United States
Environmental Protection
Agency
Office of
Research and Development
Washington, DC 20460
EPA/600/1 -90/005a
September 1990
&EPA Methods for the
Investigation and
Prevention of
Waterborne Disease
Outbreaks
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EPA/600/1 -90/OOSa
September 1990
Methods for the Investigation
and Prevention of
Waterborne Disease Outbreaks
Edited by:
Gunther F. Craun
Health Effects Research Laboratory
U.S. Environmental Protection Agency
Cincinnati, OH 45268
Printed on Recycled Paper
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We have learned that many of our water sources are inadequately
protected and treated to prevent the transmission of giardiasis. The
waterborne transmission of this disease was first reported in the
United States in 1965 and has increasingly been reported since 1971.
Giardia is now the most commonly identified pathogen in waterborne
outbreaks. Contaminated surface water is a significant source of
infection for giardiasis, and almost half of the outbreaks of waterborne
giardiasis have occurred in surface water systems where the only
treatment was disinfection. In addition to Giardia, a number of new
etiologic agents have been recognized in recent years and as laboratory
analysis and investigation procedures improve, more agents are likely
to be identified. Some of the newly recognized pathogens include
Cryptosporidium, Campylobacter, Yersinia, and Norwalk agent. Also
described is a chronic diarrhea for which an agent has yet to be
identified despite extensive laboratory analyses. In a recently
investigated waterborne outbreak in Missouri during December 1989-
January 1990, hemorrhagic E. coli serotype 0157:H7 was identified as
the etiologic agent.
Only a fraction of the waterborne outbreaks that occur in the
United States are recognized, investigated, and reported, and in only
half of the reported waterborne outbreaks was an etiologic agent
identified. We must improve the investigation and surveillance of
waterborne disease to identify the causes of these outbreaks and the
etiologic agents. Are outbreaks of acute gastroenteritis of undefined
etiology caused by unrecognized viral, protozoan, or bacterial agents or
by pathogens that could not be determined only because of delayed
investigation or inadequate laboratory analyses? It is necessary to
obtain more information on the pathogens causing these outbreaks in
order to evaluate the effectiveness of drinking water regulations,
surveillance activities, water treatment technologies and source water
protection policies. The identification of Giardia as an important
waterborne pathogen stimulated the research that has identified the
types of filtration, operating parameters, and disinfection levels
necessary for removal and inactivation of this pathogen. Water
treatment research is now being conducted to obtain similar
information for Cryptosporidium.
Regulations have been found insufficient to prevent the
waterborne transmission of infectious disease, as outbreaks have
occurred in systems that have not exceeded standards for coliforms and
turbidity. Data from outbreaks clearly show that disinfection has not
been effective as the only treatment for surface water sources;
filtration, including any required pretreatment, may be required in all
but exceptional situations to ensure the removal and inactivation of
waterborne pathogens, such as Giardia and Cryptosporidium. Criteria
IV
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have now been proposed for the filtration and disinfection of surface
water supplies, and regulations are being revised for coliforms
Outbreaks have also occurred in filtered water systems where facilities
have been improperly designed or have been operated in a poor or
simply casual manner, emphasizing the need to pay greater attention
to providing and maintaining effective, continuous water treatment.
All of the waterborne pathogens are transmitted through the fecal-
oral route of exposure, and human sewage is an important source of
water contamination. A review of the important waterborne diseases
since the turn of the century indicates that new strategies are needed
to prevent waterborne outbreaks. The important waterborne diseases
in the earlier part of this century were typhoid fever and cholera.
Although filtration played an important role in reducing the
waterborne transmission of these diseases in areas where water
sources were heavily contaminated, certain characteristics of these
pathogens allowed development of water sources with minimal
treatment. These diseases are transmitted exclusively among humans
and waterborne transmission could be prevented by protecting sources
from sewage contamination. Since these pathogens are quite
susceptible to disinfection, chlorination was found to be adequate
treatment where water sources could be protected from discharges of
human sewage. The more recently identified waterborne diseases such
as giardiasis and cryptospordiosis are caused by protozoa which are
much more resistant to disinfection. These protozoa can also cause
infection at a much lower dose than S. typhi and V. cholera. In addition
to being transmitted through human sewage, it has been found that
wild and domestic animals are important primary or intermediate
sources of infection for these protozoa and for other newly recognized
pathogens as well. Since it is impossible to exclude animals from
watersheds, greater emphasis must be placed on water treatment
barriers. Although the protection of raw water quality is still
important, it can only be considered one barrier to the transmission of
waterborne disease, especially with increasing evidence of the
importance of animals in the transmission of waterborne disease The
increasing potential for contamination of surface water sources
warrants the maintenance of additional barriers - filtration and
disinfection - to ensure adequate margins of safety.
Public health officials are committed to the multiple barrier
concept which provides for sewage treatment, protection of water
sources, and multiple water treatment processes/The validity of this
concept has evolved from water treatment experiences and if properly
applied can significantly reduce the risk of waterborne transmission of
infectious disease. Outbreak data, however, indicate that more
emphasis must be placed on the design and operation of filtration
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facilities if they are to be effective as part of the multiple barrier
concept. Many years ago I recommended the disinfection of untreated
ground water to reduce the occurrence of waterborne outbreaks. I am
still of the opinion that this recommendation is appropriate,
particularly for small systems where wells or springs may be subject to
intermittent contamination that cannot be readily determined or
prevented. This does not suggest a decreased emphasis on protection of
ground-water sources from contamination and proper construction
techniques for developing these sources, but instead would provide an
additional barrier for an increased level of protection.
Almost half of the reported outbreaks were caused by
contaminated, untreated, or inadequately treated ground water and
one fourth were caused by untreated or inadequately treated surface
water. Adequate, continuous disinfection of drinking water must
continue as the final barrier against waterborne disease, but the
question remains whether chlorine will continue to be the primary
disinfectant. The concerns over the human health effects that may be
associated with long-term exposures to chlorine or chlorinated by-
products may require changes in disinfection techniques or the use of
disinfectants other than chlorine. These concerns must be tempered
with considerations of the benefits provided by chlorination. As Dr.
Wolman has so aptly stated many times, the chlorination of water
supplies has saved numerous lives. Reducing the levels of chlorine
added to water or using alternate disinfectants may be appropriate to
reduce or prevent the formation of by-products, but the effects of these
actions on the occurrence of waterborne disease must be carefully
weighed. Additional research is required. Little is known about
potential by-products and adverse effects of the other disinfectants,
and unresolved questions remain about the significance of the
relatively low cancer risks reported from the epidemiologic studies of
chlorinated water systems. Although the risks of chlorination are not
as well recognized as the benefits, the prudent public health practice at
this time should be to minimize exposure to chlorine and chlorinated
by-products without significantly increasing infectious disease risks.
Chlorination may be the final barrier against transmission of
waterborne pathogens, but it must not be the only barrier. Properly
designed and operated filtration plants can make disinfection more
effective by removing turbidity and some substances tha^t exert
chlorine demand and by reducing microbiological contamination.
When disinfection is only one part of the multiple barrier approach and
not relied upon so heavily, lower concentrations of chlorine can be
used, thereby lowering the levels of chlorinated by-products produced.
G.F.C.
VI
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Contents
Preface •... , _ yj
Acknowledgments . ix
I. Introduction
Review of the Causes of Waterborne Disease 1
The Safe Drinking Water Act and the Regulation of
Microorganisms in Drinking Water 23
II. Disease Surveillance
Waterborne Disease Outbreak Surveillance:
Federal Requirements and Responsibilities 29
Surveillance for Waterborne Illness and
Disease Reporting: State and Local Responsibilities 39
HI. Investigation of Waterborne Disease Outbreaks
Differences between Outbreak Investigation
, and Research Epidemiology 45
Epiderniologic Principles for the Study of "' '
Waterborne Outbreaks 55
Data Analysis: Estimating Risk 65
Avoiding Bias: Systematic and Random Error ....... 75
Predicting Exposure to Water Contaminants in
Distribution Systems 83
Treatment Plant Evaluation
During a Waterborne Outbreak 127
IV. Engineering and Water Quality Concerns
Surface Water Source Protection 157
Principles of Water Filtration '.'.'.'.'.'.'.'. 169
Principles of Drinking Water Disinfection '
for Pathogen Control 135
Determination of C-T Values 193
Distribution Systems: Treated Water Quality Versus
Coliform Noncompliance Problems 207
vn
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V. Sampling and Analytical Methods
Environmental Sampling for Waterborne Pathogens:
Overview of Methods, Application Limitations,
and Data Interpretation 223
Bacteriologic Analysis of Clinical Specimens in Waterborne
Disease Outbreaks 235
Clinical Laboratory Diagnosis of Enteric Viral Diseases .. 243
Clinical Diagnosis of Enteric Protozoans 247
Analysis of Water Samples for Bacterial Pathogens 249
Virological Analysis of Environmental Water Samples ... 275
Field Method for Concentrating Viruses
from Water Samples 285
Analysis of Water Samples for Protozoans 297
Epilogue 317
Vlll
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Acknowledgments
This volume is a series of articles based on selected presentations
made at the U.S. Environmental Protection Agency (EPA) and
Association of State Drinking Water Administrators Workshop on
Methods for Investigation of Waterborne Disease Outbreaks held on
October 11-13, 1988, in Denver, Colorado. Articles were selected to
provide background on the etiologies and causes of previous outbreaks,
various aspects of epidemiologic methods, disease surveillance and
reporting, and laboratory analysis. This is intended to serve as a
handbook to assist in the investigation and prevention of waterborne
outbreaks. A number of previously published articles have been
reprinted in a separate volume to supplement information contained in
the handbook. These reprints provide examples of outbreak
investigations and surveillance activities and illustrate principles
discussed in the handbook. The contributions of the authors of both
handbook and journal articles are gratefully acknowledged.
Many of the authors of articles in the handbook provided
substantial peer review assistance. In addition, Dr. Dennis Juranek,
Centers for Disease Control, Atlanta, Georgia; Mr. Peter Karalekas,
Jr., Water Supply Section, EPA, Boston, Massachusetts; and Dr.
Vincent P. Olivieri, Johns Hopkins School of Hygiene, Baltimore,
Maryland, served as peer reviewers for the handbook.
Gunther Craun (EPA Health Effects Research Laboratory,
Cincinnati, Ohio) selected the subject matter, edited and compiled the
manuscripts, and provided substantive guidance for preparation of the
articles. Susan Richmond of Eastern Research Group, Inc., Arlington,
Massachusetts, provided editorial assistance. Carol Legg and Steve
Wilson (EPA Center for Research Information, Cincinnati, Ohio)
provided production assistance and figure preparation.
This report has been reviewed by the U.S. Environmental
Protection Agency and approved for publication.
IX
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I. Introduction
Review of the Causes of Waterborne Outbreaks
by: Gunther F. Craun
Coordinator, Environmental Epidemiology Program
U.S. Environmental Protection Agency
26 West Martin Luther King Drive
Cincinnati, Ohio 45268
(513) 569-7422
Introduction
On a national basis, the reporting of waterborne disease outbreaks
is voluntary. Information on waterborne outbreaks is obtained from
the scientific and medical literature and through the assistance of
State and local health officials, epidemiologists, and engineers. The
U.S. Environmental Protection Agency (EPA), Cincinnati, Ohio, and
the Centers for Disease Control (CDC), Atlanta, Georgia, have
cooperated in the reporting of waterborne outbreaks since 1971 and
have made this information available annually (1-14). Reviews of the
causes of waterborne outbreaks have been published for various time
periods (15). This analysis updates previous information and includes
outbreaks reported through 1985.
Reported waterborne outbreaks are those in which drinking water
was implicated epidemiologically as the vehicle of illness
transmission. In most of the outbreaks, water was found to be
bacteriologically or chemically contaminated, but in only a few
outbreaks was the etiologic agent isolated from drinking water. To be
considered an outbreak, at least two cases of a disease must be reported
so that a common source can be noted and investigated. Except in
unique circumstances, such as a case of chemical poisoning in which
the chemical is identified, a single case of illness cannot be recognized
as having been caused by drinking water Single cases of infantile
methemoglobinemia associated with high nitrate concentrations in
water have been included in this reporting system since 1979.
Outbreaks are primarily associated with water used or intended
for drinking or domestic purposes, but outbreaks associated with
ingestion of contaminated lakes, springs, creeks, and other sources of
nonpotable water are also included. Excluded from this analysis are
waterborne outbreaks occurring in cruise ships operating from U.S.
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ports and outbreaks among swimmers, bathers, and hot-tub users
where ingestion of contaminated water was not the route of exposure.
For analysis, water systems are classified as community or
noncommunity according to definitions contained in the Safe Drinking
Water Act (SDWA). Two additional classifications, individual and
recreational water systems, are not covered by SDWA. Individual
water systems are used by residents in areas without community
systems or persons traveling outside of populated areas who may
consume water from nonpotable sources (e.g., backpackers, campers).
Water systems involved in outbreaks where contaminated water was
ingested while swimming, diving, bathing, etc., are classified as
recreational.
I recently reviewed individual reports of waterborne outbreaks
reported during 1971-1985 to ensure the accuracy of information on
this subject. This review resulted in minor changes to data previously
reported (1-13), including changes to dates, water system
classifications, and deficiencies responsible for outbreaks. Because of
reporting delays, some outbreaks had been identified previously by
date of report rather than date of occurrence. Changes in water system
classifications were required so that definitions would correspond to
those in the Safe Drinking Water Act. Additional information obtained
on water system deficiencies also resulted in some changes. These
revisions were reported to CDC and have recently been published
(14,16). Because these data are maintained and shared by CDC and
EPA for periodic evaluations, it is important that the reported
information be complete and accurate.
During the period 1971-1985, 502 waterborne outbreaks and
111,228 cases of illness were reported to CDC and EPA.l Although
more outbreaks were reported during 1971-1985 than any previous 15-
year period since 1920 (Figure 1.1.1), the number of reported
waterborne outbreaks has declined since 1981. The number of cases
reported during the most recent 15-year period is exceeded only by the
number reported during the time periods 1936-1950 and 1920-1935
(Figure 1.1.2). The average number of cases per waterborne outbreak
has fluctuated from a high of 572 during 1926-1930 and 681 during
1961-1965 to less than 200 cases per outbreak during six time periods,
including 1981-1985. The incidence of waterborne disease in the U.S.
population has also declined from approximately 8 cases of illness per
100,000 person-years during 1920-1940 to 4 cases per 100,000 person-
years during 1971-1985 (15). With a few notable exceptions, reported
waterborne outbreaks now tend to occur in small water systems,
1 The 502 outbreaks and 111,228 cases in this analysis include the 485 outbreaks
(110,359 cases) reported in Reference-14 (also used by Tauxe in a subsequent article in
this volume) and 17 outbreaks (869 cases) related to ingestion of contaminated water
while swimming or other recreational activity. All of these outbreaks were reported in
previous CDC publications (1-14).
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affecting fewer people. The number of deaths associated with
waterborne outbreaks has decreased dramatically since 1920 (Table
1.1.1), and no deaths were reported during 1981-1985. Most previous
deaths were due to typhoid; however, the primary causes of death in
recent waterborne outbreaks have been shigellosis and chemical
poisoning.
196
202
176
138
Figure 1.1.1. Number of waterborne disease outbreaks, 1920-1985.
During 1920-1935 waterborne outbreaks were more frequently
reported in community systems, whereas in most succeeding years,
outbreaks were more frequently reported in noncommunity systems
(Figures 1.1.3 and 1.1.4). However, during the most recent 5-year
period, more outbreaks were reported in community systems than in
noncommunity systems. During 1971-1985, 42 percent of all reported
waterborne outbreaks and 68 percent of the cases of illness occurred in
community water systems; 43 percent of the outbreaks and 31 percent
of the cases occurred in noncommunity systems (Table 1.1.2).
It is difficult to determine whether the decline in the reported
number of waterborne outbreaks during 1981-1985 is due to the
occurrence of fewer outbreaks or less active surveillance and reporting
(Tauxe discusses this further in the article, "Waterborne Disease
Outbreaks Surveillance: Federal Requirements and Responsibilities,"
on p. 29). The most dramatic decrease occurred during 1984 and 1985
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Thousands
82.1
67.0
50.6
Figure 1.1.2. Cases of waterborne disease, 1920-1985.
Table 1.1.1. Illness and Deaths Associated with Waterborne
Outbreaks, 1920-1985
Deaths
Time Period
Cases per Outbreak
1920-25
1926-30
1931-35
1936-40
1941-45
1946-50
1951-55
1956-60
1961-65
1966-70
1971-75
1976-80
1981-85
254
572,
100
504
206
115
125
101
681
110
224
251
186
435
234
238
82
55
6
3
7
13
7
2
1
0
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105
84
83
58
63
40
26
24
20 26 31
25 =,30 35
36
40
41
45
46
50
51
55
56
60
61
65
66 .71
70 75
76
80
I
81
85
Figure 1.1.3. Number of waterborne disease outbreaks in community water
systems, 1920-1985.
when the number of outbreaks reported was 27 and 18, respectively,
compared with 48 reported in 1983. The decline in reported waterborne
outbreaks was observed primarily in noncommunity systems. The
number of outbreaks in noncommunity systems decreased from a total
of 92 reported during 1976-1980 to 55 during 1981-1985, whereas the
number of outbreaks reported in community systems was similar for
each of these periods (Figures 1.1.3 and 1.1.4). Annually, the number
of outbreaks reported in noncommunity systems decreased from 18 in
1981 to 8 in 1983, 5 in 1984, and 9 in 1985. In community systems, the
number of reported outbreaks increased from 14 in 1981 to 30 in 1983,
but then decreased to 12 in 1984 and 6 in 1985.
Outbreaks in community water systems, which number about 59,000
and serve about 180 million people, are probably the most likely to be
reported. Because less active surveillance and reporting should
continue to recognize and investigate potential outbreaks in at least
the larger community water systems, perhaps the decrease in
outbreaks observed in 1985 is a result of improvements in these water
systems. Additional data for 1986-1988 are required before a trend can
be established. Outbreaks in noncommunity systems, which number
about 240,000 and serve about 20 million people, primarily transients,
are the next most likely to be reported. However, an active
surveillance program would be required to continue to detect these
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92
82
68
13
Figure 1.1.4. Number of waterborne disease outbreaks in noncommunity water
systems, 1920-1985.
Table 1.1.2. Waterborne Outbreaks by Type
of Water System, 1971-1985
Community
Noncommunity
Individual
Recreational
Totals
211
216
59
16
502
75,754
33,958
719
797
111,228
outbreaks, especially those involving travelers. Therefore, the drastic
decline in outbreaks observed for noncommunity water systems since
1981 may be primarily the result of less active reporting and may not
reflect an actual decrease in outbreak occurrence. Outbreaks in
individual and recreational water systems are the least likely to be
reported, and their recognition is, therefore, most susceptible to
surveillance trends.
During the previous 15-year period, the number of reported
outbreaks increased most dramatically during 1979-1983; it could also
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be argued that this large increase was primarily the result of improved
surveillance and reporting, while the decrease in reported outbreaks
observed since 1983 reflected a less active, more typical surveillance
system. The numbers of reported outbreaks in 1984 and 1985 are
comparable to the number of outbreaks reported each year during the
period 1967-1975. An annual average of 22.5 outbreaks was reported
during 1984-1985 compared with 21.3 during 1967-1975.
Many factors influence the degree to which outbreaks are
recognized, investigated, and reported in any single year, including
interest in the problem and the capabilities for recognition and
investigation at the state and local level. For example, officials in
Pennsylvania, Colorado, and Washington generally increased their
waterborne disease surveillance activities during the 1970s; together
these states reported 31 percent of all waterborne outbreaks during the
period 1971-1980. During the previous 10-year period, only 8 percent
of all waterborne outbreaks were reported by these states (15).
Examination of the period 1981-1985 shows that these three states
reported 44 percent of all waterborne outbreaks during 1981-1983 but
only 20 percent of all waterborne outbreaks during 1984-1985. When
three states report almost one third to one half of all waterborne
outbreaks during a period of time, it is difficult not to attribute an
increased number of outbreaks primarily to improved surveillance
activities. It will be interesting to see if decreases continue to be
observed in both reported outbreaks and the total proportion of
outbreaks reported by these states.
A similar situation occurred during 1941-1950, when the large
number of reported waterborne outbreaks was attributed primarily to
an active surveillance program in a single state (15). New York
reported 46 percent of all waterborne outbreaks during 1941-1950
compared with 18 percent during the previous 21-year period and 7
percent during the subsequent 30-year period. One cannot be certain
whether the changes in the number of waterborne outbreaks reported
in these states over various time periods reflect an actual decrease or
increase in outbreaks or merely differences in surveillance and
reporting activities. However, the magnitude of the changes observed
in outbreaks reported by these few states suggests the importance of
surveillance differences during the various time periods, and one
should be cautious about ascribing increases or decreases in reported
waterborne outbreaks to the deterioration or improvement of water
systems.
While it is generally agreed that waterborne outbreak reporting is
incomplete, it is difficult to estimate the number of outbreaks that may
go undetected or unreported. One estimate, based on data collected
from 1946 to 1970, suggested that about one half of the waterborne
outbreaks in community water systems and about one third of those in
noncommunity systems are detected, investigated, and reported (17).
During a 3-year period of intensive waterborne disease surveillance in
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Colorado from 1980 to 1983, 18 waterborne outbreaks were
documented compared to 6 waterborne outbreaks during the previous
3-year period when the health department had a passive waterborne
outbreak surveillance program (18,19). A study of the occurrence of
foodborne outbreaks in Washington State indicated that prior to
initiation of improved surveillance and investigation only 1 foodborne
outbreak in 10 had been recognized and reported (20). Although this
study considered only the reporting of foodborne outbreaks, the results
may be applicable to waterborne outbreaks, especially those that occur
in noneommunity water systems because of the similarities in methods
of recognition and investigation of these kinds of outbreaks. This
estimate could represent the maximum number of unreported
waterborne outbreaks in small water systems.
