepatitis

Shigellosis

Typhoid fever

ViralGastroenteritis

MicrobialAgent

Disease General Symptoms

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Secondary T

reatment

RawWastewater

PrimarySludge

Grit

SecondarySludge

To furthertreatment ordischarge

Prim

ary Treatm

ent

Figure 2.Simplified flowchartof typicalwastewatertreatmentprocesses.

Pretreatment

Sediment-ation

BiologicalTreatment

Sediment-ation

proper drinking water qualitythrough monitoring, and providepublic notification of contaminationproblems.

Relating to prevention of water-borne disease, the SDWA requiredEPA to:

1) set numerical standards,referred to as Maximum Con-taminant Levels (MCLs — thehighest allowable contaminantconcentrations in drinking water)or treatment technique require-ments for contaminants in publicwater supplies;

2) issue regulations requiringmonitoring of all regulated andcertain unregulated contami-nants, depending on the numberof people served by the system,the source of the water supply,and the contaminants likely to befound;

3) set criteria under which sys-tems are obligated to filter waterfrom surface water sources; itmust also develop procedures forstates to determine which sys-tems have to filter;

4) develop disinfection rules forall public water supplies; and

5) require all states to developWellhead Protection Programsdesigned to protect from sourcesof contamination areas aroundwells that supply public drinkingwater systems.

Through the Surface WaterTreatment Rule (SWTR), EPA hasset treatment requirements to controlmicrobiological contaminants inpublic water systems using surfacewater sources (and ground-watersources under the direct influence of

In 1974, Congress passed theSafe Drinking Water Act (SDWA)setting up a regulatory programamong local, state, and federal agen-cies to help ensure the provision ofsafe drinking water in the U.S. Thestates are expected to administer andenforce these regulations for publicwater systems (systems that eitherhave 15 or more service connectionsor regularly serve an average of 25 ormore people daily for at least 60 dayseach year). Public water systemsmust provide water treatment, ensure

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surface water). These requirementsinclude the following:

1) treatment must remove orinactivate at least 99.9% ofGiardia lamblia cysts and99.99% of viruses;

2) all systems must disinfect, andare required to filter if certainsource water quality criteria andsite-specific criteria are not met;

3) the regulations set criteria fordetermining if treatment, includ-ing turbidity (suspended particu-late matter) removal and disin-fection requirements, is adequatefor filtered systems; and

4) all systems must be operatedby qualified operators as deter-mined by the states.

Current EPA Research –Barriers to Contamination

Although water treatment anddisinfection techniques are quiteeffective at microbe reduction,finished drinking water is not sterile.Survival and regrowth of microor-ganisms in drinking water distribu-tion systems can lead to the deterio-ration of water quality and even non-compliance of a supply. Regrowthhas largely been associated withheterotrophic bacteria (i.e., thosebacteria – including pathogens – thatrequire preformed organic com-pounds as carbon and energysources). Bacterial growth occurs onthe walls of the distribution system(referred to as “biofilms”) and in thewater either as free living cells orcells attached to suspended solids. Amulti-faceted phenomenon, bacterialregrowth is influenced primarily bytemperature, residence time in mainsand storage units, the efficacy ofdisinfection, and nutrients.

Currently, it isestimated thatthere will be over100,000violations of theSDWA annually.Nearly half ofthese will be forMCL violations.Of these, themajority will bemicrobiologicalviolations bysmall systems.

Single stepmembrane filterprocedure forenumerating E. coliin recreationalwaters. The yellowcolonies are E. coliwhile the blue, redand purple coloniesare other coliforms.

Assimilable organic carbon(AOC) is the portion of the totalorganic carbon (TOC) dissolved inwater that is easily used by microor-ganisms as a carbon source (i.e.,nutrients). ORD researchers arecurrently investigating treatmentprocesses to control AOC. Onepromising process is biologicallyactive filtration wherein bacterialcommunities are intentionally estab-lished in the filters to use up, orbiodegrade, the AOC as it passesthrough. This treatment process mustbe employed before final disinfectionso that bacteria escaping from thefilter can be properly controlled. Asdescribed in Figure 1, most waterutilities do not disinfect with chlorineuntil late in the treatment train. Thislimits the formation of disinfectionby-products (i.e., those compoundslike chloroform produced whenchlorine reacts with naturally occur-ring organic carbon).

