United States
Environmental Protection
Agency
Office of Water (4601M)
Office of Ground Water and Drinking Water
Distribution System White Paper
Health Risks from Microbial Growth and Biofilms
in Drinking Water Distribution Systems
June 17, 2002
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PREPARED BY:
U.S. Environmental Protection Agency
Office of Ground Water and Drinking Water
Standards and Risk Management Division
1200 Pennsylvania Ave., NW
Washington DC 20004
Background and Disclaimer
The USEPA is revising the Total Coliform Rule (TCR) and is considering new possible
distribution system requirements as part of these revisions. As part of this process, the
USEPA is publishing a series of issue papers to present available information on topics
relevant to possible TCR revisions. This paper was developed as part of that effort.
The objectives of the issue papers are to review the available data, information and
research regarding the potential public health risks associated with the distribution
system issues, and where relevant identify areas in which additional research may be
warranted. The issue papers will serve as background material for EPA, expert and
stakeholder discussions. The papers only present available information and do not
represent Agency policy. Some of the papers were prepared by parties outside of EPA;
EPA does not endorse those papers, but is providing them for information and review.
Additional Information
The paper is available at the TCR web site at:
http://www.epa.gov/safewater/disinfection/tcr/regulation revisions.html
Questions or comments regarding this paper may be directed to TCR@epa.gov.
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JUNE 17, 2002
Health Risks From Microbial Growth and Biofilms in Drinking Water Distribution Systems
I. Purpose of the Document
This document is one of a series of papers intended to review what is known about the
health risks associated with several distribution system issues, and where relevant, identify areas in
which additional research may be warranted. The issues were selected based on the input of
distribution system experts. The distribution system issues of concern are: Growth/Biofilms, Cross-
Connections, Intrusion, Aging Infrastructure, Decay of Water Quality over Distribution System
Residence Time, Contamination During Infrastructure Repair and Replacement, Nitrification,
Covered Storage, and Corrosion, Permeation and Leaching.
The goal of this document is to review existing literature, research and information on the
potential public health implications associated with the survival and/or growth of pathogens, as well
as with the presence of microbial metabolic products in the biofilms of drinking water distribution
systems. More specifically, the goal of this document is to review what we know regarding:
Microbes in or associated with biofilms that may present a public health risk in the
distribution system;
the types of disease each pathogen or metabolic product cause;
Routes through which pathogens can enter the distribution system;
factors that influence survival and growth of pathogens within the distribution system;
the effects of biofilms on some other health-related issues associated with the distribution
system;
suitable measures for controlling biofilm development; and
possible indicators of the presence of a biofilm problem and the effectiveness of control
measures.
II. Executive Summary
Many different microbes have demonstrated the ability to survive in the distribution system,
with some possessing the ability to grow and/or produce biofilms. Some of these organisms may be
primary pathogens (i.e., those that cause disease in healthy individuals), while others may be
opportunistic pathogens (i.e., those that cause disease in individuals with underlying conditions that
may facilitate infection). Microbes can enter distribution systems through a wide range of avenues,
including treatment processes or through deficiencies of the distribution system infrastructure.
Microbial presence in the distribution system can result in colonization of the distribution system
infrastructure. Once biofilm development begins, subsequent material, organisms and contamination
1
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JUNE 17, 2002
introduced to the distribution system can become entrained in the biofilm. The biofilm can protect
microbes from disinfection and allow microbes injured by environmental stress and disinfectants to
recover and grow. In addition, biofilms may increase pipe corrosion, adversely affect pipe
hydraulics and reduce the utility of total coliforms as indicator organisms. Microbial growth in
biofilms may result in deterioration of water quality, generation of bad tastes and odors, and
proliferation of macro in vertebrates.
Contamination and material in the biofilm may subsequently be released into the flowing
water under various circumstances. As a result, biofilms can act as a slow-release mechanism for
persistent contamination of the water. The organisms and their products may decrease disinfectant
levels (by increasing disinfectant demand), pose a direct public health risk, or create taste and odor
problems. Biofilms likely exist in all distribution systems, and are recognized as a normal part of the
distribution system.
III. Definitions
Drinking water in the distribution system is not sterile, regardless of the degree to which the
water is treated. The water contains microbes that survive the treatment process or enter the
distribution system through the pipe network. Many of these microbes can attach to the pipe wall
and become part of a biofilm.
Several definitions for biofilms have been published in the literature (LeChevallier, 1999a;
Berger et al., 1993; Characklis and Marshall, 1990; Characklis, 1981). There is not one universally-
recognized definition for biofilms; however, common among the definitions is that a water
distribution system biofilm is a complex mixture of microbes, organic and inorganic material
accumulated amidst a microbially produced organic polymer matrix attached to the inner surface of
the distribution system. The inner surface of a water pipe may have a continuous biofilm, but
usually biofilms are quite patchy (Walch, 1992; van der Wende and Characklis, 1990).
Under certain circumstances regrowth events can occur. The term "regrowth" is not
precisely defined in the literature. Some use "regrowth" to refer to any growth that occurs in the
distribution system; others restrict the meaning to the recovery and growth of environmentally- or
disinfectant-stressed microbes. This document will use the term "growth" as referring to both the
recovery and growth of injured microbes, as well as the growth of non-injured microbes.
IV. Microbes that May Present a Public Health Risk in the Distribution System
This section of the paper will discuss the potential public health concern that arises when
certain microbes and their products become a component of the distribution system biofilm. While
some potential health effects are listed in the tables herein, additional health effects are provided in
tables on the EPA Office of Ground Water and Drinking Water website. The organisms and toxins
discussed are:
Bacteria
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JUNE 17, 2002
Viruses
Protozoa
Invertebrates
Algae and algal toxins
Fungi
Microbial toxins
A number of technical reviews of the literature have been published on biofilm organisms in
the water distribution system and factors that influence their survival and growth (Geldreich and
LeChevallier, 1999; Geldreich, 1996; van der Wende and Characklis, 1990; LeChevallier, 1989a;
LeChevallier et al., 1990a; 1990b; 1999b; Costerton and Lappin-Scott, 1989; Marshall, 1992;
Mittelman, 1991; USEPA, 1992b; NRC, 1982).
Any microbe (including some pathogens) present in water may attach, or become enmeshed,
in the biofilm. Primary pathogens, which cause disease in healthy humans, may survive for a time in
the biofilm. However, the survival time for many pathogens in biofilms is uncertain and likely
varies depending on the organism For some pathogens, the distribution system is a physical,
chemical, and biological environment unsuited for their growth. However, pathogens may
accumulate in the biofilm, and the biofilm may extend the survival of primary pathogens by
protecting them from disinfectants. These pathogens may be sloughed from the biofilm into the
water column due to changes in the flow rate. The persistence of waterborne disease, or of
microbial contamination in a distribution system, long after the cause of the distribution system
problem has apparently been corrected suggests that there may be an isolated pocket of static or
slow-flowing water or biofilm erosion or sloughing is occurring (i.e. the slow-release mechanism).
In contrast to enteric primary pathogens (i.e., those which inhabit the gastrointestinal tract),
aquatic microbes are well-adapted to the low nutrient level and cool water temperature of the
distribution system, especially in the biofilm. Select aquatic microorganisms may be responsible for
the majority of infections and illnesses in humans and other animals. Some aquatic microbes may
cause disease in humans under certain circumstances, especially in individuals with a weakened
immune system or other major underlying conditions that facilitates infection. These microbes are
referred to as opportunistic, or secondary, pathogens. Opportunistic pathogens include Pseudomonas
aeruginosa, Legionella pneumophila, and the Mycobacterium avium complex (MAC).
Most waterborne pathogens both primary and opportunistic also have routes of
transmission other than water. Many of these microbes, especially the primary bacterial pathogens,
are important agents of foodborne outbreaks (Schaechter et al., 1998). Direct person-to-person
spread is common, especially for the viral and protozoan agents. The importance of water relative
to other sources of transmission depends upon the organism and other factors, and is often
uncertain.
A. Bacteria
The primary intestinal bacterial waterborne pathogens include Shigella, Salmonella, Yersinia
3
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JUNE 17, 2002
enterocolitica, Campylobacter jejuni, and Escherichia coli 0157 (Table 1). The potential for them to attach
to biofilms exists, and limited growth in some circumstances cannot be ruled out. One primary
pathogen that may be waterborne, Helicobacter pylori, was found to survive at least 192 hours on
stainless steel coupons (inserts used to monitor biofilm buildup) in a chemostat (Mackay et al.,
1998). Park, et al., (2001) have also noted the presence of H. pylori in biofilms of drinking water
mains. In another study, two non-pathogenic E. coli strains injected into a pilot distribution system
with a biofilm (20°C) grew slightly in the biofilm before eventually dying out (Fass et al., 1996). The
building plumbing system may act either as a direct conduit for pathogens sloughed off from
distribution system biofilms or as an amplifier of these pathogens. A waterborne disease outbreak
caused by E. coli 0157 persisted for weeks after the suspected source contaminated water meters
and main breaks were replaced or repaired (Swerdlow et al., 1992). Although a biofilm was not
implicated, the potential exists for biofilms to prolong the survival of some microbes. In another
study, Salmonella typhimurium was able to grow for a short time at 24°C in non-sterile tap water
(Armon et al., 1997).
Table 1: Primary Bacterial Pathogens Capable of Causing Waterborne Disease
()rif;inisni
Major Disease'
Primary Source
\\ I5DO'
CCL2
Salmonella typhi
typhoid fever
human feces
X
Salmonella paratyphi
paratyphoid fever
human feces
X
Salmonella typhimurium
gastroenteritis
human/animal feces
X
Other Salmonella sp.
gastroenteritis (salmonellosis)
human/animal feces
X
Shigella
bacillary dysentery
human feces
X
Vibrio cholerae
cholera
human feces, coastal
X
Enterovirulent E. coli
gastroenteritis
human feces
X
Yersinia enterocolitica
gastroenteritis
human/animal feces
X
Campylobacterjejuni
gastroenteritis
human/animal feces
X
Legionella pneumophila
Legionnaires Disease, Pontiac fever
warm water
X
Helicobacterpylori
peptic ulcers
saliva, human feces?
X
1
Documented waterborne disease outbreak in U.S.
2 Pathogen is on EPA's Contaminant Candidate List (CCL) of March 1998
3 Disease symptoms described in Benenson (1995)
Much more information is available on the presence of opportunistic bacterial pathogens.
Table 2 lists some of the aquatic and soil bacteria that have been associated with both distribution
system biofilms and disease. However, strain variation exists within each of the listed bacteria.
4
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JUNE 17, 2002
Purely environmental strains and clinical strains of the same genus or species may be present in a
distribution system. The clinically significant strains are opportunistic pathogens. Infective dose
studies on healthy individuals and animals, using the oral or intranasal route, demonstrate that very
high doses (106-1010 cells) are needed for infection or disease, at least for healthy individuals (Rusin
et al., 1997). In one study (Olson, 1982), Arinetobacter was detected on the surface layer of a mortar-
lined pipe at levels up to 109/cm2. While these studies focused on healthy individuals and animals,
little infective dose data are available for more susceptible populations. The clinically significant
strains of bacteria listed in Table 2 may cause disease ranging from mild to severe, including
pneumonia and septicemia (invasion of the blood). Outcomes are sometimes fatal (Toder, 1998;
Inderlied et al., 1993; Jarvis et al., 1985; Pier, 1998; Hardalo and Edberg, 1997; Thomas et al., 1977).
Since opportunistic pathogens affect sensitive individuals, such as some hospitalized
individuals, the percent of hospital-acquired (nosocomial) infections caused by these organisms may
provide some insight on the effects on sensitive individuals in general. The percent of nosocomial
infections caused by various opportunistic bacterial pathogens common in biofilms is given in Table
3. The CDC data (Table 3) indicate that these bacteria cause about 25% of the nosocomial
infections (CDC, 1996). There are several sources through which sensitive individuals can come
into contact with these bacteria, with some cases being linked to drinking water. Given that a large
number of sensitive individuals exist (Table 4), even a very small percentage contribution from
drinking water may represent a sizeable number of people.
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JUNE 17, 2002
Table 2: Opportunistic Bacterial Pathogens Detected in the Distribution System and/or Biofilms
()pporiunisiic
pathogens
Health lilTccis
Inl'oniiaiion on:
\\ 15 L)()
i
CCL2
Disease
Presence in L)S
liiolllni Presence
Acinetobacter
calcoaceticus
pneumonia, meningitis, infections of urinary
tract, septicemia
Davis et al., 1973, Horan et
al, 1988
Geldreich, 1990
LeChevallier et al.,
1987; Geldreich,
1990
Aemmonas hydrophila
sepsis, gastrointestinal illness, respiratory
tract infections
Davis et al., 1973
Geldreich, 1990
Reasoner, 1991,
van der Kooij and
Hijnen, 1988
X
Citrobacter spp.3
septicemia, pneumonia
Keusch and Acheson, 1998
Geldreich, 1990
Geldreich, 1990
E nterobacter spp.3
septicemia, pneumonia
Keusch and Acheson, 1998
Geldreich, 1990
Geldreich, 1990
Flavobacterium spp.
septicemia, meningitis
Davis et al., 1973
Geldreich, 1990
Geldreich, 1990
Klebsiella pneumoniae 3
septicemia, pneumonia
Keusch and Acheson, 1998
Geldreich, 1990
Geldreich, 1990
Moraxella spp.
pneumonia, conjunctivitis, septicemia, otitis,
urethritis, meningitis, bronchitis, sinusitis
Benenson, 1995, Davis et al.,
1973, Walker, 1998
LeChevallier, 1987
LeChevallier, 1987
M. avium complex
chronic diarrhea, chronic lung disease
Schaechter et al. 1998
Geldreich, 1990
Norton et al., 2000
X
X
Pseudomonas cepacia
foot infections
Tally, 1998
Geldreich, 1990
LeChevallier et al.,
1987
Pseudomonas aeruginosa
infections when severe burns, cancer
patients, lungs when cystic fibrosis,
pneumonia, meningitis, others
Toder, 1998
Geldreich, 1990
Geldreich, 1990
Serratia marcescens3
septicemia, pneumonia
Schaechter et al. 1998
Geldreich, 1990
1
Documented waterbome disease outbreak in U.S.
2 Pathogen is on EPA's Contaminant Candidate List (CCL) of March 1998
3 Some species are coliforms
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JUNE 17, 2002
Table 3: Nosocomial Infections1, 1/90-3/96
()rif;inisni
IVrceni
E. coE
12
Enterococci
10
Pseudomonas aeruginosa
9
Enterobacter spp.
6
Klebsiella pneumoniae
5
Acinetobacter spp.
1
Serratia marcescens
1
Citrobacter spp.
1
Other Klebsiella spp.
1
Other Enterobacteriaceae
4
Staphylococci
24
Yeast and fungi
7
All others
19
1 from CDC's Nat'l Nosocomial Infections Surveillance System (5/96)
Note 1: About 5% of all patients develop an infection while in the hospital
Note 2: In bold are the opportunistic pathogens that occur naturally in water
Table 4: Immunocompromised Sub-Populations in U.S.
Immunocompromised Suh-popul;uion
Number
AIDS patients
274,0001
Cancer patients on immuno-suppressive therapy
4.3 million (est.)
People with organ transplants
195,5612
Diabetics
8 million3
Hospitalized burn patients
75,000 /year4
Cystic fibrosis
30,000
through 12/98 (CDC HIV/AIDS Surveillance Report vol 10, no. 2)
2 1988-1998 (Scientific Registry, United Network for Organ Sharing, 1999)
3 CDC, National Center for Health Statistics, data for 1995
4 J. Burn Care & Rehab., May/June 1992.
Populations susceptible to opportunistic bacterial pathogens that are common in biofilms
include infants, young children, pregnant women, the very elderly, and those who have a severely
7
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JUNE 17, 2002
weakened immune system or other major impairment of the host defense. Table 4 identifies the
immunocompromised populations in the U.S., and their numbers, that are susceptible to these
pathogens. Cystic fibrosis is included although, strictly speaking, the immune response is normal;
however, antibodies cannot reach the bacteria embedded in the thick sticky mucus in these patients
(Pier, 1998). Table 4 includes only those groups that are more susceptible to opportunistic pathogens
due to being immunocompromised (with the exception of cystic fibrosis), and not those that are more
susceptible and not classified as immunocompromised, such as individuals with prolonged antacid or
antibiotic use or heavy smokers.
The opportunistic bacterial pathogens common in biofilms that are of most concern include
Lpneumophila, MAC, and P. aeruginosa, although there are others. Depending on the organism,
waterborne disease and injury may result from exposure through ingestion, inhalation of aerosols, and
by dermal exposure (e.g., through wounds). The link between nosocomial or community infections
caused by these organisms and drinking water is summarized below.
1. Legionella pneumophila
At least 39 species of Legionella have been identified, and a substantial proportion can cause a
type of pneumonia called Legionnaires disease. L pneumophila accounts for 90% of the cases of
Legionnaires disease reported to CDC (Breiman, 1993). According to CDC (MMWR Summary of
Notifiable Diseases, 12/31/99), 1355 cases of legionellosis (includes Legionnaires Disease and a
milder non-pneumonic illness known as Pontiac Fever) were reported to CDC in 1998; however,
CDC believes a majority of these illnesses are not reported, and estimates that 8,000 to 18,000
legionellosis cases occur in the U.S. annually fwww.cdc.gov/nciod/dbmd/diseaseinfo). The
underestimate is probably due to the fact that Legionella testing is not routinely performed, possibly
because the organism is so difficult to culture and identify (Stout and Yu, 1997).
