Preventing Waterborne Disease a Focus on EPA's Research

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United States Office of Research and EPA/640/K-93/001 Environmental Protection Development April 1993 Agency Washington, DC 20460 Preventing Waterborne Disease

A Focus on EPA’s Research EPA’s Office of Research and Development

The Office of Research and Development (ORD) conducts an integrated program of scientific research and development on the sources, transport and fate processes, monitoring, control, and assessment of risk and effects of environmental pollutants. These activi- ties are implemented through its headquarters offices and National Research Laboratories and Centers. The research focuses on key scientific and technical is- sues to generate knowledge supporting sound decisions today, and to anticipate the complex challenges of tomorrow. With a strong, forward-looking research program, less expensive more effective solu- tions can be pursued and irreversible damage to the environment can be prevented.

Front cover photo by Lang Photography. “The reported case total for the epidemic nears three-quarters of a million. Since the beginning of the epidemic in January 1991, the total number of reported cases is 746,968 with 6,448 deaths.” (Cholera Epidemic in the Americas, CDC Update, February 11, 1993)

Although the above-listed statis- prevent water contamination by tics are alarming, the risk that exten- harmful microorganisms. From sive outbreaks of waterborne cholera monitoring our nation’s ground will occur in the United States is water systems for viral pathogens...to minimal. Effective treatment of developing more effective technol- drinking water and sewage, coupled ogy for large and small systems...to with adequate personal hygiene providing other nations with critical habits, has contributed to a success- technical assistance, ORD scientists ful line of defense against the spread and engineers continue their mission of cholera in the U.S. Still, the ease to ensure safe waters. As the focus of Researcher isolating of international travel has guaran- our efforts adjusts to deal with infectious bacteria in teed the import of a wide variety of emerging challenges, past and cur- one of ORD’s diseases not generally considered to rent successes add to our scientific pathogen be native to North America. Addi- arsenal against disease. containment suites. tionally, although fatalities caused by waterborne diseases have de- clined dramatically in the U.S. during this century, annual reports of water-related, microorganism- induced disease continue to number in the thousands. Just one waterborne outbreak of cryptosporidiosis in western Georgia (1987), for example, affected an estimated 13,000 people. In the “colonias” (poor settlements along the Texas- Mexico border), high levels of disease have been associated with the lack of public water supplies and inadequate waste treatment. While the words “typhoid fever” fade from our vocabulary, such terms as “Giar- dia,” “Legionella,” and “Norwalk virus” are becoming more familiar. The United States Environmen- tal Protection Agency (U.S. EPA), through its Office of Research and Development (ORD), is conducting research to better understand and

Printed on Recycled Paper Microorganisms Associated with Waterborne Disease

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

Bacteria. Bacteria are the most widely distributed life forms. Pathogenic bacteria range in length from approximately 0.4 to 14 µm (a µm or “micrometer” equals one one-thousandth of a millimeter) and 0.2 to 1.2 µm in width. Key bacterial pathogens responsible for waterborne disease include Legionella, Salmonella typhi, Shigella, and Vibrio cholerae.

Viruses. Viruses are inactive when outside of a living host cell. Viruses linked to waterborne disease have protein coats that provide protection from environmental hazards and range in size from 0.02 to 0.09 µm. Unlike bacteria and protozoa, they contain only one type of nucleic acid (RNA or DNA). Key pathogens include hepatitis A and Norwalk virus.

Protozoa. Protozoa, common in bodies of water, are much larger than bacteria and viruses. To survive harsh environmental conditions, some species can secrete a protective covering and form a resting stage called a “cyst.” Encystment can protect protozoa from drinking water disinfection efforts and facilitate the spread of disease. Key protozoa being studied as agents of waterborne disease include Giardia and Cryptosporidium.