To accomplish disinfectionearlier in treatment, some waterutilities employ ozonation. Whileozone is a very strong disinfectant, italso converts a portion of the TOCinto AOC. ORD researchers areexamining the advantages (e.g.,disinfection of bacteria, viruses andprotozoan cysts, control of color,control of taste and odor, enhance-

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drinking water exposure risk assess-ments, and calibrating networkhydraulic models. It can provideinsight into how changes in watersource utilization, pumping waterstorage levels, use of satellite treat-ment, and targeted pipe cleaning andreplacement would affect drinkingwater quality.

In support of small communityand non-community (less than 3300people) drinking water treatmentsystems, ORD researchers are de-signing, modifying and testing“Hybrid Drinking Water TreatmentPackage Plants.” These packageplants are factory-built, skid-mounted, and ready to be operated inthe field with minimal site prepara-tion. They exhibit lower capital costthan custom designed facilities builtonsite and can incorporate anydrinking water treatment process.Promising technologies being con-sidered for incorporation includemembranes, advanced oxidation, bagfilters, and photocatalytic oxidation.By merging, modifying, and adapt-ing conventional treatment trainswith innovative treatment technolo-gies, a broader variety of contami-nants (including pathogens) can beremoved and SDWA compliance canbe facilitated.

Concern has recently mountedover the ability of certain pathogenicprotozoan (Cryptosporidium) cyststo survive treatment processes andenter the distribution system. ORD,in its project entitled “Evaluation ofParticulate Removal Processes,” isdesigning and testing the most effec-tive filtration techniques to physi-cally remove the cysts. Being studiedare slow sand (see Figure 3), diatom-aceous earth, and coagulation/rapidsand filtration processes. Results ofthis research will build upon earlierwork with filtration of Giardialamblia.

In situ cytotoxicity test for heterotrophic bacteria found indrinking water. Heterotrophs recovered from drinking waterform individual yellow colonies (left) that can be transferred to atissue cell culture (right). Formation of plaques (i.e., clear areascaused by destruction of infected cells) in the tissue culture canindicate virulence and signal the need for further action.

ment of coagulation, and partialoxidation of the naturally occurringorganic carbon that reacts withchlorine) and disadvantages of ozone(e.g., enhancement of AOC, conver-sion of bromide to bromate, andformation of its own disinfection by-products like formaldehyde).

The project entitled “EPANET”involves the development and testingof a water quality model for drinkingwater distribution systems. TheEPANET model is a computerprogram that performs extendedperiod simulation of hydraulic andwater quality behavior within waterdistribution networks. It tracks theflow of water in each pipe, the pres-sure at each pipe junction, the heightof water in each tank, and the con-centration of a contaminant through-out the network during a multipletime period simulation. Water ageand source tracing can also be simu-lated.

EPANET can be useful foranalyzing the loss of disinfectantresidual, designing water qualitysampling programs, performing

In the lesserdevelopedcountries,waterbornedisease is still amajor problem.The WorldHealthOrganization hasestimated thatmore people dieannually ofwater-relateddiarrhealillnesses than ofcancer or AIDs.

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Erlandsen et al., 1990.

Figure 3. Basicelements of a slowsand filter.

Active BiologicalLayer

RawWater

FlowMeasurement

Filter Effluent

Ventilation

V-Notch Weir

SupernatantWater

OverflowFlow

Control

In Minnesota, 100%of the muskrats and7% of the beaversexamined werepositive for Giardia.In fourNortheastern states(Maine, NewHampshire, NewYork, andVermont), thecorrespondingfigures were 94%for muskrats and17% for beavers.

parts of the country. Seasonal andgeographic differences have beenrecognized and data on concentra-tions in various waters have beencollected.

Work on cross-species infectiv-ity of animal and human cysts hasestablished that beavers and musk-rats may at least be secondary reser-voirs for giardiasis. Also, while itappears that avian cysts are notinfective for mammals, we cannotnow distinguish avian and mamma-lian cysts in a water sample. Thegoal of an ORD project entitled“Development of Gene Probes forSpeciation of Giardia” is to developand test the application of geneticand molecular probe methods toallow classification of detectedGiardia species.