L. pneumophila is a naturally occurring and widely distributed organism. In one study, it was
isolated from all samples taken in a survey of 67 rivers and lakes in the United States. Higher
recoveries occurred in warmer waters (Fliermans et al., 1981). L pneumophila has been found in the
biofilms of water mains, although they may not proliferate therein to any extent (States et al., 1990;
Armon et al., 1997). Various plumbing materials support the growth of Legionella, including latex,
ethylene-propylene, polypropylene, polyethylene, polyvinylchloride (PVC) and steel (Rogers et al.,
1994). Legionella proliferation is facilitated within Acanthamoeba and other aquatic amoeba (States et al.,
1990; Kwaik et al., 1998), and virulence may be enhanced by their interaction within the amoeba, or
may even be a necessary condition for virulence (Cirillo et al., 1999). Small numbers of Legionella can
occur in the finished waters of systems, including those employing conventional treatment. These
organisms can colonize hot water plumbing systems, and aerosols from fixtures, such as showerheads,
may cause disease via inhalation (U.S. Environmental Protection Agency, 1989; Tobin et al., 1981).
Aerosols from cooling towers, hot tubs, and whirlpools containing Legionella have also been implicated
as a route of infection (Moore et al., 1993). It is likely that drinking water is an important, if not the
primary source, of Legionella that seed hot water plumbing systems and cause outbreaks (Schaechter et
al., 1998). Especially vulnerable to outbreaks caused by Legionella are hospitals and other large
institutions that have an extensive hot water plumbing system and cater to susceptible subpopulations.
8
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JUNE 17, 2002
Several reports have been published on the presence of Legionella in the biofilm of dental-unit water
systems (Walker et al., 2000; Atlas et al., 1995).
Another indirect effect is primarily associated with the concern about Legionella proliferation in
the hot water plumbing systems of hospitals and other health care institutions. This concern has
prompted some hospitals to increase their hot water temperatures to levels that inhibit Legionella
growth (about 60°C). The higher temperatures have led to an increase in accidental patient scalding.
2. Mycobacterium avium complex (MAC)
MAC is a small group of bacteria in the genus Mycobacterium, primarily consisting of M. avium
and M. intracellulars. MAC is common in the environment and colonizes water systems and plumbing
systems (du Moulin and Stottmeier, 1986; du Moulin et al., 1988; Schulze-Robbecke et al., 1992;
Norton and LeChevallier, 2000). Mycobacterium species are common in pipe biofilms (Kubalek et al.,
1995; Schulze-Robbecke et al., 1992). Schulze-Robbecke et al., (1992) detected mycobacteria in 90%
of 50 biofilm samples, usually at levels between 103-l04 colonies/cm2. MAC is relatively resistant to
chlorine disinfection (Pelletier et al., 1988). MAC causes chronic lung disease in the
immunocompromised population, especially in those receiving cancer chemotherapy, but chronic
diarrhea is the major symptom caused by these organisms in AIDS patients (Inderlied et al., 1993;
Singh and Yu, 1994). About 40-60% of late-stage AIDS patients suffer from MAC-caused chronic
diarrhea. Infrequently, MAC causes disease in otherwise healthy individuals, especially older women
(Prince et al., 1989; Inderlied et al., 1993). Clinically important strains have been found in distribution
systems (Squier et al., 2000; Aronson et al., 1999), and drinking water has been epidemiologically
linked to MAC infections in hospital patients (du Moulin and Stottmeier, 1986).
3. Pseudomonas aeruginosa
P. aeruginosa is widespread in environmental waters, especially in those waters associated with
human activity. The organism is often found in finished waters and in pipe biofilms. Although P.
aeruginosa has not been conclusively implicated in a reported waterborne disease outbreak, it has a
significant role in nosocomial illness, including outbreaks. It is a pathogen of concern for people with
severe burns and wounds, diabetes, and is the primary cause of injury and death in people with cystic
fibrosis (Toder, 1998). Some strains cause pneumonia in general intensive care units (ICUs) and
pediatric ICUs. Clinically significant strains have been found in the hospital plumbing system,
suggesting that drinking water may contribute to nosocomial infections. A list of nosocomial
outbreaks associated with P. aeruginosa in contaminated drinking water appears in Highsmith et al.
(1986). However, the linking between the water distribution system (as opposed to the hospital
plumbing system) and the presence of clinically important strains of P. aeruginosa in the nosocomial
setting is still open to question (Samadpour, 2001).
B. Viruses
Viruses need a specific host (e.g., humans) to proliferate, therefore, they may accumulate, but
not grow, in the biofilm. One pilot-scale distribution system study demonstrated that more poliovirus
9
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JUNE 17, 2002
1 were recovered from the biofilm than from the water column (Quignon et al., 1997). In the
presence of chlorine there were ten-fold more viruses in the biofilm than in the water flow; without
chlorine there were two-fold more (Quignon et al., 1997). It is likely that biofilms may harbor other
viruses. Vanden Bossche and Krietemeyer (1995) detected coxsackievirus B in biofilms in water
mains exiting a treatment plant. Table 5 presents viruses associated with waterborne disease that
could potentially become entrained in the biofilm matrix for later release. The biofilm may protect the
viruses against disinfectants (Quignon et al., 1997) and thus allow them to survive for a time.
Table 5: Viral Fecal Pathogens Capable of Causing Waterborne Disease
()rif;inisni
Major Disease'
Primary Source
\\ I5L)()'
CCL2
Poliovirus
poliomyelitis
human feces
X
Coxsackievirus
upper respiratory disease
human feces
X
Echovirus
upper respiratory disease
human feces
X
Rotavirus
gastroenteritis
human feces
Norwalk and other Caliciviruses
gastroenteritis
human feces
X
X
Hepatitis A virus
infectious hepatitis
human feces
X
Hepatitis E virus
hepatitis
human feces
Astro virus
gastroenteritis
human feces
Enteric adenoviruses
gastroenteritis
human feces
X
1
Documented waterborne disease outbreak in U.S.
2 Pathogen is on EPA's Contaminant Candidate List (CCL) of March 1998
3 Disease symptoms described in Benenson (1995)
C. Protozoa
A few studies have also examined the presence of protozoa (i.e, unicellular animals), in the
distribution system or in pipe biofilms. A diverse flora of free-living aquatic microbes exist in the pipe
biofilm and pipe sediment, and protozoa are a natural part of that community. Ciliates, thecamoebae,
amoebae, and flagellates have been detected in the biofilm of pilot distribution systems (Sibille et al.,
1998; Block et al., 1993; Pedersen, 1990). Sibille et al. (1998) found an average protozoal count of 103
cells/cm2 in the biofilm. Amoeba were observed in a hospital plumbing system (Michel et al., 1995).
Because many protozoa feed on bacteria, it is likely that the protozoan population in the biofilm
correlates with bacterial density.
Table 6 presents some primary protozoal pathogens. Cryptosporidium, Giardia, Toxoplasma,
Cyclopora and other primary human pathogenic protozoa are present in natural water in a non-
reproductive protective stage (e.g., cyst, oocyst). Available data suggest that these organisms may
10
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JUNE 17, 2002
attach to, and accumulate within, the pipe biofilm and persist. For example, in a laboratory
experiment, Piriou et al., (2000) placed Cryptosporidium oocysts in a recirculated biofilm reactor (PVC
pipes) containing glass beads with biofilm, and found that a sizeable fraction of the oocysts became
associated with the biofilm. Rogers et al., (1996) also found that Cryptosporidium oocysts attached
rapidly to biofilms on glass tiles in a chemostat. Although protozoan cysts/oocysts may attach to, and
accumulate within, the pipe sediment and biofilm, these organisms do not likely proliferate in such
environments. They need a suitable warm-blooded host for this purpose. Reports of ongoing
recoveries of oocysts from the distribution system water after switching to an alternate source
provided evidence of biofilm involvement following a cryptosporidiosis outbreak (Howe et al., 2002).
Table 6: Protozoa Capable of Causing Waterborne Disease
()ru;aiiisni
Major Disease'
Primary Source
\\ I5D()'
CCL2
Giardia lamblia
giardiasis (gastroenteritis)
human & animal feces
X
Cryptosporidium parvum
cryptosporidiosis (gastroenteritis)
human & animal feces
X
Entamoeba histolytica
amoebic dysentery
human feces
X
Cyclospora cayatanensis
gastroenteritis
human feces
X
Microspora
gastroenteritis4
human feces
X
Acanthamoeba
eye infection
soil and water
X
Toxoplasma gondii
similar to infectious mononucleosis
cats
Naegleria fowleri
primary amoebic meningoencephalitis
soil and water
1
Documented waterborne disease outbreak in U.S.
2 Pathogen is on EPA's Contaminant Candidate List (CCL) of March 1998
3 Disease symptoms described in Benenson (1995), except as noted
4 Weber et al, 1994
Several free-living protozoa have been implicated in waterborne disease, especially some
Acanthamoeba species and Naegleriafowleri. Acanthamoeba is common in soil and water, including
drinking water and home plumbing systems (Sawyer, 1989; Gonzalez de la Cuesta et al., 1987; Seal et
al., 1992). Some A canthamoeba species are pathogenic, and can cause corneal inflammation, especially
in wearers of soft or disposable contact lenses (Seal et al., 1992). They have also been reported to
cause chronic encephalitis and skin lesions in the immunocompromised (Kilvington, 1990; Torno et
al., 2000).
Because A canthamoeba have been found in aquatic sediments, they may also be present in the
sediment of the water distribution system. These sediments provide a surface for biofilm
development. An additional public health concern related to A canthamoeba is the ability of Legionella
and M. avium, after ingestion by A canthamoeba and other aquatic protozoa, to multiply and become
more virulent within these protozoa (Steinert et al., 1998; Cirillo et al., 1997). In addition, the
11
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JUNE 17, 2002
protozoa may protect these ingested pathogens from disinfectants (King et al., 1988). Other bacterial
pathogens may be protected by Acanthamoeba against disinfection, including E. coli 0157 (Barker et al.,
1999), P. aeruginosa (Burghardt and Bergmann, 1995), and possibly Vibrio cholerae (Thorn et al., 1992).
N. fowkri is a free-living amoeba in soil, water, and decaying vegetation. Though it is common
in many warm surface waters, it rarely causes disease. The disease it does cause, primary amoebic
meningoencephalitis, is invariably fatal, with death occurring within 72 hours after symptoms appear
(CDC, 1992). All disease incidents to date have been associated with swimming in warm fresh waters;
however, transmission via drinking water cannot be ruled out. The route of infection is via inhalation,
not ingestion. Although N. fowleri has not been reported in the distribution system, other Naegleria
species have been found, including some in the biofilm (Rivera et al., 1979; Block et al., 1993).
D. Invertebrates
Invertebrates include all animals lacking a spinal column. Loosely defined, macroinvertebrates
include the larger invertebrates such as insects and worms. A number of macroinvertebrates may
colonize the distribution system, including tiny nematodes (worms), mites, insect larvae, rotifers, and
tiny crustaceans (Levy, 1990; Geldreich, 1996). These organisms can enter the distribution system
through the treatment plant, broken mains, backsiphonage, taps and hydrants (van Lieverloo et al.,
1997). However, only some taxa can survive in mains, with these organisms being dependent on the
presence of bacteria as a food source (van Lieverloo et al., 1997). Water mites, cladocerans, copepods
(and their larvae), oligochaetes and asellids have been observed in water flushed from distribution
system lines (van Lieverloo et al., 1997). These have not been implicated in waterborne disease
outbreaks, however. Various aquatic bacteria have also been found within the gut of nematodes
collected from natural water, and laboratory feeding studies have demonstrated that nematodes can
ingest bacterial pathogens and protect them from water disinfectants, and enhancing their survival in
biofilms and through treatment processes (Levy et al., 1986). The most recognized problem is that
macroinvertebrates occasionally occur in the tap water, or discolor the water, causing frequent
consumer complaints (Levy, 1990).
E. Algae and algal toxins
A few algal species, primarily cyanobacteria, or blue-green algae, produce algal blooms in
fresh waters, which can result in elevated toxin levels. The toxins, which include hepatotoxins and
neurotoxins, may be sufficiently potent to kill an animal within minutes (USEPA, 1992a; Yoo et al.,
1995). Numerous reports, summarized by Yoo et al. (1995), show that cyanobacteria blooms can kill
large animals such as cattle, sheep, horses, pigs, and dogs within a few minutes or hours after ingesting
lake water containing an algal bloom. An outbreak of waterborne gastrointestinal illness in the U.S.
was associated with an algal bloom in an uncovered finished water reservoir (Lippy and Erb, 1976). In
addition to acute effects, cyanobacterial toxins have been shown to be mutagenic, i.e., cause mutations
to DNA (Falconer and Humpage, 1996), and epidemiological data suggests a link between an algal
hepato toxin and liver cancer (Ueno et al., 1996).
A search of the published literature found information from two studies about the presence of
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algae or their toxins in pipe biofilm. These studies, using scanning electron microscopy on a pipe
surface, found diatom and other algal fragments or "microfossils" embedded in the biofilm (Nagy and
Olson, 1986; Allen et al., 1979; 1980). Other authors have found that some algae can grow
heterotrophically in the dark (Berger et al., 1979), and cyanobacteria have been found at low levels in
groundwater (Sinclair, 1990). In addition, algal toxins are relatively stable in the dark (Jones and
Sivonen, 1997), and may last at least one week in water (Drikas et al., 2001).
F. Fungi
Fungi are categorized as molds if they have branching, threadlike filaments, and yeasts if they
are single-celled organisms that reproduce by budding. Fungi are ubiquitous in the environment. A
diverse group of fungi has been found in water distribution systems (Rosenzweig et al., 1986; Niemi et
al., 1982; Zacheus and Martikainen, 1995; Frankova and Horecka, 1995; Nagy and Olson, 1982;
Geldreich and LeChevallier, 1999). Several studies report that filamentous fungi and yeast are
common on water pipe surfaces, even in the presence of free chlorine residuals (Nagy and Olson,
1986; Doggett, 2000). Elevated open storage tanks and fire hydrants may be a significant source of
fungi (Rosenzweig and Pipes, 1988; Rosenzweig and Pipes, 1989). Low flow rates are frequently
maintained in these structures, which can enhance biofilm development (Geldreich, 1990).
Table 7: Pathogenic Fungi Found in Water Distribution Systems and Biofilms
Winn*
Si>nie ilisoasos/synipumis
Roloroncos 1 ¦ >r
Disease
Frosonco in liii>l'ilms
Aspergillusfumigatus
pulmonary disease, allergies
Benenson, 1995
Rosenzweig et al., 1983; 1986
Aspergillusflavus
pulmonary disease, allergies
Benenson, 1995
Doggett, 2000; Rosenzweig et al., 1986
Aspergillus niger
ear infection
Benenson, 1995
Rosenzweig et al., 1983; 1986
Cyptococcus neoformans
meningitis, lung infections
Benenson, 1995
genus: Doggett, 2000; Rosenzweig and
Pipes, 1988,1989
Candida albicans
vaginal, urinary, and esophageal
infections, thrush
Benenson, 1995
genus: Rosenzweig and Pipes, 1988,1989
Mucor
thrombosis, infarction, nasal or
paranasal sinus infections, GI
disorders
Benenson, 1995
Rosenzweig & Pipes, 1988,1989;
Doggett, 2000; Nagy & Olson, 1986
Vetriellidium bqydii
(Pseudallescheria bojdii)
extracutaneous infections,
brain abscess, systemic central
nervous system infection
Fisher et al.,
1982
Roesch and Leong, 1983
Sporothrix schenkii
skin infection
(dermatomycoses)
Benenson, 1995
genus: Doggett, 2000
Stachybotys chartarum
infant pulmonary
hemosiderosis
Jarvis, 2002
Doggett, 2000
Trichophyton
scalp infections
Benenson, 1995
Frankova and Florecka, 1995
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A few studies have identified the fungi present in the distribution system (Doggett, 2000). A
few fungi are known to cause disease, primarily in immunocompromised individuals. Those fungi are
listed in Table 7. Of the listed fungi, those species that have been observed in the pipe biofilm are
Aspergillus flavus, Stachybotrys chartarum, and Pseudallescheria boydii. In addition, the fungal genera Mucor,
Sporothri-x, and Cryptococcus have been detected in the biofilm. These genera include pathogenic species,
but apparently speciation was not performed.
Although fungi have been found in drinking water distribution systems and biofilms, fungi
have not been conclusively implicated in waterborne disease. Those pathogenic fungi that have been
detected in the distribution system are opportunistic and infrequently cause illness, whatever the route
of infection. However, A .flavus and several other Apergillus species detected in distribution systems
produce potent toxins (mycotoxin), including aflatoxins. Also, a pathogenic yeast, Candida albicans
(candidiasis), can establish itself in the gastrointestinal tract (Schaechter et al., 1998) and thus could
potentially be spread by the fecal-oral route. It has been found in seawater and in sand at marine
beaches. C. albicans and other important fungal pathogens are also associated with soil. Thus, the
potential for waterborne disease caused by fungi in the biofilm exists, but the significance is unknown.
G. Microbial toxins
A number of human enteric pathogens produce a variety of toxins to facilitate their entry and
proliferation within their human hosts. In contrast to the enteric pathogens, many microbes adapted
to the aqueous environment release toxins as a survival mechanism or during cell autolysis.