2 Why Can’t Waterborne waterborne outbreaks is recognized, It is estimated Pathogens Be Eliminated? reported and investigated. Of these, that swimmers the pathogenic agent is identified Microorganisms are present and waders may only half of the time. Additionally, everywhere in our environment. ingest from 0.3 to experts believe that some food- Invisible to the naked eye, vast 1.7 ounces of related disease outbreaks may origi- numbers of these microbes can be water per outing. found in soil, air, food and water. nate with an initial infection (e.g., of Although humans are essentially a restaurant worker) caused by free of microorganisms before birth, contaminated drinking water. constant circumstances of exposure Bacteria, viruses and protozoa (e.g., breathing, eating, and drink- are the microorganism groups con- ing) quickly allow the establishment taining pathogens of primary concern of harmless microbial flora in our in the study of waterborne diseases. bodies. To eliminate these pathogens from Microbial pathogens (microor- our water, especially from our drink- ganisms capable of causing disease), ing water, seems theoretically however, can and often do harm straightforward. Simply mix in a those who become infected. More- disinfectant, allow adequate contact over, diseases that healthy individu- time to assure inactivation (rendering als “weather” well may prove fatal the microbes unable to produce to individuals with compromised disease), and pump the water into the Cl Cl immune systems. In some cases, an distribution lines. infection can persist to create a In reality, many conditions “carrier state” where a disease- render the above scenario unwork- able. The physical characteristics of causing agent is harbored by the P body (and spread) without any the water, primarily represented by B dissolved and suspended solids apparent symptoms. B S Waterborne diseases are typi- content, can affect the disinfection V cally considered to be those diseases process. The chemical content, both resulting from ingestion of contami- naturally occurring and anthropo- nated water. Additional pathways of genic (i.e., generated by humans), can also interfere with the chemical infection being studied by EPA Cl include inhalation of water vapors as reactions desired during treatment well as body contact during bathing and disinfection. Finally, pathogens associated (i.e., imbedded in or (opportunistic pathogens) in the To kill or inactivate clumped) with higher organisms hospital environment. drinking water (e.g., algae, rotifers, worms) may be Since voluntary water ingestion contaminants such (drinking water) and bathing are protected from the action of disinfec- as bacteria (B), universal practices and accidental tants. protozoa (P), and ingestion during recreational activi- To overcome these obstacles to viruses (V), ties (e.g., swimming, water skiing, disinfection, successful treatment of adequate contact time with the wading) is common, inadequate drinking and waste water generally includes a series of steps. The flow- disinfectant protection of water integrity could (chlorine or Cl in charts in Figures 1 and 2 depict the lead to widespread outbreaks (the this representation) Centers for Disease Control defines steps involved in typical drinking must be allowed. an outbreak to be two or more cases and waste water treatment processes. Adsorption to and of illness that can be traced to a In the case of drinking water clumping of solid common source). Because symp- disinfection, once the impurities have particles (S) can toms can be mild and short-lived, it been removed, enough disinfectant is inhibit the disinfec- is estimated that only a fraction of added to inactivate pathogens. Addi- tion process. 3 tionally, a residual level of disinfec- upon by other communities presents Raw tant must be maintained throughout a significant health risk. Source Water the distribution system to guard waters heavily loaded with disease- against potential problems (e.g., causing microorganisms can reduce microorganisms entering through the effectiveness of “downstream” breaks in distribution lines or re- drinking water treatment processes. Screening growth). Such advances as ultraviolet light Proper distribution system opera- disinfection systems, initially inves- tion and maintenance practices are tigated as a wastewater disinfection essential deterrents of pathogen option several years ago, are pres- Coagulation/ entry, recovery and survival. These ently becoming more widely ac- Rapid Mixing practices (according to Geldreich et cepted and reliable with recent al., 1992) include: design enhancements. This technol- ogy has been demonstrated to be • Systematic flushing of the entire Flocculation capable of meeting existing disinfec- distribution system “to get more tion criteria without the release of movement of the chlorine residual dangerous disinfection by-products. into all parts of the pipe network...to remove static water What Progress Has Been from slow-flow sections, deadends Made? Sediment- and stratified water in storage tanks ation on a periodic basis;” Early in this century, the waterborne diseases of chief concern in • Effecting repairs and replacement the U.S. were typhoid fever and of distribution line components amebiasis. Of the 1,087 deaths (e.g., broken mains and service associated with waterborne out- Filtration meters) in a sanitary manner (i.e., breaks between 1920 and 1991, 943 soil-free replacement parts, were attributed to typhoid fever disinfection and flushing of while 102 were caused by amebiasis. repaired lines, valves and fittings); Disinfection Overall, 83% of the deaths occurred prior to 1936 and less than 1% • Preventing pathogens from being occurred after 1970. Additionally, drawn into the distribution system the number of outbreaks in commu- by maintaining continuous positive nity water systems since 1945 is pressure and preserving barriers be- Clear about half as great as the number tween public water supplies and Holding documented during the first half of sewage or storm water drainage; Tank this century. The reduction in fatali- • Varying the sample sites during ties and number of outbreaks indi- routine monitoring to produce data cates that progress has been made in more representative of the entire the prevention of certain waterborne To Distribution system. diseases. Much of the progress has System been the result of increased imple- While the importance of source mentation of important treatment water treatment to ensure safe drink- practices (e.g., filtration, disinfec- ing water seems obvious, the need to tion, sewage treatment). Although devote equal effort to pathogen progress has been significant, the Figure 1. Simplified reduction in wastewater is not al- nation’s water continues to be a flowchart of drinking ways recognized. The release of source of disease. It must be rigor- water treatment untreated or inadequately treated ously monitored for indicators of processes. wastewater into source waters drawn fecal contamination.

4 Some Waterborne Diseases of Concern in the United States

Disease Microbial General Symptoms Agent

Amebiasis Protozoan Abdominal discomfort, fatigue, (Entamoeba histolytica) diarrhea, flatulence, weight loss

Campylo- Bacterium Fever, abdominal pain, diarrhea bacteriosis (Campylobacter jejuni)

Cholera Bacterium Watery diarrhea, vomiting, occasional (Vibrio cholerae) muscle cramps

Cryptospor- Protozoan Diarrhea, abdominal discomfort idiosis (Cryptosporidium parvum)

Giardiasis Protozoan Diarrhea, abdominal discomfort (Giardia lamblia)

Hepatitis Virus Fever, chills, abdominal discomfort, (hepatitis A) jaundice, dark urine

Shigellosis Bacterium Fever, diarrhea, bloody stool (Shigella species)

Typhoid fever Bacterium Fever, headache, constipation, appetite (Salmonella typhi) loss, nausea, diarrhea, vomiting, appearance of an abdominal rash