Gene sequences have beenmapped in species of Giardia shedby animals (e.g., herons and mice)and compared with correspondinggene sequences in human-hostedGiardia. Preliminary results indicatethat through these mapped se-

Current EPA Research —Pathogenic IntestinalProtozoa

During the last 20 years, signifi-cant improvements have been madein the quantitative methods fordetecting pathogenic intestinalprotozoa (particularly Giardia andCryptosporidium) in water. Addi-tionally, there has been progress instandardizing these methods. Currentmethods, however, are still time-consuming and skill-intensive (re-quiring highly trained analysts), andlack the ability to indicate viability(whether the cyst is dead or alive) orinfectivity. The latter item hasclouded the development of quantita-tive risk assessments.

The cosmopolitan nature ofintestinal protozoa, and the certaintythat all surface water supplies mustbe contaminated with these organ-isms, has been established throughstudies in animals (beavers, musk-rats, and birds) and by occurrencestudies in sewage throughout all

SandFilterbed

Underdrains

9

would allow assessing the signifi-cance of positive reports and mayallow establishment of numericalstandards under the SDWA. Theobjectives of the projects “MolecularProbes for the Detection of Proto-zoan Parasites” and “Induction ofStress Proteins as a Measure ofGiardia Cyst Viability” are to dis-cover, separate and amplify specificgenetic sequences (DNA or RNA)associated with viable Giardia cysts.If these specific sequences can beidentified, probes can be developedto allow testing for viable cysts only.

Practical methods for isolation,identification and quantification ofwaterborne pathogens such as Giar-dia are not yet available. Isolationand identification methods areneeded before control methods canbe evaluated and regulatory deci-sions can be made regarding requiredtreatment processes and MCLs. Thegoal of ORD’s project entitled “Im-munological Methods for Detectionof Etiological Agents of WaterborneDisease” is to develop innovativeimmunologic methods for the detec-tion, identification and enumerationof pathogenic microorganisms.Immunologic methods may providethe sensitivity and specificity neededfor detection since low numbers oftarget organisms may be present inlarge volumes of water along withhigh numbers of the normal flora andfauna.

To accomplish this, the patho-genic agents will be isolated andtheir antigens (proteins that stimulatethe body to produce antibodies) willbe used to produce specific antiserafor immunologic tests (e.g., immun-ofluorescent assay, enzyme immuno-assay, radioimmunoassay).

Because standardized proceduresfor detecting pathogenic protozoa donot now exist, confusion in theinterpretation of results obtained by

quences, once labelled with a detect-able probe, human type Giardia canbe differentiated from bird andmouse Giardia. Probes have beensynthesized and experiments showthat one reacts with 10 different G.lamblia human isolates but not withG. psittaci (associated with birds) orG. muris (associated with mice). It ishoped that progress in speciation ofGiardia can be applied to the studyof Cryptosporidium.

Current Giardia detection meth-ods are unable to distinguish viablefrom nonviable cysts. A practicaldetection method for viable cysts

Two-stage life cycleof Giardia lamblia:the activetrophozoite stageand theenvironmentally-resistant, restingcyst stage. Cellularcomponents shownabove includenuclei (blue),axonemes (red),and median bodies(green).

Cyst

Trophozoite

10

different laboratories occurs. Thegoal of the project entitled “Stan-dardized Methods for DetectingPathogenic Protozoa in Water” is todevelop standardized methods fordetecting Giardia and Cryptospo-ridium in water. These methods willalso assist EPA in assessing researchfindings relevant to regulatory activi-ties under the SDWA.

Cryptosporidium is the onlymicroorganism on the Office ofGround Water and Drinking Water’slist of contaminants to be addressedin the next round of regulations.Quantitative risk assessment for thisorganism is hampered by the un-availability of any human infectiousdose data and by the scarcity ofanimal dose data. Additionally, verylittle is known of the longevity andprotective ability of the body’simmune response to Cryptospo-ridium infection.