In Colorado, increased disease surveillance and follow-up were
found to be more effective in detecting waterborne outbreaks than
increased surveillance and follow-up of water quality problems (18,19).
Activities important for effective surveillance included educational
outreach programs to local health agencies, physicians, and the public
and the designation of one individual to whom all water-related
complaints and health department inquiries were directed.
A waterborne outbreak (21) in a residential community of 6,500
persons in Florida in 1974 is a good example of how an active disease
surveillance program can help detect outbreaks. Initially, only 10
cases of shigellosis were recognized by health authorities, but further
investigation detected an additional 1,200 illnesses. If local health
authorities had not been conducting shigellosis surveillance, the
initial 10 cases might never have been recognized as an unusual
occurrence, and an outbreak of waterborne disease as large as this
might have gone undetected.
Causes of Outbreaks
The use of contaminated, untreated, or inadequately disinfected
ground water was responsible for 49 percent of the waterborne
outbreaks (Figure 1.1.5) and 47 percent of the cases of illness (Figure
1.1.6) reported during 1971-85. The use of contaminated, untreated, or
inadequately treated surface water was responsible for 24 percent of
the waterborne outbreaks and 32 percent of the cases. The remaining
outbreaks were caused by contamination of the distribution system
and miscellaneous deficiencies, which include use of water not
intended for consumption, contaminated ice, and ingestion of
contaminated water while swimming. Also included under
migcellanepus deficiencies were 15 outbreaks where data were
insufficient to determine the source of contamination.
When waterborne outbreaks.caused by Giardia are analyzed
separately, it becomes clear that water supply deficiencies responsible
for giardiasis are different from the deficiencies responsible for other
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Untreated or in-
adequately disin-
fected or filtered
surface water
24%
Miscellaneous
11%
Untreated or
inadequately
disinfected
groundwater
49%
Distribution or storage
deficiencies
16%
Figure 1.1.5. Causes of 502 waterborne disease outbreaks, 1971-1985.
diseases (Figure 1.1.7). Over 70 percent of the waterborne outbreaks of
giardiasis during 1971-1985 were attributed to no or inadequate
treatment of surface water; 20 percent of the giardiasis outbreaks were
attributed to use of contaminated, untreated ground water, inadequate
or interrupted disinfection of ground water, cross-connections, and
other distribution system deficiencies. For all other waterborne
diseases reported during 1971-1985, only 14 percent of the outbreaks
were attributed to no or inadequate treatment of surface water; the
majority (74 percent) were attributed to contaminated, untreated, and
inadequately disinfected ground water, cross-connections, and other
distribution deficiencies.
Causes of Outbreaks in Ground-Water Systems
Contaminated ground water has consistently been responsible for
more waterborne outbreaks than contaminated surface water. In each
time period since 1930, between 42 and 56 percent of all outbreaks
were caused by use of contaminated ground water; 14 to 27 percent of
all outbreaks were caused by contaminated surface water.
During 1971-1985, the use of untreated or inadequately treated
ground water resulted in 243 outbreaks and 52,091 cases of illness. A-
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Untreated
quately diiinfeeted or til
47%
figure 1,1,8, Causes of 111,881 waterberne disease outbreaks, 1971-1BSS.
seasonal distribution of outbreaks in these systems is apparent; 71
'
As a cause of outbreaks, use of contaminated, untreated ground
water has decreased in important while inadequate or interrupted
disinfection of ground water has increased (Figure 1,1.1), For example,
use of contaminated, untreated ground water was responsible for 53
percent of all waterborne outbreaks during 1 S314940 but only 29
percent during 19814885, Inadequate or interrupted disinfection
caused 21 percent of all outbreaks during 19714980 compared with 2
percent during 19204930, The increased use of disinfection, especially
In situations where little or no effort has been made to reduce ground--
water contamination, and the lack of attention to maintaining
adequate, continuous disinfection have likely been responsible for the
increased outbreaks in disinfected ground=water systems, Most of the
outbreaks in disinfected ground waters were the result of Improper
chlorlnatlon; however, several outbreaks during 19714985 resulted
f>-om Inadequate or Interrupted iodine disinfection in noneommunlty
systems,
Some 23 percent of the outbreaks in untreated ground*water
systems during 11714985 were caused by the overflow or seepage of
sewage into wells and springs; II percent were caused by surface
runoff or flooding from contaminated streams; 8 percent were caused
10
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A - Bislnfgetien • enly nurfaee water
of ground water
F = Gfeii=eerm§etien§
Figure 1,1.7, Major water tupply deflolcnoict reipontlble for waterborne dlteate
by chemical contamination; and 5 percent were caused by
contamination through limestone or fissured, rock. Improper system
construction was identified in 3 percent of the outbreaks, and there
were insufficient data to classify the remaining outbreaks in untreated
ground-water systems. Contaminants detected in ground waters in
cases of chemical poisoning were nitrate, benzene, phenol, selenium,
trichloroethylene, polychlorinated biphenyls, oil, and gasoline.
Causes of Outbreaks in Surface Water Systems
One hundred twenty-three waterborne outbreaks during 1971-
1985 were attributed to the use of contaminated, untreated, or
inadequately treated surface water. A seasonal distribution of
outbreaks in these systems is also apparent; 60 percent occurred
11
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53%
Inadequate or
interrupted disinfection
Contaminated,
untreated
i
2%
r~
t2%
4v
s-"\"
? *
3%
r~
:
>
5%
3UYo
'
| -
OU /o
5%
\'
45%
6%
/
32%
21%
"
29%
13%
'j
20
30
31
40
41
50
51
60
61
70
71
80
81
85
Figure 1.1.8. Waterborne outbreaks in untreated and disinfected only ground-
water systems (wells and springs), 1920-1985.
during May through September. The numbers of outbreaks occurring
in untreated, disinfected only, and filtered surface water systems were
also examined separately to determine the relative importance of each
of these deficiencies over the years. The increased use of disinfection
and filtration for surface waters since 1920 is the likely explanation for
the decreased importance of contaminated, untreated surface water in
the transmission of waterborne disease (Figure 1.1.9). For example, 19
percent of waterborne outbreaks during 1920-1930 were attributed to
untreated surface water compared with 3 percent during 1981-1985.
Until 1971, more outbreaks were attributed to the use of
contaminated, untreated surface water than to deficiencies in the
treatment of surface water. Now, outbreaks in surface water systems
occur primarily because of inadequate or interrupted disinfection,
especially in systems which provide disinfection as the only treatment
(Figure 1.1.9). Thirteen percent and 14 percent of waterborne
12
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Percent of All Outbreaks
I I Inadequate or
Interrupted Disinfection
19%
Contaminated,
Untreated
10%
13%
14%
10%
4%
9% 9% 9% ,
7%
3%
3%
8%
3%
'20
30
31
40
41
50
51
60
61
70
71
80
81
85
Figure 1.1.9. Waterborne outbreaks in untreated and disinfected only surface
water systems, 1920-1985.
outbreaks during 1971-1980 and 1981-1985, respectively, were
attributed to inadequate or interrupted disinfection of disinfected only
surface water; previously only 3 to 7 percent of waterborne outbreaks
had been attributed to this deficiency since 1931. In recent years, an
increased number of waterborne outbreaks of giardiasis has occurred
in unfiltered surface water sources in which disinfection was
inadequate or interrupted. Disinfection with chlorine can inactivate
pathogens including Giardia cysts only if several conditions are
maintained:
• The water consistently has low concentrations of substances
which can cause turbidity, create a chlorine demand, or
interfere with the disinfection process.
• A sufficient concentration of disinfectant and contact time, as
determined from laboratory or field studies, are provided
according to various operating parameters, especially water
temperature and pH.
• The chlorine residual is not depleted for any reason.
13
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• Heavy or overwhelming contamination does not occur either
briefly or intermittently.
In the giardiasis outbreaks reported in unfiltered systems having
inadequate disinfection, one or more of these conditions were not met.
In most outbreaks, the concentration of chlorine and/or contact time
provided was insufficient; in all outbreaks disinfection was ineffective
for the inactivation of pathogens in the raw water. In several
outbreaks, it was noted that during the period of suspected
contamination, chlorine residual levels were reduced to nil. In
outbreaks where disinfection was interrupted, chlorine equipment
failed or the chlorine supply was exhausted.
In filtered surface water systems, the number of outbreaks
attributed to inadequate or interrupted disinfection has remained
relatively stable over the years, causing 1 to 2 percent of waterborne
outbreaks. During 1981-85, a dramatic increase in outbreaks was
noted for filtered surface water supplies; 8 percent of all waterborne
outbreaks during that period were attributed to inadequate control of
filtration or pretreatment. This increased number of outbreaks in
filtered water systems serves as a reminder that treatment not only
must be appropriately designed to deal with varying source water
quality but also must be properly operated and maintained to provide
continuous and effective removal and inactivation of pathogens.
Data have recently been made available on the number of
communities and populations served by filtered and unfiltered surface
water sources (16). These data were combined with the data on
waterborne outbreaks to obtain outbreak and disease rates for various
water sources and treatment. Comparison of these rates can provide an
indication of the susceptibility of water systems. Outbreak and disease
rates were calculated using the waterborne outbreaks reported during
1971-1985 in community water systems with surface water sources
(Figures 1.1.10 and 1.1.11). Outbreaks caused by contamination of the
distribution system and miscellaneous deficiencies in surface water
systems were excluded, since their occurrence is unrelated to source
water contamination and treatment. The reported chemical poisonings
caused by deficiencies in chemical feed were also excluded. Because the
reporting of waterborne outbreaks is voluntary, the rates should be
used primarily for comparison purposes. For identifying water systems
with the greatest potential for failure, the outbreak rate (Figure
1.1.10) is less variable and represents a more stable rate for
comparison purposes. However, the disease rate (Figure 1.1.11) may
have more public health significance because the prevention of large
outbreaks can result in a dramatically lower disease rate.
Approximately 4,611 community water systems serve filtered and
disinfected surface water to a population of some 133,560,000. These
systems experienced 23 outbreaks and 9,450 cases of illness for a rate
14
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Outbreaks per 1000 Systems
40.5
32.5
Untreated
Disinfected
Only
Filtered and
Disinfected
Figure 1.1.10. Waterborne outbreak rates attributed to source contamination and
treatment inadequacies in community systems using surface water
of 5.0 waterborne outbreaks per 1,000 facilities served during 1971-
1985 and 4.7 cases of waterborne illness per million person-years.
Community water systems that provide disinfection as the only
treatment for surface water experienced an outbreak rate of 40.5/1,000
facilities (8 times the rate for filtered surface water) and a disease rate
of 66.3/million person-years (14 times the rate for filtered surface
water). The rates for community water systems using untreated
surface water are also much higher than the rates for communities
with filtered surface water; however, these rates are based on only five
outbreaks and 612 cases of illness in 154 water systems servine
110,000 people.
Etiologic Agents
An etiologic agent was determined in only 50 percent of all
waterborne outbreaks during 1971-85; the remaining outbreaks were
categorized as acute gastrointestinal illness of unidentified etiology
(Figure 1.1.12). In ground-water systems, an agent was identified in
only 38 percent of the outbreaks; in surface water systems, an agent
was identified in 62 percent of the outbreaks. In many of the outbreaks
of undetermined etiology, the search for an etiologic agent included
15
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Illness per Million Person Years
370.9
66.3
4.7
Untreated
Disinfected
Only
Filtered and
Disinfected
Figure 1.1.11.Waterborne disease rates attributed to source contamination and
treatment inadequacies in community systems using surface water
sources, 1971-1985.
only stool cultures for Salmonella or Skigella; in others, the
investigation and collection of clinical specimens were delayed, and
the etiologic agent could not be identified because samples were not
collected in a timely manner. It is suspected that the gastroenteritis
outbreaks represent a combination of viral, bacterial, and parasitic
etiologies.
In recent years waterborne viral gastroenteritis caused by
Norwalk agent or rotavirus, bacterial gastroenteritis caused by
Campylobacter and Yersinia, giardiasis, and cryptosporidiosis have
been reported in both surface and ground-water systems. During a
waterborne outbreak, each of these agents should be considered,
depending upon the symptomatology, and appropriate clinical testing
should be performed to identify the etiologic agent. Only by identifying
the etiologic agents of waterborne outbreaks can researchers ascertain
whether current surveillance activities, regulations, and water
treatment techniques are adequate to prevent the waterborne
transmission of specific infectious diseases.
Although each of these etiologic agents could be transmitted
through either contaminated ground-water or surface water systems,
Giardia lamblia was found to be the most commonly identified
16
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Undefined AGI
Giardiasis
Chemical poisoning
Shigellosis
Hepatitis A
Viral AGI
Campylobacterosis
All Others
50%
2%
4%
Figure 1.1.12. Etiology of waterborne outbreaks, 1971-1985.
pathogen responsible for waterborne outbreaks in surface water
systems. Shigella and hepatitis A virus were the two most commonly
identified pathogens in ground-water outbreaks. Almost all of the
outbreaks of hepatitis in ground-water systems were caused by sewage
contamination of untreated wells and springs, while shigellosis
outbreaks m ground-water systems were caused both by sewage
contamination of untreated water sources and interrupted or
inadequate disinfection of contaminated wells and springs. Outbreaks
of chemical poisoning occurred when ground-water sources were
contaminated and through cross connections and inadequate control of
chemical feed in surface water systems. Of the 50 reported outbreaks of
chemical poisonings, 13 resulted from contamination of wells and
springs; 12 from cross connections; 12 from corrosion or other
distribution deficiencies; and 8 from deficiencies in the feeding of
chemicals during treatment.
Giardiasis was the disease most often transmitted because of
ineffective filtration or pretreatment of surface water and inadequate
disinfection of surface water when disinfection was the only treatment.
These two deficiencies were responsible for 56 percent of all cases of
illness reported in surface water systems. While a significant number
of giardiasis outbreaks occurred in untreated surface water systems,
tjie systems affected were small and only 322 cases of giardiasis were
reported. Although contamination of source waters by human sewage
has caused waterborne outbreaks of giardiasis, numerous wild and
domestic animals, especially beavers, have been implicated as primary
17
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or intermediary sources of infection and also important in wattrborne
transmission (22,23). Because animals cannot be completely excluded
from the watershed, all surface water supplies are at risk of
contamination with Giardia. Restricting discharges of human sewage
on the watershed may be insufficient protection for surface water
systems that only disinfect, and additional treatment barriers may be
needed to prevent the waterborne transmission of giardiasis. Outbreak
rates have shown the importance of surface water filtration, but
greater emphasis is also needed on the design, installation, operation,
and maintenance of filtration facilities. Giardiasis outbreaks have also
occurred in ground-water systems where human sewage or
contaminated surface water entered improperly constructed or located
wells Six giardiasis outbreaks were reported in untreated ground-
water systems and four were reported in disinfected ground-water
systems.
Only one outbreak of cryptosporidiosis was reported during 1971-
1986. This outbreak occurred in a ground-water system, but
Cryptosporidium could be an important contaminant of surface water
systems as well. The occurrence of the protozoan is widespread, and
animals, as well as humans, are involved in the waterborne
transmission of this organism (24,25). Little is known about the
inactivation of Cryptosporidium oocysts by chemical disinfectants, but
the available data suggest the organism may be more resistant to
chlorination than Giardia (26). Thus, it may be necessary to remove
the oocysts from water by filtration rather than depend upon
inactiv&tion by disinfection.
A second large waterborne outbreak of several thousand cases of
cryptosporidiosis occurred in a filtered surface water system in
Carrollton, Georgia, during January and February 1987. Water
treatment in this system included coagulant feed, mechanical rapid
mix flocculation, sedimentation, and filtration through anthracite
sand filters (2 gallons per minute per square foot) (27). Turbidity was
not routinely monitored at each filter effluent, but was measured
during the outbreak investigation. Analysis of the filtered water
turbidity of each niter's effluent suggested that the practice of stopping
and later restarting the flow of water through some filters without
backwashing could result in passage of alum floe and contaminants
through the filters into the finished water. Earlier EPA research
indicated that turbidity breakthrough, or passage of floe through the
filter, could be accompanied by contaminants such as Giardia cysts.
Cryptosporidium may have entered the system because filters were not
backwashed prior to placing them in service.
Summary
Waterborne outbreaks continue to occur, and the largest number
in any 15-year period since 1920 was reported during 1971-1985.
However, the reporting of waterborne outbreaks has declined since
18
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1080, Since outbreak! now oeeur primarily in small community and
K0lT^nifcy,flf«irA8,y8tim8J the inci{tenee of waterborne disease has
declined iinet 1020. Almost three fourths of the outbreaks in the past
1.5 years were eaustd by contaminated, untreated, or inadequately
treated ground water and surface water, «««»equi»eiy
t,. bwd in8ufflcisnt to prevent the waterborne
transmission of infectious disease, as waterborne outbreaks have
occurred in systems which had not exceeded current EPA regulations
for eoliforms and turbidity. In addition to the protection of source
water quality, adequate, continuous disinfection is a necessary final
barrier to prevent the transmission of waterborne disease. Disinfection
has been inadequate as the only treatment for surface water. In all but
exceptional iituations, effective filtration must also be provided
ineluding any required pretreatment) to ensure the removal and
inaetivation of waterborne pathogens, especially Oiardia and
gp^J"n
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References
1. Foodborne Outbreaks Annual Summary 1972, Centers for Disease
Control, Department of Health, Education and Welfare,
Publication No. (CDO74-8185,1973.
2. Foodborne and Waterborne Disease Outbreaks Annual Summary
1973 Centers for Disease Control, Department of Health,
Education and Welfare, Publication No. (CDC)75-8185,1974.
3. Foodborne and Waterborne Disease Outbreaks Annual Summary
1974 Centers for Disease Control, Department of Health,
Education and Welfare, Publication No. (CDC)76-8185,1976.
4. Foodborne and Waterborne Disease Outbreaks Annual Summary
1975, Centers for Disease Control, Department of Health,
Education and Welfare, Publication No. (CDO76-8185,1976.
5. Foodborne and Waterborne Disease Outbreaks Annual Summary
1976 Centers for Disease Control, Department of Health,
Education and Welfare, Publication No. (CDO78-8185,1977.
6. Foodborne and Waterborne Disease Surveillance Annual
Summary 1977, Centers for Disease Control, Department of
Health, Education and Welfare, U.S. Government Printing Office
640-010-3610,1979.
7 Water-Related Disease Outbreaks Surveillance Annual Summary
1978 Centers for Disease Control, Health and Human Services
Publication No. (CDQ80-8385,1980.
8. Water-Related Disease Outbreaks Surveillance Annual Summary
1979, Centers for Disease Control, Health and Human Services
Publication No. (CDC)81-8385,1981.
9 Water-Related Disease Outbreaks Surveillance Annual Summary
1980, Centers for Disease Control, Health and Human Services
Publication No. (CDC)82-8385,1982.
10 Water-Related Disease Outbreaks Surveillance Annual, Summary
1981 Centers for Disease Control, Health and Human Services
Publication No. (CDQ82-8385,1982.
11. Water-Related Disease Outbreaks Surveillance Annual Summary
1982, Centers for Disease Control, Health and Human Services
Publication No. (CDO83-8385,1983.
12. Water-Related Disease Outbreaks Surveillance.Annual Summary
1983 Centers for Disease Control, Health and Human Services
Publication No. (CDQ84-8385,1984.
13. Water-Related Disease Outbreaks Surveillance Annual Summary
1984 Centers for Disease Control, Health and Human Services
Publication No. (CDO99-2510,1985.
20
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14. CDC Surveillance Summaries, Morbidity and Mortality Weekly
Report, Health and Human Services Publication No
8017,1988. .
15'
/'*1' Statistics of waterborne outbreaks in the U.S. (1920-
Craun (e
-------
28. Drinking water: National primary drinking water regulations;
filtration, disinfection; turbidity, Giardia lamblia, viruses,
Legionella, and heterotrophic bacteria. Proposed rule. 40 CFR
parts 141 and 142, Fed. Reg. 52(212):42718,1987.
29. National primary drinking water regulations: total coliforms.
Proposed Rule. 40 CPR parts 141 and 142. Fed. Reg.
52(212):42224,1987.
30. Logsdon, G.S. and Hoff, J.C. Barriers to the transmission of
waterborne disease. In: G.P. Craun (ed.), Waterborne Diseases in
the United States. CRC Press, Inc., Boca Raton, FL, 1986, p. 255.
31. Logsdon, G.S., Thurman, V.C., Frindt, E.S., and Stoecker, J.G.
Evaluating sedimentation and various filter media for removal ol
Giardia cysts. J. AWWA. 77:61,1985.
32. Logsdon, G.S. and Rice, E.W. Evaluation of sedimentation and
filtration for microorganism removal. In; Proceedings Annual
Conference, American Water Works Association, Denver, CO,
June 23-27,1985, p. 1177,
22
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The Safe Drinking Water Act and the Regulation of
Microorganisms in Drinking Water
by: Paul S. Berger and Stig Regli
Office of Drinking Water
U.S. Environmental Protection Agency
Washington, DC 20460
(202) 382-3039
(202) 382-7379
Requirements of the Safe Drinking Water Act
The Safe Drinking Water Act (SDWA) requires the U.S.
Environmental Protection Agency (EPA) to publish National Primary
Drinking Water Regulations (NPDWRs) which set either a maximum
contaminant level (MCL) or a water treatment technique requirement
for contaminants that may have an adverse health effect. The SDWA
also requires EPA to specify monitoring and reporting requirements
for each regulated contaminant.