Viral Viruses Fever, headache, gastrointestinal Gastroenteritis (Norwalk, rotavirus and discomfort, vomiting, diarrhea other types)

5 proper drinking water quality Raw through monitoring, and provide Wastewater public notification of contamination problems. Relating to prevention of water-

borne disease, the SDWA required Primary Treatment Grit Pretreatment EPA to:

1) set numerical standards, referred to as Maximum Con- taminant Levels (MCLs — the Primary Sediment- highest allowable contaminant Sludge ation concentrations in drinking water) or treatment technique requirements for contaminants in public water supplies;

2) issue regulations requiring monitoring of all regulated and Biological Secondary Treatment certain unregulated contami- Treatment nants, depending on the number of people served by the system, the source of the water supply, and the contaminants likely to be found; Secondary Sediment- Sludge ation 3) set criteria under which systems are obligated to filter water from surface water sources; it must also develop procedures for states to determine which systems have to filter; To further treatment or 4) develop disinfection rules for discharge all public water supplies; and

Figure 2. In 1974, Congress passed the 5) require all states to develop Simplified flowchart Safe Drinking Water Act (SDWA) Wellhead Protection Programs of typical setting up a regulatory program designed to protect from sources wastewater among local, state, and federal agen- of contamination areas around treatment cies to help ensure the provision of wells that supply public drinking processes. safe drinking water in the U.S. The water systems. states are expected to administer and enforce these regulations for public Through the Surface Water water systems (systems that either Treatment Rule (SWTR), EPA has have 15 or more service connections set treatment requirements to control or regularly serve an average of 25 or microbiological contaminants in more people daily for at least 60 days public water systems using surface each year). Public water systems water sources (and ground-water must provide water treatment, ensure sources under the direct influence of 6 surface water). These requirements Assimilable organic carbon include the following: (AOC) is the portion of the total Currently, it is organic carbon (TOC) dissolved in estimated that 1) treatment must remove or water that is easily used by microor- there will be over inactivate at least 99.9% of ganisms as a carbon source (i.e., 100,000 Giardia lamblia cysts and nutrients). ORD researchers are violations of the 99.99% of viruses; currently investigating treatment SDWA annually. processes to control AOC. One Nearly half of 2) all systems must disinfect, and promising process is biologically these will be for are required to filter if certain active filtration wherein bacterial MCL violations. source water quality criteria and communities are intentionally estab- Of these, the site-specific criteria are not met; lished in the filters to use up, or majority will be microbiological 3) the regulations set criteria for biodegrade, the AOC as it passes through. This treatment process must violations by determining if treatment, includ- small systems. ing turbidity (suspended particu- be employed before final disinfection late matter) removal and disin- so that bacteria escaping from the fection requirements, is adequate filter can be properly controlled. As for filtered systems; and described in Figure 1, most water utilities do not disinfect with chlorine 4) all systems must be operated until late in the treatment train. This by qualified operators as deter- limits the formation of disinfection mined by the states. by-products (i.e., those compounds like chloroform produced when chlorine reacts with naturally occur- Current EPA Research – ring organic carbon). Barriers to Contamination To accomplish disinfection Single step Although water treatment and earlier in treatment, some water membrane filter disinfection techniques are quite utilities employ ozonation. While ozone is a very strong disinfectant, it procedure for effective at microbe reduction, enumerating E. coli also converts a portion of the TOC finished drinking water is not sterile. in recreational Survival and regrowth of microor- into AOC. ORD researchers are waters. The yellow ganisms in drinking water distribu- examining the advantages (e.g., colonies are E. coli tion systems can lead to the deterio- disinfection of bacteria, viruses and while the blue, red ration of water quality and even non- protozoan cysts, control of color, and purple colonies compliance of a supply. Regrowth control of taste and odor, enhance- are other coliforms. has largely been associated with heterotrophic bacteria (i.e., those bacteria – including pathogens – that require preformed organic 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. 7 drinking water exposure risk assessments, and calibrating network hydraulic models. It can provide insight into how changes in water source utilization, pumping water storage levels, use of satellite treatment, and targeted pipe cleaning and replacement would affect drinking water quality. In support of small community and non-community (less than 3300 people) drinking water treatment systems, ORD researchers are designing, modifying and testing In situ cytotoxicity test for heterotrophic bacteria found in “Hybrid Drinking Water Treatment drinking water. Heterotrophs recovered from drinking water Package Plants.” These package form individual yellow colonies (left) that can be transferred to a plants are factory-built, skid- tissue cell culture (right). Formation of plaques (i.e., clear areas mounted, and ready to be operated in caused by destruction of infected cells) in the tissue culture can the field with minimal site prepara- indicate virulence and signal the need for further action. tion. They exhibit lower capital cost than custom designed facilities built ment of coagulation, and partial onsite and can incorporate any oxidation of the naturally occurring drinking water treatment process. organic carbon that reacts with Promising technologies being con- chlorine) and disadvantages of ozone sidered for incorporation include (e.g., enhancement of AOC, conver- membranes, advanced oxidation, bag sion of bromide to bromate, and filters, and photocatalytic oxidation. formation of its own disinfection by- By merging, modifying, and adapt- products like formaldehyde). ing conventional treatment trains The project entitled “EPANET” with innovative treatment technolo- involves the development and testing gies, a broader variety of contami- of a water quality model for drinking nants (including pathogens) can be water distribution systems. The removed and SDWA compliance can In the lesser EPANET model is a computer be facilitated. developed program that performs extended Concern has recently mounted countries, period simulation of hydraulic and over the ability of certain pathogenic waterborne water quality behavior within water protozoan (Cryptosporidium) cysts disease is still a distribution networks. It tracks the to survive treatment processes and major problem. flow of water in each pipe, the pres- enter the distribution system. ORD, The World sure at each pipe junction, the height in its project entitled “Evaluation of Health of water in each tank, and the con- Particulate Removal Processes,” is Organization has centration of a contaminant through- designing and testing the most effec- estimated that out the network during a multiple tive filtration techniques to physi- more people die time period simulation. Water age cally remove the cysts. Being studied annually of and source tracing can also be simu- are slow sand (see Figure 3), diatom- water-related lated. aceous earth, and coagulation/rapid diarrheal EPANET can be useful for sand filtration processes. Results of illnesses than of analyzing the loss of disinfectant this research will build upon earlier cancer or AIDs. residual, designing water quality work with filtration of Giardia sampling programs, performing lamblia.