Virtually all gram-negative bacteria, which represent the vast majority of bacteria in water,
release a complex lipopolysaccharide known as endotoxin upon their death and autolysis. Endotoxin
is capable in sufficient quantities of causing a non-specific response in humans such as fever. Using
the Umulus lysate procedure, which is sensitive to picogram levels of endotoxin, various investigators
have found this substance in the drinking water of most cities investigated (Diluzio and Friedmann,
1973; Jorgensen et al., 1976). Jakubowski and Ericksen (1980) discussed the health significance of the
levels found in water, and determined that additional data were needed to provide a clear answer.
V. Effects on Other Health Related Issues Associated with the Distribution System
Some health issues associated with biofilms are indirect. Biofilms may compromise the use of
total coliforms as drinking water indicators or, by corroding pipes, weaken pipe integrity. Although
aesthetic problems may not directly represent a public health risk, the appearance of aesthetic
problems may signal pipe deterioration, heavy biofilm, or other flaw that may represent, or lead to, a
health concern. The indirect health issues include:
Microbially-induced corrosion
Loss of indicator organism utility
Taste, color and odor problems
Others
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A. Microbially-induced corrosion
Pipe material invariably erodes with time. The rate of this process is affected by the
composition of the pipe, the corrosivity of the water within the pipe and the soil and water external to
the pipe, the microbial activity in the pipe bio film, and other factors (Geldreich, 1996). Over time,
corrosion may become serious enough to restrict water passage, produce taste and odor problems,
cause pipe breaks, and accelerate biofilm development (Geldreich, 1996).
Pipe corrosion may be caused by non-biological factors such as rapidly flowing water (causes
erosion corrosion and impingement attack) or chemical oxidation processes (Schock, 1999).
However, microbes in the biofilm can also play an important role in pipe corrosion. Ultimately,
corrosion can lead to pipe leaks, creating a pathway for pathogen intrusion into the drinking water.
Biofilms on the inner surface of the pipe represent a complex and dynamic ecosystem that collectively
influences the pipe corrosion process. The microbes most closely identified with pipe corrosion are
the iron and sulfur bacteria. Iron bacteria such as Gallionella oxidize soluble reduced iron (Fe2+) in the
pipe and water to the insoluble oxidized form (Fe3+), which precipitates (AWWA, 1995). Microbes
involved in the oxidation of iron and steel surfaces can deposit oxides of iron and manganese in raised
hard outgrowths from the pipe known as tubercles (Walch, 1992). Sulfur-oxidizing microbes such as
Thiobacillus generate sulfate and hydrogen ions, which lowers the pH, often resulting in a highly acidic
environment that pit and gouge metal. More important, sulfur-reducing microbes can generate
hydrogen sulfide gas, which has a rotten egg odor, and which can accelerate corrosion (AWWA,
1995). Nitrifiers may decrease the pH by oxidizing ammonium to nitrate and other nitrogen
compounds, and thus corrode copper and other pipe material (Schock, 1999). Other bacteria produce
polymers that may complex with pipe material or change the redox potential of the pipe surface,
accelerating corrosion (Schock, 1999).
Higher corrosion rates are associated with both high-flow and low-flow areas; warmer, poorly
buffered water; presence of high levels of iron, sulfur, and chlorides; and a well-developed biofilm.
Factors affecting corrosion are reviewed in detail by Schock (1999). Microbially-induced corrosion
may penetrate 5/8-inch steel within six months (Costerton and Lappin-Scott, 1989).
B. Loss of indicator organism utility
An extensive, well-developed pipe biofilm may compromise the effectiveness of total
coliforms as an indicator of drinking water quality in two major ways. Firstly, a high level of
heterotrophic bacteria in the pipe biofilm and sediments may interfere with the analysis of total
coliforms. This may occur when high levels of heterotrophic bacteria detach from the biofilm and
enter the water flow. As a result, water samples collected for the analysis of total coliforms may
contain a large number of heterotrophic bacteria that, by competitive inhibition for nutrients and
production of various toxins, may prevent the growth, and thereby detection, of coliforms with at
least some normally used analytical media. This phenomenon has been examined by Geldreich et al.
(1978) and Seidler et al. (1981), among others, and reviewed in EPA's Drinking Water Criteria
Document for Heterotrophic Bacteria (USEPA, 1984). The problem is partially obviated by using the
presence or absence of coliforms in a sample, rather than a density measurement.
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Secondly, the conditions that facilitate microbial growth on pipes can result in the growth of
coliforms as part of the biofilm. Instances of coliform proliferation in pipe biofilms are well-
documented in the literature, as reviewed by LeChevallier (1990) and Geldreich (1996). Biofilm
coliforms may detach into the flowing water and result in coliform-positive samples. For some
systems this phenomenon reduces the usefulness of the coliform test for detecting problems in water
treatment or distribution system integrity. However, a coliform-positive test resulting from coliform
growth in the biofilm may represent a distribution system deficiency because the conditions that
permit its proliferation in the biofilm may also permit the growth of many other microbes as well,
including opportunistic pathogens. In addition, an extensive coliform biofilm could reflect a high
degree of pipe corrosion and deterioration, as well as water system operational problems.
C. Taste, color and odor problems
Aesthetic concerns such as discoloration of the water and taste and odor problems may
result from a number of processes, some of which are microbially-mediated. Microbes most often
linked to aesthetic problems in drinking water are the actinomycetes, iron and sulfur bacteria, and
algae, especially the blue-green algae (Cohn et al., 1999; AWWA, 1995; Burlingame and Anselme,
1995). Many algal species and some actinomycetes produce geosmin and 2-methylisoborneol, both of
which produce earthy-musty odors. Some pseudomonads can also produce foul-smelling sulfur
compounds. The bacteria in the genus Hyphomicrobium, when sloughed off a biofilm, can cause
episodes of black water (van der Wende and Characklis, 1990).
In many cases, the microbially-produced metabolites that produce objectionable aesthetic
effects enter the distribution system and accumulate in static water areas or stratified storage tanks.
This is especially true for the algal metabolites. However, the microbes indicated above, with the
possible exception of the algae, are common in the pipe biofilm (Geldreich, 1996).
The percentage of complaints to water suppliers associated with aesthetic concerns is often
high and may change with the season. According to a national survey, 60% of responding utilities
reported taste and odor to be their most common water quality problems, with red water ranking
second with 47.7% (O'Conner and Banerji, 1984).
D. Others
Biofilms react with chemical disinfectants, thereby reducing the level available for inactivating
pathogens in the water (Berger et al., 2000). An extensive biofilm may reduce the disinfectant levels to
a point that may increase the public health concerns. If a system counters this problem by raising the
initial disinfectant dose, the level of disinfectant byproducts generated by this process may become
notable.
VI. Routes Through Which Pathogens Can Enter the Distribution System
Pathogens can enter the distribution system via a variety of pathways and become entrained in
the biofilm for later release. The pathways discussed in this section are:
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Treatment breakthrough
Leaking pipes, valves, joints, and seals
Cross-connections and backflow
Finished water storage vessels
Improper treatment of materials, equipment, or personnel before entry
Inadequate distribution system security
A steady inflow of bacteria, fungi, protozoa, algae, nematodes, and other microorganisms enter
the distribution system (Sibille et al., 1998). In general, pathogens may enter the distribution system
either through the source water, or at any point within the distribution system (Ratnayake and
Jayatilake, 1999). Treated water has ample recontamination possibilities based on the construction
characteristics, operation, and maintenance of the water distribution system (Berger et al., 1993).
Additional pathways include contamination through uncovered storage facilities, penetrations in
covered storage facilities, water main installation and repair sites, cross-connections or during
transitory contamination events, i.e. low pressure events leading to intrusion or backflow (Kirmeyer et
al., 2001). The severity of the potential health consequences that may result from contamination in
the distribution system depends, in part, on the route of entry. An expert panel recently ranked
various routes of pathogen entry into distribution systems by the potential health consequences, which
took into account the severity of disease, probability ofwaterborne disease outbreak, volume
contaminated and frequency of intrusion (Kirmeyer et al., 2001). These are presented in Table 8.
While potential sources of contaminant entry are known, the route of entry of microbes present in
distribution systems is still poorly understood (Gauthier et al., 1999).
Table 8: Some Pathways Through Which Pathogens Can Enter the Distribution System
Risk lc\cl
Pathway
High
Treatment breakthrough, intrusion, cross-connections, main repair/break.
Medium
Uncovered water storage facilities.
Low
New main installation, covered water storage facilities, growth and resuspension, purposeful
contamination.
Generated based on information from expert panel ranking in Kirmeyer et al., 2001.
A. Entry through the source water (e.g., treatment breakthrough)
It has been shown that the majority of organisms that colonize the pipe materials in
distribution systems can be found in the system's source water (Camper, 1996). Some organisms will
break through the treatment barriers (Schaule and Fleming, 1997), particularly following rainfall events
(USEPA, 1992b). The likelihood of filtration breakthrough depends on several factors, including the
condition of the filter media. Filter breakthrough may also lead to coliform episodes (Characklis,
1988).
The principal cause of growth is the failure of primary disinfection and loss of disinfectant
residual (Trussell, 1999). Klebsiella pneumoniae (a coliform, a few strains of which are opportunistic
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pathogens) is protected from disinfectants by several means, including their attachment to carbon
fines used to control odor and taste (Morin et al., 1996). Ineffective treatment may also allow fungi
and planktonic diatoms to enter the distribution system (Doggett, 2000). Breakthrough due to
inadequate treatment may have been responsible for elevated coliform counts in a distribution system
in Springfield, Illinois (Hudson et al., 1983).
B. Entty through broken or leaking pipes, valves, joints and seals
Broken or leaking pipes, or leaking valves, joints or seals provide a pathway for potential entry
of microbes which can then become entrained in the biofilm. Even in systems using good sanitary
practices, main breaks may result in contaminant entry (LeChevallier, 1999b). Aging infrastructure
contributes to main leaks and breaks, and may be a significant cause of drinking water contamination.
It is estimated that 42-69% of drinking water pipes in the United States are greater than 20 years old,
the percentage being dependent on the size of the system (Haas, 1999). Some systems are still using
distribution system piping that is up to 140 years old (Haas, 1999). Based on data from a recent
survey by AWWA on the distribution systems of 20 cities, 75% of transmission and distribution piping
were 20 years of age or more (AWWA, 1999). The main break frequency per system varies by size,
ranging from 1.33 breaks/year for systems serving fewer than 500 people to 488 breaks/year for
systems serving more than 500,000 people (Haas, 1999). A recent survey of 300 public water systems
showed 58 systems that responded to the survey had an average of 2,146 main breaks annually (37
breaks per system) that resulted in reduced distribution system pressures (ABPA, 2000).
Temperature effects can also cause thermal contraction and expansion can lead to main
breaks, and therefore, microbial contaminant entry. In northern states a seasonal variation in main
breaks is observed (O'Day et al., 1986). Breaks in the pipe was a contributing factor in the Cabool,
Missouri outbreak of 1989-1990. This outbreak, which occurred during unusually cold weather, was
caused by contamination that entered the distribution system through two major pipe breaks and 45
service meter failures (Swerdlow et al., 1992).
Intrusion may result from water pressure fluctuations in pipes. Transient negative pressure
can draw leaked water back into the pipe at any point where water is leaking out of the system
(LeChevallier et al., 1999). Even in well-operated systems leakage may represent 10-20 percent of the
water produced (LeChevallier, 1999b). Once these leaks or breaks occur, any microbial contamination
in the vicinity of the break or leak can potentially enter the distribution system given the pressure
changes that occur during breaks or leaks. One major fecal source are nearby sewer lines, which are
notorious for leaking (LeChevallier et al., 1999). This may have been a contributing factor in an
outbreak of E. coli 0157:H7, in Cabool, Missouri (Swerdlow, et al., 1992), which caused 243 cases of
illness and four deaths. An August, 2000 cryptosporidiosis outbreak in Northern Ireland resulting
from ingress of sewage from a septic tank into the drinking water distribution system caused at least
117 cases of illness (Glaberman et al., 2002). Kirmeyer, et al., (2001) found that 42.8% of 32 water
samples immediately next to water mains from six states were fecal coliform positive, while 12.5%
were positive for culturable enteric viruses using a cell culture assay. Main breaks can also introduce
high concentrations of injured coliform bacteria (undetectable by standard coliform techniques) into
the distribution system (LeChevallier, 1999b). In addition to contamination in the vicinity of mains
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and main breaks, pressure transients may also be common (LeChevallier et al., 1999). Some causes of
pressure transients are pump startup and shutdown, flushing operations, opening and closing fire
hydrants, sudden change in demand, feed tank draining, power failure, main breaks, altitude valve
closure, malfunctioning air-release, vacuum and pressure valves, air-valve slam, surge tank draining,
and resonance (LeChevallier, 1999b). In addition to these pressure transient causes, observed causes
of pressure reductions include fire flow, elevation changes, service line breaks and main installation
(ABPA, 2000). Additional information related to intrusion is being addressed in a separate paper
entitled "The Potential for Health Risks from Intrusion of Contaminants into the Distribution System
from Pressure Transients".
C. Entry through cross-connections and backflow
Cross-connections have a significant potential to introduce microbial contamination to the
distribution system when the cross-connections are not protected by properly operating backflow
preventers, and when a pressure change is experienced by the distribution system, particularly when
the pressure drops to subatmospheric. Microbes introduced to the distribution system as a result of
cross-connections and backflow can become part of the biofilm matrix, and may be released at a later
time. Entry of contamination through cross-connections is a major contributor to waterborne disease
outbreaks (Geldreich, 1996). Of 57 waterborne disease outbreaks related to backflow events
identified in CDC outbreak data from 1971-1994, 20 were associated with microbial contamination
(USEPA, 1999). It has been estimated that, at most, 10% of cross-connection incident reports
nationwide are submitted to the University of Southern California's Foundation for Cross-Connection
Control and Hydraulic Research (USEPA, 1995) in part due to systems' concerns about potential
liabilities arising from distribution system contamination. It is likely many more go unrecognized
given the transient nature of many pressure fluctuations, understaffmg of local cross-connection
personnel, and the lack of recognition of actual cross-connections due to their transient nature.
An M-DBP Federal Advisory Committee concluded that cross-connections and backflow pose
a significant health risk (US EPA, 2000). Although some feel the probable occurrence of cross-
connections is low in systems with a vigilant cross-connection control program (LeChevallier, 1999b),
rarely do all service connections in a system have backflow prevention devices (LeChevallier, 1999b).
Drinking water contamination from backflow events may have caused more waterborne disease
outbreaks in the US than any other cause (Kirmeyer et al., 2001). Worldwide, the most common
sources of contamination result from inadequate pressure and backsiphonage (Geldreich and
LeChevallier, 1999). An expert panel convened at a recent workshop regarded cross-connections
(Table 8) among the entry pathways of highest risk (Kirmeyer et al., 2001). More details on cross-
connections and backflow are being addressed in a separate cross-connection control and backflow
prevention paper.
D. Entry through contamination of finished water storage vessels
Both covered and uncovered finished water reservoirs provide opportunities for microbial
contamination of the distribution system, and the subsequent inclusion in distribution system biofilms.
Contaminated stored water can enter water distribution pipes when the water is drawn from the
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vessels for distribution. Contamination introduced through earlier points in the distribution system
may be amplified during storage (e.g., biofilm growth). Storage vessels may accumulate sediment,
enhancing the ability of microbes to thrive during storage.
Microbial contaminants can enter open storage reservoirs by natural phenomena, animals or
humans. Birds and other animals can introduce microbial contaminants through their feces, or
through general contact with the finished water. Some open finished water reservoirs may also be
subject to surface runoff which may be contaminated. The Interim Enhanced Surface Water
Treatment Rule (IESWTR) requires that all newly constructed finished water reservoirs, holding tanks
and other facilities constructed for surface water systems or ground water systems under the direct
influence of surface water serving 10,000 or more people, be covered (Federal Register, December 16,
1998). The Long Term 1 Enhanced Surface Water Treatment Rule (LT1) extended this requirement
to surface water systems or ground water systems under the direct influence of surface water serving
fewer than 10,000 people (Federal Register, April 10, 2000).
Inadequately secured covered finished water storage vessels may allow microbial
contamination to enter the distribution system. When air is drawn through air vents to replace water
leaving the vessel, contamination in the air can enter (USEPA, 1992b). Humans and animals can enter
inadequately protected covered finished water vessels and introduce contamination. Underground
basins are susceptible to bird, animal and human contamination (USEPA, 1992b), while ground level
and elevated finished water storage tanks can also become contaminated by humans and birds. A S.
typhimurium outbreak in Gideon, Missouri, which caused over 400 cases of illness and seven deaths,
was likely caused by bird feces contaminating an elevated storage tank (Clark et al., 1996). More
information on contamination of storage vessels is addressed in a separate paper on covered storage.
E. Entry through Improper Treatment of Materials, Equipment or Personnel in Contact
with Finished Water
Materials, equipment and personnel introduced to the distribution system also provide
pathways for microbial contaminants to enter bio films. The materials can include filter materials,
piping, sealing vials and others (Schaule and Fleming, 1997). Personnel in contact with the water can
provide a pathway for contaminant introduction (Schaule and Fleming, 1997) by introducing
contaminants during maintenance or repairs of the distribution system or storage vessels. Equipment
placed inside water distribution systems, such as tank cleaning equipment or video equipment used to
inspect pipelines, can introduce contaminants if not decontaminated prior to use.