The SDWA requires EPA to set both a maximum contaminant
level goal (MCLG) and a NPDWR for each contaminant. The MCLG is
a nonenforceable health goal that is set at a level at which no known or
anticipated adverse health effects occur and which allows an adequate
margin of safety. In contrast, the MCL, if set, is an enforceable
standard which is set as close to the MCLG as is feasible, taking cost
and other factors into account.
According to the SDWA, EPA is to set an MCL for a contaminant if
it is economically and technologically feasible to ascertain the level of
the contaminant. If not, EPA is to set a water treatment requirement.
Some of the factors EPA considers in making this judgment include
method reliability, laboratory experience with available methods,
method detection limits, the ability to relate the measurement to the
determination of health risk significance, and cost of analysis.
EPA currently regulates two indicators of microbiological
drinking water quality: total coliforms and turbidity. The 1986
Amendments to the SDWA require EPA to regulate Giardia lamblia,
viruses, Legionella, and heterotrophic bacteria, as well as total
coliforms and turbidity. They also require EPA to publish regulations
specifying criteria under which filtration is required as a treatment
technique for public water systems supplied by surface water sources.
In addition, the 1986 amendments require the Agency to publish
23
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regulations requiring disinfection as a treatment technique for all
public water systems (except those which meet certain criteria
allowing them to avoid this requirement).
EPA will comply with the above requirements of the 1986
amendments by publishing three rulemaking packages: the revised
total coliform rule, the surface water treatment requirements (SWTR),
and the ground-water disinfection rule. As of the date of publication of
these proceedings, EPA has published the revised total coliform rule
and the SWTR. The Agency believes that, collectively, the
requirements in these three packages will minimize occurrence of
waterborne disease in the United States.
Surface Water Treatment Requirements (SWTR)
(54 FR 27486-27541, June 29, 1989)
Under the SWTR, all public water systems using surface water or
ground water under the direct influence of surface water must provide
disinfection. Systems must also filter the water unless they meet
specific conditions relating to source water quality. The SWTR will
also regulate Giardia lamblia, turbidity, viruses, heterotrophic
bacteria, and Legionella in surface waters.
The SWTR will regulate Giardia lamblia, turbidity, viruses,
heterotrophic bacteria, and Legionella by establishing treatment
technique requirements rather than maximum contaminant levels.
Under the rule, all systems using surface water must achieve at least
99.9 percent removal/inactivation of Giardia lamblia cysts and 99.99
percent removal/inactivation of viruses.
Criteria Systems Must Meet to Avoid Filtration
To avoid filtration, systems must meet the following criteria:
• Measure turbidity levels every 4 hours. The turbidity level
must not exceed 5 NTU.
• Test the source water for either total coliforms or fecal
coliforms at least once per week to as much as to 5 per
week (depending on population served). Also, during days
that the turbidity exceeds 1 NTU in the raw water, at least one
total or fecal coliform measurement must be made. Total
coliforms must not exceed 100/100 mL or fecal coliforms must
not exceed 20/100 mL in more than 10 percent of the
measurements for the previous 6 months, calculated each
month.
• Maintain a disinfectant residual concentration of at least
0.2 mg/L in water entering the distribution system, as
demonstrated by continuous monitoring. Systems serving
3,300 persons or less can take one to four grab samples
(depending on population served) instead of continuous
24
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monitoring. Provide disinfection to ensure that the 99.9
percent and 99.99 percent removal/inactivation of Giardia
lamblia and viruses, respectively, are being achieved as
demonstrated by meeting C-T values in the rule (C-T is the
product of residual concentration in mg/L and disinfectant
contact time in minutes, measured at peak hourly flow).
• Maintain a detectable disinfectant residual in the
distribution system. Disinfectant residuals cannot be
undetectable in more than 5 percent of the samples each
month, for 2 consecutive months. Samples must be taken at the
same frequency as total coliform samples. A system may
measure heterotrophic bacteria (HPC) in lieu of disinfectant
residual. If the HPC is less than 500 colonies/mL, the site is
considered to be equivalent to having a detectable disinfectant
residual.
• Maintain a watershed control program that will minimize
the potential for contamination by human enteric viruses
and Giardia lamblia cysts.
• Have had no waterborne disease outbreaks in its present
configuration. If an outbreak occurs, system must either
filter or correct the deficiency to the state's satisfaction to
prevent another such outbreak.
• Be in compliance with the MCL for total coliforms during
at least 11 of 12 consecutive months, unless the state
determines the violations are not due to a treatment
deficiency.
• Be in compliance with the MCL for total trihalomethanes.
Currently, this requirement only pertains to systems serving
greater than 10,000 people. When the regulations for
disinfection by-products are promulgated, EPA will require
compliance for all system sizes as a condition for avoiding
filtration.
Criteria Filtered Systems Must Meet
Filtered systems must measure turbidity levels every 4 hours or by
continuous monitoring. The state may reduce the number of grab
samples to once per day for some systems. For systems using
conventional filtration or direct filtration, the turbidity levels in
filtered water must never exceed 5 NTU and not exceed 0.5 NTU in
more than 5 percent of the samples collected each month. (The state
may allow a level up to but less than 1 NTU under certain conditions.)
For systems using slow sand filtration or diatomaceous earth, the
turbidity level in the filtered water must always be less than 5 NTU
and not exceed 1 NTU in more than 5 percent of the samples collected
25
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each month. (The state may allow a system using slow sand filtration
to exceed 1 NTU under certain conditions.)
A filtered system must also monitor disinfectant residuals at the
point of entry to the distribution system at the same frequency and
locations as an unfiltered surface water system. Filtration and
disinfection combined must achieve a 99.9 percent and 99.99 percent
removal/inactivation ofGiardia lamblia and viruses, respectively. The
state defines the level of disinfection required.
Total Coliform Rule Requirements
(54 FR 27544-27568, June 29, 1989)
Maximum Contaminant Level (MCL)
The MCL is based on the percentage of samples collected during a
month which contains any total coliforms, not on the density of total
coliforms in a sample. For systems analyzing at least 40
samples/month, no more than 5.0 percent of the monthly samples may
be total coliform-positive. For systems analyzing fewer than 40
samples/month, no more than one sample/month may be total coliform-
positive.
Routine Monitoring
Each public water system must sample according to a written
sample siting plan. The state must establish a process that ensures the
adequacy of the sample siting plan for each system.
The monitoring frequency for community water systems is based
upon the population served, as is the case under the current total
coliform rule, but with only 34 rather than 84 population categories.
For noncommunity water systems the monitoring frequency is as
follows:
• A system using surface water, or ground water under the
direct influence of surface water, must monitor at the same
frequency as a like-sized community water system.
• A system serving more than 1,000 persons during any month
must monitor at the same frequency as a like-sized community
water system. The state may reduce the monitoring frequency
for any month the system serves 1,000 persons or fewer.
• No system may monitor less than annually after June 1994.
Monitoring After a Total Coliform-Positive Sample
If a system has a total coliform-positive sample, it must collect a
set of repeat samples within 24 hours after being notified. Systems
collecting one routine sample/month or fewer must collect at least four
repeat samples; all other systems must collect at least three repeat
samples. Repeat samples must be taken 1) at the same tap as the
26
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original total coliform-positive sample, 2) at a tap within five service
connections upstream, and 3) at a tap within five service connections
downstream.
If any repeat sample is total coliform-positive, the system must
collect another set of repeat samples, as before, unless the MCL has
been violated and the system has notified the state.
If a system collects fewer than five routine samples/month, it must
not only take a set of repeat samples within 24 hours, but also at least
hve routine samples the next month it provides water to the public
The state may waive the requirement for a system to collect five
routine samples the next month under certain circumstances.
All total coliform samples - routine and repeat - count toward
compliance, except those that are invalidated by the state (or, for total
coliform- negative samples, by the laboratory).
If a routine or repeat sample is total coliform-positive, the system
must analyze that total coliform-positive culture to determine if fecal
cohforms or Escherichia coli are present. If present, the system must
notify the state by the end of the next business day. If any repeat
sample is fecal coliform- or E. coft-positive, or if a fecal coliform- or E
coh-positive routine sample is followed by a total coliform-positive
repeat sample, the system is in violation of the MCL for total coliforms
When the MCL is violated under these circumstances, the system must
notify the public via the electronic media.
Invalidation of Samples
A state may only invalidate a total coliform-positive sample under
one of the following circumstances:
• The laboratory acknowledges it performed the analysis
improperly.
• The system determines that the contamination is a
nondistribution plumbing problem on the basis that any repeat
sample taken at the same tap as the original total coliform-
positive sample is also total coliform-positive, and all other
repeat samples taken at nearby locations are total coliform-
negative.
• The state has substantial grounds to believe that a coliform-
positive result is due to some circumstance or condition not
related to the quality of drinking water in the distribution
system. In this case, the state must explain this judgment in
writing, the supervisor of the state official who draws this
conclusion must sign the document, and the document must be
made available to EPA and the public. The written
documentation must state the specific cause of the total
27
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coliform-positive sample, and what action the system has
taken, or will take, to correct this problem.
Laboratories must invalidate a total coliform-negative sample if
there is evidence that high levels of heterotrophic bacteria interfered
with the total coliform analysis.
Sanitary Surveys
All systems that collect fewer than five samples/month must
conduct a periodic sanitary survey. The responsibility for conducting
the sanitary survey is with the system, not the state.
A community water system must have a sanitary survey by June
29,1994, and every 5 years thereafter. A noncommunity water system
must have a sanitary survey by June 29, 1999, and every 5 years
thereafter or, for systems that use protected and disinfected ground
water, every 10 years thereafter.
Analytical Methodology
In conducting coliform monitoring, laboratories may use one or
more of the following analytical methods: 10-tube multiple tube
fermentation technique, membrane filter technique, presence-absence
(P-A) coliform test, and minimal medium ONPG-MUG test. In
addition, a 100-mL sample volume must be used in analyzing for total
coliforms, regardless of the analytical method used.
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II. Disease Surveillance
Waterborne Disease Outbreaks Surveillance:
Federal Requirements and Responsibilities
by: Robert V. Tauxe, M.D., M.P.H.
Enteric Diseases Branch
Division of Bacterial Diseases
Center for Infectious Diseases
Centers for Disease Control
Atlanta, Georgia 30333
(404) 639-2888
Introduction
The current collaborative surveillance for waterborne disease
outbreaks began at the federal level in 1971. Since then the U S
Environmental Protection Agency (EPA) and Centers for Disease
Control (CDC) have collected reports of waterborne disease outbreaks
and have summarized them annually. The purposes of national
.surveillance of waterborne disease outbreaks have been 1) to
determine general trends in the frequency of waterborne disease
outbreaks, 2) to characterize the epidemiologic patterns of waterborne
diseases, 3) to disseminate information on waterborne diseases, and 4)
to provide data for evaluating disease control efforts.
For purposes of surveillance, a waterborne disease outbreak is
defined as an acute illness affecting two or more persons with similar
symptoms that is epidemiologically associated with ingestion of water
or some other exposure to water intended for drinking. Single well-
documented cases related to toxic exposures such as methemoglobin-
emia are also included. Reporting for all federal disease surveillance
systems occurs as the result of a voluntary effort supported by the
Council of State and Territorial Epidemiologists. Reporting of specific
diseases is a result of legislation in each state concerning notifiable
diseases; reporting of all diseases to the federal government is
voluntary. In addition to outbreaks associated with water intended for
drinking, CDC has also collected information on illness related to
recreational water uses, and included these in annual summaries. The
annual summaries have also included outbreaks of gastroenteritis on
cruise ships, whether or not they were shown to be associated with
water exposure. Outbreaks on cruise ships and outbreaks associated
with ingestion of water while swimming have been excluded, however,
from the statistics reported here.
29
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Recent Trends
Between 1971 and 1985, 485 outbreaks of water-related disease
were reported, involving 110,359 cases (Figure 2.1.1). This is an
annual mean of 32.3 outbreaks per year, affecting a mean of 7,357
persons per year. The mean size of outbreaks is 228 cases, and the
median size is 35 cases. Several important trends can be observed from
the statistics. First, the number of reported outbreaks increased fairly
steadily through 1983 with some year-to-year variation. Second, the
number of reported outbreaks dropped sharply in 1984 and 1985; in
1985, the number of reported outbreaks was lower than at any point
since surveillance began. Preliminary results for 1986 indicate that
this recent decrease persists. The large peaks in the actual number of
reported cases (Figure 2.1.2) are related to large outbreaks of
undetermined etiology, the so-called acute gastrointestinal illness
(AGI). In 1984 and 1985, the reported numbers of cases were the lowest
since the inception of this surveillance system.
The recent and marked decrease in the reported number of
outbreaks was a major focus of discussions at the recent EPA
Workshop on Methods for Investigating Waterborne Disease
Outbreaks. Possible explanations for this decrease include changes in
surveillance itself and changes in the underlying frequency of
waterborne disease outbreaks. To be reported, an outbreak must first
be identified and investigated. If this decrease is the result of changes
in surveillance, we must presume that waterborne disease outbreaks
are still occurring in the states, but are not being recognized,
investigated, or reported as frequently as they were 5 to 10 years ago.
To use the "tip of the iceberg" metaphor, these explanations would
suggest that the iceberg is as big as ever, but that we see less of it
because it is sinking. It is important to remember, however, that
substantial effort and resources have been devoted to improving the
nation's water supply in recent years, including efforts to modernize
treatment facilities, add filtration units, and educate the operators. If
these efforts have been well-directed, we should expect the actual
number of outbreaks to decrease, particularly those involving
community water supplies. As a consequence, the number of reported
outbreaks might also reasonably be expected to decrease. If these
explanations are correct, they would suggest that we see less of the
iceberg because it is in fact melting, not sinking.
Overall, the number of outbreaks related to community water
supplies during the 15-year period 1971 to 1985 almost equals the
number of outbreaks related to noncommunity water supplies (Table
2.1.1). Outbreaks related to community water supplies, however, affect
many more people on average than do the noncommunity outbreaks.
Between 1983 and 1985, the number of outbreaks related to
community water supplies dropped sharply, while the number related
to noncommunity water supplies remained essentially unchanged.
Over the same 15-year period, the most frequent type of deficiency
30
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Outbreaks
60 r-
50
40
30
20
10
J L
1970
1975
1980
1985
Figure 2.1.1. Reported outbreaks of waterborne disease, by year, United States, 1971-
1985.
causing the outbreak of waterborne disease was treatment deficiency
as measured by both outbreaks and cases. Use of untreated ground
water was the second most frequent cause in terms of number of
outbreaks; defects in the distribution system was the second most
frequent cause in terms of number of cases.
We can also describe the waterborne disease outbreaks by the
etiologic cause of the outbreak. Between 1971 and 1985, 245 (50%) of
the 485 outbreaks with reported cause were AGI; that is, a specific
diagnosis was not reached. Ninety outbreaks (19%) were caused by
parasites, 59 outbreaks (12%) by bacteria, 40 outbreaks (8%) by
viruses, and 51 outbreaks (11%) by chemicals. The number of
outbreaks reported each year by agents during the 15 years since
surveillance began shows three distinct trends (Table 2.1.2). First, the
31
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Cases
(Thousands)
22 (-
1970
Figure 2.1.2.
1975
. - '1980 ••'.='• '-• , ; 1985
Reported cases of illness associated with outbreaks of waterborhe
disease, by year, United States, 1971-1985. __','•-
undiagnosed (AGI) outbreaks have varied in number considerably
from one year to the next and account for much of the total variation in
the number of outbreaks. Second, the number of reported parasitic
outbreaks began to increase in the late 1970s and by 1983 accounted
for a substantial fraction of all outbreaks. All but two of these parasitic
disease outbreaks were caused by the parasitic pathogen Giardia
32
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Table 2.1.1. Reported Waterborne Disease Outbreaks, by Year and Type of
Water Supply Systems, and Median Size of Outbreaks, United
States, 1971-1985
Community Noncommunity Individual Total Total Cases Median Size
1971
1972
1973
1974
1975
1976
1977
1978
1979
1980
1981
1982
1983
1984
1985
Total
8
9
6
11
6
9
14
10
24
26
14
26
30
12
6
211
8
19
16
9
16
23
18
19
13
20
18
15
8
5
9
216
4
2
3
5
2
3
2
3
7
7
4
3
4
8
1
58
20
30
25
25
24
35
34
32
44
53
36
44
42
25
16
485
5,184
1,650
1,762
8,356
10,879
5,068
3,860
11,435
9,769
20,045
4,537
3,588
20,923
1,742
1,561
110,359
58
26
44
. 20
84
30
46
95
49
40
36
31
35
20
31
lamblia. Finally, in 1984 and 1985, steep declines in parasitic, viral,
and undiagnosed (AGI) outbreaks have been seen.
Trends can also be seen over time in the specific causative agents. I
have already commented on the rise of giardiasis outbreaks. Among
the bacterial pathogens, the most common agent has been Shigella. In
1971 to 1973, Shigella represented over 70% of all bacterial disease
outbreaks. The more recently discovered pathogen, Campylobacter,
first appeared in 1977 as a waterborne pathogen and since then has
accounted for a steadily increasing fraction of all bacterial waterborne
disease outbreaks. In the period 1983 to 1985, Campy lobacter
accounted for more than 50% of water-related bacterial disease
outbreaks. Salmonella is an infrequent cause of waterborne disease
outbreaks; and, in particular, only two outbreaks caused by
Salmonella typhi, the causative agent of typhoid fever, were traced to
water exposures during this 15-year period. Among chemical
outbreaks the most frequently reported chemical was copper sulfate
(11 outbreaks), followed by hydrocarbons (7), fluoride (7), pesticide or
herbicide (6), nitrates (3), and lead (3); 13 outbreaks were caused by
other chemical or toxic agents. All reported viral disease outbreaks for
the first 6 years of the surveillance were caused by hepatitis A virus.
Since then, the Norwalk-like agents have accounted for more than 50%
of viral disease .outbreaks. Given the considerable technical difficulty
33
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Table 2.1.2. Reported Waterborne Disease Outbreaks, by Type of
Agent, United States, 1971-1985.
Outbreaks
Year
1971
1972
1973
1974
1975
1976
1977
1978
1979
1980
1981
1982
1983
1984
1985
Bacterial
3
5
6
5
2
3
3
7
3
3
4
3
4
4
4
59
Parasitic
0
4
4
4
1
3
4
4
7
8
11
12
18
7
3
90
Viral
6
5
2
0
1
0
1
3
3
6
1
7
3
2
0
40
Chemical
2
3
0
5
3
3
6
2
7
7
5
3
1
3
1
51
AGIa
9
13
13
11
17
26
20
16
24
29
15
19
16
9
8
245
Total
20
30
25
25
24
35
34
32
44
53
36
44
42
25
16
485
a AGI = Acute gastrointestinal illness.
of diagnosing infections caused by these viral agents, they are likely to
represent a larger fraction than these figures would suggest.
Thus, the general trends in pathogens reported as causes of
waterborne disease outbreaks in this 15-year interval are as follows:
• The number of outbreaks caused by classic waterborne agents
such as S. typhi, hepatitis A virus, and Shlgella has decreased.
• Newly recognized pathogens emerging as important
waterborne disease agents include Giardia, Campy lobacter,
Norwalk virus, and most recently Cryptosporidia.
These trends indicate the increasing importance of chlorine-resistant
organisms.
To return to the issue of the recent decrease in the number of
reported outbreaks: this drop occurred most sharply after 1983. This
decrease probably reflects both a real decrease in the frequency with
which outbreaks occur and some decrease in the proportion that are
investigated. The resources available to devote to waterborne disease
outbreak investigation and surveillance have decreased overall in the
last 5 years. State health departments have had to cope with the
tremendous challenge posed by the acquired immunodeficiency
34
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syndrome (AIDS) epidemic, often without increased resources. The
resources available within EPA for advising and assisting in the
evaluation of potential outbreaks of waterborne diseases decreased
during this time. At CDC, the techniques of surveillance remained
constant during the period 1981 to 1985. During the time of greatest
decline, between 1983 and 1984, the surveillance system was
maintained by the same individuals who applied equal effort in both
years. Since 1985, the effort at CDC has decreased, and until 1990 a
surveillance summary had not been published since that covering the
year 1985.
Conclusions
In the last 2 years covered by published surveillance data, 1984
and 1985, the number of outbreaks was small. Preliminary data from
1986 through 1988 suggest this trend will continue. Further
surveillance data will help to understand the causes for the decline.
Several pieces of evidence, however, suggest that the decrease in
reported outbreaks may be real. First, the reported outbreaks have
tended to be smaller in recent years, indicating that the system is not
becoming less sensitive to small outbreaks. The median size of
outbreaks in the late 70s was 46 to 95, compared to the median size of
20 to 35 in the period 1983 to 1985 (Table 2.1.1). Secondly, the decrease
in outbreaks has been particularly marked in community water
systems where efforts to improve water supplies have been
concentrated; these outbreaks are also most likely to be detected and
investigated. The decrease has affected the parasitic and viral as well
as undiagnosed diseases, suggesting it is not simply a decrease in
specific diagnostic capacities.
A second issue before us is the effect of this decline on efforts to
maintain and improve the quality of drinking water in the United
States. Large waterborne disease outbreaks increase the attention
given to the general problem of waterborne diseases in communities,
states, and federal agencies. In the past, we have tended to depend on
the acute outbreak as the primary mechanism for stimulating interest
in this problem. Constant efforts are needed to continue to improve
water-supply systems and to maintain the improvements in systems
that have already been upgraded. We can no longer depend on the
acute waterborne disease outbreak as a primary tool for educating the
public or the government about the importance of these efforts.