8 Flow Overflow Control

Supernatant Water Raw Ventilation Water Active Biological Layer V-Notch Weir

Sand Filterbed

Underdrains

Filter Effluent Flow Measurement

Current EPA Research — parts of the country. Seasonal and Figure 3. Basic Pathogenic Intestinal geographic differences have been elements of a slow Protozoa recognized and data on concentra- sand filter. During the last 20 years, signifi- tions in various waters have been cant improvements have been made collected. in the quantitative methods for Work on cross-species infectiv- detecting pathogenic intestinal ity of animal and human cysts has protozoa (particularly Giardia and established that beavers and musk- Cryptosporidium) in water. Addi- rats may at least be secondary reser- tionally, there has been progress in voirs for giardiasis. Also, while it standardizing these methods. Current appears that avian cysts are not methods, however, are still time- infective for mammals, we cannot In Minnesota, 100% consuming and skill-intensive (re- now distinguish avian and mamma- of the muskrats and quiring highly trained analysts), and lian cysts in a water sample. The 7% of the beavers lack the ability to indicate viability goal of an ORD project entitled examined were (whether the cyst is dead or alive) or “Development of Gene Probes for positive for Giardia. Speciation of Giardia” is to develop infectivity. The latter item has In four and test the application of genetic Northeastern states clouded the development of quantita- and molecular probe methods to (Maine, New tive risk assessments. allow classification of detected Hampshire, New The cosmopolitan nature of Giardia species. York, and intestinal protozoa, and the certainty Gene sequences have been Vermont), the that all surface water supplies must mapped in species of Giardia shed corresponding be contaminated with these organ- by animals (e.g., herons and mice) figures were 94% isms, has been established through and compared with corresponding for muskrats and studies in animals (beavers, musk- gene sequences in human-hosted 17% for beavers. rats, and birds) and by occurrence Giardia. Preliminary results indicate Erlandsen et al., 1990. studies in sewage throughout all that through these mapped se-

9 would allow assessing the significance of positive reports and may allow establishment of numerical standards under the SDWA. The objectives of the projects “Molecular Probes for the Detection of Proto- zoan Parasites” and “Induction of Stress Proteins as a Measure of Giardia Cyst Viability” are to discover, separate and amplify specific genetic sequences (DNA or RNA) associated with viable Giardia cysts. If these specific sequences can be identified, probes can be developed Trophozoite to allow testing for viable cysts only. Practical methods for isolation, identification and quantification of waterborne pathogens such as Giar- dia are not yet available. Isolation and identification methods are needed before control methods can be evaluated and regulatory decisions can be made regarding required treatment processes and MCLs. The goal of ORD’s project entitled “Im- munological Methods for Detection of Etiological Agents of Waterborne Disease” is to develop innovative immunologic methods for the detection, identification and enumeration of pathogenic microorganisms. Immunologic methods may provide Cyst the sensitivity and specificity needed for detection since low numbers of Two-stage life cycle quences, once labelled with a detect- target organisms may be present in of Giardia lamblia: able probe, human type Giardia can large volumes of water along with the active be differentiated from bird and high numbers of the normal flora and trophozoite stage mouse Giardia. Probes have been fauna. and the synthesized and experiments show To accomplish this, the patho- environmentally- that one reacts with 10 different G. genic agents will be isolated and resistant, resting cyst stage. Cellular lamblia human isolates but not with their antigens (proteins that stimulate components shown G. psittaci (associated with birds) or the body to produce antibodies) will above include G. muris (associated with mice). It is be used to produce specific antisera nuclei (blue), hoped that progress in speciation of for immunologic tests (e.g., immun- axonemes (red), Giardia can be applied to the study ofluorescent assay, enzyme immuno- and median bodies of Cryptosporidium. assay, radioimmunoassay). (green). Current Giardia detection meth- Because standardized procedures ods are unable to distinguish viable for detecting pathogenic protozoa do from nonviable cysts. A practical not now exist, confusion in the detection method for viable cysts interpretation of results obtained by