F. Entty through inadequate distribution system security
Lack of proper security may result in microbe entry, followed by incorporation of the
microbial contaminants into the distribution system biofilm. This may result from intentional security
breaches, such as vandalism or terrorism. Also, unintentional contamination can result from
unauthorized users tapping into the distribution system and swimmers using storage vessels or
reservoirs. Distribution systems can have many miles of pipe, and many storage tanks and
interconnections. Because of this, systems can be susceptible to tampering, allowing contamination
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(LeChevallier, 1999b).
VII. Factors that Influence Pathogen Survival and Growth in the Distribution System
A variety of physical, chemical, and biological factors affect pipe biofilm development. For a
particular system, the interplay among these factors is complex and variable, often making predictions
precarious. A number of investigations have shed light on these factors and interactions, and several
comprehensive review articles have been published (Geldreich, 1996; Geldreich and LeChevallier,
1999; LeChevallier 1989a;1990a,b). Most of the data on the factors that influence biofilm
development are based upon changes in the total viable counts (e.g., heterotrophic plate count) or on
changes in the growth of specific coliforms. This document addresses the following factors, below:
Environmental factors
Presence of nutrients
Microbial interactions
Distribution system materials
System hydraulics
Presence of distribution system residual
Sediment accumulation
A. Environmental Factors
Water temperature affects the microbial growth rate, disinfection efficiency, pipe corrosion
rates, and other phenomena associated with biofilm development (LeChevallier, 1989a), as well as
opportunity for microbes to enter the distribution system. Where nutrients are adequate, microbes
generally grow more rapidly at warmer water temperatures than at colder temperatures (Donlan and
Pipes, 1988; LeChevallier et al., 1996). Thus, warmer temperatures likely facilitate the growth of
opportunistic pathogens in the biofilm. Predictions are somewhat complicated by the fact that
disinfectants are less efficient at lower temperatures in inactivating microbes.
Generally, fecal pathogens can survive longer in colder waters because metabolic processes
slow (Atlas and Bartha, 1993). Some may also survive longer in very warm waters with a high organic
load. In tropical and subtropical regions, the higher temperatures and organic loading of water is
more similar to that of the gut of humans and other warm-blooded animals. In these waters, E. coli
can survive and even grow (Solo-Gabriele et al., 2000; Jimenez et al., 1989). The density of S.
typhimurium declined by 90% in 28.8 hours in a temperate source water versus 131 hours in a tropical
source water (Hazen and Toranzos, 1990). In contrast, the survival of enteroviruses, Cryptosporidium,
and Giardia decreases with increasing water temperatures (O'Brien and Newman, 1977; Fayer et al.,
1998; DeRegnier et al., 1989).
Environmental factors other than temperature that affect biofilm development include
finished water turbidity and water pH. Water pH affects the effectiveness of disinfection residuals,
and a low pH water is aggressive (Geldreich, 1996). The water turbidity level may interfere with
disinfection and turbidity particles can protect pathogens adsorbed to them from disinfection
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(Geldreich, 1996).
The release of coliforms from biofilms may be caused by seasonal changes in the water pH.
In Wilmette, Illinois, apparently as the result of the near-shore turnover of bottom water, the water
pH increased from 7.7 during the summer to 8.2 in December, and then quickly decreased to 7.4 in
January (Geldreich, 1996). This resulted in repeated coliform problems. This problem was resolved
by adjusting the water to pH 8.3 and slowly adding sufficient lime to develop a more stable coating on
the pipe walls (Geldreich, 1996).
B. The Presence of Nutrients
For growth and energy, heterotrophic microbes need a supply of biodegradable organic
material (known as assimilable organic carbon, AOC) and sufficient phosphorus, ammonium, and
other essential nutrients either from the biofilm or the water (LeChevallier, 1989a; 1990a). These
nutrients tend to concentrate at the solid-liquid interface, creating a favorable environment for
biofilm growth (LeChevallier, 1989a). The bacterial exopolysaccharide matrix that is part of a mature
biofilm can trap and concentrate nutrients (Costerton and Lappin-Scott, 1989). Higher nutrient levels
may also facilitate the recovery of disinfectant-stressed microbes (Watters and McFeters, 1990).
Frequently, the AOC level controls the rate and extent of biofilm development. Based on a
study of Dutch water systems, many of which do not use chemical disinfectants, maintaining the AOC
level below 10 |j,g C/L was sufficient to limit increases in the density of heterotrophic bacteria, as
measured by the HPC (van der Kooij et al., 1999). Many Dutch water systems prefer the use of AOC
control over disinfectant residuals to avoid disinfection byproducts, to reduce taste and odor
complaints and to reduce costs (van der Kooij et al., 1999). In another study of systems in the U.S., an
AOC level less than 50 |-ig/L was needed to control coliform growth in the distribution system
(LeChevallier et al., 1991). In contrast, Gibbs et al. (1993) found no correlation between AOC levels
and spatial and seasonal variations in plate counts within a distribution system (conventional treatment
with post-chloramination). Rainfall events may increase organic levels in the source water, and
thereby increase biofilm growth (LeChevallier et al., 1989a).
Phosphorus and ammonium are sometimes limiting with regard to microbial growth in the
distribution system (LeChevallier, 1989a; 1990b). Some forms of phosphorus do not support
microbial growth. For example, phosphate-based corrosion inhibitors were found to have an
insignificant influence on the growth of some coliform bacteria (Rosenzweig, 1987), while zinc
orthophosphate inhibited some coliforms (USEPA, 1992b). Studies investigating the ability of some
nitrogenous compounds (nitrate, nitrite, ammonia, organic nitrogen) to stimulate microbial growth
have met with mixed results. None of the nitrogen compounds examined by Donlan and Pipes (1988)
affected the attached microbial population density, suggesting that nitrogen was not a growth-limiting
factor under the experimental conditions. Bacterial survival and growth is frequently supported by
ammonia concentrations in groundwater supplies according to Rittman and Snoeyink (1984). When
systems use chloramines for disinfection, the ammonium added can also be a source of nitrogen,
which may support bacterial growth and biofilm formation. The phenomenon is addressed in more
detail in a separate paper on nitrification. Other potential growth-limiting nutrients may be highly site-
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specific. Iron can stimulate coliform growth (Victoreen, 1980).
The presence or absence of specific nutrients can also select for the microbial population
present, including pathogens. However, some pathogens, such as the opportunistic pathogen, P.
aeruginosa, are especially versatile in the types of organic nutrients they can use. The proportion of
multiple antibiotic-resistant bacteria in drinking water was found to increase due to increased copper
levels from corrosion (Armstrong, 1981).
C. Microbial Interactions
A microbe faces stiff competition from other microbes for the limited supply of nutrients.
Moreover, some aquatic bacteria may produce substances that inhibit other organisms (Waksman,
1941), at the same time they produce extrapolysaccharide and other material that support the growth
of these organisms. Protozoan grazing is a major factor in the decrease of enteric bacteria in natural
water (Gonzalez et al., 1992; Chao et al., 1988), and possibly in the distribution system as well.
However, some opportunistic pathogens thrive in the distribution system environment (Geldreich,
1996).
Some opportunistic pathogens such as L. pneumophila, M. avium, and primary pathogens such as
V. cholerae, and E. coli 0157:H7 survive and even grow within certain common amoeba (Barker and
Brown, 1994; Barker et al., 1999; Wadowsky et al., 1991; Cirillo et al., 1997; Thom et al., 1992), and
may be protected from disinfection. Some of the biofilm organisms may even supply an essential
nutrient to facilitate the growth of an opportunistic pathogen. In one study, Legionella only grew near
colonies of the bacterium Flavobacterium breve on an L-cysteine-deficient medium (Wadowsky and Yee,
1983).
D. Distribution System Materials
Some types of pipe and appurtenance materials are especially prone to biofilm development.
The materials may include the pipes, valves, joints, fittings or joint-packing material. The Netherlands
tests materials for biofilm growth potential, and the utilities use approved construction materials and
appendages (van der Kooij et al., 1999). Pipe material may be more influential than the level of
organic matter in the system (Voik and LeChevallier, 1999). Some materials provide the microbes a
protective niche where growth can occur, while some provide nutrients to support microbial growth.
The bacterial levels on disinfected iron pipes generally exceed those on disinfected PVC pipes (Norton
and LeChevallier, 2000). Biofilms also develop more rapidly on iron pipes, even with corrosion
control (Haas et al., 1983; Camper, 1996). In addition, iron pipes support a more diverse microflora
compared to PVC pipes (LeChevallier, 1999b). Iron pipes facilitate the development of tubercles,
which are primarily iron oxides (Tuovinen et al., 1980), and these tubercles can adsorb organic
material (Geldreich, 1996; Geldreich and LeChevallier, 1999). In this manner, the level of corrosion
and tuberculation (i.e., buildup of corrosion pitting products) affect biofilm development. Sloughing
of biofilms into the water column can also occur as a result of elevated biofilm levels on iron pipes
(Norton and LeChevallier, 2000). Biofilm problems also tend to increase when systems have iron pipe
that is predominantly 50 years old or more (Geldreich, 1996). The position (e.g., top versus bottom)
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of the biofilm on the internal circumference of cast iron pipes can influence the extent of biofilm
growth (Holden et al., 1995). In an additional study, Rogers et. al., 1994, found that elastomeric
materials supported more abundant biofilms and higher levels of L. pneumophila than did glass or steel.
The relative efficiency of disinfection varies with pipe material. With iron pipes, even low
corrosion levels can interfere with chlorine disinfection, but not with monochloramine (LeChevallier
et al., 1993). A free chlorine residual of 3 mg/L may not be effective in controlling biofilms on iron
pipes (LeChevallier, 1990). In contrast, low chlorine or chloramine levels (1 mg/L) can control the
development of biofilms on galvanized, copper, or PVC pipes (Lund and Ormerod, 1995).
Some materials used for pipe gasket seals and other pipe appurtenances that come into contact
with water are susceptible to biofilms, and some have been found to support the growth of Legionella
(Rogers et al., 1994), including rubber gaskets and washers (Colbourne et al., 1984). Materials that
support microbial growth include rubber, silicon, PVC, polyethylene and bituminous coatings
(Schoenen and Scholer, 1985; Frensch et al., 1987; Schoenen and Wehse, 1988). Besides pipe
appurtenances, material added to the infrastructure, such as lining materials which may contain
additives, solvents, or monomers, can support microbial growth (Rigal and Danjou, 1999).
When corrosion is severe additional problems may result. Corrosion of pipes can lead to
pipeline breaks and leaks in pipelines, valves, joints and seals. Corrosion can occur internal or external
to the pipe, with each being influenced by a variety of factors, including the water chemistry, presence
of iron and sulfur-oxidizing bacteria for internal corrosion, and the soil corrosivity, water table, and
electrical grounding for external corrosion. Corrosion will be discussed in more detail in a separate
paper.
E. System Hydraulics
The hydraulic characteristics of the distribution system is one of several factors that may be
more influential than the levels of organic matter in regulating the biofilm's biological activity (Voik
and LeChevallier, 1999). A variety of hydraulic conditions, such as long residence times due to low
flow rates or dead ends, high flow rates, or fluctuating flow rates can influence the survival and growth
of microbes in biofilms. A simple relationship between the hydraulic effects and microbial growth in
biofilms does not exist.
Among the many factors that can influence flow rates are pipe layout, condition and size,
demand, pump operation, and elevation. High water velocities may increase the level of nutrients and
disinfectants in contact with the biofilm, and cause greater shearing of biofilms that may contain
pathogens from the pipe surface. The reversal of flow caused by backflow can also shear biofilms
(USEPA, 1992b), which may result in the release of microbes entrained in the biofilm and lead to their
accumulation in low flow areas such as dead ends. Biofilm debris can accumulate in the periphery of
distribution systems, leading to sediment accumulation and microbial proliferation (van der Kooij,
2000).
Interrupted or pounding water flows (i.e., water hammer), or reversal of water flows, may
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dislodge tubercles and shear bio films (LeChevallier, 1990) that accumulate in these low flow areas,
resulting in release of elevated levels of the contaminants to the water column. Opheim et al., (1988)
found, the starting and stopping of flow increased bacterial levels in an experimental pipe ten fold.
Low velocities may result in stagnant water and a loss of disinfectant residual, which facilitates
microbial growth. Some factors contributing to low flow include mains not arranged in a looped
fashion, storage used only for high demand, oversized pipelines and lack of valve exercise. Complex
structures in the pipe network may also reduce flows and thereby support biofilm development
(Geldreich and LeChevallier, 1999). In addition, the distribution system design may influence the
formation of biofilms in finished water storage, as the design and operation of tanks, and the presence
of oversized lines, dead ends, and closed valves can lead to growth and a lack of a residual due to long
residence times (Crozes and Cushing, 2000). According to one study, the level of colonization in a
cast iron coupon biofilm was negatively correlated with water velocity (Donlan and Pipes, 1988).
However, this relationship may vary with the system and type of pipe, due to the complex interplay
between water velocity, the rate of nutrient and disinfectant transport to the biofilm, the concentration
of nutrients and biofilm detachment rate (NRC, 1982).
F. Disinfectant Types and Residuals
Organisms attached to biofilms are more resistant to disinfection than are planktonic cells, i.e.
those in the water (Berger et al., 1993; LeChevallier 1990b; Crozes and Cushing, 2000). The rates of
disinfectant diffusion, reaction and sorption to the biofilm, and the disinfectant demand are important
factors in the control of biofilms using disinfectants. In addition, microbes that have sloughed off the
biofilm are often aggregated and surrounded in biofilm glycocalyx, which makes inactivation by
disinfectants more difficult (Carlson et al., 1975).
Biofilm accumulation, the extent of biofilm development, and the microbial population can be
influenced by the chlorine concentration (Characklis, 1988; Holden et al., 1995). With chlorine, a
gradient exists between biofilm level and chlorine residual, with the effect that biofilms are more
highly developed downstream, where chlorine concentrations are lower (Characklis, 1988). Excessive
biofilm growth can result from the loss of the disinfectant residual (Crozes and Cushing, 2000).
Maintenance of a free chlorine residual does not necessarily control biofilms (Wierenga, 1985;
LeChevallier, 1990b). Once a biofilm is established, it may take a high level of chlorine residual (much
greater than 0.2 mg/L) to reduce microbial levels significantly (LeChevallier, 1989b). Maintenance of
high chlorine levels may be complicated by a need to control disinfection byproducts and pipe
corrosion, and to minimize taste and odor problems. Chloramine, being less reactive, tends to
penetrate into the biofilm to a greater extent than chlorine (Jacangelo et al., 1987; LeChevallier
1990b;de Beer et al., 1994), although the relative efficiencies of chlorine and chloramines for
controlling biofilms are not consistently clear-cut (Stewart et al., 1984). Chloramine is more effective
than chlorine for controlling Legionella in hospital plumbing systems (Kool et al., 1999). Resistance to
chlorine increases with biofilm age and low nutrient levels (LeChevallier et al., 1988). In contrast,
resistance to monochloramine is not affected by the nutrient level (LeChevallier et al., 1988).
Treatment with ozone before water enters the distribution system increases the level of easily
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degradable organic compounds in the water, and is associated with greater densities and variety of
biofilm organisms (Lund and Ormerod, 1995).
G. Sediment Accumulation
Significant microbial activity may occur in accumulated sediment (USEPA, 1992b). Organic
and inorganic sediments can also accumulate in low-flow areas of the distribution system, and enhance
microbial activity by providing protection and nutrients (USEPA, 1992b). Biofilms that slough can
accumulate in the periphery of distribution systems leading to sediment accumulation and the
proliferation of some microorganisms (van der Kooij, 2000). Sediments may be an important source
of nutrients in open finished water reservoirs, by accumulating slowly biodegrading materials which
are then broken down and released into the water column (LeChevallier, 1999b). The opportunities
for biofilm development may be more abundant in storage tanks than in distribution system piping.
Frequently, water is drawn from storage tanks only when water demand is high, such as during
drought, fire flow, and flushing operations. This intermittent use results in prolonged storage times
that may lead to increased sediment accumulation and lack of a disinfectant residual in the finished
water storage vessel. Biological and aesthetic effects can be observed following the release of
accumulated sediments from low flow areas of the distribution system (Geldreich, 1990).
Many studies have identified microbes in accumulated sediments, including both pathogens
and non-pathogens. These include bacteria, viruses, protozoa, algae, fungi and invertebrates.
Opportunistic pathogens that have been detected, and can multiply in sediments, include Legionella and
mycobacteria (van der Kooij, 2000). Some primary pathogens can also survive for some time in
sediments. Hepatitis A virus survived more than four months in sediments at both 5°C and 25°C
(Sobsey et al., 1986). Other opportunistic pathogens found in sediments include Pseudomonasfluorescens
and Flavobacterium spp. (Berger et al., 1993). Sediments can also release nutrients into the water which
stimulate biofilm growth downstream (LeChevallier, 1999b).