Maintaining high standards of water treatment needs to become a goal
in and of itself. An appropriate analogy may be a successful vaccine
against an infectious disease. When the disease is epidemic, the need
for widespread use of an effective vaccine is immediately apparent to
everyone. Once the vaccine has been successfully deployed and the
disease becomes rare, fear of the disease itself no longer propels
parents to have their children immunized. Other mechanisms must be
set in place to insure high levels of vaccination in the population The
most useful measure of success of a vaccination program then becomes
35
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the proportion of the target population that is vaccinated, rather than
documentation of the continued absence of the disease.
Similarly, for waterborne disease, program-based endpoints other
than the number of waterborne disease outbreaks may be more useful
measures of drinking water safety in the future. These endpoints may
be related to the success of education programs (for instance, measures
of the knowledge and practices of water treatment plant operators), to
the success of efforts to upgrade treatment facilities .(for instance,
measures of the proportion of those drinking surface water who are
served by water that is both chlorinated and filtered), or to the utility
of the hazard analysis-critical control point approach to water quality
testing (for instance, tests of water quality parameters during and
after the critical steps of filter backwash, routine maintenance
shutdown, or simulated chlorinator failure in a sample of plants).
The Future of Waterborne Disease Outbreaks and Surveillance
With some trepidation, let us gaze into the crystal ball. What do
the observed trends suggest for the future of waterborne disease
outbreaks? We can expect new pathogens to emerge that will evade
current water treatment efforts. Chlorine-resistant organisms will
represent a greater fraction of waterborne disease pathogens than in
the past. Past experience shows that there is a particular risk of
outbreaks occurring while a water plant is under repair or being
improved. As our existing infrastructure of water treatment plants
ages, it will require maintenance and replacement. It is critical that
these repairs be done safely. We can expect more intense stress on
available drinking water sources as the population increases, and we
can also expect an increasing fraction of the population to be
immunosuppressed by HIV infection, so that the presence of low
numbers of pathogens in drinking water may be of more concern.
As we move into the 1990s, the federal government has several key
roles to play in maintaining surveillance and expertise in the area of
waterborne disease outbreaks. These include the following:
• Maintaining- a national surveillance system with which to
monitor new trends, assess the results of the control measures
implemented, and provide feedback to the states. Maintenance
of this system requires resources at the federal level that have
been extremely limited in recent years.
« Maintaining and promoting expertise in epidemiologic
investigation of waterborne outbreaks, including
characterization of illness, technical assessment of water-
treatment facilities, and use of epidemiologic methods to define
the association of outbreaks with water sources. This includes
providing technical support in outbreak investigations^ in
assessment of water-treatment facilities and water quality,
36
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and in diagnosis of illness; and epidemiologic assistance to
state health departments.
• Developing better diagnostic tools with which to assess the
importance of newly described pathogens and diseases, for
example, the Norwalk-like viruses and the chronic diarrhea
syndrome. This requires support for the expensive laboratory
procedures involved and for the search for less expensive
' diagnostic tools.
• Assisting in development of programmatic endpoints with
which to monitor the success of drinking water purity
programs.
37
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Surveillance for Waterborne Illness and Disease
Reporting: State and Local Responsibilities
by: Laurence R. Foster
State Epidemiologist
Oregon Health Division
Portland, Oregon 97201
(503) 229-5552
Introduction
The division of responsibilities between state and local public
health agencies for disease surveillance and outbreak investigation
differs in each state. For example, in Oregon, the local health
departments are autonomous and responsible for local surveillance
activities. The state health agency provides consultation, technical
support, and centralized computer analysis of surveillance data. In
contrast, all public health activities in New Mexico are directly
supervised by the state public health agency.
No matter how the delivery of these services is organized in a state,
there is a common set of basic tasks to be carried out for effective
waterborne disease surveillance. For purposes of this discussion, these
tasks will be considered without regard to whether the work should be
done by a state or a local agency.
In order for waterborne illness surveillance to be successful, the
traditional approaches must be rigorously maintained. Additional
outreach, however, must be used to supplement these traditional
approaches.
The first purpose of surveillance for waterborne disease is
immediate prevention of further illness once an outbreak has started.
The surveillance system should be sensitive enough to detect
outbreaks early in their course. Such detection should trigger
epidemiological and environmental investigation of a suspected
outbreak. If such investigation determines drinking water to be the
source of illness in an outbreak, control measures can be rapidly
initiated to prevent further illnesses. Such control measures include
short- term solutions such as boil water notices, increased disinfection,
or recommending bottled water or alternative water sources. They also
include long-term measures such as disconnecting a cross connection,
39
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improving water treatment practices, and upgrading water treatment
facilities. •>••••
Surveillance for waterborne disease includes three major
categories of tasks: case identification, case evaluation, and outbreak
investigation. Case identification involves obtaining reports of cases of
illness. Case evaluation means determining from individual cases
whether a group of illnesses may potentially be from a common so.urce
and whether drinking water is a potential source. The outbreak
investigation then determines whether there is an outbreak, and, if soj
its probable source.
Case Identification
The traditional approach to case identification is for the state
public health agency to declare that certain diseases are reportable by
law or administrative rule. Physicians, and often other health care
providers, are obligated to report individuals with certain diagnoses.
Examples of potentially waterborne diseases that are reportable in
Oregon include giardiasis, hepatitis A, campylobacteriosis,
yersihiosis, shigellosis, and salmonellosis. In Oregon, an
"extraordinary occurrence of illness" is also reportable. This means
that when a health care provider recognizes an unusual number of
cases, or a clustering of cases, of an illness that is not specifically
reportable, he or she must report that fact to the local health
department. This requirement is intended to promote identifications of
outbreaks caused by agents that are not otherwise reportable, such as
Norwalk agent and toxic substances.
Generally a case report must include the patient's name and
locating information, the doctor's name and address, and the specific
diagnosis. Suspected cases of illness, in addition to confirmed cases,
have been made reportable in Oregon to improve the sensitivity and
timeliness of the surveillance system. Reports must generally be made
within one working day of the time of exposure, but extraordinary
occurrence of illness is to be reported immediately, day or night.
Oregon also requires that clinical laboratories report the
identification of certain disease-causing agents or markers for
infection by those agents. For example, positive cultures for
Campylobacter jejuni and Shigella species, positive stool examination
for Giardia lamblia and Entamoeboa histolytica, and a positive IgM
antihepatitis A virus antibody test are reportable. The Oregon Health
Division has found that this greatly enhances the completeness of
reporting.
To further enhance reporting, we have discovered that we must go
beyond the traditional approach to surveillance described above. This
necessity has been demonstrated by the fact that for most of Oregon's
documented waterborne disease outbreaks, the initial case report came
from a private citizen. In fact, all of Oregon's larger waterborne
40
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outbreaks have come to our attention that way. This may be because
Oregon's larger waterborne disease outbreaks have occurred in
communities heavily frequented by tourists. Initial reports from these
outbreaks have come from visitors to the outbreak communities who
have become ill after returning to their homes.
These experiences tell us that public health agencies should seek
reports 'of suspected waterborne illness from the general public.
Although there is no simple formula for doing this successfully, it
helps to maintain a high public profile for epidemiology and disease
control activities generally. Significant events related to outbreaks
and disease control should be actively reported to the news media,
within the limits of confidentiality and professional .propriety, of
course. Epidemiology staff should always be willing to help reporters
get the information they need, again within the limits of
confidentiality and propriety. The epidemiology unit should publish a
high profile newsletter directed toward health care providers; but also
sent to the news media. This newsletter should be clinically relevant,
concise, and informative, and its topics should be timely relative to
news releases. All of these efforts should promote an image of the
health agency as concerned and responsive, thereby encouraging
citizens to report to the agency when they develop an illness and
suspect it to be from drinking water.
Another useful outreach focus to supplement traditional'
approaches to surveillance is the water system ope.rator. These officials
regularly .receive complaints from their customers, and those
complaints sometimes are about illnesses suspected to have been
caused by the drinking water. In Oregon, the Water Quality Program
has developed a standardized complaint form to help system operators
collect systematic information from complainants. When an illness is
part of the complaint, the operator can then turn the information 'over
to.the public health agency for further investigation.
The surveillance component for case'identification should,
therefore, include requirements for reporting of specific diseases and
extraordinary occurrences of illness by health care providers,
encouraging reporting by private citizens, and developing specific
mechanisms to improve reporting of customer complaints by water
system operators.
Case Investigation
When an individual case of a reportable disease is reported, health
agency staff carry out an investigation of that case to identify the
possible source of infection, as well as to prevent further spread. For
example, when a case of hepatitis A is reported, the health department
epidemiologist tries to determine the probable source, such as a family
member with recent hepatitis, day care, foreign travel, a restaurant, or
drinking water potentially contaminated with human feces. Possible
41
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sources to be sought by interviewing a giardiasis case would include
water from a mountain stream, a community drinking water system,
or a day care.
The epidemiologist usually cannot draw a conclusion about a
common source, such as a community water supply, on the basis of a
single case investigation. However, the investigator may suspect
community drinking water to be the source of infection for an
individual, if an alternative source of infection cannot be identified,
and if the individual drank water from a system with inadequate
treatment. This suspicion may seem to be confirmed particularly if the
relative timing between an individual's drinking of the water and the
onset of illness fits the known incubation period for the particular
disease.
The epidemiologist, however, usually suspects a community
drinking water supply to be the source of infection only when a cluster
of cases is identified among people who drank the water and who have
similar dates of onset, and no alternative sources of infection are
identified. Detecting such clusters of cases requires alertness on the
part of the investigator to recognize when an unusual number of cases
of a particular illness is occurring. This recognition requires a
standardized approach to individual case investigation, as well as
thorough training and a high degree of motivation.
Computer review of the number of cases reported each week is also
useful in detecting unusual clusters of cases in jurisdictions with
sufficient population to make computer review more efficient than
manual review. In Oregon, the number of cases of each reportable
disease reported each week is tabulated by computer. A computer
program has been developed to identify those counties that have
experienced a number of cases for a particular disease that exceeds the
99 percent confidence interval for number of cases expected for that
week. This 99 percent confidence interval is based upon the weekly
average over an 8-week time period during the same season of the
previous year.
One way to screen for a possible waterborne outbreak is to tabulate
reported cases within various drinking water systems to determine if
the expected number of cases is exceeded. However, in Oregon, we
have found that routinely tabulating reported cases of illness by
drinking water system or source was not feasible due to the many
different sources of water in the state, the use of more than one water
source by most individuals, and the lack of a standardized
nomenclature for recording an individual's drinking water sources.
The cost effectiveness of investigating individual cases of
reportable illness should be evaluated for each disease and in each
state. Although such cost effectiveness has not been rigorously studied
42
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in Oregon, experience has shown that routine individual case
investigation may not be worth the effort for some diseases.
In 1981, campylobacteriosis and giardiasis were made reportable
in Oregon. Local health departments were required to perform
individual case investigations and to complete standard interview
forms for every case. The resulting workload was immense. The two
diseases quickly became the most commonly reported diseases in the
state, second only to gonorrhea. Therefore, the number of required
individual investigations was large. The extensive effort required was
not productive; only two small community waterborne disease
outbreaks were identified with 3 years of such effort. These outbreaks
could have been identified simply by observing that an unusual
number of cases were occurring in the affected communities. The
individual case investigations did not enhance the sensitivity or
timeliness of simple case count tabulation for identifying the
occurrence of these outbreaks.
Because of this finding, individual cases of campylobacteriosis and
giardiasis are no longer investigated in Oregon unless the number of
cases in a county exceeds the threshold limit. In other words,
individual case investigations are initiated only when an unusual
number of reported cases suggest a common source outbreak may be
occurring.
Outbreak Investigation
Once the epidemiologist suspects, on the basis of individual case
investigations, that a waterborne outbreak may be occurring, a full-
fledged outbreak investigation should be started immediately. Such an
investigation should first seek to determine whether drinking water
really is the source of the outbreak. If it is, the second stage of this
investigation is to help end the outbreak. Outbreak investigations are
discussed in more detail in another chapter.
Conclusion
Surveillance for waterborne disease at the state and local level
must include case identification, individual case investigation, and the
carrying out of an outbreak investigation. In addition, public health
agencies should continually evaluate the effectiveness and efficiency of
their surveillance systems in order to seek improvements.
Although public health agencies should emphasize the value of an
effective surveillance system and maintain constant vigilance for
waterborne disease, they must not promote the mentality that
"everything is okay" if no outbreaks are identified. Our real emphasis
must be on prevention by improving currently inadequate drinking
water systems and by attending to the proper long-term maintenance
and operation of those systems that are currently good ones.
43
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III. Investigation of Waterborne Disease Outbreaks
Differences Between Outbreak Investigation and
Research Epidemiology
by: Neal D. Traven
Department of Epidemiology
Graduate School of Public Health
University of Pittsburgh
Pittsburgh, Pennsylvania 15261
(412) 624-0097
Introduction
Epidemiology, in one form or another, has been practiced for
thousands of years. For example, in the treatise Airs, Waters, Places,
Hippocrates recognizes the importance of environmental changes and
behavioral patterns in the study of human disease patterns (1). The
Old Testament book of Daniel (Chapter 1, Verses 1-15) recounts the
tale of an experiment comparing two dietary regimens; in the end,
Daniel's diet is found to be more healthful than the king's (2).
The origins of modern epidemiology can be traced from John
Graunt's 1662 studies of mortality patterns in London, through the
1747 demonstration by James Lind of the efficacy of citrus fruits in
combatting scurvy, to the appointment of William Farr as the first
Registrar-General of England's General Registry Office in 1839 (3).
Farr and his colleagues founded the London Epidemiological Society in
1850.
Among those colleagues was the eminent physician John Snow,
already well known in London for having administered chloroform to
Queen Victoria during childbirth. Snow investigated a series of
cholera outbreaks in London during the period 1848-1854 (4).
At that time, two companies supplied water to most of the homes in
a section of London south of the Thames River; their systems were so
intertwined that adjoining buildings might be served by different
suppliers. Though both companies took their water from the sewage-
laden Thames, one had recently moved its intake to a less
contaminated section of the river. By comparing the addresses on
water bills to those on cholera death certificates (provided by Farr's
General Registry Office), Snow demonstrated a nearly ninefold
increase in cholera mortality in homes supplied by one of the
companies (see Table 3.1.1). Snow's conclusion that something in
Southwark and Vauxhall's water supply caused cholera was so
45
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persuasive that all of London's suppliers were required to filter their
water by 1857, just 2 years after he published his findings.
Table 3.1.1. Cholera Deaths per 10,000 Houses by Water Supply, London, 1854*
Water Supply Number of Houses Deaths from Cholera Deaths per 10,000 Houses
Southwark and
Vauxhall Company
Lambeth Company
Rest of London
40,046
26,107
256,423
1,263
98
1,422
315
37
59
"Adapted from (4).
It should be noted that Snow's work, as well as that of
Semmelweiss on puerperal fever, Budd on typhoid fever, and Panum
on measles, predated the germ theory promulgated by Pasteur and
others. Indeed, the cholera vibrio was not identified until 1883, by
Robert Koch.
Outbreak Investigation or Epidemiology?
The late 19th century saw an explosion of public health action
against many of the diseases that had plagued mankind throughout
history. In rapid succession, the infective agents and modes of
transmission of malaria, tuberculosis, diphtheria, scarlet fever,
typhus, tetanus, rabies, smallpox, and many others were uncovered. In
many cases, researchers devised vaccines capable of preventing the
occurrence of the diseases; for other infectious diseases, improvements
in medical care and the advent of antibiotics could cure those who
contracted them.
Underlying these monumental achievements was a newly
developed fundamental concept of the mechanisms of infectious
diseases. Koch's postulates (Table 3.1.2) were formulated by the
German pathologist-microbiologist around 1890. These axioms define
a strong causal relationship between microorganisms and specific
diseases (5). In the century since the advent of microbiology, it has
been recognized that Koch's postulates overstate the causal association
between microorganisms and diseases; later research in immunology,
microbiology, and related fields has unearthed the complexities of
infectious diseases. The eminent American infectious disease epidemi-
ologist Alfred Evans has since developed an extended set of postulates
based on Koch's (Table 3.1.3) (6).
Mankind's victory over the plagues of yesteryear, capped by the
total eradication of smallpox from the planet, means that we are now
only rarely subjected to epidemics of infectious diseases. In
46
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Table 3.1.2. Koch's Postulates
• The agent must be shown to be present in every case of the disease by isolation in
pure culture.
• The agent must not be found in cases of other disease.
• Once isolated, the agent must be capable of reproducing the disease in experimental
animals.
• The agent must be recovered from the experimental disease produced.
Table 3.1.3. Evans' Postulates*
• Prevalence of the disease should be significantly higher in those exposed to the
hypothesized cause than in controls not so exposed.
• Exposure to the hypothesized cause should be more frequent among those with the
disease than in controls without the disease - when all other risk factors are held
constant.
• Incidence of the disease should be significantly higher in those exposed to the
hypothesized cause than in those not so exposed, as shown by prospective studies.
• The disease should follow exposure to the hypothesized causative agent with a
distribution of incubation periods on a a bell-shaped curve.
• A spectrum of host responses should follow exposure to the hypothesized agent along a
logical biological gradient from mild to severe.
• A measurable host response following exposure to the hypothesized cause should have
a high probability of appearing in those lacking this before exposure, or should increase
in magnitude if present before exposure. This response pattern should occur infrequently
in those not so exposed.
• Experimental reproduction of the disease should occur more frequently in animals or
humans appropriately exposed to the hypothesized cause than in those not so exposed;
this exposure may be deliberate in volunteers, experimentally induced in the laboratory,
or may represent a regulation of natural exposure.
• Elimination or modification of the hypothesized cause should decrease the incidence of
the disease (i.e., attenuation of a virus, removal of tar from cigarettes).
• Prevention or modification of the host's response on exposure to the hypothesized
cause should decrease or eliminate the disease (i.e., immunization, drugs to lower
cholesterol, specific lymphocyte transfer factor in cancer).
• All of the relationships and findings should make biological and epidemiologic sense.
"Adapted from (6).
contemporary industrial and post-industrial societies, the few
outbreaks of acute infectious disease are investigated rapidly.
Waterborne disease outbreaks are usually found to result from a
breakdown in the mechanical systems that ordinarily protect us from
waterborne pathogens (e.g., drinking water or sewage treatment
47
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plants). These breakdowns are nearly always amenable to engineering
solutions. Outbreak investigation can be likened to "firefighting" -
when an outbreak flares, investigators rush to the scene, assess the
damage, find its cause, correct the problem, and return the system to
its normal state.
On the other hand, today's plagues are largely chronic
degenerative conditions like heart disease, cancer, pulmonary disease,
arthritis, and many others. With these diseases, strong relationships
with specific causative agents are observed very rarely. Chronic
diseases are associated with complex interactions within the
"epidemiologic triad" of host, agent, and environment. For example,
insulin-dependent diabetes mellitus may be triggered by a disordered
host response to viral infections in persons with a genetic
predisposition to the disease. The infection may lead to diabetes,
however, only if the individual is in a particular susceptible age range
(generally, under age 20). But even in the presence of susceptible age,
viral infection, and genetic predisposition, there is no guarantee that
diabetes will result; nor does Type I diabetes occur only in persons with
such a profile. In addition, the risk factors most strongly associated
with contracting a disease may differ greatly from those related to its
progression and outcome'. Finally, a disease may itself be a risk factor
for other diseases; for example, Type I diabetes mellitus greatly
increases the risk of coronary heart disease in young men.
Even where a disease-pathogen relationship exists (e.g., AIDS and
HIV), then, epidemiologic research no longer studies disease
causation; these multifactorial diseases do not have single causes.
Instead, epidemiologists search for risk factors of disease. The presence
of risk factors, such as those described above for diabetes mellitus,
increases the probability of disease, but does not cause it.
Contemporary epidemiology is a probabilistic endeavor in that it
often boils down to a search for personal behaviors, genetic
propensities, and/or environmental exposures that may affect the
likelihood of disease occurrence or progression. These risk factors may
have occurred in the remote past. Epidemiologists seek "associations"
between disease states and risk factors, taking into account the
possible independent and interactive effects of other potential risk
factors. Rather than Koch's deterministic postulates, contemporary
epidemiologists apply criteria such as those put forward by Evans or
Bradford Hill (7) to assess evidence about association between diseases
and potential risk factors (see Table 3.1.4).
It should be noted that these postulated criteria for the conduct of
epidemiologic research overlap significantly. For example, it is
difficult to differentiate between Bradford Hill's criteria of biological
plausibility and coherence. Taken as broad guidelines, however, they
provide a valuable underpinning for epidemiologic researchers. The
concepts of specificity of effect, dose-response relationship,
48
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Table 3.1. 4. Assessment of Epidemiologic Evidence About Associations with
Potential Risk Factors*
~* o;ons/sfe/?cy -tne association is consistent if the results are replicated when studied in
different settings and by different methods.
• Strength - an expression of the disparity between the frequency with which a factor is
found in the disease and the frequency with which it occurs in the absence of the
GISGclSG.
• Specificity - established with the limitation of the association to a single putative cause
• and single effect.
• Dose-response relationship - established when an increased risk or severity in disease
occurs with an increased quantity ("dose") or duration of exposure to a factor.
• Temporality - the exposure to a putative cause always precedes, never follows the
outcome. '
• Biological plausibility - it is desirable that the association agree with current
understanding of the response of cells, tissues, organs, and systems to stimuli.
• Coherence - associations should not conflict with the generally known facts of the
natural history and biology of disease.
• Experiment - it may be possible to appeal to experimental, or quasi-experimental
evidence, e.g., an observed association leads to some preventive action.
"Adapted from (7).
temporality, and biological plausibility remain essential to
investigators of waterborne diseases.