10 different laboratories occurs. The collected. Through the goal of the project entitled “Stan- project entitled “Cyst and dardized Methods for Detecting Oöcyst Levels in the Ohio Cell Pathogenic Protozoa in Water” is to River,” ORD is monitor- develop standardized methods for ing monthly one raw detecting Giardia and Cryptospo- water sample (collected Chromosome ridium in water. These methods will from the river) plus also assist EPA in assessing research samples from five differ- findings relevant to regulatory activi- ent points in the drinking ties under the SDWA. water treatment process. Cryptosporidium is the only Although current meth- microorganism on the Office of ods are based on micro- Gene (Linear stretch of DNA) Ground Water and Drinking Water’s scopic examination of list of contaminants to be addressed concentrated samples obtained from in the next round of regulations. large volumes of water, immunofluo- Quantitative risk assessment for this rescence membrane assays and gene organism is hampered by the un- probe techniques are being used for availability of any human infectious this project. Findings from this dose data and by the scarcity of project will also be used in a nation- animal dose data. Additionally, very wide survey for occurrence of these little is known of the longevity and organisms in water supplies. protective ability of the body’s In the early 1980s, a waterborne immune response to Cryptospo- disease study in Washington State ridium infection. suggested that certain elements were The objective of the project required for a good waterborne entitled “Cryptosporidium Infectious disease surveillance and investiga- Dose and Immune Response” is to tion program. Since that time, com- determine Cryptosporidium infec- puter hardware and software have tious dose and the associated im- been introduced which may increase mune response in human volunteers. the potential for improved efficiency Organisms known to of disease reporting. Although be infectious for cryptosporidiosis humans will be out- obtained from infected calves From 1986 through 1990, and administered 20 waterborne outbreaks due to in drinking water intestinal protozoa were reported in the U.S.; to the volunteers. these outbreaks occurred in 10 states and Conclusions drawn affected more than 15,000 people. from this project could help shape future maximum contaminant level regulations. In preparation for development breaks of disinfection and disinfection by- have been associated product rules, information on the with drinking water, the relative occurrence of Giardia cysts and significance of drinking water in the Cryptosporidium oöcysts in source transmission of this disease is un- waters and throughout the drinking known. The project entitled “Surveil- water treatment process must be lance of Waterborne Disease/

11 Cryptosporidiosis Epidemiological more information on the nature and An epidemiological Feasibility Study” is underway to: 1) extent of viral contamination in our investigation systematically evaluate waterborne nation’s waters. The objective of involves the study disease strategies, computer software ORD’s project entitled “Practical and occurrence of and educational programs in local Methods for Monitoring Viral Patho- disease within a and state health departments, and 2) gens in Surface and Groundwater population. In the design an epidemiological study to Source Waters to Define Level of study of address the significance of drinking Treatment” is to develop improved waterborne water in the transmission of methods for detection of waterborne disease, cryptosporidiosis. Products from viruses. In addition to supporting epidemiological these efforts could shed light on the EPA’s risk assessment efforts relat- data can indicate a understanding and tracking of water- ing to water quality, these methods need for additional borne disease outbreaks throughout will provide the means to support the drinking water the world. establishment of new virological treatment (e.g., standards and to permit the formation filtration). of effective options for regulatory Current EPA Research — decisions. Viruses This project will focus on the Traditionally, methods for detect- development of biotechnology meth- ing and identifying viruses have ods based on recognition of viral- relied on slow cell culture methods. specific nucleic acids within infected Existing methods may underestimate cell systems. The use of a biotechnol- the quantity of viruses present or ogy approach that employs DNA alternatively produce false negatives probes to detect the presence of when viruses are actually present in viruses is faster, less expensive and sampled water. Some viruses (e.g., easier to perform than traditional hepatitis A and Norwalk) simply plaque assay methods. cannot be detected by the commonly The Science Advisory Board's used cell culture/plaque assay meth- (SAB) Reducing Risk report to EPA ods. Given the health risks presented describes pollutants in drinking water by viruses, it is essential to develop as one of the four highest risks to