The objective of the projectentitled “Cryptosporidium InfectiousDose and Immune Response” is todetermine Cryptosporidium infec-tious dose and the associated im-mune response in human volunteers.Organisms known tobe infectious forhumans will beobtained frominfected calvesand administeredin drinking waterto the volunteers.Conclusions drawnfrom this projectcould help shapefuture maximum contami-nant level regulations.

In preparation for developmentof disinfection and disinfection by-product rules, information on theoccurrence of Giardia cysts andCryptosporidium oöcysts in sourcewaters and throughout the drinkingwater treatment process must be

collected. Through theproject entitled “Cyst andOöcyst Levels in the OhioRiver,” ORD is monitor-ing monthly one rawwater sample (collectedfrom the river) plussamples from five differ-ent points in the drinkingwater treatment process.Although current meth-ods are based on micro-scopic examination ofconcentrated samples obtained fromlarge volumes of water, immunofluo-rescence membrane assays and geneprobe techniques are being used forthis project. Findings from thisproject will also be used in a nation-wide survey for occurrence of theseorganisms in water supplies.

In the early 1980s, a waterbornedisease study in Washington Statesuggested that certain elements wererequired for a good waterbornedisease surveillance and investiga-tion program. Since that time, com-puter hardware and software havebeen introduced which may increasethe potential for improved efficiencyof disease reporting. Although

cryptosporidiosisout-

breakshave been associated

with drinking water, the relativesignificance of drinking water in thetransmission of this disease is un-known. The project entitled “Surveil-lance of Waterborne Disease/

From 1986 through 1990,20 waterborne outbreaks due tointestinal protozoa were reported in the U.S.;

these outbreaks occurred in 10 states andaffected more than 15,000 people.

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Gene (Linear stretch of DNA)

Chromosome

Cell

more information on the nature andextent of viral contamination in ournation’s waters. The objective ofORD’s project entitled “PracticalMethods for Monitoring Viral Patho-gens in Surface and GroundwaterSource Waters to Define Level ofTreatment” is to develop improvedmethods for detection of waterborneviruses. In addition to supportingEPA’s risk assessment efforts relat-ing to water quality, these methodswill provide the means to support theestablishment of new virologicalstandards and to permit the formationof effective options for regulatorydecisions.

This project will focus on thedevelopment of biotechnology meth-ods based on recognition of viral-specific nucleic acids within infectedcell systems. The use of a biotechnol-ogy approach that employs DNAprobes to detect the presence ofviruses is faster, less expensive andeasier to perform than traditionalplaque assay methods.

The Science Advisory Board's(SAB) Reducing Risk report to EPAdescribes pollutants in drinking wateras one of the four highest risks to

Cryptosporidiosis EpidemiologicalFeasibility Study” is underway to: 1)systematically evaluate waterbornedisease strategies, computer softwareand educational programs in localand state health departments, and 2)design an epidemiological study toaddress the significance of drinkingwater in the transmission ofcryptosporidiosis. Products fromthese efforts could shed light on theunderstanding and tracking of water-borne disease outbreaks throughoutthe world.

Current EPA Research —Viruses

Traditionally, methods for detect-ing and identifying viruses haverelied on slow cell culture methods.Existing methods may underestimatethe quantity of viruses present oralternatively produce false negativeswhen viruses are actually present insampled water. Some viruses (e.g.,hepatitis A and Norwalk) simplycannot be detected by the commonlyused cell culture/plaque assay meth-ods. Given the health risks presentedby viruses, it is essential to develop

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

Percentage of State Populations Served by Ground Water for Domestic Supply

An epidemiologicalinvestigationinvolves the studyand occurrence ofdisease within apopulation. In thestudy ofwaterbornedisease,epidemiologicaldata can indicate aneed for additionaldrinking watertreatment (e.g.,filtration).

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LEGENDPercent of Population

25-4950-7475-100

0-24

human health. With over 50% of theU.S. population relying on groundwater as their primary source ofdrinking water, the need for ground-water resource protection, includingprotection from pathogens, is clear.