Epidemiologic Studies - Design and Evaluation
Study Designs
The two primary epidemiologic study designs are case-control and
cohort studies. In the former, subjects are grouped according to their
disease status; their prior behaviors or exposures are investigated
retrospectively. The latter type of study follows subjects who are
initially free of the disease, determines their exposure or behavior and
examines their disease status after a (usually lengthy) period of time.
The history of case-control studies has been traced by Lilienfeld
and Lilienfeld (3) to the French physician-mathematician P.C A Louis
and his 1825 paper on tuberculosis. The first modern case-control
study, on breast cancer, was published by J.E. Lane-Claypon in 1926
In the contemporary era, since World War II, case-control studies have
become increasingly prevalent in the epidemiologic literature. In
recent years, much methodologic research in the field has been
concentrated on improving the sophistication of case-control analysis.
Each of the major types of epidemiologic study has its advantages
and its disadvantages. Cohort studies are extremely expensive and
time consuming, since the disease of interest may take decades to
49
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develop; for example, the Framingham Heart Study has been
underway for over 40 .years. They also require a large number of
subjects, since even an extremely common disease may affect only 1 in
10 or 20 participants. The extended follow-up period is likely to result
in attrition problems, and changes in standard medical practice may
invalidate study criteria and methods (8). Conversely, only in, a
prospective cohort study can one determine incidence rates and
relative risks."Also, the temporal sequence of risk factor and disease
can be demonstrated convincingly only with the cohort design.
Case-control studies require many fewer subjects than cohort
studies. With rare diseases (and fortunately, most diseases do occur
infrequently), the case-control study is the only practical design,
though it can be applied to the analysis of any disease. Case-control
studies can usually be completed more quickly than cohort studies,
taking years rather than decades. Due to their retrospective nature,
however, case-control studies are particularly susceptible to biased
recall of events in the remote past and to incomplete information.
Statistically, they yield only odds ratios, though this quantity is often
an excellent estimate of the true incidence rate (9). The primary
difficulty in designing a case-control study, however, is the selection of
appropriate control subjects. Often, controls are matched with cases on
a number of variables in order to increase their comparability. Many
epidemiologic studies have been subjected to criticism regarding the
appropriateness of the selected control groups and potential problems
in generalizing study results.
Evaluation of Epidemiologic Data
Such basic tools for evaluating epidemiologic results as incidence
rate, rate differences, relative risk, and odds ratio are described in
another paper in the proceedings (10). As noted there, the odds ratio
has become the measure of choice used by epidemiologists to
investigate associations between risk factors and diseases. In any
epidemiologic study, it is always necessary to address potential biases
in sample selection, questionnaire design, recall by subjects, and a host
of other sources; these subjects are dealt with in a paper by Richard
Vogt, also in these proceedings.
Another issue to be faced in the analysis of epidemiologic data is
confounding by other variables. A confounder is an extraneous
variable which can account for some portion of the apparent effect of
the risk factor of interest. As described by Rothman (11), a confounder
is itself a disease risk factor which is associated with the study
variable but is not a consequence of it. For example, in a study of the
effects of alcohol consumption on throat cancer, cigarette smoking
could confound the relationship under investigation; smoking is also a
risk factor for throat cancer and is itself associated with alcohol use.
50
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The effects of confounding variables on the association of interest
can be negative or positive, large or small. In other words, a
confounder may mask a real association (yielding a spuriously
nonsignificant odds ratio), or it may produce a falsely significant
relationship. The epidemiologist, then, must always be aware of
putative risk factors other than those in which he or she is interested,
and should ensure that questionnaires are written to address those
issues.
The effects of potential confounders can be removed or controlled
by choosing the appropriate study design. In a matched design, control
subjects are chosen with foreknowledge of their status on selected
factors. By ensuring that cases and controls do not differ with respect
to those factors, this design eliminates the potential confounding effect
of the matching variables. The disadvantages of this approach are that
the matching variables cannot be used in the analysis and that it may
be difficult to find close matches (particularly when matching
simultaneously on several variables) unless the pool of potential
control subjects is very large.
An alternative strategy is to use epidemiologic methods to
evaluate and statistically control for confounding. The two techniques
most widely used to control for confounding are stratification and
multivariate analysis.
In stratified analysis, the basic fourfold table calculation of the
odds ratio is carried out for each of several levels or strata of the
potential confounder. For instance, one might determine the odds ratio
within each of three or four age strata. These separate stratum-specific
odds ratios can be combined into a single summary odds ratio
(controlled for levels of the potential confounder) using the procedure
outlined by Mantel and Haenszel in their seminal 1959 paper (12).
Mantel-Haenszel stratified analysis, also provides a one degree-of-
freedom summary chi-square for the overall association between the
study variable and the disease.
The Mantel-Haenszel technique can be extended to account for
several potential confounders. But unless the original sample is very
large, one quickly finds many cells with very small numbers of
subjects. In addition, the Mantel-Haenszel technique may require the
researcher to impose artificial stratification or arbitrarily chosen
cutpoints on some potential confounders.
The other method to control for confounding in epidemiologic
analysis is the multivariate technique known as logistic regression In
this procedure, the data are fitted to the model:
P(y|x) =
..
51
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where P(y|x) is the probability of disease and the tys are regression
coefficients for the predictor variables (the x(s), among which is the
study risk factor. In this model, the predictor variables may be discrete
or continuous. Under appropriate conditions, logistic regression
coefficients are algebraically identical with the logarithm of the odds „
ratio (13), so the logistic model is used to determine an odds ratio for
the variable of interest while simultaneously controlling for -many
potential confounding factors. ,
In most cases, results from the Mantel-Haenszel procedure and the
multiple logistic model are quite similar. In their recent textbook on
epidemiologic statistics, Kahn and Sempos (13) illustrate the near-
equivalence of these methods applied to Framingham data.
Summary
Much of the early history of epidemiology, such as Snow's work on
cholera, revolved around investigation of outbreaks of acute infectious
diseases. Investigation of waterborne disease outbreaks still owes
much to these methodologies developed in the 19th and early 20th
centuries. Improvements in sanitation and drinking'water
purification, advances in microbiologic identification of pathogens,
and the wide availability of vaccines, antibiotics, and medical care
have vastly decreased the number and scope of such outbreaks in
industrial and postindustrial societies. Since World War II, then, the
research focus of epidemiologists has turned increasingly to
investigation of such chronic conditions as heart disease, cancer,
diabetes mellitus, and a host of others.
In a research environment where Koch's postulates are of
diminished usefulness, epidemiologic study designs and analytic
methodologies have been developed to examine such multifactorial
disorders. Instead of "causes," epidemiologists now search for "risk
factors" for diseases. Much attention is paid to associations between
diseases, the influence of health behaviors, environmental and'
occupational exposures, genetic predisposition, and many other
confounding or interacting factors. Selection of appropriate study
subjects is of overwhelming importance in such research. In addition,
the purview of epidemiologic methodology has now expanded to
encompass such fields as controlled clinical trials and evaluation of
health programs.
Research Opportunities
As indicated earlier, investigation of waterborne disease
outbreaks is largely a matter of identifying the pathogen, determining
where an equipment failure has permitted it to enter the water supply,
and repairing the damage.' The sophisticated methodologies of
contemporary epidemiology have not supplanted the traditional
52
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techniques deriving from the days of William Farr and John Snow in
such short-term problem resolution.
Nothing said in this chapter should be taken as a "put-down" of the
methods used in the epidemiologic investigation of waterborne disease
outbreaks. In a recent paper (14), Kuller elucidates the relationship
between chronic and infectious disease epidemiology, placing special
emphasis on the similarities between them. As he points out, all
epidemiplogic studies strive to identify the determinants of diseases in
order to decrease the societal burden of morbidity and mortality they
carry. The traditional goals of infectious disease epidemiology -
recognition of an agent, incubation period, mode of transmission, and
so forth - have proved extremely difficult for chronic disease
investigators to achieve. Because the incubation period of a chronic
disease is so long, and the spectrum of disease is so broad, these
traditional approaches have (perhaps unfairly) been superseded by
complex multivariate statistical techniques and the concept of
multifactorial etiology of disease.
The methodologies of chronic disease epidemiology certainly have
a place when considering the possible long-term effects of drinking
water consumption. Craun (15) has reviewed the evidence that
chlorination by-products, particularly trihalomethanes, might be a
risk factor for gastrointestinal and genitourinary cancers, as well as
for.cardiovascular diseases. In addition, some studies have found
associations between hard water and decreased risk of cardiovascular
diseases, and others have examined the possible effects of ingestion of
sodium or other salts on blood pressure and other physiologic func-
tions, •.--• ' • •'•-••
The seroepidemiology approach developed by Albert Evans may be
a valuable avenue for collaboration between infectious disease and
chronic disease epidemiologists. Evans advocates using serum banks
to investigate disease risk among persons seronegative or seropositive
to a particular organism, or to evaluate potential associations between
seroconversion and subsequent risk of disease (16). As new serologic
tests are developed, one might, for example, be able to estimate
baseline population exposure to waterborne pathogens. As new
pathogens/are characterized, stored,sera could be examined for
presence of antibodies to the organism. This method has already been
used to characterize a "Pontiac fever" outbreak (which occurred before
isolation of Legionella in 1976) as due to that organism, and to trace
the introduction of the HIV virus into the United States.
References
1. Hippocrates, The Genuine Works of Hippocrates. Translated
from the Greek by Francis Adams. Williams and Wilkens
Baltimore, Maryland, 1939. p. 19. '
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2. Slotki, J.J. Daniel, Ezra, Nehemiah. Hebrew Text and English
Translation with Introductions and Commentary. The Soncino
Press, London, England, 1951. p. 3.
3. Lilienfeld, A.M. and Lilienfeld, D.E. Foundations of
Epidemiology. Second edition. Oxford University Press, New
York, New York, 1980.
4. Snow, J. On the Mode of Communication of Cholera. Second
edition. Churchill, London, England, 1855. Reprinted as Snow on
Cholera. The Commonwealth Fund, New York, New York, 1936.
5. Last, J.M. A Dictionary of Epidemiology. Second edition. Oxford
University Press, New York, New York, 1988..
6. Evans, A.S. Causation and disease: The Henle-Koch postulates
revisite'd. Yale J. Biol. Med. 49:175,1976.
7. Bradford Hill, A. The environment and disease: Association or
causation. Proc. Roy. Soc. Med. 58: 295,1965.
8. Mausner, J.S. and Bahn, A.K. Epidemiology: An Introductory
Text. W.B. Saunders, Philadelphia, Pennsylvania, 1974.
9. Schlesselman, J.J. Case-Control Studies: Design, Conduct,
Analysis. Oxford University Press, New York, New York, 1982.
10. Traven, N.D. Data analysis: Estimating risk. In: Gr.F. Craun (ed,)
Methods for Investigation of Waterborne Disease Outbreaks,
Report No. EPA/600/l-90/005a, U.S. Environmental Protection
Agency, Cincinnati, OH, 1990, pp 65-74..
11. Rothman, K.J. Modern Epidemiology. Little, Brown and
Company, Boston, Massachusetts, 1986.
12. Mantel, N. and Haenszel, W. Statistical aspects of the analysis of
data from retrospective studies of disease. J. Natl. Cancer Inst.
22:719,1959.
13. Kahn, H.A. and Sempos, C.T. Statistical Methods in
Epidemiology. Oxford University Press, New York, New York,
1989.
14. Kuller, L.H. Relationship between acute and chronic disease
epidemiology. Yale J. Biol. Med. 60: 363,1987.
15. Craun, G.F. Surface water supplies and health. J. AWWA. 80(2):
40,1988.
16. Evans, A.S., Kirchhoff, L.V., Pannuti, C.S., and Carvalho, R.P.S.
A case-control study of Hodgkin's disease in Brazil, ll.
Seroepidemiologic studies in cases and family members. Am. J.
Epidemiol. 112: 609,1980.
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Epidemiologic Principles for the Study of
Waterborne Outbreaks
by:
Richard L. Vogt, M.D.
Vermont State Epidemiologist
Vermont Department of Health
Burlington, Vermont 05401
(803) 863-7240'
Introduction
There are at least three goals for investigating an outbreak
potentially caused by a waterborne agent. The first goal of any
Waterborne disease investigation is to protect the health of people at
risk by stopping the waterborne transmission of the agent. Early in the
investigation, this may require identifying another water source or
recommending boiling water that will be consumed if waterborne
disease is strongly suspected. The next goal is to determine the cause of
the outbreak in order to correct any problems that may be contributing
to contamination of the water system. This should provide longer term
protection against waterborne illnesses for the community. Finally, a
third goal is to learn about new aspects of waterborne disease that may
apply to other communities (1).
Outbreak Determination
Waterborne illness comes to the attention of a public health
agency through several mechanisms. Information may be obtained
frorii a variety of sources including health care providers, water supply
operators, town or local health officials, 'health department
surveillance reports, laboratory reports, the general public, and the
news media. Reports can vary from a single case of cholera, to a report
that a whole town appears to be ill. Frequently, the earliest challenge
is to determine if an outbreak has occurred.
After a health department receives a report of an Unusual
occurrence of disease that may be a waterborne outbreak, an
investigation is undertaken. The investigation may or may not be
carried out to completion, depending upon what is determined in the
early stages of the study. First, it is important to verify an outbreak
and determine the diagnosis of the ill persons, if possible. Next, health
55
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officials need to establish the extent of the problem by searching
reliable sources of data, including records in physicians' offices,
emergency rooms, and laboratories.
If a likely outbreak is identified, investigators must gather
demographic information and attempt to summarize the reports of
illness by three variables: person, place, and time. This
characterization will assist in testing scientific hypotheses about the
cause and possibly the effect of illness.
In order to organize the illness reports by person, place, and time,
investigators need to develop a case definition. The case definition is
determined by first identifying the predominant signs and symptoms
of ill persons in the outbreak and developing inclusion criteria. These
criteria serve to identify persons with illness that is characteristic of
the outbreak and exclude those who are well or ill with different
symptoms. Developing a case definition will enable the investigator to
specifically categorize those surveyed as "cases" or "noncaseS."
Investigators can then graph the numbers of cases of illness over time
to develop an epidemic curve.
The known incubation periods for commonly identified waterborne
pathogens are shown in Figure 3.2.1. Figure 3.2.2 shows an example of
a point source outbreak; that is, an outbreak caused by a single source
of contamination occurring over a brief period of time. Using an
epidemic curve that shows a pattern.typical of a point source outbreak,
one can determine either the incubation period of the agent or the
likely date of contamination if the other variable is known. This is
because the interval between the time of contamination and the peak
of the outbreak will approximate the incubation period of the illness.
Testing a Waterborne Hypothesis
If the early steps of the .investigation suggest that waterborne
transmission is likely, it is important to clearly establish drinking
water as the source responsible for the illness. Agents that can cause
waterborne illness may be transmitted through food and person-to-
person contact, as well. ,
Contaminated water can be ingested through many possible
vehicles, simultaneously. As Table 3.2.1 demonstrates, there is no
single source for infection from contaminated water. Combining all
exposures to unheated tap water may combine unequal risks and will
reduce the ability to determine the likely vehicle of infection.
To specifically test a hypothesis that water has caused illness,
there are several study designs that can assist the investigator. If the
outbreak was identified through health department reports of
reportable diseases, one can use the information to determine if there
is an excess of disease within a specific water delivery area. This
analysis can be useful if an outbreak seems to be continuing over a
56
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Organism
Campylotacter
Salmonella
Shigella
Giardia
Hepatitis A
Norwalk
Week
23456
Figure 3.2.1. Incubation periods of waterborne microbial agents.
long period of time. If during the process of an outbreak investigation
the causative organism is identified, it may be helpful to analyze
disease surveillance reports by geographic area. Information on
previous illness can serve as a comparison for the current case reports
or can tell the investigator whether the illness is a new or old problem
in that particular geographic area. Unfortunately, it may take some
time for reports to be received by the health department. As a result
health departments may not be able to create timely information
through this data source to assist in the investigation of possible
waterborne illness.
To test the hypothesis that water may have caused illness
investigators may conduct one of two more labor-intensive studies- the
follow-up or case-control study. A follow-up study first identifies a
community that has both individuals who are exposed and unexposed
to the probable vehicle of infection. Illness is then ascertained for each
of the exposed and unexposed groups of individuals and rates of illness
are calculated. An example of a group that could be used for a follow-up
study would be church members who drank water from a church well
that was contaminated with sewage. First, it is determined which
individuals did and did,not drink from the church water fountain and
subsequently, which individuals developed illness. '
Investigators may also use a case-control study design to test the
waterborne illness hypothesis. Cases are persons who have already
been identified by the "case definition" as having illness that may have
been caused by contaminated water. Controls are the noncases who are
57
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Number
of Case
14 _
13 -
12 -
11 -
10 -
9 -
8 -
7 -
6 _
5 -
4 -
3 -
2 -
1 -
S
I
I I
I
I
Average
Incubation
Period
First E
Exposure
r
I I
I
I
I I I I
-
—
-
—
—
—
-
—
-
i —
I
AM PM AM PM AM PM AM PM AM PM AM PM
12 13 14 15 16 17
January
Figure 3.2.2. Cases of gastrointestinal illness by date of onset.
in the same catchment area as cases. Example of cases could be persons
diagnosed with shigellosis from one doctor's office; controls could be a
sample of noncases taken from a list of all patients in the same doctor's
office. After cases and controls are selected for study, their exposure to
drinking water is determined. For example, a history of attendance at
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Table 3.2.1. Possible Vehicles for Waterborne Illness
Unheated Tap Water
Drinking Water
Water Used for Brushing Teeth
Unheated Tap Water Beverages
Orange Juice
Instant Iced Tea
Kool-Aid
Lemonade
Milk Made with Powdered Milk
•__• Ice
one church would be obtained and, even more specifically
consumption of water at the church water fountain would be
determined for each patient. The challenge with the case-control
method is to choose controls that have an equal likelihood of becoming
cases except for exposure to the agent in question. If cases were
identified through physicians' logs, controls should probably be
selected through this mechanism as well.
A questionnaire is administered to a survey population with both
study designs to obtain information on exposures and illness. With the
lollow-up study design, random-digit telephone or door-to-door
household surveys are two ways to administer the questionnaire to test
a hypothesis that water has caused illness. If people in either large or
small groups, such as persons in camps, those attending a family
gathering, or employees in an industry, can be identified as having
been exposed to contaminated water, the entire group can be studied If
sufficient numbers of individuals did and did not consume water it can
be determined whether contaminated water caused illness The ability
to statistically determine whether water caused illness is markedly
diminished if the vast majority of the group was exposed to the vehicle
In addition, if only a small group or sample of a large group is studied
it is important that the group is representative of others who may be
exposed to the contaminated water. Otherwise, it will be difficult to
generalize the results of the epidemiologic investigation beyond the
sample. .
Using the follow-up study design, Figure 3.2.3 shows how illness
rates, also called attack rates, are calculated for those who are and are
not exposed to contaminated water. If the rates are. higher for those
who are exposed compared to those who are unexposed, water may
have caused illness. One further calculation can be made with the
comparison- of illness rates, the calculation of the relative risk The
relative risk is defined as the illness rate of those exposed divided by
59
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the illness rate of those unexposed and indicates the magnitude of risk
for waterborne exposure (Figure 3.2.4). The resultant expression is a
ratio that compares the relative likelihood of becoming ill after
exposure to the likelihood of illness for those unexposed. Illness rates
and relative risks cannot be calculated in the case-control study. An
estimate of risk called the relative odds, also called the odds ratio (OR),
is calculated instead. The OR ratio is interpreted in the same way as
the relative risk.
Exposed
Illness Rate (Exposed) = A/(A + B) (%)
Illness Rate (Unexposed) = C/(C + D) (%) '
Figure 3.2.3. Calculation of illness rates in a follow-up study.
If an investigator can show a positive exposure-response
relationship (a greater likelihood of illness with more reported
consumption of water), a greater assurance is provided that water was
the cause of that illness. A positive exposure-response relationship
tends to reinforce other significant findings of an investigation. An
example of a positive exposure-response relationship is shown in
Figure 3.2.5.
Laboratory Analyses
In addition to' administering the questionnaire and gathering
other data on potential exposures that may assist in determining
whether illness was caused by water consumption, it is important for
the investigator to attempt to determine the agent that causes illness.
In half of the waterborne outbreaks reported between 1971- 1985, no
causative agent was identified (2). Good laboratory support and timely
investigation are necessary to identify etiologic agents.
At the Vermont Department of Health, fecal and sera samples are
collected in most investigations of outbreaks that could be caused by a
waterborne agent. Stool samples or rectal swabs are collected and
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Exposed
Relative Risk
(A + B)
(C + D)
Odds Ratio =
AD
BC
Figure 3.2.4. Calculations for measurement of risk.
transported in Gary, Blair media. In addition, stool samples are
collected in sterile containers that will be refrigerated at 5°C for
possible virus identification. Finally, stool samples preserved in
formalin are collected if parasites are considered a possible agent of
illness. Acute and convalescent sera are drawn for antibody tests for
the Norwalk-like viruses and other experimental antibody tests that
are being developed for Giardia, Campylobacter^ and other agents At
least 12 stool swabs, 12 stool samples, and 12 acute and convalescent
sera samples are usually collected from both ill and well persons in an
outbreak investigation. The Vermont Department of Health is capable
of analyzing stool samples for bacterial and parasitic agents. Other
diagnostic work is sent to laboratories outside of the state.
Investigation Caveats
There are many challenges to the investigation of waterborne
outbreaks. Researchers have been justifiably concerned about
exposure information collected from questionnaires. Specifically the
ability to accurately recall any event, such as water consumption will
decay over time. It is best to minimize this decay of information by
surveying the population as soon after an outbreak as possible It is
recognized that responses provide, at best, estimates of water
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Illness Rate (%)
Glasses of Water per Day
Figure 3.2.5. Exposure-response relationships in an outbreak of waterborne
illness.
consumption and that answers are estimates and not precise measures
of water exposure.