VIII. Suitable Measures for Controlling Biofilm Development
Biofilm control is one of the important objectives for ensuring that water delivered to the
consumer is of high quality. Many different methods have been used to control biofilms, and some
of the most important are included in Table 9. Biofilm control often requires the use of a variety of
tools, rather than a single "best" tool, and the relative effectiveness of a control practice may be site-
specific. Most biofilm organisms, including opportunistic bacterial pathogens, are indigenous to the
aquatic environment. This is not the case with primary fecal pathogens. This section will discuss the
following measures for controlling biofilms:
Nutrient control
Control of contamination from materials and equipment
Control and mitigation of system hydraulic problems
Cross-connection control and backflow prevention
Disinfectant residuals
Corrosion control
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Infrastructure replacement and repair
Security
Storage vessel management and alteration
The presence of biofilms and the factors that contribute to biofilm development is often
determined through sanitary surveys. A sanitary survey that includes the distribution system can often
reveal the existence of a biofilm problem and the factors that promote it (Kirmeyer et al., 2001). This
can be the first step in identifying the cause of biofilm problems so systems can implement the
appropriate measures to control or correct the biofilm problems. It may be necessary to apply specific
testing techniques to determine the extent of growth, the water's potential to support or promote
growth, and the most effective means for controlling the growth (Crozes and Cushing, 2000).
Distribution system sanitary surveys, along with cross-connection control programs and the
maintenance of sanitary conditions during main repair, are three areas that must be improved to
prevent pathogen intrusion (Kirmeyer et al., 2001). Utilities should focus their attention on their
ability to maintain distribution system barriers at critical points, including repair and construction sites,
valves, cross-connections and storage facilities (Kirmeyer et al., 2001). It has been estimated that
pipeline repair or damage, along with cross-connections, are associated with 15% of all cases of
giardiasis in the United States (Craun, 1986).
Table 9: Methods to Control or Mitigate
Siofilms in Distribution Systems
Control or Miiiiruiion Measure
.\uihor(s)
Main flushing, pigging and cleaning
Costello (1984), Bergpr et al (1993), USEPA (1992b), Trussell
(1999), Van der Kooij et al (1999)
Disinfectant residual
Trussell (1999), Geldreich and LeChevallier (1999)
Main repair and replacement
Costello (1984), USEPA (1992b), Kirmeyer et al, (2001)
Minimization of dead ends/flow management
Costello (1984), Geldreich and LeChevallier (1999)
Corrosion control program
Berger et al (1993), Volk et al (2000), Trussell (1999), Geldreich
and LeChevallier (1999)
Proper Storage Tank/Reservoir O&M
USEPA (1992b), Geldreich and LeChevallier (1999)
Control and mitigation of system hydraulic
problems
USEPA (1992b), Van der Kooij et al (1999)
Nutrient suppression
Volk et al (2000), Trussell (1999), Van der Kooij et al (1999),
Geldreich and LeChevallier (1999)
Cross-connection control
Kirmeyer et al (2001), Van der Kooij et al (1999)
A. Nutrient Control
Nutrient control is recognized as one of the most effective methods for controlling microbial
growth and biofilm formation. This can be accomplished by controlling the source of carbon or
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other nutrients (e.g., phosphorus, nitrogen), depending on the growth-rate limiting nutrient for the
specific system. Control of nutrients for the subsequent control of growth in the distribution system
is one of the main reasons that systems apply biological treatment (Norton and LeChevallier, 2000).
Some methods for nutrient control include biological treatment, coagulation, membrane filtration, and
granular activated carbon (GAC). Alternative source waters have also been suggested (Crozes and
Cushing, 2000).
While control of nutrient levels is the most direct method of controlling biofilm growth, it is
also the most difficult (USEPA, 1992b). Biofilm control through nutrient reduction may not be
immediate. Several months were required for biological filtration to have an observable impact on
bacterial water quality, according to one study (Voik and LeChevallier, 1999). Careful disinfectant
selection is also necessary. Oxidation with chlorine or ozone may increase the amount of
biodegradable organic matter in the finished water (Joret and Prevost, 1992; LeChevallier et al., 1992).
Since organic carbon is the primary carbon and energy source for much of the distribution
system microbial activity, the rate and extent of biofilm formation can be minimized by control of the
organic carbon concentration (Characklis, 1988). To control the influent organic carbon, treatment
for control of AOC is applied at the treatment plant. Control of the AOC concentration has proven
so effective at controlling bacterial survival and growth that some systems have discontinued applying
secondary disinfection (Schellart, 1986; van der Kooij, 1987).
Biological treatment is one technology for systems to control AOC. Biological treatment uses
microbial activity at the point of treatment to reduce the AOC concentration in the water entering the
distribution system, thereby reducing the rate and extent of microbial growth and biofilm formation.
Preozonation is commonly used, followed by biological filtration with GAC (Morin et al., 1996).
Preozonation oxidizes organic matter to a more readily degradable form prior to biological treatment.
When using biological treatment it is important to encourage the growth of bacteria in the GAC filter.
Several instances have been noted in which biofilms accumulated in systems where bacterial growth in
the GAC filter was hindered. In addition, changes in the disinfectant type and dose, and point of
disinfectant application can impact AOC levels in finished drinking water (LeChevallier, 1999a).
Activated carbon, as both GAC and the powdered form (PAC), can effectively remove AOC
from drinking water prior to distribution. GAC and PAC filter out the AOC by sorption. However,
research suggests coliforms and opportunistic pathogens can be associated with GAC particles
released from GAC filters (Camper et al., 1986; Stewart et al., 1990). The GAC and PAC processes
require careful control, as well as careful monitoring of breakthrough of GAC particles containing
organisms. The controls may include preozonation (Morin, et al., 1996).
AOC is mainly low molecular weight non-humic substances that are difficult to remove by
coagulation (Volk and LeChevallier, 1999). However, coagulation has been effective at removing
dissolved organic carbon (DOC) and biodegradable dissolved organic carbon (BDOC) (Volk and
LeChevallier, 1999).
Both nanofiltration (NF) and reverse osmosis (RO) have been suggested for the removal of
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nutrients (Crozes and Cushing, 2000). In addition to decreasing the concentration of organic matter
from drinking water, NF and RO are also effective at removing microbes (Sibille, 1998).
Control of finished water phosphorus concentrations may also help limit microbe survival and
growth and biofilm formation where phosphorus is the limiting nutrient. Often, systems with high
organic carbon levels have phosphorus as the growth-limiting nutrient (Miettinen et al., 1997).
Phosphorus limitation has been shown to be site-specific within the distribution system (Herson et al.,
1984). This emphasizes the importance of understanding site-specific characteristics of the system, as
treatment for non-growth-limiting nutrients will not control growth. In general, for most systems,
phosphorus is not the growth limiting factor (Donlan and Pipes, 1988). Some common technologies
that are used for reducing phosphate levels in wastewater treatment are biological treatment, and
precipitation using lime or metal salts such as ferric chloride, or aluminum sulfate (alum).
Because organic carbon is usually the growth-limiting substrate, control of nitrogen levels may
be ineffective at controlling microbial growth and biofilm formation. According to LeChevallier et al,
(1987), AOC concentrations decreased during travel through a distribution system, whereas nitrogen
compound concentrations remained adequate to maintain balanced growth (Camper, 1996). Careful
control of the correct ratio of chlorine to ammonia when practicing chloramination can minimize the
stimulation of microbes (LeChevallier, 1999b). Systems can remove ammonia from drinking water by
biological removal associated with GAC (Dahab and Woodbury, 1998; Snoeyink, 1990). In some
systems with poor water quality, a process of superchlorination-dechlorination can be used to oxidize
ammonia (Haas, 1990). Ion exchange, biological denitrification, reverse osmosis, electrodialysis and
distillation can be used for nitrate removal (Dahab and Woodbury, 1998). Ion exchange and RO are
listed as best available technologies for both nitrate and nitrite removal in drinking water systems,
while electrodialysis is also listed for nitrate removal. However, most bacteria use nitrogen in a
reduced form (e.g., ammonia).
B. Control of Contamination from Materials and Equipment
Control of contamination from materials and equipment can reduce the subsequent
contamination of the distribution system. When microbial contamination is present on materials or
equipment used in distribution system maintenance, the microbes can become a part of the microbial
community of the distribution system (i.e. provides a biological seed to the distribution system). By
reducing the biological seed entering the distribution system, biofilm problems can be reduced
(Trussell, 1999). Poor operations and maintenance procedures facilitate pathogen entry into the
distribution system (Berger et al., 1993). Therefore, good distribution system maintenance techniques
are viable alternatives for biofilm control (Camper, 1996). Equipment may have been idle for a long
time or may not have been adequately cleaned following previous usage, and may, therefore, harbor
microbial contaminants. Disinfection of equipment and a high pressure flush of various tools with tap
water could reduce the pathogen seed.
When replacing or repairing infrastructure, systems can take steps to prevent microbial
contaminant entry when the system returns to service. Thorough disinfection and flushing is
important before placing the system back into service. (To reduce biofilm sloughing, the most
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effective type of flushing is unidirectional flushing.) Following a main break systems usually isolate
the section of the pipe to be repaired before superchlorinating and flushing the section (LeChevallier,
1999b). Sanitary conditions are difficult to maintain, and quality control procedures are to follow,
given that water main repairs often involve working in wet environments (Kirmeyer et al., 2001).
System maintenance of sanitary conditions during main repair is one of three areas (along with cross-
connection control and sanitary surveys) in which systems need to improve for preventing pathogen
intrusion (Kirmeyer et al., 2001).
C. Control and Mitigation of System Hydraulic Problems
System hydraulic control is important in controlling microbial contamination in the
distribution system. Among the several measures systems can use to control the hydraulic condition
of the pipelines and biofilms is to flush and/or pig (use of a water-propelled device) the pipeline at
regular intervals. Flushing and pigging can remove the biofilm, sediments (Crozes and Cushing,
2000), and tuberculation, improving the system hydraulics. Where tuberculation is severe flushing
may not suffice, and pigging, or even the use of cable-drawn devices may be necessary (AWWA,
1987). Routine systematic flushing is a primary component of proper distribution system maintenance
(USEPA, 1992b). Drinking water systems should also thoroughly flush the distribution system
following a contamination event (LeChevallier, 1999a). Flushing, along with valve-turning proved
effective in helping a system in Washington, D.C. regain compliance following violations of the Total
Coliform Rule (TCR) inl996 (Clark et al., 1999). Flushing was one of two interim measures used to
control biofilm containing high coliform levels in a system in Connecticut (CDC, 1985).
Distribution system cleaning practices, such as flushing and pigging will not prevent
recolonization (Walker and Morales, 1997). Therefore, flushing and pigging are measures that many
systems routinely conduct. Furthermore, flushing and/or pigging the entire distribution system at
frequencies necessary to maintain low biofilm densities may not be cost effective. Although repeated
flushing and/or pigging are effective for localized contamination (USEPA, 1992b).
The elimination of low-flow areas and dead ends can improve system hydraulics (Camper,
1996), thereby reducing microbe survival and biofilm formation. Dead ends (causing excessive
residence times) can be eliminated by valve exercising, and eliminating excess storage, while low flow
areas can be eliminated by line resizing (Crozes and Cushing, 2000). Some of the practices that can
lead to changes in water velocity are routing flow to fire hydrants, and proper pipe network design and
pipe size which can eliminate low flow areas (Smith et al., 1989; Geldreich, 1988). Systems should
avoid sudden flow increases (USEPA, 1992b) or hydraulic disturbances (Characklis, 1988). These can
cause accumulated biofilm to shear or slough, resulting in release to the water column.
Continual positive pressure throughout the distribution system is recommended (Kirmeyer et
al., 2000), and is a best available technology (BAT) in the Total Coliform Rule (USEPA, 1992b). Lack
of positive pressure may commonly occur, as circumstances producing transient pressure waves may
be common to every water system (LeChevallier et al., 1999), and occur frequently in many water
distribution systems. One modeling study of three distribution systems, saw low or negative pressure
from loss of pumping power, loss of flow, fire flow or main breaks in 13 to 31% of pressure model
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nodes (Kirmeyer et al., 2001). Systems should also avoid extreme changes in pressure gradients
(Kroon, 1984), and consider surge control devices as part of the design (Kirmeyer et al., 2001).
Pressure monitoring in all parts of the distribution system can ensure that positive pressure is
maintained by alerting operators to the need to manage pumps and other components of the
distribution system. However, negative pressures may last for only a few seconds, and systems may
not detect them using conventional pressure monitoring (LeChevallier, 1999b). Record keeping of the
events that contribute to pressure changes may aid systems in minimizing such events.
D. Cross-Connection Control andBackflow Prevention
Backflow prevention devices are an important barrier against entry of contaminated water
(LeChevallier, 1999b). Installation of backflow prevention assemblies or devices are also frequently
accompanied by the regular inspection of the assemblies and devices, as well as regular testing of the
assemblies. Kirmeyer et al., (2001) recommends annual inspection and testing. Where backflow is a
problem cross-connection control has been identified by an expert panel as one area of improvement
necessary to prevent pathogen intrusion (Kirmeyer et al., 2001). A US General Accounting Office
report on the review of 200 sanitary surveys and a nationwide questionnaire of States identified
inadequate cross-connection control programs as the most common deficiency (USGAO, 1993). The
specific methods for controlling contamination due to backflow are being addressed in a separate
paper on cross-connection control and backflow prevention.
E. Disinfectant Residuals
Systems provide disinfectant residuals throughout the distribution system (i.e., secondary
disinfection) for protection of the finished water from microbial contamination in the distribution
system. Among the reasons for secondary disinfection are the inactivation of coliforms and pathogens
entering through cross-connections and line breaks, and the suppression of bacterial growth and
biofilm in static water areas (Geldreich, 1996; Trussell, 1999). Secondary disinfection also protects
against reinoculation of the flowing water by microbes trapped in the biofilm (Haas, 1999), which can
occur through sloughing or erosion of the biofilm. Disinfectant residuals can also reduce the amount
of viable organisms available to become adsorbed to the biofilm. Although contamination from
cross-connections and backflow may be controlled by a disinfectant residual (Snead et al., 1980), some
water supply professionals believe a disinfectant residual is not effective when cross-connections result
in massive contamination (LeChevallier, 1999b; Snead et al., 1980).
The residual disinfectant concentration is important in determining biofilm bacterial density
and composition (Norton and LeChevallier, 2000). Bacterial growth can be controlled with adequate
disinfectant residuals (Morin et al., 1996), and bacterial density will remain low (Berger et al., 1993).
However, many factors influence the concentration of the disinfectant residual in the distribution
system, and therefore the ability of the residual to control microbial growth and biofilm formation.
These factors include the AOC level, the type and concentration of disinfectant, water temperature,
pipe material, and system hydraulics. Because of these many factors, preventing pathogen survival and
growth requires strict attention to the residual disinfectant throughout the system (Trussell, 1999).
Disinfectant residual penetration into biofilms can also be inhibited by corrosion products due to the
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reactions between the residuals and the corrosion products (LeChevallier et al., 1993; LeChevallier et
al., 1996).
Various studies have obtained different results with respect to the ability of chlorine to control
biofilms, depending on the chlorine level used and other factors. At higher levels (1 mg/L), free
chlorine or chloramine was effective in disinfecting biofilms on galvanized iron, copper or PVC pipes
(LeChevallier et al., 1990b). However, some investigators have found that significant biofilms can
develop in the presence of low levels of residual free chlorine, and even at high levels, biofilms are not
eliminated (Characklis, 1988). According to Nagy and Olson (1986), bacterial densities in biofilms
were unaffected by the presence of free chlorine residuals. Camper (1996) noted that free chlorine
residuals were ineffective in controlling coliform occurrences when the AOC concentrations are
greater than 100 |Jg/L. Characklis (1988) noted that biofilm sloughing has not been documented in
the presence of chlorine. Some research has shown that most of the free chlorine is depleted before
penetrating the biofilm (Haas et al., 1991). In contrast to biofilms, chlorine controls the level of
heterotrophic bacteria and viruses in water (Characklis, 1988; Quignon et al., 1997).
Following TCR violations in Washington, D.C., in 1996, an increase in the residual chlorine
level, along with a close monitoring of the temperature, was one of three factors that helped the
system return to compliance (Clark et al., 1999). Increased chlorination was also used to control high
total coliform counts in a system in Connecticut (CDC, 1985) and in Springfield, Illinois (Hudson et
al., 1983).
Utilities can also use chloramines for controlling microbial growth and biofilm formation in
the distribution system. Chloramines may be preferred over free chlorine when the disinfecting
objective is biofilm control (Trussell, 1999), as chloramines may be more effective than free residual
chlorine at controlling biofilm formation. In support of this position, LeChevallier (1999a) found that
in filtered systems, coliforms were present in 0.97 percent of systems using free chlorine, but only 0.51
percent of systems using chloramines. While chloramines can be used to retard biofilm formation, no
disinfectants completely eliminate biofilms. Mycobacteria were frequently detected in eight well-
characterized systems in the presence of a chloramine residual (LeChevallier, 1999a). Water treatment
plant operators can more effectively control the concentration of the disinfectant in distant reaches of
the distribution system due to the increased stability of chloramines over free chlorine (LeChevallier,
1999b). The increased effectiveness of chloramines over free chlorine for biofilm control is more
pronounced in systems using iron pipes (LeChevallier et al., 1990b). However, chloramine
disinfection was not able to control coliform organisms in a system in Springfield, Illinois, following
an increase in coliform organisms, whereas chlorine was (Hudson et al., 1983).
Like free chlorine, the chloramine concentration impacts the ability of chloramines to control
biofilm formation. Monochloramine concentrations below 0.5 mg/L in distribution system dead
ends, result in more coliform occurrences than when higher monochloramine concentrations are
present (LeChevallier et al., 1996). The types of chloramines present also influence their effectiveness,
with monochloramines being less effective than dichloramines, but more effective than nitrogen
trichloride (trichloramines). Monochloramines are more widely used, however, as dichloramines have
a lower odor threshold, are more corrosive, and decrease in predominance at pH values of 7-8. One
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study found that neither chlorine residuals nor chloramines residuals alone were able to control
biofilm development, however when used in combination biofilms were controlled (Momba and
Binda, 2002).