“Monitoring of Ground Waterfor Human Enteric Viruses” is acurrent project to address the man-date of the SDWA that EPA estab-lish treatment requirements andcriteria for ground water systems. Avirus occurrence survey of vulner-able ground water systems is beingperformed to support requirementsfor minimum levels of virus inactiva-tion and ultimately a ground waterdisinfection rule. A number of publicground water systems will be identi-fied and ranked according to vulner-ability to fecal contamination. Ofthese, 25 systems will be monitoredfor the presence of viruses throughtissue culture methods and geneprobe techniques.

Norwalk and Norwalk-likeviruses cause viral gastroenteritis(the second leading cause of illnessin the U.S.) in consumers of con-taminated water and food. Sincethese viruses cannot be grown andidentified in tissue cultures, theycannot be detected in water samplesby current monitoring techniques. Asmall amount of Norwalk virus isavailable for studies. This viruspreparation has been isolated fromstool samples of infected individualsand used in an enzyme immunoassayfor the detection of Norwalk virus.Because immunologic methodsrequire a high virus concentration,the etiologic agent responsible formost waterborne outbreaks of gastro-enteritis usually is not determined.Since the viral density in environ-mental samples is normally too lowfor direct immunologic detection andsince there is no known cell culturemethod for this virus, the genetic

material of the Norwalk virus par-ticle must currently be amplifiedusing a biotechnology approachcalled polymerase chain reaction(PCR).

Although known to be highlyinfectious, the infectious dose forNorwalk virus is unknown. The onlysafety-tested virus inoculum (amicroorganism-containing specimenthat has been shown to be free ofother pathogens) available cannot beused for infectious dose studiesbecause it is not possible to deter-mine the virus concentration. Theproject entitled “Develop a Dose-

Labelledgeneticprobe

Target DNA sequence

Once virus particlesinfect cells in asingle layer tissueculture, cellulardamage (clear areasor “plaque-formingunits” in the brownagar depicted at left)becomes apparent.The plaque assay isused foridentification,counting, andpurification ofviruses.

Hybridized probe (inblue) binds withtarget geneticsequence (in red) tomake it detectableby radiation orcolor.

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during feeding. Since shellfish areoften eaten raw or insufficientlycooked, subjecting shellfish waters tohuman wastes constitutes a publichealth risk.

Because there are more than 100waterborne virus types that maycause clinical outbreaks in humans,monitoring efforts are essential toensure the virological safety ofwaters and particularly the reliabilityof water and wastewater treatmentpractices. This information can onlybe provided through increased moni-toring and assessment programs ofeach major pathway leading to thedeposition of human enteric virusesinto the nation’s waters. These vi-ruses are responsible for seriousillnesses ranging from hepatitis tomyocarditis to central nervous sys-tem disorders to acute gastroenteritis.The general recommendation hasbeen that drinking water should befree of human enteric viruses and thatrecreational water viral limits be set.The goal of the project entitled“National Monitoring and Assess-ment Program: Status and Long-Term Trends in Human Enteric VirusPollutants in the Aquatic Ecosys-tems” is to establish a national viralsurvey program focusing on thefollowing five factors: 1) selection ofmonitoring sites based on those mostlikely to have broad public healthimportance; 2) field sampling thatwill result in the collection of ad-equate and representative samplevolumes to safeguard against falsenegatives; 3) concentration proce-dures to increase the density ofviruses so that they can be effectivelyassayed; 4) standard protocols forviral detection using both gene probeand classical plaque assay tech-niques; and 5) parallel biological andchemical analysis that will serve todetermine the quality of the watersource.

Although the rate ofwater transportthrough their gillsvaries greatly,oysters have beenfound to filter asmany as 154gallons per day. Inwaters exposed tohuman sewage,these shellfish canfilter out andconcentratepathogens as wellas food.

Response Curve for Norwalk Virus”is approaching this problem in fourphases: 1) cell cultures capable ofgrowing the Norwalk virus will bedeveloped; 2) Norwalk viruses willbe grown in cell culture, purified andsafety-tested for use in volunteerstudies; 3) a measure of the numberof total and infectious virus particlesin the purified sample will be estab-lished; and 4) a human volunteerstudy will be initiated to determinethe amount of virus particles re-quired to cause disease.