Diarrhea can be another confounding factor for an epidemiologic
investigation. It is quite possible that investigators are not really
testing the hypothesis that water consumption caused diarrhea as
much as showing that ill people tend to drink more water than those
who are well, to replace fluids. Questions concerning water
consumption need to be specific about water exposure prior to the onset
of the current illness.
Secondary person-to-person transmission may cloud an association
between illness and the consumption of water. To analyze data in this
situation, it may be advantageous to study only early outbreak cases if
the agent is known to be spread easily from person to person. In
addition, since many investigations really sample households through
random-digit and door-to-door surveys, data should be analyzed by
affected households instead of affected persons.. This analysis will
minimize the possible confounding effect of person-to-person
transmission within households.
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Although it may seem attractive to select persons in one township
as a control for a township that has been affected by consuming
contaminated water, there can be problems with this approach The
populations between the two towns should be similar except for
exposure to contaminated water. If the comparison township has fewer
children in day-care or has residents who are in a higher socioeconomic
status, it may not be suitable as a comparison for the affected town
Any differences in illness rates between these two townships may be
due to these possibly confounding factors instead of exposure to
contaminated water. Similarly, if the comparison township has a
greater number of children in day-care or has a lower socioeconomic
status one may have difficulty determining whether illness was
caused by contaminated water in the affected township. The infectious
agent could have been transmitted from person to person through
affected day-cares instead of through contaminated water
Fortunately there are now techniques to evaluate and control some
forms of confounding after studies are conducted (3).
Conclusion
_ In an investigation of a potential waterborne outbreak it is
important to follow these steps:
• Obtain information about the nature and extent of illness
• Determine if water is a likely hypothesis
• Conduct an epidemiologic study to test the hypothesis
• Conduct an environmental study of the water
• Analyze the data
• Develop conclusions
• Institute control measures at appropriate points in the
investigation
Thorough epidemiologic and environmental investigations are
necessary for collecting scientific evidence that will show that
contaminated water caused an outbreak of illness.
References
1. Rosenberg M L^ A guide to the investigation of waterborne
outbreaks. J. AWWA^ 69: 4106-4110,1977.
2.
IQQC *,~~\~j.—r Contro1- Water-related disease outbreaks,
1985. Morbidity Mortality Weekly Report CDC Surveillance
Summaries. 37(55-2): 15-24,1988.
3. Rothman, K. J. Stratified analysis. In: Modern Epidemiology
Little, Brown and Company, Boston, Massachusetts, 1986. p. 177.
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Data Analysis: Estimating Risk
by:
Neal D. Traven
Department of Epidemiology
Graduate School of Public Health
University of Pittsburgh
Pittsburgh, Pennsylvania 15261
(412) 624-0097
Introduction
After investigators have amassed information about a suspected
waterborne disease outbreak; these data must be analyzed statistically
in order to test hypotheses about the data under study. Statistical
analysis of questionnaire data collected from persons who may have
been exposed to microorganisms suspected of causing disease is an
essential component of the investigation of waterborne disease
outbreaks.
Guidebooks for outbreak investigators often include such
procedures as determination of the incubation period and graphing of
the epidemic curve in their section on statistical analysis (1). Those
techniques, however, are useful largely in identification of common
source outbreaks and their possible causes rather than in evaluating
statistical associations between the disease and environmental risk
factors.
This chapter assumes that a disease-causing organism has been
characterized and that the outbreak has been identified as arising
from a common source. It also assumes that the collected data are
accurate and unbiased, and that the questions asked of interviewees
are appropriate and well-designed. Our focus is instead on the
assessment of associations between the disease and other factors. The
statistical tests discussed below can assist the investigator in
estimating the strength of suspected associations with environmental
exposures.
Among the topics covered in this report are point estimates of risk
(rate difference, relative risk, and odds ratio) and the associated
confidence intervals of these risk estimates. Equations for calculating
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these quantities are given, along with information which may aid the
investigator in deciding which statistics to calculate in the analysis of
a data set. Where appropriate, illustrative examples are presented
from data taken from an actual waterborne disease outbreak
investigation. In addition, the two types of statistical error will be
defined. The interrelationship between sample size and statistical
power, which is a function of Type II error, will be described and
characterized.
Definitions and Notation
Before discussing epidemiologic data analytic methodologies, it is
necessary to present some general notation that will be used
throughout this chapter. Underlying much of epidemiologic data
analysis is the fourfold table shown in Figure 3.3.1. In this generic
form, both the disease and the environmental exposure under
investigation are dichotomized. The letters a, b, c, and d represent the
number of responses in each cell; the values HI, n%, mi, and m% are the
marginal totals for each category; and N is the total number of
respondents in the entire analytic table.
Exposed
Yes
No
Disease
Yes No
a
c
b
d
Figure 3.3.1. Epidemiologic data analysis: generic 2x2 table notation.
The essence of epidemiologic data analysis is comparison. To
assess the significance of an epidemiologic association, one must
establish a reference level with which to compare the exposure of
interest. For example, nearly all heroin addicts drank milk in
childhood, but it would be incorrect to conclude from this fact that
drinking milk is associated with heroin addiction. One would probably
find a similar milk consumption history among nonaddicts as well,
In making epidemiologic comparisons, it is essential to place the
quantities being compared on an equal footing. Looking only at the
raw numbers of diseased and healthy individuals, for example, can be
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misleading. Instead, epidemiologic comparisons are often drawn
between rates, defined as the number of persons with a certain
characteristic divided by the total population, in a specified time
period (2). Such comparisons may also be drawn on proportions, in
which the value in the numerator is included in the denominator (3).
One can carry out both difference and ratio comparisons of rates; both
forms offer important information about the magnitude of risk.
In these comparisons, the best single value estimate of the effect in
question is called a point estimate. While the point estimate indicates
the magnitude of the comparison, it does not take into account the
degree of random variability in the observations. To allow for this
variability, the researcher can estimate the effect using a range of
values. This range is called the confidence interval, and the process of
calculating it is interval estimation (4). The width of the confidence
interval depends on both the variability of the data and an arbitrarily
chosen level of confidence. A general formulation for any confidence
interval is:
(point estimate) + (z-score)-(std dev)
where z-score is the value of the Gaussian normal distribution
corresponding to a preselected confidence level, and std dev is the
standard deviation of the point estimate (the standard deviation, of
course, is a measure of variability).
To apply the equations in this chapter, examples will be presented
using data derived from an actual investigation of a waterborne
disease outbreak. In this 1974 study (5), the causative agent was
Shigella sonnei. A community whose well was contaminated with the
organism was compared with a neighboring community served by an
uncontaminated well. The fourfold table from which these examples
are derived is displayed in Figure 3.3.2. The interval estimates in
these numerical examples will all use 95 percent confidence intervals
(z-score 1.96).
Epidemiologic Study Design
The hallmark of the epidemiologic study is analysis of the
relationship between groups of persons with the disease condition
under study and other persons, otherwise comparable to those persons,
who are free of the disease. Figure 3.3.3 presents a typology of
epidemiologic study designs proposed by Lilienfeld and Lilienfeld (6).
Experimental studies and trials are usually carried out to test the
safety, efficacy, and utility of pharmaceutical products or other
therapeutic modalities. Though experimental trials have been
employed to determine infectious dose of some pathogenic organisms,
this form of epidemiologic study will not be discussed in this chapter.
Most epidemiologic research studies are observational in design.
The two major types of observational designs are cohort and case-
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Affected Community Yes
Control Community No
Diarrheal Disease
Yes No
67
16
286
276
353
292
83 562 645 ' ,:
Figure 3.3.2. Epidemiologic data analysis: numerical example (S. sonnei).
Epidemiologic Studies
Random
Assignment
Clinical Trial
Sampling by
Disease
X-secf/
refrospecf/ve
Figure 3.3.3. The Uilienfelds' typology of epidemiologic studies.
Adapted from (6).
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control studies. These designs differ with respect to the theoretical
populations from which subjects are drawn.
In a cohort study, participants are selected based on their risk
factor status, then followed through time to determine their disease
status at some future date; hence its alternative title, "prospective"
study. At the beginning of a cohort study, all participants are free of
observable disease.
In contrast, a case-control study.samples diseased persons and
comparable persons without the disease, then examines their personal
histories to determine whether prior risk factor differences are
associated with their current disease status. Case-control studies have
also been called "retrospective" studies. Cross-sectional studies, in
which the disease and the risk factor are examined at the same time,
are analytically similar to the case-control design.
The Lilienfelds' typology is just one of several such frameworks put
forward by epidemiologists. One might, for example, establish a cohort
based on common exposure in the past, then follow this historical
cohort into the present; this design is used regularly by occupational
epidemiologists. The nested case-control design draws its cases and
controls from the population base of a preexisting cohort study. In an
ecologic study, the unit of measurement and.analysis is aggregate data
rather than information on specific individuals.
Statistical Tests
Chi-Square Test of Association
The standard statistical test used to analyze data in a fourfold
table is the chi-square test of association. In this procedure, the sample
chi-square is calculated and compared with the entries in a table of
standard chi-square values^ Using the notation introduced in Figure
3.3.1, chi-square can be computed by substituting into the following
equation:
(ad - be) • N
(1 degree of freedom [d.f.])
iy n2- ny m2
Algebraically equivalent formulas for computation of chi-square
are available in any statistical textbook; suggested sources include
Snedecor and Cochran (7) or Fleiss (8). All are variations on the
concept of squaring the difference between the observed and
expected" values, and dividing by the expectation. It should be noted
that the above chi-square equation differs from those found in most of
these statistical texts in that it does not include the "correction for
continuity." This quantity was introduced by Yates (9), and has been
the subject of controversy in the statistical community for decades. In
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large studies, the Yates correction makes very little difference in the
calculated chi-square statistic.
In the numerical example (Figure 3.3.2) the calculated chi-square
is 25.98, indicating that the disease is strongly associated with the
contaminated well (with the Yates correction the value is 24.79, very
slightly lower than the uncorrected result).
Rate Differences
Outbreak investigators should be familiar with this statistical
measure, because tables of "attack rates" are nothing more than sets of
rate difference comparisons. The point estimate of the attack rate
difference (RDa) is calculated by simply subtracting the disease rate in
the reference group from the disease rate in the affected population:
RDa = (a/mi) - (c/m2)
Because it is a function of two proportions, the RD statistic ranges
between -1 and +1; its null value is zero. Its distribution is symmetric
about the null value.
For calculating the interval estimate of attack rate differences, the
standard deviation term is derived as the sum of two binomial
variances (3):
RD + 1.96-V[(ab/m13) + (Cd/m23)]
It should be noted that there are methods other than the standard
deviation approach for calculating interval estimates. Among them
are Miettinen's test-based method (10) and the iterative method
developed by Cornfield (11). The test-based method is easiest to
calculate, but it has been shown to be unreliable in situations where
the statistic is very distant from the null hypothesis value (12).
Cornfield's method is more accurate, but its computational complexity
requires the use of a computer and specialized software. The standard
deviation method given in this chapter, then, is a good compromise
choice between those of Miettinen and Cornfield.
In the numerical example, the calculated difference between the
disease attack rates of the study communities is (0.190 - 0.055) =
+ 0.135. The 95 percent confidence interval estimate covers the range
(+0.086, +0.184). Because this interval does not contain zero, the null
value under the hypothesis of no association, it is reasonable to
conclude that there is a significant difference between the incidence
rates in the two communities and that this difference is approximately
135 per 1,000 residents.
Relative Risk
While the RD statistic describes the difference between incidence
in the exposed and control populations, the relative risk statistic (RR)
consists of the ratio of those incidence rates. Its equation is:
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a/m
RR=
Relative risk takes values in the range from zero (when a is zero) to
positive infinity (when c equals zero). The null hypothesis value is
unity, at which point the incidence rates in the exposed and unexposed
populations are equal.
In contrast to the rate difference, the distribution of the relative
risk is asymmetrical. It is necessary to transform the statistic in some
manner in order to calculate a standard deviation. After this step, one
must then perform the inverse transformation to produce the
confidence limits for the interval estimate of relative risk. These limits
are symmetric about the point estimate in the transformed scale but
are not symmetric after retransformation to the original scale.
The appropriate transformation in this situation is logarithmic.
The natural log of the relative risk, or ln(RR), is a linear function
ranging from negative infinity to positive infinity with a mean of zero.
Thus, Rothman's equation for calculating the interval estimate of the
relative risk (3) is.
exp{ln(RR) ± 1.96-V[(b/ann) + (d/cm2)]}
In the numerical example, the point estimate of the relative risk is
3.46; the incidence rate in the affected community is about three and
one half times that of the unaffected community. The researcher
calculates the standard deviation of ln(RR), computes the interval
estimate of the transformed variable, and then takes the exponential
of the confidence limits. This procedure results in a 95 percent
confidence interval for relative risk with the range (2.05, 5.84). This
interval does not include the null hypothesis value of unity, so one can
conclude that there is a significantly increased risk of the disease in
the study community with contaminated water.
Odds Ratio
In many situations, it is not appropriate to calculate the relative
risk. The primary obstacle is that incidence rates cannot be calculated
in cross-sectional or case-control studies, in which sampling is carried
out based on disease status. In such studies, the sampling fractions for
cases and controls are necessarily quite divergent; one always samples
a far higher proportion of all diseased persons than of nondiseased
persons (13).
The odds ratio (OR), defined below, is an extremely useful
alternative to the relative risk. For rare diseases, it closely
approximates RR. It can be determined from samples based on either
disease status or exposure status, as long as it can be assumed that the
study cases are representative of the diseased population, and that the
controls represent the nondiseased general population (6).
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OR=
This statistic has gained favor in epidemiologic data analysis for a
number of reasons. In addition to those outlined above, it is easy to'
compute, the variance of its logarithm is appealingly simple (see.
equation below), and the coefficients in logistic regressionr;'ah
important multivariate statistical technique, are equivalent to, odds
ratios. The interval estimate of the OR is: , , • , : .
exp{ln(OR) ± 1.9
The odds ratio for the numerical example equals 4.04. The 95
percent confidence interval, or interval estimate of the OR, covers. the •
range (2.29, 7.15). Once again, this confidence interval does not
contain the null hypothesis value, which is 1.00 for the odds' ratio.
Therefore, one concludes that the odds of diarrheal disease are
significantly higher in the affected community than in the comparison
community. The best estimate is that the probability of disease in the
affected community is four times as high as in the unaffected
community.
Sample Size and Statistical Power
An epidemiologic study should include enough subjects to control
for two sources of statistical error. Type I or a-error occurs when one
claims that an association exists between two factors (in this case,
exposure and disease), when no such association truly exists. In Type II
or 0-error, the researcher claims that there is no association between
the factors, when in actuality there is a real association. Of course, it is
impossible to know the true state of association between the factors (if
it were possible, there would be no reason to do the study!).
Standard statistical hypothesis testing is concerned with
estimating the degree of a-error. In part, this concentration on. Type I
error is an artifact of the historical development of statistical theory.
The turn-of-the-century founders of modern statistics (R.A. Fisher,
Karl Pearson, and others) worked in agricultural research, where Type
I error was far more important than Type II error; serious food
shortages might result if the claim that a new fertilizer would improve
crop yields turned out to be incorrect.
Statistical power is defined as the quantity, 1 - p. If a true
association does exist in the population, then the power of the study is
the probability of finding a sample odds ratio significantly different
from the null value of unity.
Several equations are available for calculating the power of a
sample (the details are beyond the scope of this chapter). The
unknowns in such equations are a-error, {5-error, minimum detectable
odds ratio, and sample size. By fixing three of these quantities and
solving for the fourth, the researcher could: 1) determine the required
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sample size for a given level of a-error, 0-error, and minimum odds
ratio; 2) calculate the power of the study to detect a given odds ratio at
a selected level of a-error; or 3) estimate the smallest detectable odds
ratio for a study with given a-error, £5- error, and sample size.
Discussion
Choosing the right statistical tests for comparing and estimating
disease risks is more an art than a science. In general, the odds ratio is
preferable to the relative risk, since it is a valid statistic for both
disease-based and exposure-based study designs. Its simplicity and its
extendibility to multivariate analysis increase its utility in
epidemiolbgic research.
In situations where it is appropriate to calculate disease incidence
rates (i.e., when sampling with respect to the exposure variable), both
the rate difference and either the relative risk or the odds ratio are
appropriate statistical measures. In the numerical example in this
chapter, sampling was undertaken based on the community of
residence; the researchers were evaluating disease rates in areas with
different water supply systems. Hence, there was justification for
calculating both the RD and RR statistics in addition to the odds ratio
(OR).
These methods for analyzing fourfold tables can be extended in
many ways. There are epidemiologic methods for analyzing variables
with more than two levels, for investigating the simultaneous effects of
many variables, for studying interactions among variables, for
matched analysis, and many more.
References
1. Bryan, F.L. Epidemiologic procedures for investigation of
waterborne disease outbreaks. In: G.F. Craun (ed.),
,'. , Waterborne Diseases in the United States. CRC Press, Boca
• Raton, Florida, 1986. p. 171.
2. Mausner, J.S. and Bahn, A.K. Epidemiology: An Introductory
Text. W.B. Saunders Co., Philadelphia, Pennsylvania, 1974.
3. Last, J.M. A Dictionary of Epidemiology. 2nd edition. Oxford
University Press, New York, New York, 1988.
4., Rothman, K.J. Modern Epidemiology. Little, Brown and
Company, Boston, Massachusetts, 1986.
5. Weissman, J.B., Craun, G.F., Lawrence, D.N., Pollard, R.A.,
Saslaw, M.S., and Gangarosa, E.J. An epidemic of
gastroenteritis traced to a contaminated public water supply.
Am. J. Epidemiol. 103: 391,1976.
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6. Lilienfeld, A.M. and Lilienfeld, D.E. Foundations of
Epidemiology. 2nd edition. Oxford University Press, New
York, New York, 1980.
7. Snedecor, G.W. and Cochran, W.G. Statistical Methods. 7th
edition. The Iowa State University Press, Ames, Iowa, 1980.
8. Fleiss, J.L. Statistical Methods for Rates and Proportions. 2nd
edition. John Wiley & Sons, New York, New York, 1981.
9. Yates, F. Contingency tables involving small numbers and the
chi-squaretest. JRoy. Stat. Soc. Suppl. 1: 217,1934.
10. Miettinen, O.S. Estimability and estimation in case-referent
studies. Am. J. EpidemioL 103: 226,1976.
11. Cornfield, J. A statistical problem arising from retrospective
studies. In: Proceedings of the Third Berkeley Symposium,
Volume IV. University of California Press, Berkeley,
California, 1956. p. 135.
12. Gart, J.J. and Thomas, D.G. The performance of three
approximate confidence limit methods for the odds ratio. Am.
J. EpidemioL 115: 453,1982.
13. Schlesselman, J.J. Case-Control Studies: Design, Conduct,
Analysis. Oxford University Press, New York, New York,
1982.
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Avoiding Bias: Systematic and Random Error
by: Patricia A. Murphy
Health Effects Research Laboratory
Human Studies Division
U.S. Environmental Protection Agency
26 West Martin Luther King Drive
Cincinnati, OH 45268
, (513) 569-7226
Introduction
The investigation and control of communicable disease epidemics
has long been considered the paradigm of epidemiologic study. Recent
methodologic developments and a greater emphasis on the study of
chronic, noninfectious diseases have helped to force a change in
epidemiology. Along with this shift there has been a trend to analyze
data from an epidemic investigation using some of the more
sophisticated statistical procedures developed for the study of chronic
disease. Data that may be appropriate for confirming the existence of
an outbreak, however, are.not always adequate for further robust
statistical analysis. As this trend in analysis becomes increasingly
common, it is imperative to be aware of the potential limitations of any
inferences made in epidemiologic investigation where findings are
sometimes based on a small, biased sample of the population.
It is well recognized that investigations of waterborne disease
transmission almost always take place in the public eye. The
epidemiologist must make rapid decisions regarding control of an
epidemic, and the decisions may later affect inferences that can be
drawn from the available data. In particular, the expedient collection
of information may result in certain biases or error. Although all
outbreak situations have unique characteristics, certain information
must always be collected. Investigators must begin to develop a
standardized way to collect this information (for example, amount of
water consumed) and minimize measurement error in epidemiology
studies wherever possible. Etiologic research standards must be
applied to all data, including those from outbreak investigations, that
are analyzed and presented as if they were originally collected for that
purpose.
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All epidemiologic studies, whether basic etiologic research,
compilations of descriptive statistics, or outbreak- investigations,
require some type of quantitative measurement. For example, the
purpose may be to define the prevalence of a gastrointestinal illness in
residents of a nursing home or to determine the amount of
contaminated drinking water consumed by persons with and without a
particular illness. A major goal of most epidemiologic investigations is
to make a quantitative statement about the relationship between a
particular exposure and a particular disease in a population. In order
to draw accurate and valid conclusions from this work, investigators
should strive to make these measurements as error-free as realistically
possible in any given situation. This goal will be realized only through
awareness of the major sources of error that exist, the likelihood of
their occurrence, and the manner in which measurements may be
affected. This article presents an overview of some of the major sources
of measurement error that can bias epidemiologic study results and
interpretation.