F. Corrosion Control
The ability of corrosion control to inhibit biofilm formation is widespread in the literature
(Smith et al., 1990; LeChevallier et al., 1990a; Smith et al., 1990). Corrosion control may be better in
controlling biofilm activity than reducing organic matter levels (Voik and LeChevallier, 1999), and may
be the most important factor for control of biofilm development (Voik et al., 2000). Corrosion
control may include the inhibition of biofilm formation or the prevention of biofilm sloughing by
coating the biofilm (Berger et al., 1993). Corrosion is among the fundamental factors leading to the
release of biofilms into the water (LeChevallier et al., 1998). The effectiveness of zinc orthophosphate
or polyphosphate in reducing biofilm densities has been shown in one study (Abernathy and Camper,
1997), while the South Central Connecticut Regional Water Authority saw a long term decrease in
coliform occurrences after increasing the zinc metaphosphate mixture concentration (Smith et al.,
1989). However, according to Rosenzweig (1987), the growth of some coliform bacteria are not
significantly influenced by the presence of phosphate-based corrosion inhibitors.
Several factors influence the effectiveness of corrosion inhibitors for controlling microbes and
biofilm formation. One of the factors includes high concentrations of organic material (Voik et al.,
2000). Corrosion control can also impact disinfection effectiveness on biofilms, with an increase in
free chlorine disinfection effectiveness being observed when using corrosion control on iron pipes
(LeChevallier et al., 1990b; Lowther and Moser, 1984; Martin et al., 1982). However, using corrosion
inhibitors can be detrimental at excessive concentrations. A legionellosis outbreak in Lanzarote,
Canary Islands in 1993 may have been amplified due to excessive polyphosphate concentrations
(Crespi and Ferra, 1997).
G. Infrastructure Replacement and Repair
In some cases where growth is severe, replacement of pipe sections may be necessary
(USEPA, 1992b). The temporary fixes (e.g., where flushing may break corrosion-weakened pipes) may
be more costly in the long run than the more permanent solutions of pipeline rehabilitation or
replacement (USEPA, 1992b). Main reconditioning or replacement is a practice recommended for the
control of microbial growth (Costello, 1984), and is listed as a Best Available Technology (BAT) under
the Total Coliform Rule (TCR) (USEPA, 1992b). When systems have frequent contamination
problems resulting from deficiencies in or due to the nature of the infrastructure, the system may opt
to initiate a repair or replacement program. This may enable the system to resolve existing problems
without catastrophic failures or the associated costs. Although in many cases problems may be more
severe in older pipes, this is not always true. In general, pipe replacement should target sections of the
system experiencing the greatest number of leaks (Kirmeyer et al., 2001). However, mains are not the
only infrastructure whose failure can lead to contamination. Failure of other appurtenances , such as
isolation valves, air valves and surge control devices may lead to contamination (Kirmeyer et al., 2001).
33
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JUNE 17, 2002
Some materials used in drinking water distribution systems may provide limiting nutrients to
biofilm organisms and enhance microbial growth. In some instances, replacement of these materials
may be a viable solution. These materials may include the pipes, valves, joints, fittings or joint-packing
material. Bacteria can colonize piping, pipe joints, valves, elbows, tees, and other fittings due to the
changing water movement and stagnant areas of the distribution system (Berger et al., 1993). Studies
have also found that iron pipes require higher monochloramine concentrations than other pipe
materials, and that monochloramines were more effective than free chlorine in eliminating surface-
associated bacteria on iron pipe (LeChevallier et al., 1990b).
H. Adequate Security
The issue of adequate water system security has recently become highly visible, and marked
attention has been directed to this theme. This paper will not address intentional sabotage of the
distribution system, except to state that adequate security includes fencing, alarms, and regular
surveillance. Unintentional breeches should also be minimized.
I. Proper Storage Vessel Management and Alteration
Proper storage vessel management and alteration, when necessary, can prevent contamination
of the distribution system. Following TCR violations in 1996 in Washington D.C., one measure that
proved effective in bringing the system back into compliance was the cleaning, inspection and
disinfection of storage tanks and reservoirs (Clark, et al., 1999). To reduce pathogen presence and
biofilm development, systems should have a scheduled program to rehabilitate all water storage
facilities (USEPA, 1997). Proper operation and maintenance of storage tanks and reservoirs is listed
as a BAT in the TCR (USEPA, 1992b). Storage tanks and standpipes should be pressure flushed or
steam cleaned, then disinfected before returned to service (USEPA, 1992b), preferably with a
disinfectant solution. This may not only remove microbial contamination from the vessel's inner
surface, but also nutrients that may be present. Proper operation of storage vessels can also reduce
excessive residence times, which can lead to microbial survival and growth, and biofilm formation.
Properly designed inlets and outlets, and the overall system design can improve problems caused by
dead ends (Trussell, 1999). Pathogen contamination due to air introduction can be reduced by
installing air filters to guard against pollution entering covered water reservoirs (USEPA, 1992b).
Covering finished water reservoirs can protect against contamination from airborne sources, surface
runoff, accidental spills and animals, such as insects and birds (USEPA, 1992b). EPA's Uncovered
Finished Water Reservoirs Guidance Manual describes recommended contamination control measures
related to birds and other animals, human activity, algal growth and insects and fish (USEPA, 1999b).
An understanding of the storage hydraulics and operation is important in reducing contamination of
the finished water.
Proper turnover of the water in finished water storage facilities eliminates what amounts to
dead ends and can reduce the extent to which biofilms develop, minimize nutrient availability and
prevent the accumulation of sediments. To accomplish this systems can exercise valves to reduce
stagnation, and eliminate excess storage (Crozes and Cushing, 2000).
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JUNE 17, 2002
Systems can exercise additional control over biofilm accumulation and microbial growth in
finished water storage vessels by preventing sediment accumulation. This can be accomplished
through periodic flushing (Crozes and Cushing, 2000) and cleaning. Pigging of pipes to reduce the
biofilm seed that leads to sediment accumulation in storage vessels may help.
IX. Indicators that Signal the Presence of Biofilm Problems
Several indicators have been mentioned as a potential means of signaling the presence of a
significant biofilm problem. One such indicator is a high level of heterotrophic bacteria (Geldreich,
1996; Carter et al., 2000). Microbial density in a pipe biofilm has been estimated in several ways.
Some investigators removed a small section of pipe, or a pipe coupon, and examined it under
epifluorescent or scanning electron microscopy. In other studies, biofilm material was scraped from a
pipe with a spatula or brush, and cultured.
In addition, where other problems are not evident (e.g., cross-connections, treatment upsets), a
persistent coliform problem may indicate extensive growth problems (Crozes and Cushing, 2000).
Several species of coliforms that can grow in pipe biofilms, including Enterobacter cloacae, Klebsiella spp.,
Citrobacterfreundii and Enterobacter agglomerans (Geldreich, 1996). Another potential indicator is the loss
of disinfectant residual (Snead et al, 1980)
X. Additional Research Needs
As with most areas, further opportunities exist for research to result in greater certainty of the
health impacts associated with drinking water distribution systems. To better control pathogen
survival and growth in the biofilm and other public health problems associated with the biofilm in the
distribution system, the following areas of research are important:
Link between organisms present in distribution system biofilms and human health impacts
Effectiveness of potential indicators of extensive biofilm growth, including loss of disinfectant
residual, high AOC levels, pipe corrosion, and the presence of red or black water
The relative effectiveness of preventive and corrective measures
Potential problems created by cleaning deteriorated pipes
Level of public health protection provided by adding disinfectant residuals to the distribution system
Some specific research opportunities related to drinking water distribution systems are
outlined in two reports being prepared for EPA as part of Comprehensive Drinking Water Research
Strategy and the Microbial/Disinfection Byproducts (M/DBP) Research Council.
XI. Summary
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JUNE 17, 2002
A wide range of primary and opportunistic pathogens have demonstrated the ability to survive,
if not grow, in biofilms. These pathogens are of both fecal and non-fecal origin, and have a multitude
of pathways through which they can enter the distribution system. Some of the pathogens identified
as growing or potentially surviving in biofilms include Legionella, Mycobacterium avium complex,
Pseudomonas aeruginosa, poliovirus 1, coxsackievirus B and several species of fungi. Microbes can enter
the distribution system through inadequate treatment, cross-connections, leaking pipes and
appurtenances, as well as other means. Once becoming established as part of the biofilm, pathogens
can be protected from disinfection. Systems may select from a range of measures which can limit the
growth of microbes in the biofilm, prevent microbes from entering the distribution system, and/or
minimize the number of microbes present. Some of these measures are most effective if performed
regularly. Systems can also choose from a range of methods to detect biofilm problems.
36
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JUNE 17, 2002
Bibliography
Abernathy, CG, and A Camper. 1997. Interactions between pipe materials, disinfectants, corrosion inhibitors, organics
and distribution system biofilms. AWWA Water Qual. Tech. Conf. (Denver, 1997).
ABPA. 2000. American Backflow Prevention Association. 2000 Cross-connection control survey of public water systems.
Unpublished data.
Allen, MJ, EE Geldreich, and RH Taylor. 1979. The occurrence of microorganisms in water main encrustations, pp. 113-
135. AWWA Water Qual. Tech. Conf. (Denver, 1979).
Allen, MJ, RH Taylor, and EE Geldreich. 1980. The occurrence of microorganisms in water main encrustations. J.
Amer. Water Works Assoc. 72:614-625.
Armon, R, J Starosvetzky, T Arbel, and M Green. 1997. Survival of Legionella pneumophila and Salmonella typhimurium in
biofilm systems. Water Sci. Technol. 35(ll-12):293-300.
Armstrong, JL, JJ Calomiris, DS Shigeno, and RJ Seidler. 1981. Drug resistant bacteria in drinking water, pp. 263-276.
AWWA Water Quality Tech. Conf. (Seattle, 1981).
Aronson, T, A Holtzman, N Glover, M Boian, S Froman, OG Berlin, H Hill, and G Stelma. 1999. Comparison of large
restriction fragments of Mycobacterium avium isolates recovered from AIDS and non-AIDS patients with those of isolates
from potable water. J. Clin. Microbiol. 37:1008-1012.
Atlas, RN, and R Bartha. 1993. Microbial Ecology: Fundamentals and Application. Benjamin/Cummings Publishing Co.
Redwood City, CA.
Atlas, RN, JF Williams, and MK Huntington. 1995. Legionella contamination of dental-unit waters. Appl. Envir. Microbiol.
61: 1208-1213.
AWWA, American Water Works Association. 1999. 20 City Survey.
AWWA. American Water Works Association. 1995. Problem organisms in water: identification and treatment. AWWA
M7. Denver, CO.
AWWA. American Water Works Association. 1987. Cleaning and lining water mains. AWWA M28. Denver, CO.
Barker, J, TJ Humphrey, and MWR Brown. 1999. Survival of Escherichia coli 0157 in a soil protozoan: implications for
disease. FEMS Microbiol. Letters. 173:291-295.
Barker, J, and MRW Brown. 1994. Trojan horses of the microbial world: protozoa and the survival of bacterial pathogens
in the environment. Microbiology. 140(6):1253-1259.
Benenson, AS. 1995. Control of communicable diseases manual. 16th ed. APHA. Washington, DC.
Berger, PS, RM Clark, and DJ Reasoner. 2000. Water, Drinking. In: Encyclopedia of Microbiology. 2nd Edition. Vol.
4:898-913.
Berger, PS, RM Clark, and DJ Reasoner. 1993. Water, Drinking. In: Encyclopedia of Microbiology. Vol. 4:385-398.
Berger, PS, J Rho, and HB Gunner. 1979. Bacterial suppression of Chlwella by hydroxylamine production. Water Res.
37
-------
JUNE 17, 2002
13:267-273.
Block, J C, K Haudidier, JL Paquin, J Miazga, and Y Levi. 1993. Bio film accumulation in drinking water distribution
systems. Biofouling. 6:333-343.
Brazos, BJ, JT O'Connor, and S Abcouwer. 1985. Kinetics of chlorine depletion and microbial growth in household
plumbing systems, pp. 239-274. AWWA Water Quality Tech. Conf. (Houston, 1985).
Breiman, RF. 1993. Modes of transmission in epidemic and nonepidemic Legionella infection: directions for further study.
In: Legionella: Current Status and Emerging Perspectives. Barbaree, JM, RF Breiman, and AP Dufour (eds.) Pp. 30-35.
American Society for Microbiology. Washington, DC.
Burghardt, MR, and H Bergmann. 1995. Acanthamoeba, naturally intracellularly infected with Pseudomonas aeruginosa, after
their isolation from a microbiologic ally contaminated drinking water system in a hospital.
Zentralbl. Hyg. Umweltmed. 196(6):532-44. German.
Burlingame, G.A., and C. Anselme. 1995. Distribution system tastes and odors. IN: Advances in Taste-and-Odor
Treatment and Control, pp. 281-320. AWWARF/Lyonnaise des Eaux publication. American Water Works Association
Research Foundation, Denver, CO.
Camper, AK. 1996. Factors limiting growth in distribution systems: laboratory and pilot-scale experiments. AWWARF.
Denver, CO.
Camper, AK, MW LeChevallier, SC Broadway, and GA McFeters. 1986. Bacteria associated with granular activated
carbon particles in drinking water. Appl. Environ. Microbiol. 52:434-438.
Carlson, S, U Hasselbarth, and R Langer. 1975. Water disinfection by means of chlorine: killing of aggregate bacteria.
Zentralbl. Bakteriol. 161:233-247. German.
Carter, JT, EW Rice, SG Buchberger, and Y Lee. 2000. Relationships between levels of heterotrophic bacteria and water
quality parameters in a drinking water distribution system. Water Res. 34(5): 1495-1502.
Centers for Disease Control. 1992. Primary amebic meningoencephalitisNorth Carolina, 1991. Morbidity and Mortality
Weekly Report. 41:437-440. (June 26, 1992).
Centers for Disease Control. 1985. Detection of elevated levels of coliform bacteria in a public water supply -
Connecticut. Morbidity and Mortality Weekly Report. 34:142-144. (March 15,1985).
Chao, WL, RS Chen, and CL Tai. 1988. Factors affecting the survival of pathogenic bacteria in subtropical river water.
ChineseJ. Microbiol. 21:85-92.
Characklis, WG, and KS Marshall, eds. 1990. Biofilms: a basis for an interdisciplinary approach, pp. 3-15. In: Biofilms.
Characklis, WG, and KS Marshall (eds.). J. Wiley and Sons. New York, NY.
Characklis, WG. 1988. Bacterial Regrowth in Distribution Systems. AWWARF. Denver, CO.
Characklis, WG. 1981. Fouling biofilm development: a process analysis. Biotechnol. Bioengin. 23:1923-1960.
Cirillo, JD, SL Cirillo, L Yan, LE Bermudez, S Falkow, and LS Tompkins. 1999. Intracellular growth in Acanthamoeba
castellanii affects monocyte entry mechanisms and enhances virulence of Legionella pneumophila. Infection and Immunity.
67:4427-4434.
38
-------
JUNE 17, 2002
Cirillo, JD, S Falkow, LS Tompkins, and LE Bermudez. 1997. Interaction of Mycobacterium avium with environmental
amoebae enhances virulence. Infec. and Immunity. 65:3759-3767.
Clark, RM, GS Rizzo, JA Belknap, and C Cochrane. 1999. Water quality and the replacement and repair of drinking water
infrastructure: the Washington, DC case study. J Water SRT - Aqua. 48(3):106-114.
Clark, RM, EE Geldreich, KR Fox, EW Rice, CH Johnson, JA Goodrich, JA Barnick, F Abdesaken, JE Hill, and FJ
Angulo. 1996. A waterborne Salmonella tjphimurium outbreak in Gideon, Missouri: results of a field investigation. Intl. J.
Environ. Health Res. 6:187-193.
Cohn, PD, M Cox, and PS Berger. 1999. Health and aesthetic aspects of water quality, Chapter 2. pp. 2.1-2.86. In: Water
Quality and Treatment (5th ed.). Letterman, RD (ed.). McGraw-Hill, Inc. New York, NY.
Colbourne, JS, MG Smith, SP Fisher-Hoch, and D Harper. 1984. Source of Legionella pneumophila infection in a hospital
hot water system: materials used in water fittings capable of supporting L. pneumophila growth, pp. 305-307. In: Legionella:
Second International Symposium, American Society for Microbiology. Thornsberry, C (eds.). Washington, DC. (Atlanta,
1983).
Costello,JJ. 1984. Postprecipitation in distribution systems. J. Amer. Water Works Assoc. 76(11):46-49.
Costerton, JW, and HM Lappin-Scott. 1989. Behavior of bacteria in biofilms. ASM News. 55:650-654.
Craun, GF. 1986. Statistics of waterborne disease outbreaks in the U.S. (1920-1980). pp. 73-160. In: Waterborne diseases
in the United States. Craun, GF (ed.). CRC Press. Boca Raton, FL.
Crespi, S, and J Ferra. 1997. Outbreak of legionellosis in a tourist complex in Lanzarote concomitant with a treatment of
the water system with megadoses of polyphosphates. Wat. Sci. Tech. 35(ll-12):307-309.
Crozes, GF, and RS Cushing. 2000. Evaluating biological regrowth in distribution systems. AWWARF. Denver, CO.