The Clean Water Act stipulatesthat the nation’s rivers, lakes, andcoastal waters be swimmable andfishable. Water quality standardsbased on established criteria toachieve this goal must be developed.The project entitled “Shellfish Meth-ods and Exposure Response Assess-ment/Viruses” is being conducted todevelop methods for detecting andenumerating contamination of shell-fish and shellfish growing waters byhuman enteric viruses. Shellfishgrowing in polluted waters areknown to concentrate these viruses

Preparing stocktissue cell culturesfor isolation andidentification ofviruses in watersamples.

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Current EPA Research —Bacteria

The new National PrimaryDrinking Water Regulations requirethat all drinking water samplestesting positive for total coliforms befurther tested for the presence ofeither fecal coliforms or E. coli.There is a method currently availablethat allows the simultaneous detec-tion of total coliforms and E. coli ina broth medium in 24 hours; how-ever, there is no equivalent methodfor use with membrane filters. De-velopment of such a method willallow those who prefer to obtaincounts of these organisms in theirdistribution systems to use a mem-brane filter method and to haveresults within the 24-hour timeframe. Through the project “Devel-opment of a Membrane Filter Me-dium for the Simultaneous Detectionof Total Coliforms and E. coli,” amembrane filter medium on whichboth total coliforms and E. coli canbe distinguished from noncoliformswill be developed and patented.

E. coli are fecal organisms thatwhen present in drinking water areindicative of fecal pollution. Logisti-cal concerns in sample handling andholding require evaluation of condi-tions for optimizing sample stabilityand longevity. No current regulationsexist for handling samples for analy-sis of E. coli. Through the projectentitled “Optimal Sample HoldingConditions for Analysis of Fecal E.coli in Drinking Water,” sampletemperature and holding time will bedetermined for E. coli or fecal colif-orm analysis methods (i.e., Colilertand M-FC agar). Relative recoveryof methods and storage conditionswill be assessed for optimal E. colirecovery.

The requirement (through theSDWA amendments) to test allcoliform-positive drinking water

In the U.S., thepresence ofcoliform bacteriain drinking wateris used as anindicator ofpossiblemicrobiologicalcontamination.When totalcoliforms aredetected, fecalcoliform or E. colianalysis must beperformed.

samples for either fecal coliforms orE. coli is new. Data from availablemethods for detecting chlorine-damaged E. coli in drinking waterare limited. The objective of theproject entitled “Detection of LowNumbers of Chlorine-Stressed E.coli in Drinking Water” is to evalu-ate and compare the abilities of acommercial method (Colilert) and astandard coliform method (EC-MUG) to recover low numbers ofchlorine-stressed E. coli from po-table water. Pure cultures of E. coliwill be washed, nutrient-stressed infinished drinking water, and treatedwith chlorine. The chlorine-stressedE. coli will then be enumerated,diluted to levels that would be foundin marginally unsafe drinking waterandassayedin mul-tipletubes bythe threemethods.Theseexperi-mentswill berepeatedusingnaturallyoccurring E. coli from diluted humanfecal specimens, contaminatedsource waters and effluents.

The infectious bacterial agentidentified from the stools of choleravictims is Vibrio cholerae. Theepidemic in Latin America hasprompted a renewed interest incontrol measures for this disease.Through the project entitled “Inacti-vation of Vibrio cholerae Biotype ElTor and Biotype Classical by Chlori-nation,” it has been determined thatthe strain responsible for the epi-demic in Peru is capable of revertingto a variant which is more resistant

The golden metallicsheen of thecolonies at leftindicate thepresence of totalcoliforms and thepossibility that thesampled watersupply iscontaminated.

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nia are usually spread via finisheddrinking water. Certain free livingamoebae (protozoa) support themultiplication of L. pneumophila indrinking water systems. These amoe-bae may also be responsible forenhancing the virulence (capacity ofa microorganism to cause disease) ofthe Legionellae and for protectingthem from adverse environmentalfactors such as high temperature andchlorine disinfection. The projectentitled “Multiplication ofLegionellae in Amoebae and Assess-ment of Virulence” will determinethe effect of intracellular growth ofLegionella in amoebae on virulenceand as protection against chlorineand high temperature. To accomplishthis, a method will be established tostudy the ability of various types ofamoebae to provide a protectiveniche for the multiplication ofLegionellae under adverse environ-mental conditions. Combinations ofLegionella isolates and specificamoebae that result in high yields ofLegionella after intracellular growthwill be used to study the effects ofintracellular growth on virulence.Preliminary studies on the ability ofamoebae to supply iron toLegionellae growing intracellularlyshowed no obvious associationsbetween growth and iron concentra-tion.