Percentage of State Populations Served by Ground Water for Domestic Supply

LEGEND Percent of Population 75-100 50-74 25-49 0-24

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

12 human health. With over 50% of the Once virus particles U.S. population relying on ground infect cells in a water as their primary source of single layer tissue drinking water, the need for ground- culture, cellular damage (clear areas water resource protection, including or “plaque-forming protection from pathogens, is clear. units” in the brown “Monitoring of Ground Water agar depicted at left) for Human Enteric Viruses” is a becomes apparent. current project to address the man- The plaque assay is date of the SDWA that EPA estab- used for lish treatment requirements and identification, criteria for ground water systems. A counting, and virus occurrence survey of vulner- purification of able ground water systems is being viruses. performed to support requirements for minimum levels of virus inactiva- material of the Norwalk virus par- tion and ultimately a ground water ticle must currently be amplified disinfection rule. A number of public using a biotechnology approach ground water systems will be identi- called polymerase chain reaction fied and ranked according to vulner- (PCR). ability to fecal contamination. Of Although known to be highly these, 25 systems will be monitored infectious, the infectious dose for for the presence of viruses through Norwalk virus is unknown. The only tissue culture methods and gene safety-tested virus inoculum (a microorganism-containing specimen probe techniques. Hybridized probe (in that has been shown to be free of Norwalk and Norwalk-like blue) binds with viruses cause viral gastroenteritis other pathogens) available cannot be target genetic (the second leading cause of illness used for infectious dose studies sequence (in red) to in the U.S.) in consumers of con- because it is not possible to deter- make it detectable taminated water and food. Since mine the virus concentration. The by radiation or these viruses cannot be grown and project entitled “Develop a Dose- color. identified in tissue cultures, they cannot be detected in water samples by current monitoring techniques. A small amount of Norwalk virus is available for studies. This virus preparation has been isolated from stool samples of infected individuals Labelled genetic and used in an enzyme immunoassay probe for the detection of Norwalk virus. Because immunologic methods require a high virus concentration, the etiologic agent responsible for most waterborne outbreaks of gastroenteritis usually is not determined. Since the viral density in environmental samples is normally too low Target DNA sequence for direct immunologic detection and since there is no known cell culture method for this virus, the genetic

13 during feeding. Since shellfish are often eaten raw or insufficiently cooked, subjecting shellfish waters to human wastes constitutes a public health risk. Because there are more than 100 waterborne virus types that may cause clinical outbreaks in humans, monitoring efforts are essential to ensure the virological safety of waters and particularly the reliability of water and wastewater treatment practices. This information can only be provided through increased monitoring and assessment programs of each major pathway leading to the deposition of human enteric viruses into the nation’s waters. These viruses are responsible for serious illnesses ranging from hepatitis to myocarditis to central nervous sys- Preparing stock Response Curve for Norwalk Virus” tem disorders to acute gastroenteritis. tissue cell cultures is approaching this problem in four The general recommendation has for isolation and phases: 1) cell cultures capable of been that drinking water should be identification of growing the Norwalk virus will be free of human enteric viruses and that viruses in water developed; 2) Norwalk viruses will recreational water viral limits be set. samples. be grown in cell culture, purified and The goal of the project entitled safety-tested for use in volunteer “National Monitoring and Assess- studies; 3) a measure of the number ment Program: Status and Long- of total and infectious virus particles Term Trends in Human Enteric Virus in the purified sample will be estab- Pollutants in the Aquatic Ecosys- lished; and 4) a human volunteer tems” is to establish a national viral study will be initiated to determine survey program focusing on the the amount of virus particles re- following five factors: 1) selection of quired to cause disease. monitoring sites based on those most Although the rate of The Clean Water Act stipulates likely to have broad public health water transport that the nation’s rivers, lakes, and importance; 2) field sampling that through their gills coastal waters be swimmable and will result in the collection of ad- varies greatly, fishable. Water quality standards equate and representative sample oysters have been based on established criteria to volumes to safeguard against false found to filter as achieve this goal must be developed. negatives; 3) concentration proce- many as 154 The project entitled “Shellfish Meth- dures to increase the density of gallons per day. In ods and Exposure Response Assess- viruses so that they can be effectively waters exposed to ment/Viruses” is being conducted to assayed; 4) standard protocols for human sewage, develop methods for detecting and viral detection using both gene probe these shellfish can enumerating contamination of shell- and classical plaque assay tech- filter out and fish and shellfish growing waters by niques; and 5) parallel biological and concentrate human enteric viruses. Shellfish chemical analysis that will serve to pathogens as well growing in polluted waters are determine the quality of the water as food. known to concentrate these viruses source.

14 Current EPA Research — samples for either fecal coliforms or Bacteria E. coli is new. Data from available In the U.S., the methods for detecting chlorine- presence of The new National Primary coliform bacteria Drinking Water Regulations require damaged E. coli in drinking water are limited. The objective of the in drinking water that all drinking water samples is used as an testing positive for total coliforms be project entitled “Detection of Low Numbers of Chlorine-Stressed E. indicator of further tested for the presence of possible either fecal coliforms or E. coli. coli in Drinking Water” is to evaluate and compare the abilities of a microbiological There is a method currently available contamination. that allows the simultaneous detec- commercial method (Colilert) and a standard coliform method (EC- When total tion of total coliforms and E. coli in coliforms are a broth medium in 24 hours; how- MUG) to recover low numbers of chlorine-stressed E. coli from po- detected, fecal ever, there is no equivalent method coliform or E. coli for use with membrane filters. De- table water. Pure cultures of E. coli will be washed, nutrient-stressed in analysis must be velopment of such a method will performed. allow those who prefer to obtain finished drinking water, and treated counts of these organisms in their with chlorine. The chlorine-stressed distribution systems to use a mem- E. coli will then be enumerated, brane filter method and to have diluted to levels that would be found results within the 24-hour time in marginally unsafe drinking water frame. Through the project “Devel- and opment of a Membrane Filter Me- assayed in mul- dium for the Simultaneous Detection The golden metallic tiple of Total Coliforms and E. coli,” a sheen of the membrane filter medium on which tubes by colonies at left both total coliforms and E. coli can the three indicate the be distinguished from noncoliforms methods. presence of total will be developed and patented. These coliforms and the E. coli are fecal organisms that experi- possibility that the sampled water when present in drinking water are ments will be supply is indicative of fecal pollution. Logisti- contaminated. cal concerns in sample handling and repeated holding require evaluation of condi- using tions for optimizing sample stability naturally and longevity. No current regulations occurring E. coli from diluted human exist for handling samples for analy- fecal specimens, contaminated sis of E. coli. Through the project source waters and effluents. entitled “Optimal Sample Holding The infectious bacterial agent Conditions for Analysis of Fecal E. identified from the stools of cholera coli in Drinking Water,” sample victims is Vibrio cholerae. The temperature and holding time will be epidemic in Latin America has determined for E. coli or fecal colif- prompted a renewed interest in orm analysis methods (i.e., Colilert control measures for this disease. and M-FC agar). Relative recovery Through the project entitled “Inacti- of methods and storage conditions vation of Vibrio cholerae Biotype El will be assessed for optimal E. coli Tor and Biotype Classical by Chlori- recovery. nation,” it has been determined that The requirement (through the the strain responsible for the epi- SDWA amendments) to test all demic in Peru is capable of reverting coliform-positive drinking water to a variant which is more resistant