Dahab, MF, and BL Woodbury. 1998. Biological treatment options for nitrate removal from drinking water. AWWA
Inorganic Contam. Workshop. San Antonio, TX.
Davis, BD, R Dulbecco, HN Eisen, HS Ginsberg, and WB Wood, Jr. 1973. Microbiology. 2nd ed. Harper and Row.
Hagerstown, MD.
De Beer, D, R Srinivasan, and PS Stewart. 1994. Direct measurement of chlorine penetration into biofilms during
disinfection. Appl. Environ. Microbiol. 60:4339-4344.
DeRegnier, DP, L. Cole, D.G. Schupp, and S.L. Erlandsen. 1989. Viability of Giardia cysts suspended in lake, river and tap
water. Appl. Environ. Microbiol. 55:1223-1229.
DiLuzio, NR, and TJ Friedmann. 1973. Bacterial endotoxins in the environment. Nature. 244:49-51.
Doggett, MS. 2000. Characterization of fungal biofilms within a municipal water distribution system. Appl. Environ.
Microbiol. 66(3):1249-1251.
Donlan, RM, and WO Pipes. 1988. Selected drinking water characteristics and attached microbial population density. J.
Amer. Water Works Assoc. 80:70-76.
Drikas, M, CWK Chow, J House, and MD Burch. 2001. Using coagulation, flocculation, and settling to remove toxic
cyanobacteria. J. Amer. Water Works Assoc. 93:100-111.
39
-------
JUNE 17, 2002
Du Moulin, GC, I
-------
JUNE 17, 2002
Glaberman, S, J.E. Moore, C.J. Lowery, R.M. Chalmers, I. Sulaiman, K. Elwin, P.J. Rooney, B.C. Millar, J.S.G. Dooley, A.
Lai and L. Xiao. 2002. Three Drinking-Water-Associated Cryptosporidiosis Outbreaks, Northern Ireland. In: Centers for
Disease Control. Emerging Infectious Diseases. http://www.cdc.gov/ncidod/EID/vol8no6/01-0368.htm.
Gonzalez, JM, J Iriberri, L Egea, and I Barcina. 1992. Characterization of culturability, protistan grazing, and death of
enteric bacteria in aquatic ecosystems. Appl. Environ. Microbiol. 58:998-1004.
Gonzalez-de-la-Cuesta, N, M Arias-Fernandez, E Paniagua-Crespo, and M Marti-Mallen. 1987. Free-living amoebae in
swimming pool waters from Galicia (Spain). Rev. Iber. Parasitol. 47:207-210. Abstract in English only; text in Spanish.
Haas, CN. 1999. Benefits of using a disinfectant residual. J. Amer. Water Works Assoc. 91(l):65-69.
Haas, CN. 1990. Disinfection. IN: Water Quality and Treatment, 4th ed. (FW Pontius, ed.), Chapter 14. McGraw-Hill,
New York.
Haas, CN, MA Meyer, and MS Paller. 1983. The ecology of acid-fast organisms in water supply, treatment and
distribution systems. J. Amer. Water Works Assoc. 75:139-144.
Hardalo, C, and SC Edberg. 1997. Pseudomonas aeruginosa, assessment of risk from drinking water. Crit. Rev. Microbiol.
23:47-75.
Hazen, TC, and GA Toranzos. 1990. Tropical source water, pp. 32-53. In: Drinking Water Microbiology. McFeters, GA
(ed.). Springer-Verlag. New York, NY.
Herson, DS, DR Marshall, and HT Victoreen. 1984. Bacterial persistence in the distribution system. J. Amer. Water
Works Assoc. 76:309-322.
Highsmith, AK, TG Emori, SM Aguero, MS Favero, and JM Hughes. 1986. Heterotrophic bacteria isolated from hospital
water systems. International Symposium on Water-Related Health Issues, pp. 181-187. American Water Resources
Association.
Holden, B, M Greetham, BT Croll, and J Scutt. 1995. The effect of changing inter process and final disinfection reagents
on corrosion and biofilm growth in distribution pipes. Wat. Sci. Tech. 32(8):213-220.
Horan, T, D Culver, WJarvis, G Emori, S Banerjee, W Martone, and C Thornsberry. 1988. Pathogens causing
nosocomial infections: preliminary data from the National Nosocomial Infection Surveillance System. In: The antimicrobic
newsletter. Vol.5. No. 9. Sept. 1988. Pp. 65-68. Elsevier Science Publishing Co., Inc. New York, NY.
Howe, AD, S Forster, S Morton, R Marshall, K Osborn, P Wright, and PR Hunter. 2002. Cryptosporidium oocysts in a
water supply associated with a cryptosporidiosis outbreak. CDC. In: Emerging Infectious Diseases. Vol. 8. No. 6.
http://www.cdc.gov/ncidod/EID/vol8no6/01-0127.htm.
Hudson, LD, JW Hankins, and M Battaglia. 1983. Coliforms in a water distribution system: a remedial approach. J. Amer.
Water Works Assoc. 75:564-568.
Inderlied, CB, CA Kemper, and LEM Bermudez. 1993. The Mycobacterium avium complex. Clin. Microbiol. Rev. 6:266-
310.
Jacangelo, JG, VP Olivieri, and K Kawata. 1987. Mechanism of inactivation of microorganisms by combined chlorine.
AWWARF. Denver, CO.
Jakubowski, W, and TH Ericksen. 1980. Health significance of bacterial endotoxins in drinking water, pp. 245-260.
41
-------
JUNE 17, 2002
AWWA Water Qual. Technol. Conf. (Miami Beach, FL, 1980).
Jams, BB. 2002. Chemistry and toxicology of molds isolated from water-damaged buildings. Adv. Exp. Med. Biol.
504:43-52.
Jarvis, WR, et al. 1985. The epidemiology of nosocomial infections caused by Klebsiella pneumoniae. Infection Control.
6:68-74.
Jimenez, L, I Muniz, GA Toranzos, and TC Hazen. 1989. Survival and activity of Salmonella typhimurium and Escherichia coli
in tropical freshwater. J. Appl. Bacteriol. 67(l):61-69.
Jones, G, and K Sivonen. 1997. Fate of cyanotoxins persistence, removal, degradation and bioaccumulation. Draft
WHO Guidelines for Drinking Water Quality, Series on Protection and Control of Water Quality: Cyanobacteria, Their
Toxins, Water, and Flealth. World Flealth Organization.
Joret, JC, and M Prevost. 1992. (eds). Biodegradable organic matter in drinking water.
Jorgensen, JH, JC Lee, and FIR Pahren. 1976. Rapid detection of bacterial endotoxins in drinking water and renovated
wastewater. Appl. Environ. Microbiol. 32:347-351.
Keusch, GT and DWK Acheson. 1998. Enteric Bacteria: "Secretory" (Watery) Diarrhea, pp. 176-184. In: Mechanisms of
Microbial Disease (3rd ed.). Schaechter, M, NC Engleberg, BI Eisenstein, and G Medoff (eds.). Williams & Wilkins.
Baltimore, MD.
Kilvington, S. 1990. Activity of water biocide chemicals and contact lens disinfectants on pathogenic free-living amoebae.
Intl. Biodeterior. 26:127-138.
King, CH, EB Shotts, RE Wooley, and KG Porter. 1988. Survival of coliforms and bacterial pathogens within protozoa
during chlorination. Appl. Environ. Microbiol. 54:3023-3033.
Kirmeyer, GJ, M Friedman, KD Martel, D Howe, M LeChevallier, M Abbaszadegan, M Karim, J Funk and J Harbour.
2001. Pathogen intrusion into the distribution system. AWWARF. Denver, CO.
Kool, JL, JC Carpenter, and BS Fields. 1999. Effect of monochloramine disinfection of municipal drinking water on risk
of nosocomial Legionnaires' disease. Lancet 353:272-277.
Kroon,JR 1984. Water hammer: causes and effects. J. Amer. Water Works Assoc. 76:39.
Kubalek, I, S Komenda, and J Mysak. 1995. The spring-fall variations in the prevalence of environmental mycobacteria in
drinking water supply system. Cent. Eur. J. Public Health. 3:146-148.
Kwaik, YA, LY Gao, BJ Stone, C Venkataraman, and OS Harb. 1998. Invasion of protozoa by Legionellapneumophila and
its role in bacterial ecology and pathogenesis. Appl. Environ. Microbiol. 64:3127-3133.
LeChevallier, MW. 1999a. Biofilms in drinking water distribution systems: significance and control, Chapter 10. pp. 206-
219. In: Identifying future drinking water contaminants. National Academy Press. Washington, DC.
LeChevallier, MW. 1999b. The case for maintaining a disinfectant residual. J. Amer. Water Works Assoc. 91(l):86-94.
LeChevallier, MW, MR Karim, M Abbaszadegan, JE Funk, and M Friedman. 1999. Pathogen intrusion into potable water.
AWWA Water Qual. Tech. Conf. (Tampa, 1999).
42
-------
JUNE 17, 2002
LeChevallier, MW, CD Norton, A Camper, P Morin, B Ellis, W Jones, A Rompre, M Prevost, J Coallier, P Servais, D Holt,
A Delanoue, and J Colbourne. 1998. Microbial impact of biological filtration. AWWARF. Denver, CO.
LeChevallier, MW, NJ Shaw, and DB Smith. 1996. Factors limiting microbial growth in distribution systems: full-scale
experiments. AWWARF. Denver, CO.
LeChevallier, MW, CD Lowry, RG Lee, and DL Gibbon. 1993. Examining the relationship between iron corrosion and
the disinfection of biofilm bacteria disinfecting biofilms in a model distribution system. J. Amer. Water Works Assoc.
85:111-123.
LeChevallier, MW, WC Becker, P Schorr, and RG Lee. 1992. Evaluating the performance of biologically active rapid
filters. J. Amer. Water Works Assoc. 84:136-146.
LeChevallier, MW, W Schulz, and RG Lee. 1991. Bacterial nutrients in drinking water. Appl. Environ. Microbiol. 57:857-
862.
LeChevallier, MW. 1990. Coliform regrowth in drinking water: a review. J. Amer. Water Works Assoc. 82:74-86.
LeChevallier, MW, BF1 Olson, and GA McFeters. 1990a. Assessing and controlling bacterial regrowth in distribution
systems. AWWARF. Denver, CO.
LeChevallier, MW, CD Lowry, and RG Lee. 1990b. Disinfection of biofilms in a model distribution system. J. Amer.
Water Works Assoc. 82(7):87-99.
LeChevallier, MW. 1989a. Bacterial regrowth in drinking water. American Water Works Assoc. Research Foundation
report. Denver, CO. (September, 1989).
LeChevallier, MW. 1989b. Treatment to meet the microbiological MCL in the face of a coliform regrowth problem.
Amer. Water Works Assoc. Water Qual. Technol Conf. pp. 967-1008.
LeChevallier, MW, CD Cawthon, and RG Lee. 1988. Factors promoting survival of bacteria in chlorinated water supplies.
Appl. Environ. Microbiol. 54:649-654.
LeChevallier, MW, TM Babcock, and RG Lee. 1987. Examination and characterization of distribution system biofilms.
Appl. Environ. Microbiol. 53:2714-2724.
Levy, RV. 1990. Invertebrates and associated bacteria in drinking water distribution lines, pp. 224-248. In: Drinking
Water Microbiology. McFeters, GA. (ed.). Brock/Springer. New York, NY.
Levy, RV, FL Hunt, and RD Cheetham. 1986. Occurrence: public health significance of invertebrates in drinking water
systems. JAWWA. 78(9): 105-110.
Lippy, EC, and J Erb. 1976. Gastrointestinal illness at Sewickly, Pa. J. Amer. Water Works Assoc. 68:606-610.
Lowther, ED, and RH Moser. 1984. Detecting and eliminating coliform regrowth. pp. 323-336. AWWA Water Qual.
Tech. Conf. (Denver, 1984).
Lund, V, and K Ormerod. 1995. The influence of disinfection processes on biofilm formation in water distribution
systems. Wat. Res. 29:1013-1021.
Mackay, WG, LT Gribbon, MR Barer, and DC Reid. 1998. Biofilms in drinking water systems a possible reservoir for
Helicobacter pylori. Wat. Sci. Tech. 38(12):181-185.
43
-------
JUNE 17, 2002
Marshall, KC. 1992. Biofilms: an overview of bacterial adhesion, activity, and control at surfaces. ASM News. 58:202-
207.
Martin, RS, WH Gates, RS Tobin, D Grantham, P Wolfe, and P Forestall. 1982. Factors affecting coliform bacteria
growth in distribution systems. J. Amer. Water Works Assoc. 74:34-37.
Michel, R, H Burghardt, and H Bergmann. 1995. Acanthamoeba, naturally intracellularly infected with Pseudomonas aeruginosa,
after their isolation from a microbiologically contaminated drinking water system in a hospital. Zentralbl. Hyg.
Umweltmed. 196:532-544.
Miettinen, IT, T Vartiainen, and PJ Martikatnen. 1997. Phosphorus and bacterial growth in drinking water. Appl.
Environ. Microbiol. 63(8):3242-3245.
Mittelman, MW. 1991. Bacterial growth and biofouling control in purified water systems, pp. 133-154. In: Proceedings of
the International Workshop on Industrial Biofouling and Biocorrosion. (Stuttgart, Sept. 13-14,1990). Springer-Verlag.
Berlin.
Momba, MNB, and MA Binda. 2002. Combining chlorination and chloramination processes for the inhibition of biofilm
formation in drinking surface water system models. J. Appl. Microbiol. 92:641-648.
Moore, AC, BL Herwaldt, GF Craun, RL Calderon, AK Highsmith, and DD Juranek. 1993. Surveillance for waterborne-
disease outbreaks United States, 1991-1992. In: CDC Surveillance Summaries, Morbidity and Mortality Weekly Report.
42(SS-5):l-22.
Morin, P, A Camper, WJones, D Gatel, and JC Goldman. 1996. Colonization and disinfection of biofilms hosting
coliform-colonized carbon fines. Appl. Environ. Microbiol. 62:4428-4432.
Morton, LHG, and SB Surman. 1992. The role of biofilms in biodeterioration: a review. In: International symposium on
surface properties of biomaterials. West, R, and G Batts. (ed.). Manchester, UK.
Nagy, LA, and BH Olson. 1986. Occurrence and significance of bacteria, fungi, and yeasts associated with distribution
pipe surfaces, pp. 213-238. AWWA Water Qual. Tech. Confi (Houston, 1985).
Nagy, LA, and BH Olson. 1982. The occurrence of filamentous fungi in drinking water distribution systems. Can. J.
Microbiol. 28:667-671.
Neden, DG, RJ Jones, JR Smith, GJ Kirmeyer, and GW Foust. 1992. Comparing chlorination and chloramination for
controlling bacterial regrowth. J. Amer. Water Works Assoc. 84:80-88.
Niemi, RM, S Knuth, and K Lundstrom. 1982. Actinomycetes and fungi in surface waters and in potable water. Appl.
Environ. Microbiol. 43:378-388.
Norton, CD, and MW LeChevallier. 2000. A pilot study of bacteriological population changes through potable water
treatment and distribution. Appl. Environ. Microbiol. 66:268-276.
Norton C, M LeChevallier, J Falkinham, and M Williams. 2000. Recovery methods for M. avium complex in water and
biofilm samples. AWWA Water Qual. Tech. Conf. (Salt Lake City, 2000).
NRC (National Research Council). 1982. Drinking water and health, Chapter 4. (Vol. 4). National Academy Press.
Washington, DC.
O'Brien, RT, and JS Newman. 1977. Inactivation of polioviruses and coxsackieviruses in surface water. Appl. Environ.
44
-------
JUNE 17, 2002
Microbiol. 33:334-340.
O'Conner, JT, and SK Banerji. 1984. Biologically mediated corrosion and water quality deterioration in distribution
systems. U.S. Environmental Protection Agency. EPA-600/S2-84-056 (Project Summary). Cincinnati, OH.
O'Day, DK, R Weiss, S Chiavari, and D Blair. 1986. Water main evaluation for rehabilitation/replacement. AWWARF.
Denver, CO.
Olson, BH. 1982. Assessment and implications of bacterial regrowth in water distribution systems. U.S. Environmental
Protection Agency. EPA-600/S2-82-072 (Project Summary). Cincinnati, OH.
Opheim, DJ, J Growchowski, and D Smith. 1988. Isolation of coliforms from water main tubercles. N-6. Abst. Annual
Meeting. Amer. Soc. Microbiol, pp. 245.
Park, SR, WG Mackay, and DC Reid. 2001. Helicobacter sp. recovered from drinking water biofilm sampled from a water
distribution system. Wat. Res. 35(6):1624-1626.
Pelletier, PA, GC du Moulin, and KD Stottmeier. 1988. Mycobacteria in public water supplies: comparative resistance to
chlorine. Microbiol. Sciences. 5:147-148.
Pedersen, K. 1990. Biofilm development on stainless steel and PVC surfaces in drinking water. Wat. Res. 24:239-243.
Pier, GB. 1998. Pseudomonas aeruginosa. a key problem in cystic fibrosis. ASM News. 64:339-347.
Piriou, P, K Helmi, M Jousset, N Castel, E Guillot, and L Kiene. 2000. Impact of biofilm on C. parvum persistence in
distribution systems. International distribution system research symposium. Denver, CO.
Prince, DS. 1989. Infection with Mycobacterium avium complex in patients without predisposing conditions. New Engl. J.
Med. 321:863-868.