Background and Definitions
In an epidemiologic investigation, the process of quantifying the
relationship between an exposure and a disease is referred to as effect
estimation. The usual effect estimates that are calculated include the
rate ratio or relative risk (RR), the rate difference (RD), and the odds
ratio (OR). Methods for their calculation are detailed in the preceding
article by Traven (1). If the measured effect differs in some way from
its true value it is said to be biased. The primary considerations for
evaluating the accuracy of an epidemiologic measure are precision, or
lack of random error, and validity, or lack of systematic error. Within
this realm, there are many factors that may potentially, and in some
cases assuredly, lead investigators to make a biased estimate. A brief
outline of some of the recent developments in epidemiology over the
last two decades will help place in perspective the recognition of the
great role that bias may play in effect estimation.
Historically, most epidemiologic research involved investigations
of epidemics of infectious disease. Great improvements in social and
medical well-being during this century forced a shift away from the
study of infectious diseases and toward the development of the ever-
growing field of chronic diseases such as cancer and cardiovascular
disease. This shift has been instrumental in the development of newer
and more sophisticated techniques for the analysis of data generated
during an investigation. It has also contributed to a greater
understanding of the conceptual basis for specific study designs,
particularly the case-control study. Case-control studies were
employed with growing frequency, and as the popularity of this
approach to research grew, it was realized that certain features
inherent in a retrospective approach to the study of the causes of
disease created a great potential for the acquisition of biased
information. This realization led many scientists to believe that there
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was something inherently wrong or bad about retrospectively based
investigations. In fact, what should have been questioned was not the
retrospective approach in general, but rather the unfortunate lack of
attention to details of study design by persons inexperienced with the
use of this type of study. An enormous controversy grew among leading
epidemiologists, and grave reservations were expressed as to the
validity of this design option.
In an effort to openly discuss the problems and reservations about
case-control studies specifically and retrospective study designs in
general, a symposium was held in 1978, and a special issue of the
Journal of Chronic Diseases (2) was devoted to the proceedings and
discussions. One major point of contention related to the many places
in a study where some form of bias could be operating. There was also
grave concern about the scientific quality of many of the studies
appearing in the literature and the apparent lack of standards for high
quality case-control research. It was at this symposium that Sackett
(3) presented a paper on bias in analytic research, wherein he outlined
a catalogue of 35 biases that may crop up in a retrospective study. This
was the first comprehensive attempt at such an endeavor and marked
what was probably a turning point in the relatively new field of
chronic disease epidemiology. Although many investigators were
aware of the existence of and potential for bias to occur, it was
apparent that most researchers had not thought of the impact of bias
as being quite so far-reaching. It was at the same symposium that
Ibrahim and Spitzer (4) succinctly classified bias into three major
categories of systematic error: selection bias, information bias, and
confounding bias. This classification has received widespread
acceptance and most modern textbooks of epidemiologic methods
present the concept in this way (5-8). The latest edition of the
Dictionary of Epidemiology (9) lists 26 definitions of specific biases
and, like Sackett's list (3), they can all be classified into one of these
three groups. Empiric investigations of the impact of bias in
epidemiologic research have further demonstrated the need for
consideration of all sources of error in investigations (10,11). The
results from waterborne outbreak investigations can also be affected
by the same kinds of error, some of which will be considered in the next
section.
Precision and Validity
A thorough epidemiologic analysis must assess bias, confounding,
causation, and chance as explanations for the observed results (12).
Implicit is the assessment of the various components of error present in
effect estimators and occurrence measures. This section briefly reviews
the concepts of systematic and random error and their likely impact on
epidemiologic measures derived during the course of an outbreak
investigation; Whenever possible, examples are specifically geared to
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address the potential for occurrence in a waterborne disease outbreak
investigation.
Precision
Precision refers to the lack of random error in a measurement.
Random error has many components, but the major contribution comes
from the process of selecting subjects for inclusion in a study. This is
known as sampling error (6). Even if all cases of disease are included as
part of an investigation, the subjects are always considered to be a
sample and there will be some degree of sampling error present (6).
One measure of precision can be obtained by calculating a confidence
interval (CD of an arbitrarily chosen significance, usually constructed
as a 95 percent CI. A wide CI indicates a lack of precision in the
estimate. CIs are occasionally interpreted as tests of statistical
significance but their primary function, assessing measurement
variability, should not be forgotten. The p-value is usually reported in
an outbreak investigation as a measure of statistical significance.
Although often cited as evidence that a particular hypothesis has been
proved, the p-value is not the probability that the tested hypothesis is
true, but rather the probability that chance alone would produce a
difference between the compared groups at least as big as the one
observed (5). A small p-value indicates that it is unlikely that chance
or random error is the sole explanation for a study's findings, but it
reveals nothing about the role of other forms of error.
The most common way to increase the precision of a study is to
increase the sample size. Usually, this is not possible in an outbreak
investigation, as there will be only a finite number of cases that come
to medical attention and are included as study subjects. This limitation
cannot be changed; however, an investigator can make more efficient
use of the available information by either matching, increasing the
ratio of controls to cases, or appropriately restricting the study
population. These design options will help to increase the precision of
measurement when accompanied by appropriate analytic treatment.
There will always be some degree of imprecision associated with any
epidemiologic measure. An investigator should therefore provide a
quantitative estimate of this imprecision and temper all
interpretations accordingly.
Validity
When a study is considered "valid," it usually means that there is a
lack of systematic error affecting the results. It should be thought of in
terms of both internal validity and external validity, with the latter
referring to the extent to which the study results can be generalized to
another population. Before the results are projected in this way, the
internal validity must be assessed. As mentioned in the introduction,
numerous biases can occur and detract from the internal validity of a
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study; these are generally classified as selection bias, information bias
and confounding bias. '
A selection bias results from procedures used to select subjects that
lead to an effect estimate among the included subjects differing from
that occurring in the entire population of available cases (6). There are
many ways in which selection bias can occur in an epidemiologic
study. The common element underlying all selection bias is that the
relation between exposure and disease is somehow different for those
who participate and those who would be theoretically eligible for study
but do not participate. Some degree of selection bias is likely to occur in
an epidemic investigation. For example, many if not all cases have
volunteered to be included for study, simply by going to their physician
or the emergency room for treatment of their illness. Less severely ill
cases may not seek any medical care and would never be recognized as
cases. This could lead to both an underestimate of the total population
affected during an outbreak and, perhaps to a distortion of the
proportions possessing various risk factors of interest. The investigator
should anticipate the various selection biases that could be operating
m any particular study and provide an objective assessment both of the
likelihood of occurrence and the way in which results could be affected.
The second major category of bias likely to be present in any
epidemiologic study is information bias. The inherent imprecision of
the retrospective questionnaire as an instrument for data collection
can be a major cause of this form of bias. If the information obtained
from the study participants is influenced by the group to which the
participant belongs (e.g., a case or a control, exposed or not exposed)
validity will be compromised (5). Information bias can occur whenever
there are errors in the classification of study subjects, but the
consequences of the bias will be very different depending on how it
occurs. For the sake of simplicity, assume that disease classification is
perfect, i.e., all persons classified as cases for the purposes of study are
truly cases and those classified as controls truly do not have disease
and that errors occur only when assessing exposure. This is another
way of stating that the sensitivity and specificity of the disease
measure is 100 percent. Any time the sensitivity and specificity of a
measure is less than 100 percent, there will be some misclassification
If the exposure misclassification is independent of the person's disease
classification, then the misclassification is said to be nondifferential or
random. On the other hand, if one group is more likely to be
misclassified than the other, the misclassification is said to be
differential or systematic.
It is well known that random misclassification always results in
the OR being biased toward the null, resulting in an underestimate of
the association (10). When the misclassification turns out to be
systematic, however, it is not easy to predict either the magnitude or
direction of bias in the OR. There are formulas available for correcting
biased ORs, but it is necessary to know the sensitivity and specificity of
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the measure. This information generally is not available. Given the
paucity of data for correcting estimates, the superior approach is
clearly to try to anticipate and minimize misclassification.
Perhaps the most well-known type of information bias is referred
to as recall bias. Recall bias is a distinct possibility in any study where
information on past exposures is obtained. If cases are likely to recall
the same event differently than the controls, recall bias will result. It
is not hard to imagine examples of bias of this type. For example, by
the time a case-control study is conducted during an outbreak
investigation, it is likely that water has already been implicated as the
vehicle of transmission (13). Since cases know their disease is
waterborne, they may erroneously recall consuming larger quantities
of water than they actually did. This type of recall bias could result in
the appearance of a dramatic dose-response related to number of
glasses of water consumed. On the other hand, it could also mask a real
dose-response that truly does exist. It is up to the individual
investigator to assess the likelihood of any bias occurring and the most
likely way in which the results may be distorted. It is likely that any
attempt to measure retrospectively the amount of fluid consumed
during a particular point in time will be biased to some degree.
Investigators need to recognize the limitations of these kinds of data to
avoid overinterpreting findings. The situation may become further
complicated when biased exposure data are used for sophisticated
statistical modeling such as multiple logistic regression. The
application of these techniques may possibly result in a further
distortion of the effect estimate with unwarranted credence
subsequently given to the.findings. These are but a few of the ways
that misclassification can affect study results.
The third major category of bias to be considered is that of
confounding bias. The concept of confounding is an extremely
important one in modern epidemiology and requires more attention
than space permits in this chapter. The interested investigator is
referred to one of the recent textbooks in epidemiology for a complete
discussion of the issue (5-8). Rothman (6) has defined confounding as a
mixing of effects: the estimate of the effect of the exposure is distorted
because it is mixed with the effect of an extraneous factor. The
distortion can be large or small, and it can lead to both over- and
underestimation of an effect depending on the direction of the
associations the confounder has with exposure and disease. It is
possible for confounding even to change the apparent direction of an
effect (6). Investigators should always anticipate confounding and
collect appropriate data to control for its effects during the analysis if
necessary. Matching is one way to control for confounding at the
design stage but will not be appropriate or feasible in every case. In
order for the matching to be effective, investigators must also use
appropriate analysis techniques. Stratification of the data is the most
straightforward way of controlling and assessing confounding (1) in
the analysis stage, although other techniques such as multivariate
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modeling are frequently used. The appropriateness of any one means of
controlling confounding will always be study-dependent and must be
assessed on an individual basis.
Probably the most important thing to remember is that the
presence of bias in and of itself does not necessarily invalidate an
epidemiologic study. It should be incumbent on any investigator to
thoroughly examine the methods employed in the study and identify
areas where certain biases may exist. The investigator should
anticipate what biases might occur, try to prevent or control them, and
always try to estimate at least the direction of bias that might be
present. Selection and information biases always must be anticipated
and prevented since they cannot be controlled statistically during the
analysis. Confounding can be totally prevented in many instances, but
it can usually be controlled analytically if appropriate data have been
collected.
Discussion
Epidemiologic research in the midst of an outbreak situation is at
best a formidable task. Investigators of these epidemics should be
commended for the quality of the data collected in the heat of intense
public scrutiny and under extreme constraints of time. The fact that an
outbreak will only leave a certain number of cases for epidemiologic
investigation means that one must accept the fact that any measures
of disease occurrence or association (RD, RR, OR) will be somewhat
imprecise due to the limited sample size. The lack of precision,
however, should not influence unduly the credibility of the results. The
investigator should always be aware of the inherent limitations of
small sample sizes and, wherever feasible, try to make more efficient
use of the limited data points available. Some estimate of the degree of
imprecision of an OR or RR, such as a CI, should always be calculated.
A trend toward more statistically sophisticated analysis of
outbreak data has been noted and will most likely continue. The
anticipation of this type of analysis should lead to more attention being
paid to collecting all the data needed for a thorough assessment of bias
and confounding necessary to reach a valid conclusion. Only by
recognizing the potential for occurrence can flaws be either prevented
or detected and corrected so that study inferences will be on target.
Every epidemiologic study, regardless of purpose, should be held to
certain accepted standards if epidemiologists are to continue to
maintain their well-deserved place as the basic scientists of public
health.
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References
1. Traven, N.D. Data analysis: estimating risk. In: G.F. Craun
(ed.), Surveillance and Investigation of Waterborne Disease
Outbreaks. U.S. EPA, Cincinnati, OH. 1970.
2. Spitzer, W.O. (ed.). The case-control study: consensus and
controversy. J. Chron. Dis. 32:1-144,1979.
3. Sackett, D.L. Bias in analytic research. J. Chron. Dis. 32:51-
63,1979.
4. Ibrahim, M.A. and Spitzer, W.O. The case-control study: the
problem and the prospect. J. Chron Dis. 32:139-144,1979.
5. Ahlbom, A. and Norell, S. Introduction to Modern
Epidemiology. Epidemiology Resources, Inc., Chestnut Hill,
MA, 1984.
6. Rothman, K.J. Modern Epidemiology. Little, Brown and
Company, Boston, MA, 1986.
7. Kelsey, J.L., Thompson, W.D., and Evans, A.S. Methods in
Observational Epidemiology. Oxford University Press, New
York, NY, 1986.
8. Kleinbaum, D.G., Kupper, L.L., and Morgenstern, H.
Epidemiologic Research—Principles and Quantitative
Methods, Lifetime Learning Publications, Belmont, CA, 1982.
9. Last, J.M. (ed). A Dictionary of Epidemiology, 2nd edition,
Oxford University Press, New York, NY, 1988, pp. 13-16.
10. Copeland, K.T., Checkoway, H., McMichael, A.J., and
Holbrook, R.H. Bias due to misclassification in the estimation
of relative risk. Am. J. Epidemiol. 105:488-495, 1977.
11. Greenland, S. The effect of misclassification in the presence of
covariates. Am. J. Epidemiol. 112:564-69,1980.
12. Cole, P. The evolving case-control study. J. Chron. Dis. 32:15-
27, 1979.
13. Bryan, F.L. Epidemiologic procedures for investigation of
waterborne disease outbreaks. Chapter 7. In: G.F. Craun (ed.),
Waterborne Diseases in the United States, CRC Press, Boca
Raton, Florida, 1986, pp. 171-193.
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Predicting Exposure to Water Contaminants in
Distribution Systems
by: Robert M. Clark
Drinking Water Research Division
Risk Reduction Engineering Laboratory
U.S. Environmental Protection Agency
Cincinnati, Ohio 45268
(513) 569-7201
Introduction
Although the Safe Drinking Water Act (SDWA) and its
amendments clearly specify that MCLs shall be met at the consumer's
tap most regulatory concern has been focused on water as it leaves the
?nwT P Sn- e°?e enterinS the distribution system. The only
SDWA regulations that emphasize system sampling are those that
deal with microbiological contamination and total trihalomethanes.
There is, however, growing interest in determining the factors that
cause water quality variations in drinking water distribution systems
* i inished water may change in quality before it reaches the user, due to
chemical or biological transformations or due to a loss of system
integrity. Water distribution systems frequently draw water from
multiple sources, such as a combination of wells, or different surface
SO-^eSlu°r Jb.oth- Mlxing of water from different sources takes place
hvdrauS Ftr1r^SyStem> and 1S S funCti°n of ^Plex system
hydraulics. For all of these reasons the quality of water delivered to
within the
To understand water quality variations in distribution systems
requires a knowledge of several factors, including the hydraulic
behavior and the contaminant behavior of the system It is almost
impossible to study these factors directly because the scope and
comp exity of distribution systems are so great. Frequently, hydraulic
models are used to analyze water system operation. Models can also be
dKdri-t°rr ' fSPatiaLdist4butionS °f "airborne contaminants in
dstribution systems. Flow patterns in distribution systems can be
highly variable and these patterns can have significant impact on the
way contarmnants are dispersed in a network. This paper wfll discuss
the development of a set of models for pred cting propagation of
83
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contaminants in distribution systems including a steady-state model, a
contaminant-dispersion model, a travel-time diversity index, and a
dynamic water quality model for contaminant propagation. These
models will hopefully lend insight into factors affecting variations in
water quality in distribution systems. The models are developed and
verified in the context of a case study conducted by U.S. EPA with the
North Penn Water Authority (NPWA).
North Penn Water Authority
Until recently little attention has been paid to the problem of
water quality in distribution systems. However, as concern over
distribution system water quality has increased, more attention has
been paid to the factors that effect water quality variations (2).
To provide the basis for studying changes in water quality as a
result of mixing in distribution systems, a cooperative agreement was
initiated between the North Penn Water Authority, Landsdale,
Pennsylvania, and the U.S. EPA's Drinking Water Research Division.
The NPWA serves 14,500 customers in 10 municipalities with an
average of 5 million gallons (MGD) of water per day. Water sources
include a 1 MGD treated surface water source purchased from the
Keystone Water Company and 4 MGD from the 40 wells operated by
NPWA. Figure 3.5.1 is a schematic representation of the 225 miles of
pipe in the NPWA distribution system, showing the location of wells,
the Keystone "tie-in" and the three pressure zones: Souderton zone,
Lansdale low zone, and Hillcrest zone.
Surface water enters the NPWA system at the Keystone tie-in.
The rate of flow into the system is determined by the elevation of the
tank in the Keystone system, and by a throttling valve at the tie-in.
Flow is monitored continuously and is relatively constant. Water flows
into the Lansdale low- pressure zone and from there enters the Lawn
Avenue tank, from which it is pumped into the Souderton zone.
Additional water, from the Hillcrest pressure zone, enters the
Lansdale system at the Office Hillcrest transfer point (Office Stand
Pipe); this water is solely derived from wells in the Hillcrest zone.
Except for unusual and extreme circumstances, such as fire or main
breaks, water does not flow from the Souderton zone into the Lansdale
low zone, nor from Lansdale low into Hillcrest.
There are distinct chemical characteristics of Keystone water
compared with well waters. Keystone water contains total
trihalomethanes (TTHM) at significantly higher levels than well
water. Certain wells show the occurrence of trichloroethylene (TCE)
and/or cis-l,2-dichloroethylene (cis-l,2-DCE). Inorganic chemicals also
vary from well to well, and between the wells and Keystone.
Trihalomethanes are a result of the interaction of precursor material
in the raw water with chlorine. After they reach a given level they act
84
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Hilltop Standpipe
• " \ -~~~~~
. Lawn;Ave Tank «N,P21.NP30
' Souderton Zone S4-
S6/t
NP26
2 mg Tank
•,'Office Standpipe
/ Hillcrest Zone
$Hillcrest
Tank
Lansdale Law Zone
Hatfield
* Tanks ,
•Wells ' "
" ; '•• ' " NP = North Penn
... • L = Lansdale
S = Souderton
x Sampling Locations
: • -Pressure Zone Boundaries
Figure 3.5.1. North Penn Water Authority distribution system.
as conservative substances.. This is the case for TTHMs at the.
Keystone tie-in.
The NPWA distribution system was modeled in a network
representation consisting of 528 links and 456 nodes. Water demands
for modeling represented conditions during May-June 1984. The
network hydraulic model used was the WADISO Model; which
contains provisions for both steady-state and quasi-dynamic hydraulic
modeling (extended period simulation) (3). In the first phase of the
study, steady-state modeling was used, and included limited field
calibration and validation of the model. The model was employed to
study the overall sensitivity of the system to well pumpage, demand,
and other factors. This study resulted in the development of a number
of typical flow "scenarios." Researchers developed a "base" scenario,
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representing a typical situation with "wells on" and "tanks filling,"
with average demand as well as alternative scenarios which
represented other typical demand and well status scenarios.
The modeling effort revealed portions of the system which were
subject to changes in flow directions under different demand
conditions. Figure 3.5.2 depicts the spatial distribution of the
percentage of water in the Landsdale low zone from the Keystone
supply under the base scenario. ,
Steady-State Prediction of Contaminant Distribution
Engineering analysis of water distribution systems is frequently
limited to the solution of the hydraulic network problem, i.e., given the
physical characteristics of a distribution system and the demands at
nodes, the flows in links (the sections of pipe between nodes) and the
head at all nodes of the network are determined. The problem is
formulated as a set of simultaneous nonlinear equations, for which
there are a number of well-known solution methods (1,5).
In addition to the hydraulic analysis problem, however, there are
other problems of interest in water distribution systems. First, the
calculation of mixing water from different sources in the system is of
concern, particularly where sources of different water quality may
exist. Secondly, calculation of travel time from a source to any demand
node in the network may be of use in investigating waterborne disease
outbreaks (determining which populations were exposed to a higher
concentration of a contaminant), or examining time dependent quality
parameters, such as trihalomethane levels. A third area of increasing
interest is that of economic analysis and "cost of service" pricing, in
which the allocated cost of water to various points within the system is
calculated, and priced accordingly (similar in concept to the pricing of
long-distance telephone calls, or airline tickets, based on distance and
volume of use).
For each of these three types of problems, a solution variable can
be defined. For the mixing problem, the variable of interest is the
concentration of a particular constituent within the water. For the
travel time problem, the variable is the "age" of the water, assuming
that the water is of some defined age at each source in the system. For
the cost problem, the variable is unit cost (e.g., $/cubic foot/sec).
The problems are formulated by assuming that a steady-state
hydraulic solution of the system is known, i.e., all flows throughout the
system are known and satisfy a mass balance at the nodes. Then, as
each element of water proceeds from a source to a demand node, it may
undergo a transformation as it passes along each link and mixes with
water from other portions of the network at the nodes. By assuming
complete mixing at each node, a linear equation in the concentration
variable and the known flows can be written for each node, based on
conservation of mass principles. With the unknown boundary
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0%
. Predicted Percent (%) Keystone Water
Flow Direction
Pressure Zone Boundaries
Figure 3.5.2. Percent Keystone Water - average conditions scenario.
conditions at source to the network, equations in each of the unknown
nodal variables can be written and solved by standard methods.
By appropriate definition of the variables, a general formulation
for each of the three generic types of problems (concentration travel
time, and cost) can be developed. Each of the three types of problems
can be represented by similar equation forms. For the concentration
problem, there is neither a link- nor node-associated additive value.
87
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For the travel time problem, there is a link-associated added value (the
link travel time), and for the cost problem, there are both link- and
node-associated fixed cost values. Males et al. present more complete
analysis of this approach (5).