EPA is required by the SDWA toestablish appropriate controls andregulations for potable water. ORD’sproject entitled “Develop Methodsfor Identifying Potential BacterialPathogens in Drinking Water” willdevelop a data base on potentialhealth hazards (i.e., pathogenicity)associated with bacteria commonlyfound in water distribution systems.To accomplish this, three rodentspecies will be compromised usingnitrous oxides or immunosuppressiveagents, and the animals subsequently

to chlorination than the typicalsmooth variety of Vibrio cholerae.Cells of the variant appear to beimbedded in a gelatinous mucoidmaterial, facilitating the formation ofaggregates, which renders them moreresistant to disinfection. Althoughthe variant is more resistant, studieshave indicated that all strains arereadily inactivated through adequatechlorination.

The Legionella pneumophilabacterial strains that cause commu-nity- and hospital-acquired pneumo-

This one stepmethod(developed byORD) allowsenterococci (bluecolonies)enumeration in just24 hours.

A rugose (rough-surfaced) variant(above left) of Vibriocholerae 01 is ableto form aggregates.ORD studies haveindicated that thisvariant is moreresistant todisinfection than thesmooth strain(above right).

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will be chal-lenged viathe gastroi-ntestinalroute.

Althoughvirulence isusuallymeasured invivo (animalresearch), theneed forextensiveanimaltesting can besignificantly reduced by the develop-ment of a battery of in vitro (cellculture) tests for traits known to bevirulence-related. This battery can beused to predict the potential anorganism has for causing disease inexposed populations. Through theproject entitled “Develop In VitroMethods for Identifying PotentialBacterial Pathogens in DrinkingWater,” model systems will bedeveloped that can be used to deter-mine the potential pathogenicity ofbacteria found in potable waterdistribution systems. Additionally,gene probe and other assays toidentify known opportunistic patho-gens will be developed and evalu-ated.

Bacteria common to drinkingwater distribution systems colonizepoint-of-entry, granular activatedcarbon (GAC) filters where they areable to grow to very high densities.Subsequent to reaching the highdensities the bacteria begin slough-ing off the GAC filters. The numberof bacteria in the filter effluent (i.e.,water flowing out of the filter) issignificantly higher than in theinfluent water. This amplification ofbacteria in drinking water is ofconcern to EPA because GAC filtersare being considered as a substitutefor central potable (i.e., fit for drink-

ing) water treatment in small com-munities where the treatment systemhas been overwhelmed by organicsubstances that may be harmful tohuman health. EPA’s Office ofGround Water and Drinking Water(OGWDW), however, does not wantto recommend the use of these filtersif the possibility exists that their useposes an acute disease risk due tobacteria that grow on the filters. Thehealth significance of the bacteriaknown to adsorb and grow on GACfilters used in the home will beevaluated. The OGWDW will usethis information to develop appropri-ate controls and regulations for thistype of drinking water treatment asrequired by the SDWA.

The objective of ORD’s projectentitled “Health Effects Associatedwith Point-of-Entry GAC Filters” isto determine if a significant healthhazard is associated with the use ofgranular activated carbon, point-of-entry, whole house filters. To accom-plish this, a suitable study site will beselected based on the followingcriteria: 1) the water in the deliverysystem must meet EPA and localdrinking water standards; and 2) thewater distribution system shouldcontain a bacterial population whosedensity is as high as possible and stillacceptable under local regulations.

(a) (b) (c)

Two step, 48 hourmembrane filter testfor enumeratingenterococci inrecreational waters.(a) and (b) Twoperspectives ofcolonies (red)present at 24 hours.(c) At 48 hours,colonies with blackhalos are identifiedas enterococci.