Quignon, F, L Kiene, Y Levi, M Sardin, and L Schwartzbrod. 1997. Virus behavior within a distribution system. Wat. Sci.
Tech. 35(11-12):311-318.
Ratnayake, N, and IN Jayatilake. 1999. Study of transport of contaminants in a pipe network using the model EPANET.
Wat. Sci. Tech. 40(2):115-120.
Rigal, S, and J Danjou. 1999. Tastes and odors in drinking water distribution systems related to the use of synthetic
materials. Wat. Sci. Tech. 40(6):203-208.
Rittman, BE, and VL Snoeyink. 1984. Achieving biologically stable drinking water. J. Amer. Water Works Assoc.
76(10):106-114.
Rivera, F, A Ortega, E Lopez-Ochoterena, and ME Paz. 1979. A quantitative morphological and ecological study of
protozoa polluting tap water in Mexico City. Trans. Amer. Micros. Soc. 98:465-469.
Roesch, SC and LYC Leong. 1983. Isolation and identification of Petriellidium boydii from a municipal water system.
83rd annual meeting of the American Society for Microbiology. New Orleans, LA.
Rogers, J, DI Norkett, CW Keevil, and G Hall. 1996. Persistence, survival and infectivity of Cryptosporidium parvum oocysts
in biofilms in water. Abstract B-465 (p. 235). Abstracts of 96th ASM General Meeting. American Society for
Microbiology. Washington, DC.
45
-------
JUNE 17, 2002
Rogers, J, AB Dowsett, PJ Dennis, JV Lee, and CW Keevil. 1994. Influence of plumbing materials on biofilm formation
and growth of Legionellapneumophila in potable water systems. Appl. Environ. Microbiol. 60:1842-1851.
Rosenzweig, WD, and WO Pipes. 1989. Presence of fungi in drinking water, Chapter 7. pp. 85-93. In: Biohazards of
Drinking Water Treatment. Larson, RA (ed.). Lewis Publishers. Ann Arbor, MI.
Rosenzweig, WD, and WO Pipes. 1988. Fungi from potable water: interaction with chlorine and engineering effects. Wat.
Sci. Tech. 20:153-159.
Rosenzweig, WD. 1987. Influence of phosphate corrosion control compounds on bacterial regrowth. EPA CR-811613-
01-0. U.S. Environmental Protection Agency. Cincinnati, OH.
Rosenzweig, WD, H Minnigh, and WO Pipes. 1986. Fungi in potable water distribution systems. J. Amer. Water Works
Assoc. 78:53-55.
Rusin, PA, JB Rose, CN Haas, and CP Gerba. 1997. Risk assessment of opportunistic bacterial pathogens in drinking
water. Rev. Environ. Contam. Toxicol. 152:57-83.
Samadpour, M. 2001. Molecular typing of Pseudomonas aeruginosa in distribution systems. American Water Works
Association Research Foundation report 90858 (Project 268). AWWARF, Denver.
Sartory, DP, and P Holmes. 1997. Chlorine sensitivity of environmental, distribution system, and biofilm coliforms. Wat.
Sci. Tech. 35:289-292.
Sawyer, TK. 1989. Free-living pathogenic and nonpathogenic amoebae in Maryland soils. Appl. Environ. Microbiol.
55:1074-1077.
Schaechter, M, NC Engelberg, BI Eisenstein and G Medoff. 1998. Mechanisms of microbial disease. 3rd Ed. Williams
and Wilkins. Baltimore, MD.
Schaule, G, and H-C Fleming. 1997. Pathogenic microorganisms in water system biofilm need biofilm sampling.
Ultrapure Water. Corrosioneering - microorganisms in water system biofilm. http:// www.clihouston.com/microrg.htm.
April, 1997.
Schellart, JA. 1986. Disinfection and bacterial regrowth: some experiences of the Amsterdam water works before and
after stopping the safety chlorination. Wat. Supply. 4:217-225.
Schock, MR. 1999. Internal corrosion and deposition control, Chapter 17. In: Water Quality and Treatment (5th ed.).
Letterman, RD (ed.). McGraw-Hill, Inc. New York, NY.
Schoenen, D, and A Wehse. 1988. Microbial colonization of water by the materials of pipes and hoses: changes in colony
counts. Zbl. Bakt. Hyg. B186:108-117.
Schoenen, D, and HF Scholer. 1985. Drinking water materials: field observations and methods of investigation. John
Wiley & Sons. New York, NY.
Schulze-Robbecke, R, B Tanning, and R Fischeder. 1992. Occurrence of mycobacteria in biofilm samples. Tuber. Lung
Dis. 73:141-144.
Seal, D, F Stapleton, and J Dart. 1992. Possible environmental sources of Acanthamoeba spp. in contact lens wearers. Br. J.
Ophthalmology. 76:424-427.
46
-------
JUNE 17, 2002
Seidler, R, T Evans, J Kaufman, C Warvick, and M LeChevallier. 1981. Limitations of standard coliform enumeration
techniques. J. Amer. Water Works Assoc. 73:538-542.
Sibille, I, T Sime-Ngando, L Mathieu, and JC Block. 1998. Protozoan bactenvory and Escherichia colz survival in drinking
water systems. Appl. Environ. Microbiol. 64:197-202.
Sibille, I. 1998. Biological stability in drinking water distribution systems: a review. L'Annee Biologique. 37(3):117-161
Sinclair, JL. 1990. Eukaryotic microorganisms in subsurface environments, pp. 3-39 - 3-51. In: Proceedings of the First
International Symposium on Microbiology of the Deep Subsurface. Fliermans, CB, and TC Hazen (eds.). WSRC
Information Services. Aiken, SC.
Singh, N, and VL Yu. 1994. Potable water and Mycobacterium avium complex in HIV patients: is prevention possible? The
Lancet. 343:1110-1111.
Smith, DB, AF Hess, and SA Hubbs. 1990. Survey of distribution system coliform occurrence in the United States, pp.
1103-1116. AWWA Water Qual. Tech. Conf. (San Diegp, 1990).
Smith, DB, AF Hess, and D Opheim. 1989. Control of distribution system coliform regrowth. pp. 1009-1029. AWWA
Water Qual. Tech. Conf. (Philadelphia, 1989).
Snead, MC, VP Olivieri, K Kawata, and CW Kruse. 1980. The effectiveness of chlorine residuals in inactivation of
bacteria and viruses introduced by post-treatment contamination. Wat. Res. 14:403-408.
Snoeyink, VL. 1990. Adsorption of organic compounds, pp. 781-875. In: Water Quality and Treatment. (4th ed.).
Pontius, FW (ed.). McGraw-Hill. New York, NY.
Sobsey, MD, PA Shields, FH Hauchman, RL Hazard, and CW Caton III. 1986. Survival and transport of hepatitis A virus
in soils, groundwater and wastewater. Wat. Sci. Tech. 10:97-106.
Solo-Gabriele, HM, MA Wolfert, TR Desmarais, and CJ Palmer. 2000. Sources of Escherichia coli'm a coastal subtropical
environment. Appl. Environ. Microbiol. 66:230-237.
Squier, C, VL Yu, and JE Stout. 2000. Waterborne nosocomial infections. Curr. Infect. Dis. Rep. 2:490-496.
States, SJ, RM Wadowsky, JM Kuchta, RS Wolford, LF Conley, and RB Yee. 1990. Legionella in drinking water, pp. 340-
367. In: Drinking Water Microbiology. McFeters, GA. (ed.). Springer-Verlag. New York, NY.
Steinert, M, K Birkness, E White, B Fields, and F Quinn. 1998. Mycobacterium avium bacilli grow saprozoically in coculture
with Acanthamoebapoljphaga and survive within cyst walls. Appl. Environ. Microbiol. 64:2256-2261.
Stewart, PS, T Griebe, R Srinivasan, C-I Chen, FP Yu, D DeBeer, and GA McFeters. 1994. Comparison of respiratory
activity and culturability during monochloramine disinfection of binary population biofilms. Appl. Environ. Microbiol.
60:1690-1692.
Stewart, MH, RL Wolfe, and EG Means. 1990. Assessment of the bacteriological activity of associated with granular
activated carbon treatment of drinking water. Appl. Environ. Microbiol. 56:3822-3829.
Storch, GA. 1998. Pneumococcus and Bacterial Pneumonia, pp. 153-160. In: Mechanisms of Microbial Disease (3rd ed.).
Schaechter, M, NC Engleberg, BI Eisenstein, and G Medoff (eds.). Williams & Wilkins. Baltimore, MD.
Stout, JE and VLYu. 1997. Legionellosis. New Eng. J. Med. 337(10): 682-687.
47
-------
JUNE 17, 2002
Swerdlow, DL, BA Woodruff, RC Brady, PM Griffin, S Tippen, HD Donnell Jr., E Geldreich, BJ Payne, A Meyer Jr., JG
Wells, KD Greene, M Bright, NH Bean, and PA Blake. 1992. A waterborne outbreak in Missouri of Escherichia coli
0157:H7 associated with bloody diarrhea and death. Ann. Internal Med. 117:812-819.
Tally, FP. 1998. Skin and Soft Tissue, pp. 573-581. In: Mechanisms of Microbial Disease (3rd ed.). Schaechter, M, NC
Engleberg, BI Eisenstein, and G Medoff (eds.). Williams & Wilkins. Baltimore, MD.
Thom, S, D Warhurst, and BS Drasar. 1992. Association of 1Vibrio cholerae with fresh water amoebae. J. Med. Microbiol.
36:303-306.
Thomas, FE, RT Jackson, MA Melly, and RH Alford. 1977. Sequential hospitalwide outbreaks of resistant Serratia and
Klebsiella infections. Arch. Intern Med. 137:581-584.
Tobin, J.O'H., CR Bartlett and SA Waitkins. 1981. Legionnaire's Disease: Further Evidence to Implicate Water Storage
and Distribution Systems as Sources. British Medical Journal. 282:573.
Toder, DS. 1998. Pseudomonas aeruginosa, ubiquitous pathogen, pp. 199-204. In: Mechanisms of Microbial Disease (3rd
ed.). Schaechter, M, NC Engleberg, BI Eisenstein, and G Medoff (eds.). Williams & Wilkins. Baltimore, MD.
Torno, MS, R Babapour, A Gurevitch, and MD Witt. 2000. Cutaneous acanthamoebiasis in AIDS. J. Amer. Acad.
Dermatol. 42:351-354.
Trussell, RR. 1999. Safeguarding distribution system integrity. J. Amer. Water Works Assoc. 91(l):46-54.
Tuovinen, OH, KS Button, A Vuorinen, L Carlson, D Mair, and LA Yut. 1980. Bacterial, chemical, and mineralogical
characteristics of tubercles in distribution pipelines. J. Amer. Water Works Assoc. 72:626-635.
Ueno, Y, S Nagata, T Tsutsumi, A Hasegawa, MF Watanabe, H-D Park, G-C Chen, G Chen, and SZ Yu. 1996. Detection
of microcystins, a blue-green algal hepatotoxin, in drinking water sampled in Haimen and Fusui, endemic areas of primary
liver cancer in China, by highly sensitive immunoassay. Carcinogenesis. 17:1317-1321.
U.S. EPA. U.S. Environmental Protection Agency. 2000. Microbial and Disinfectant Byproduct Federal Advisory
Committee Agreement in principle. FR December 29, 2000. 83015-83024.
http://www.epa.gov/safewater/mdbp/st2aip.html
U.S. EPA. U.S. Environmental Protection Agency. 1999. Draft - Cross-connection control: an issues paper.
U.S. EPA. U.S. Environmental Protection Agency. 1999b. Uncovered finished water reservoirs guidance manual. EPA
815-R-99-011. Washington, DC.
U.S. EPA. U.S. Environmental Protection Agency. 1997. Drinking water infrastructure needs survey: first report to
Congress. Office of Ground Water and Drinking Water. EPA 812-R-97-001. Washington, DC.
U.S. EPA. U.S. Environmental Protection Agency. 1995. Office of Inspector General survey report: survey report on the
cross-connections control program. E1HWG4-01-0091-5400070. Washington, DC.
U.S. EPA. United States Environmental Protection Agency. 1992a. A status report on planktonic cyanobacteria (blue-
green algae) and their toxins. EPA/600/R-92/079. Washington, DC.
U.S. EPA. U.S. Environmental Protection Agency. 1992b. Control of biofilm growth in drinking water distribution
systems. EPA/625/R-92/001. Washington, DC.
48
-------
JUNE 17, 2002
U.S. EPA. U.S. Environmental Protection Agency. 1989. Control of Legonella in plumbing systems, pp. 79-92. In:
Reviews of Environmental Contamination and Toxicology. (Vol. 107). Ware, GW. (ed.). Springer-Verlag. New York, NY.
U.S. EPA. U.S. Environmental Protection Agency. 1984. Drinking water criteria document on heterotrophic bacteria
(Draft 5). Washington, DC.
U.S. GAO. U.S. Government Accounting Office. 1993. Drinking water: key quality assurance program is flawed and
underfunded. GAO/RCED-93-97. Washington, DC.
van der Kooij, D. 1987. The effect of treatment on assimilable organic carbon in drinking water, pp. 317-328. In:
Proceed. Second National Conference on Drinking Water. Huck, PM, and P Toft (eds.). Edmonton, Canada. Pergamon
Press. London, UK.
van der Kooij, D. 2000. The unified biofilm approach: a framework for addressing biological phenomena in distribution
systems. International Distribution Research Symposium. Denver, CO.
van der Kooij, D, JHM van Lieverloo, J Schellart, and P Hiemstra. 1999. Maintaining quality without a disinfectant
residual. J. Amer. Water Works Assoc. 91(l):55-64.
van der Kooij, D, JHM van Lieverloo, J Schellart, and P Hiemstra. 1999. Distributing drinking water without disinfectant:
highest achievement or height of folly? Aqua 48(l):31-37.
van der Kooij, D, HS Vrouwenvelder, and HR Veenendaal. 1995. Kinetic aspects of biofilm formation on surfaces
exposed to drinking water. Wat. Sci. Tech. 32(8):61-65.
van der Wende, E, and WG Characklis. 1990. Biofilms in potable water distribution systems, pp. 249-268. In: Drinking
Water Microbiology. McFeters, GA (ed.). Springer-Verlag. New York, NY.
vanden Bossche, G. and Kreitemeyer. 1995. Detergent conditioning of biofilm samples: a most sensitive method for the
detection of enterovirus infectivity. Paper presented to the IAWQ health-related water microbiology symposium,
Budapest.
Victoreen, HT. 1980. The stimulation of coliform growth by hard and soft water main deposits. AWWA Water Qual.
Tech. Conf. (Miami Beach, FL, 1980).
Volk, CJ, E Dundore, J Schiermann, and M LeChevallier. 2000. Practical evaluation of iron corrosion control in a
drinking water distribution system. Wat. Res. 34(6):1967-1974.
Volk, CJ, and MW LeChevallier. 1999. Impacts of the reduction of nutrient levels on bacterial water quality in distribution
systems. Appl. Environ. Microbiol. 65(ll):4957-4966.
Volk, CJ, C Renner, C Robert, andJCJoret. 1994. Comparison of two techniques for measuring biodegradable dissolved
organic carbon in water. Environ. Technol. 15:545-556.
Wadowsky, RM, AJ West, JM Kuchta, SJ States, JN Dowling, and RB Yee. 1991. Multiplication of Legionella spp. in tap
water containing Hartmannella vermiformis. Appl. Environ. Microbiol. 57:1950-1955.
Wadowsky, RM, and RB Yee. 1983. Satellite growth of Legionella pneumophila with an environmental isolate of
Flavobacterium breve. Appl. Environ. Microbiol. 46:1447-1449.
Waksman, SA. 1941. Antagonistic relations of microorganisms. Bact. Rev. 5:231-291.
49
-------
JUNE 17, 2002
Walch, M. 1992. Corrosion, microbial, pp. 585-591. In: Encyclopedia of Microbiology. (Vol. 1). Lederberg,J. (ed.).
Academic Press. New York, NY.
Walker, JT, and M Morales. 1997. Evaluation of chlorine dioxide (C102) for the control of biofilms. Wat. Sci. Tech.
35(ll-12):319-323.
Walker, TS. 1998. Microbiology. W.B. Saunders and Co. Philadelphia, PA.
Walker, JT, DJ Bradshaw, AM Bennett, MR Fulford, MV Martin, and PD Marsh. 2000. Microbial bio film formation and
contamination of dental-unit water systems in general dental practice. Appl. Envir. Microbiol. 66: 3363-3367.
Watters, SK, and GA McFeters. 1990. Reactivation of injured bacteria, Chapter 3. pp. 119-141. In: Assessing and
Controlling Bacterial Regrowth in Distribution Systems. LeChevallier, MW, BH Olson, and GA McFeters (eds.). AWWA
andAWWARF. Denver, CO.
Weber, R, RT Bryan, DA Schwartz, and DL Owen. 1994. Human microsporidial infections. Clin. Microbiol. Rev.
7(4):426-461.
Wierenga, JT. 1985. Recovery of coliforms in the presence of a free chlorine residual. J. Amer. Water Works Assoc.
77:83-88.
Yoo, RS, WW Carmichael, RC Hoehn, and SE Hrudey. 1995. Cyanobacterial (blue-green algal) toxins: a resource guide.
AWWARF. Denver, CO.
Zacheus, OM, and PJ Martikainen. 1995. Occurrence of heterotrophic bacteria and fungi in cold and hot water
distribution systems using water of different quality. Can. J. Microbiol. 41:1088-1094.
50
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