15 nia are usually spread via finished drinking water. Certain free living amoebae (protozoa) support the multiplication of L. pneumophila in drinking water systems. These amoebae may also be responsible for enhancing the virulence (capacity of a microorganism to cause disease) of the Legionellae and for protecting them from adverse environmental factors such as high temperature and chlorine disinfection. The project entitled “Multiplication of Legionellae in Amoebae and Assess- ment of Virulence” will determine the effect of intracellular growth of Legionella in amoebae on virulence and as protection against chlorine and high temperature. To accomplish A rugose (rough- to chlorination than the typical this, a method will be established to surfaced) variant smooth variety of Vibrio cholerae. study the ability of various types of (above left) of Vibrio Cells of the variant appear to be amoebae to provide a protective cholerae 01 is able imbedded in a gelatinous mucoid niche for the multiplication of to form aggregates. material, facilitating the formation of Legionellae under adverse environ- ORD studies have aggregates, which renders them more mental conditions. Combinations of indicated that this variant is more resistant to disinfection. Although Legionella isolates and specific resistant to the variant is more resistant, studies amoebae that result in high yields of disinfection than the have indicated that all strains are Legionella after intracellular growth smooth strain readily inactivated through adequate will be used to study the effects of (above right). chlorination. intracellular growth on virulence. The Legionella pneumophila Preliminary studies on the ability of bacterial strains that cause commu- amoebae to supply iron to nity- and hospital-acquired pneumo- Legionellae growing intracellularly showed no obvious associations between growth and iron concentration. EPA is required by the SDWA to establish appropriate controls and regulations for potable water. ORD’s This one step method project entitled “Develop Methods (developed by for Identifying Potential Bacterial ORD) allows Pathogens in Drinking Water” will enterococci (blue develop a data base on potential colonies) health hazards (i.e., pathogenicity) enumeration in just associated with bacteria commonly 24 hours. found in water distribution systems. To accomplish this, three rodent species will be compromised using nitrous oxides or immunosuppressive agents, and the animals subsequently

16 will be chal- lenged via the gastrointestinal route. Although virulence is usually measured in vivo (animal research), the need for extensive (a) (b) (c) animal testing can be significantly reduced by the developing) water treatment in small com- Two step, 48 hour ment of a battery of in vitro (cell munities where the treatment system membrane filter test culture) tests for traits known to be has been overwhelmed by organic for enumerating virulence-related. This battery can be substances that may be harmful to enterococci in used to predict the potential an human health. EPA’s Office of recreational waters. organism has for causing disease in Ground Water and Drinking Water (a) and (b) Two perspectives of exposed populations. Through the (OGWDW), however, does not want colonies (red) project entitled “Develop In Vitro to recommend the use of these filters present at 24 hours. Methods for Identifying Potential if the possibility exists that their use (c) At 48 hours, Bacterial Pathogens in Drinking poses an acute disease risk due to colonies with black Water,” model systems will be bacteria that grow on the filters. The halos are identified developed that can be used to deter- health significance of the bacteria as enterococci. mine the potential pathogenicity of known to adsorb and grow on GAC bacteria found in potable water filters used in the home will be distribution systems. Additionally, evaluated. The OGWDW will use gene probe and other assays to this information to develop appropri- identify known opportunistic patho- ate controls and regulations for this gens will be developed and evalu- type of drinking water treatment as ated. required by the SDWA. Bacteria common to drinking The objective of ORD’s project water distribution systems colonize entitled “Health Effects Associated point-of-entry, granular activated with Point-of-Entry GAC Filters” is carbon (GAC) filters where they are to determine if a significant health able to grow to very high densities. hazard is associated with the use of Subsequent to reaching the high granular activated carbon, point-of- densities the bacteria begin slough- entry, whole house filters. To accom- ing off the GAC filters. The number plish this, a suitable study site will be of bacteria in the filter effluent (i.e., selected based on the following water flowing out of the filter) is criteria: 1) the water in the delivery significantly higher than in the system must meet EPA and local influent water. This amplification of drinking water standards; and 2) the bacteria in drinking water is of water distribution system should concern to EPA because GAC filters contain a bacterial population whose are being considered as a substitute density is as high as possible and still for central potable (i.e., fit for drink- acceptable under local regulations.