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After a distribution systemmeeting the above criteria has beenfound, a volunteer population ofappropriate size will be selectedfrom among the water system cus-tomers. The selected population willbe randomly divided into GAC userand non-user groups. Point-of-entry,GAC filters will be installed in thehomes of the randomly selected usergroup. The health status of bothgroups will be monitored over apredetermined period of time andduring this time interval the bacterialpopulation in the water system andthe filter effluent will be monitoredon a routine basis. In the event of anillness where a bacterial agent isdiagnosed as the cause, the GACfilter will be removed and examinedfor the presence of the organismdetermined to be the agent of thedisease in that household unit. If anassociation between illness or dis-ease and the use of GAC filters isobserved, health advisory guidelineswill be established or processes thatwill eliminate the causative organ-isms will be developed.

Some believe that exposure tofecal pollution through recreationalwaters or ingestion of contaminatedshellfish causes greater health risks ifthe pollution is of human rather thananimal origin. Before the relativerisks of human versus animal fecalpollution can be assessed, it is neces-sary to develop a microbiologicalmethod for distinguishing humanfrom animal pollution. Currentmethods detect fecal pollution but donot reveal the source. The objectiveof the project entitled “Method toDistinguish Non-Human FecalPollution from Human Fecal Pollu-tion” is to develop a gene probespecific for E. coli that inhabit thehuman intestine for use as an indica-tor of the presence of human fecalcontamination in water. The probewill be field tested at several sites inwhich fecal pollution is exclusivelyfrom human sources, exclusivelyfrom animal sources and from mixedsources.

Shigella species are among themost common and significant patho-gens associated with wastewater and

EPA researcherusing thetransmissionelectron microscopeto detect pathogensunable to bedetected by othermethods.

Analysis ofpotable water orcooling towerwater forLegionellapneumophilarequiresapproximatelyfive to sevendays for growth ofthe organisms onthe initial isolationmedium andanother five toseven days toconfirm theidentity of theseorganisms. Geneprobe techniquescould reduceanalysis time toone day.

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sludge. Because of their low infec-tive doses, these organisms may behazardous even if present in lownumbers in wastewaters that arerecycled for potable use or sludgesthat are applied to agricultural land.Shigellae are very difficult to detectin environmental samples by conven-tional methods because of theirbiochemical similarities to E. coli.The use of current gene probe tech-nology in the project entitled “Detec-tion of Enteroinvasive Shigella inWastewaters and Sludges” shouldenable us to detect Shigellae insludges and wastewaters that wouldappear to be free of these pathogensif analyzed by conventional methods.

ConclusionThe protection and enhancement

of our nation’s water quality remainsa chief concern of the U.S. Environ-

mental Protection Agency. TheOffice of Research and Developmentis committed, through the extensivewaterborne disease research effortsearlier described, to ensure that themost effective and efficient methodsare developed to identify, detect, andinactivate/remove pathogens thatmay be present in our drinking watersupplies.

Life cycles, mechanisms ofinfection, protective or dormantstates, emergence of disinfection-resistant variants, optimal pathogenremoval techniques, regrowth indistribution lines…all are areas thatmust be investigated and understoodto afford the water quality safeguardsthat are so often taken for granted.The successes and failures of theseresearch efforts, relayed to the publicand appropriate federal, state, andlocal agencies, have helped to ensuresafe drinking water.

Human entericbacteria beingsubcultured in ananaerobic hood.

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EPA PublicationsThe 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 forEnvironmental 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 FilterProcedure, 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 LiteratureErlandsen, S.L., L.A. Sherlock, W.J. Bemrick, H. Ghobrial and W. Jakubowski. 1990. Prevalence ofGiardia 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. Searchingfor a water supply connection in the Cabool, Missouri disease outbreak of E. coli 0157:H7. Wat. Res.,26: 1127-1137.

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This publication was prepared by Patrick Burke of ORD’s NationalRisk Management Research Laboratory, Cincinnati, Ohio. Contribu-tors and reviewers include Alfred Dufour, Walter Jakubowski, RobertSafferman, Shay Fout, Gerard Stelma and Terry Covert of theNational Exposure Research Laboratory - Cincinnati, and RobertClark, Kim Fox, Edwin Geldreich, Richard Miltner, Donald Reasoner,and Eugene Rice of the National Risk Management ResearchLaboratory. Thanks to Al Lang and Jim O’Dell for photographicsupport.