17 After a distribution system Some believe that exposure to Analysis of meeting the above criteria has been fecal pollution through recreational potable water or found, a volunteer population of waters or ingestion of contaminated cooling tower appropriate size will be selected shellfish causes greater health risks if water for from among the water system cus- the pollution is of human rather than Legionella tomers. The selected population will animal origin. Before the relative pneumophila be randomly divided into GAC user risks of human versus animal fecal requires and non-user groups. Point-of-entry, pollution can be assessed, it is neces- approximately GAC filters will be installed in the sary to develop a microbiological five to seven homes of the randomly selected user method for distinguishing human days for growth of group. The health status of both from animal pollution. Current the organisms on groups will be monitored over a methods detect fecal pollution but do the initial isolation predetermined period of time and not reveal the source. The objective medium and during this time interval the bacterial of the project entitled “Method to another five to population in the water system and Distinguish Non-Human Fecal seven days to the filter effluent will be monitored Pollution from Human Fecal Pollu- confirm the on a routine basis. In the event of an tion” is to develop a gene probe identity of these illness where a bacterial agent is specific for E. coli that inhabit the organisms. Gene diagnosed as the cause, the GAC human intestine for use as an indica- probe techniques filter will be removed and examined tor of the presence of human fecal could reduce for the presence of the organism contamination in water. The probe analysis time to determined to be the agent of the will be field tested at several sites in one day. disease in that household unit. If an which fecal pollution is exclusively association between illness or dis- from human sources, exclusively ease and the use of GAC filters is from animal sources and from mixed observed, health advisory guidelines sources. will be established or processes that Shigella species are among the will eliminate the causative organ- most common and significant patho- isms will be developed. gens associated with wastewater and

EPA researcher using the transmission electron microscope to detect pathogens unable to be detected by other methods.

18 sludge. Because of their low infec- mental Protection Agency. The tive doses, these organisms may be Office of Research and Development hazardous even if present in low is committed, through the extensive numbers in wastewaters that are waterborne disease research efforts recycled for potable use or sludges earlier described, to ensure that the that are applied to agricultural land. most effective and efficient methods Shigellae are very difficult to detect are developed to identify, detect, and in environmental samples by conven- inactivate/remove pathogens that tional methods because of their may be present in our drinking water biochemical similarities to E. coli. supplies. The use of current gene probe tech- Life cycles, mechanisms of nology in the project entitled “Detec- infection, protective or dormant tion of Enteroinvasive Shigella in states, emergence of disinfection- Wastewaters and Sludges” should resistant variants, optimal pathogen enable us to detect Shigellae in removal techniques, regrowth in sludges and wastewaters that would distribution lines…all are areas that appear to be free of these pathogens must be investigated and understood if analyzed by conventional methods. to afford the water quality safeguards that are so often taken for granted. The successes and failures of these Conclusion research efforts, relayed to the public Human enteric The protection and enhancement and appropriate federal, state, and bacteria being of our nation’s water quality remains local agencies, have helped to ensure subcultured in an a chief concern of the U.S. Environ- safe drinking water. anaerobic hood.

19 EPA Publications The EPA publications listed below may provide more detailed information on the subjects discussed in this document. These references and additional copies of this bro- chure can be requested at no charge (while supplies are available) from EPA’s Center for Environmental Research Information (CERI). Once the CERI inventory is exhausted, clients will be directed to the National Technical Information Service (NTIS) where docu- ments can be purchased.

Environmental Pollution Control Alternatives: Drinking Water Treatment for Small Commu- nities, EPA/625/5-90/025.

Methods for the Investigation and Prevention of Waterborne Disease Outbreaks, EPA/ 600/1-90/005a.

Microbiological Methods for Monitoring the Environment — Water and Wastes, EPA/600/ 8-78/017.

Seminar Publication: Control of Biofilm Growth in Drinking Water Distribution Systems, EPA/625/R-92/001.

Test Methods for Escherichia coli and Enterococci in Water by the Membrane Filter Procedure, EPA/600/4-85/076.

USEPA Manual of Methods for Virology, EPA/600/4-84/013 and updates.

Waterborne Disease Outbreaks - Selected Reprints of Articles on Epidemiology, Surveil- lance, Investigation, and Laboratory Analysis, EPA/600/1-90/005b.

Center for Environmental Research Information (CERI) U.S. Environmental Protection Agency 26 W. Martin Luther King Drive Cincinnati, OH 45268 Phone: (513) 569-7562 FAX: (513) 569-7566

Cited Literature Erlandsen, S.L., L.A. Sherlock, W.J. Bemrick, H. Ghobrial and W. Jakubowski. 1990. Prevalence of Giardia spp. in beaver and muskrat populations in northeastern states and Minnesota. Appl. & Envir. Micro., 56: 31-36.

Geldreich, E.E., K.R. Fox, J.A. Goodrich, E.W. Rice, R.M. Clark, and D.L. Swerdlow. 1992. Searching for a water supply connection in the Cabool, Missouri disease outbreak of E. coli 0157:H7. Wat. Res., 26: 1127-1137.

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