The general formulation at any node i, for all three types of
problem, can be expressed as:
... P.} + dlLV..}) + {d2- NV.} + {S.- SP.}]
where the sum is taken for all j such that flow is from j to i, and:
Pi = appropriate node-related parameter
= corresponding line-associated value for link ij (cost and
travel time solution)
= corresponding node-associated value for node i (cost
solution)
S> = external source flow into node i
SP' = external boundary condition parameter value at node i
dl = 0 for concentration problem and 1 for cost and travel time
problems
d2 = 0 for concentration and travel time problem arid 1 for cost
problem , . ;
Ii = total inflow to node i
Qy = flow from node j to node i
This solution technique is called Solver (5).
Numeric Example
A simplified network can be used to demonstrate how the
equations are set up. Assume a two-source network consisting of five
links and four nodes, as shown in Figure 3.5.3. It is assumed that the
flows have previously been determined by hydraulic analysis, such
that continuity is maintained at all nodes. Figure 3.5.3 shows the link-
associated flows, flows from the two external sources, and the node
demands. The concentration case is illustrated below, assuming that
the concentration at source 1 is SCi, and the concentration at source 2
is SC2- Observing the 'flows at each node, and the associated flow
directions, the following equations can be written for each of the four
nodes:
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ci =
C2 =
C3 =
C4 =
(2)
(3)
(4)
(F4*Ci + F7*C2 + F9*Ca) (5)
F3 + F7 + Fg
(F8*Ci+-F8*Ca)
F3 + F8
(Fn*SC2)
Thus, there are four equations in four unknowns that can be solved
given the known values of the boundary conditions, SCi and SC2. In
this simplified case, these equations can be solved by direct
substitution, whereas in more complex cases, matrix methods are
applied. As mentioned earlier, once the network has been defined, the
WADISO hydraulic model can be applied under "base" conditions to
determine flows to the network links. The "Solver" component of
EPA's Water Supply Simulation Model (WSSM) was applied to the
steady-state hydraulic solution using the known concentrations in the
Keystone water sources (6). An attempt was made to "match" the
distribution of historical total TTHM data. Historical TTHM data from
April 1984 and April 1985 were used to develop average
concentrations for TTHMs as shown in Figure 3.5.4. Averages are
based on 126 sampling results from 29 sites with 5 results available at
most sites, and 52 results available at the Keystone tie-in. A TTHM
level at the Keystone supply model was set at a value equal to that
found in historical data. No bromoform was found in the TTHMs from
Keystone but was found in the distribution system samples at
approximately 10 g/L. Bromoform levels were most likely due to
bromide in the well water. Therefore TTHMs at the 10 g/L level were
assumed as coming from the wells;
Table 3.5.1 contains representative water quality values at
Keystone in the distribution system and at selected wells. In the "base"
scenario, it was assumed that water was flowing into the elevated
Chestnut Street and Lawn Avenue elevated tanks. Tanks obviously
have an impact on water quality in a distribution system because they
may tend to diffuse contaminant concentrations. This effect is
mitigated by assumption of a steady-state model but is extremely
important in a dynamic model. Predicted values in Figure 3.5.4 show
good agreement with the actual surveillance data.
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s,, sc
10
Figure 3.5.3. Example problem.
Dynamic Variations in Water Quality Data
To investigate the nature of water quality variability under
dynamic conditions within the system, a sampling program was
conducted at six sites. Sampling sites were selected based on spatial
variations determined from this historical data and modeling results.
Figure 3.5.5 shows the various sampling points used in the field study,
and Figure 3.5.6 depicts the results of the intensive sampling program
using TTHMs as a tracer. There is significant variation within the 36-
hour period for given points, and there is significant variation within
the system itself. Figure 3.5.6 also depicts the variation in hardness at
these same points (hardness is primarily associated with flow from the
wells). At the Mainland sampling point, a flushing back and forth of
water between the surface source and the well sources can be seen. The
peaks of the TTHMs at Mainland are approximately 12 hours out of
phase with the peaks from the wells, indicating that water flow at this
point is affected by the surface and ground-water sources.
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• = Average Sample Concentrations
47.3
Figure 3.5.4. Predicted versus actual trihalomethane concentrations .
(Note: Contours based on average demand with wells on [modeled conditions]).
These results point out the problems in attempting to predict a
dynamic situation using a steady-state approach. The average TTHM
data represent long-term averages taken over a number of years at
different times of the day. The pattern of flow in the NPWA
distribution system varies during the course of a day, as wells cycle off
and on..Late at night, when most wells are off, Keystone water is the
primary source of supply for the Lansdale low-pressure zone system,
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Table 3.5.1. North Penn Water Quality Based on 1984 Data
Quality
Factor (1)
Chloride
(mg/L)
Hardness
(mgA.)
Sulfate
(mg/L)
TTHM
Minimum
(2)
9
100
9
a
Maximum
(3)
152
440
242
a
Minimum
(4)
14
156
23
0.50
Maximum
(5)
93
297
125
37.9
Minimum
(6)
30
124
36
10.3
Maximum
(7)
30
190
73
131.0
•Not measured.
and this water passes into the eastern and northern portions of the
system, where it can enter the Lawn Avenue tank. During times of the
day when Lawn Avenue tank water enters the Lansdale low system,
this water can be expected to contribute TTHM (due to the origin of at
least a portion of this water from Keystone when Lawn Avenue is
filling). The pilot sampling run of November 14-15, 1985, shows a
range during the sample period from a maximum TTHM value of 36
g/L to a minimum of 13 g/L, at the inlet to Lawn Avenue. This range is
consistent with the historical data of Figure 3.5.4, where a value of
18.8 g/L is shown in the neighborhood of Lawn Avenue. Data from the
pilot sampling run, taken at the Mainland sampling site, shown on
Figure 3.5.6, shows TTHM values in the range from 9 to 34 g/L, with
significant variation. As concluded by the analysis of the pilot
sampling run, Mainland receives water from the wells in Harleysville
(NP14, NP31, NP11, NP20, NP26, NP48) during the times of day when
they are pumping, and from Keystone in the early morning hours.
Historical data shows levels of TTHM of 2 to 6 g/L in this area, as
compared to the model prediction (with wells on) of 0 g/L. The model
does show, however, that the zone of blending of Keystone and well
water is fairly close to Mainland; thus, the variations predicted by the
model are close to the actual ones. The Mainland site was selected
primarily to test this hypothesis in the pilot sampling run.
While the initial steady-state modeling did provide useful
information, so far as design of the pilot sampling study was
concerned, it was obvious that the ability to model the dynamic nature
of the complex NPWA system with a single steady-state predictive
model is limited. Accordingly, an attempt was made to extend the
steady-state predictive principles to "a sequential steady-state
modeling approach for the situations encountered in the pilot sampling
run. The following section describes the development of this approach.
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North
Keystone
Figure 3.5.5. Sampling points for field study.
Sequential steady-state modeling
The Solver algorithm used to predict concentration is steady-state
in nature, and relies upon boundary conditions and externally defined
steady-state flows. The Solver algorithm was applied to a series of
hydraulic scenarios, each representing a portion of the time period
associated with the pilot sampling run. The pattern of flow in the
Lansdale low zone was represented by three separate scenarios, each
representing a portion of time during the day. For the calculations
associated with the Solver predictions, the value of Keystone TTHM
was set to 61 g/L, the average value encountered during the field
testing. From 6:00 a.m. to noon, a scenario was selected in which a
demand factor of 2 is applied, wells are on, and water is transferred
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H/btu in ssaupjBH —
94
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from the Hillcrest zone into Lansdale. Water flows out of the elevated
tank, and into Lawn Avenue and the Chestnut Street standpipe.
Figure 3.5.7 shows the predicted TTHM contours associated with this
scenario. During this time period, with both wells on and a transfer
from Hillcrest, the Keystone water is confined in the central portion of
the pressure zone, and undergoes blending throughout this area. The
Mainland sampling site is predicted to have 100 percent well water.
The noon to midnight period was modeled using a scenario in which a
demand factor of 1 is applied, wells are on, but there is no transfer of
water from Hillcrest to Lansdale. Water flows into the Lawn Avenue,
Chestnut Street, and elevated tanks. Figure 3.5.8 shows the TTHM
contours associated with this period. Under this scenario, there is less
flow from Keystone (demand is lower) than in the 6 a.m. to noon
scenario, and there is greater flow of water into the tanks. Thus, there
is a relatively greater amount of well water in the system, and the
excursion of Keystone water northwards is somewhat suppressed, as
can be seen by comparing Figures 3.5.7 and 3.5.8. The third period,
from midnight to 6 a.m., is represented by a scenario for which wells'
are off, a demand factor of .5 is applied, and there is no Hillcrest water
transferred to Lansdale. Under this scenario, all tanks are filling, and
all water in the Lansdale low zone is derived from Keystone (all wells
are off). The steady-state analysis thus dictates the following
conclusions: the overall flow pattern is one of excursion of Keystone
water northward in the system during the period of high demand
(morning), some confinement of Keystone water in the afternoon, and
then full excursion of Keystone water into the system at the period of
low demand, when wells are off.
; The actual THM measurements made during the pilot field study
were presented in Figure 3.5.6. The most striking feature of this
display is the dramatic change in the TTHM values associated with the
Mainland sampling site, and, to a lesser degree, at the Lawn Avenue
tank inlet. The water at Mainland has the lowest TTHM values at the
1:00 p.m. and 5:00 p.m. samples (points B and C, H and I), indicating
the lowest percentage of Keystone water during that time period. This
is consistent with the overall predicted pattern that suggests that
Keystone water is most confined within the central portion of the
system during the noon to midnight period. The water then travels
throughout the system during the early morning hours when wells are
off. The specific point prediction is for 100 percent well water at the
Mainland sampling site during the two daytime prediction periods.
The model does predict the flow reversals that do take place at the
Mainland sampling site. The Lawn Avenue inlet site shows the lowest
values of TTHM at the 1 a.m. sample on November 14 (point B on
Figure 3.5.6). This is roughly consistent with the prediction of the
largest confinement of Keystone water during the noon to midnight
period. The relatively large change in TTHM from Keystone during
the sampling period complicates the analysis, and further studies are
planned, involving sampling for a longer period at shorter sampling
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--I?
TTHM Concentrations
Outside of
Zone | |
0-10
10-20
20-40
40-60 m
Demand Factors
6 AM - Noon
Demand Factor = 2
Wells On
Hillcrest to Lansdale Transfer
Pressure Zone Boundaries
Figure 3.5.7. Predicted TTHM concentrations for demand factor = 2 and "wells
on."
intervals, to better characterize the effects of the Keystone water for
the analysis.
In general, the predicted distribution is consistent with the
observed data, to the extent that general patterns of behavior are
borne out. This technique is clearly useful for modeling and tracking
the propagation of contaminants in distribution systems. ' '
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TTHM Concentrations
Outside of
CZI
Zone
0-10
10-20
20-40
40-60
Demand Factors
Noon - Mid
Demand Factor
Wells On
No Hillcrest to Lansdale Transfer
Pressure Zone Boundaries
= 1.0
60
Figure 3.5.8. Predicted TTHM concentrations for demand factor = 1.0 and "wells
on."
Contaminant Propagation in a Distribution System
The work described previously demonstrates the feasibility of
predicting general contaminant patterns in a distribution system.
However, it is also useful to be able to trace water and contaminant
movement in a system. Building on the sequential steady-state model,
97
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a microcomputer-based model has been developed for tracing the
movement of water through a distribution system under steady-state
conditions. As part of the water tracing model, the user specifies a
source node for which analysis is to be performed. The model then
traces flow from that node to all other nodes in the system and
calculates:
• The total flow entering each node
• The fraction of flow entering each node that emanated from the
designated source node
• The average travel time from the designated source node to every
other node in the system
• The minimum travel time from the designated source node to
every other node in the system
• The maximum travel time from the designated source node to
every other node in the system
• A travel time divergence index
To perform the analysis, the model establishes a nonunique
"hydraulic" ordering between all nodes and links in a distribution
system. The ordering ensures that prior to operating on a given node,
the flow from all links into that node have previously been analyzed.
Prior to operating on a given link, flow from the upstream nodes will
have been determined. Using the above ordering mechanism at each
node, the total flow entering the node is calculated as the total flow
entering from all incoming links plus the flow entering directly into
the node if the node is a source. Similarly, the flow entering the node
that originates from a designated source is calculated as the total flow
in each link that emanated from this source. The fraction of flow
entering the link emanating from the designated source is simply the
ratio of the two values.
The average travel time from source node J to node I is calculated
by the following formula:
TTu = {S[(TTkj + TTKi) * FKj * QKl]/(SFk| * QKI)} . .(6).
where :
TTjj = travel time from source node J to node I
2 = the summation over all directly upstream nodes (K)
= travel time in the link from node K to node I
= the fraction of flow at node K from source node J.,
= flow in link from node K to node I
The minimum travel time from a source node to any node is the
shortest time path between the nodes. This value is calculated by
98
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analyzing links and nodes using the previously described hydraulic
ordering. At each node, the travel time is calculated for each entering
link by adding the minimum travel time at the directly upstream node
and the travel time in the link. The entering link with the minimum
travel time is assigned to the node. A similar procedure is used to
calculate the maximum travel time for each node.
A normalized travel time divergence index is calculated for each
node using the following equation:
Divergence Index = (TTMAx - TTMIN)/TTMIN (7)
where TTMAX and TTMIN are the maximum and minimum travel
times, respectively. This index is a measure of the divergence in travel
times via alternative paths.
The water tracing model is designed for use in connection with a
hydraulic model of a distribution system. The information required for
the model corresponds to the network information used as input to a
hydraulic model supplemented by flow information generated by the
model. The input information required is listed in Table 3.5.2.
Table 3.5.2. Input Information Utilized by
Water Tracing Model
• Source node to be analyzed
• Link Information
Link number
Upstream and downstream node number
Link length
Link diameter
Flow (quality and direction)
• Source node numbers and inflows
Application of Contaminant Propagation Model
The contaminant tracing model was applied to the North Penn
distribution system under varying steady-state conditions reflective of
the differing operating conditions and demands described earlier. In
the first application, average demand was assumed and all wells were
operating at the normal supply rate. Two sources were studied for this
scenario: water supplied from the Keystone transfer (1,089 GPM) and
water emanating from well NP14 (311 GPM). The pattern of flow
emanating from the Keystone transfer is shown in Figure 3.5.9. This
figure shows the fraction of flow reaching each area originating from
Keystone. Figure 3.5.10 shows a similar pattern of flow from well
NP14. As illustrated, both supplies serve as a significant source of
99
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water for the immediate area surrounding the respective source node
and a less important source for parts of the system at a greater
distance from the supply. Even though the two sources are located far
apart, there are portions of the system that receive flow from both
sources.
L1113310
Wells on Average Demand
Fraction of Flow Originating at
Keystone Transfer
> Sources
Figure 3.5.9. Fraction of flow emanating from Keystone Transfer (average
demand conditions and wells at normal supply rates).
100
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Fraction of Flow Originating at
Well NP14
Figure 3.5.10. Fraction of flow emanating from Well NP14 (average demand
conditions and wells on normal supply rates).
Travel Time
The average travel time from the two sources to other nodes served
by the sources was calculated for the Keystone Transfer and well
NP14, respectively. As would be expected, time of travel is
approximately related to the distance from the source. In each of these
scenarios, travel times to some small dead end pipes can be very long
101
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(i.e., several days) because the average demands at these points are
very low. Figure 3.5.11 illustrates the calculation of the travel time
diversity index for flow emanating from Keystone. A higher value of
the diversity index indicates a greater normalized difference between
the maximum and minimum travel time to that node. In general, a
higher value results from multiple paths to a specific node. In Figure
3.5.11, a low diversity index «0.1) is illustrated for a corridor
connecting Keystone with the Lawn Avenue Tank in Souderton. This
can be explained by the presence of only a single major transmission
path between these two nodes. However, the diversity index is much
higher (>5) in the vicinity of Lansdale, indicating a diversity of paths
with significantly varying travel times.
Travel time plots were prepared for three other scenarios. A low
demand scenario (half of average demand) with none of the wells
operating showed that since Keystone is the only source during this
scenario (all tanks, which can also be considered sources under steady-
state conditions are filling in this scenario), the entire main pressure
zone for the system receives flow from Keystone. Travel times in excess
of 24 hours to some of the more distant parts of the system were found.
High demand (twice average demand) and very high demand (four
times average demand) scenarios were also prepared. In both cases all
wells were operating. In the very high demand scenario, all three
tanks were discharging so that Keystone water reached a smaller part
of the overall system. When the travel times corresponding to the four
scenarios are compared, the differences are relatively small and no
significant association is apparent between travel time and demand.
In Figure 3.5.12, the travel time of water from the four main
sources is plotted for the very high demand scenario. The four sources
(Keystone plus the three tanks) deliver approximately two-thirds of
the demand in this scenario. As illustrated, each of the sources supply
water to the area surrounding the source and additionally overlap with
other sources to supply areas more removed from the source. An
extension of this work is described in the following section in the
development of a dynamic water quality model.
Dynamic Water Quality Algorithm Development
The dynamic water quality model described in this paper uses a
numerical routing solution to trace water quality through a
distribution network (7). As described earlier, the distribution system
is represented by a link-node system. Demands and inflows (both
volumes and concentrations) are assumed constant over a user-defined
period and a quasi-dynamic externally generated hydraulic solution is
required for each period. Thus, the flow and velocity for each link is
known from the hydraulic solution for each time period. Each time
period is evenly divided into an integer number of computational time
steps, At. Each link is then divided into sublinks by a series of evenly
spaced subnodes (though the distance between subnodes may vary
102
-------
L1113310
Wells on Average Demand
Travel Time Diversity from
Keystone Transfer
T Max - T Min
T Min
Figure 3.15.11. Travel time diversity from Keystone Transfer to various points in
system (average demand conditions and wells on normal supply
rates).
103
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Chestnut St Standpipe
Elevated Tank
Lawn Ave.
Keystone Transfer
Figure 3.5.12. Areas receiving flow from major sources and tanks.
i
from link to link or for a link at different time periods), such that the
travel time from a subnode (or node) to the adjacent subnode (or node)
is approximately equal to At.
104
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Solution Procedure
The information required by the dynamic model may be classified
into three categories: general information, initial conditions, and
information required for each time period. Table 3.5.3 summarizes this
required information.
Table 3.5.3. Information Required by the Dynamic Water Quality Model
General Information
• Dt-Time Step
General Network Information:
• Node numbers associated with the end of each Link
Link lengths
•• Pipe diameters
• Node number associated with each source
• Node number associated with each tank
• Tank geometry
Initial Conditions:
• Concentration at each node at the start of simulation
• Volume in tank at start of simulation
Information Required for Each Period:
• Direction and flow in each link
• Velocity in each link (optionally may be calculated based on pipe diameter)
• Concentration in source flow
The solution algorithm used in the dynamic water quality model
operates sequentially by time period. During a time period, all
external forces affecting the water quality are assumed to remain
constant (i.e., demand, well pumpage, tank head, etc.).
An example of the movement of a contaminant is illustrated in
Figure 3.5.13. In this example, the relative concentration within each
subnode is illustrated by the depth of shading with black
corresponding to high concentration and white corresponding to low or
zero concentration. As illustrated, during each time step, the contents
of a subnode moves to the adjacent subnode traveling in the direction
of flow. In this example, at the end of a time period (i.e., after time step
T3), the direction of flow changes and the number of subnodes change
due to an increase in velocity. Accordingly, the high concentration
packets shift direction and move from right to left.
105
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T = 0
Direction of Flow
Direction of Flow
Direction of Flow
Direction of Flow
Direction of Flow
Direction of Flow
Direction of Flow
o
o—o—o
o o—o—o
Note: Darker Shading Indicates Higher Concentrations
Figure 3.5.13. Example movement of contaminant within link.
T = T2
T = T3
T = T4
T = T5
T = T6
T = T7
T = T8
106
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Model Application
The dynamic water quality algorithm was implemented as a
microcomputer-based dynamic water quality model written in
FORTRAN. Researchers developed versions for both the Apple
Macintosh computer and IBM-PC (or compatible) computer and
applied the model in a full-scale demonstration on the NPWA
distribution system (8).
The dynamic water quality model was used to model the
movement of constituents in the North Penn system, during a 34-hour
period of simulated conditions corresponding to those present during
the pilot level sampling program conducted on November 14-15, 1985.
Hydraulic conditions in the system were determined using the
WADISO hydraulic model. Researchers adjusted model parameters so
that predicted tank levels and flows at selected sites represented those
measured during the sampling period. Figure 3.5.14 compares
measured and modeled hydraulic conditions at three locations.
Flows in the network were aggregated into seven periods, ranging
in length from 2 to 7 hours, during which flows throughout the system
remained relatively constant. These flows were input into the dynamic
water quality model along with information on initial tank water
levels.
Three water quality constituents were modeled: TTHM,
chloroform, and hardness. Chloroform is present only in the Keystone
water source. Another component of TTHM, bromoform, is formed in
the system from precursors present in many of the wells. Based on
sampling results, a constant chloroform concentration of 38 ug/L was
introduced into the model at the Keystone import. Similarly, initial
concentrations of chloroform of 14 ug/L, 1 ug/L, and 1 ug/L in the Lawn
Avenue tank, elevated tank, and Chestnut Street standpipe,
respectively were used based on the field sampling results. The
dynamic water quality model was initially applied for a 24-hour period
and the resulting nodal concentrations used as initial conditions for
the 34-hour model application. This means of estimating initial
conditions is based on the assumption that the model will reach a
npnsteady-state equilibrium within the 24 hours and that the system
displays an approximate 24-hour periodic cycle. Examination of
modeling results and results of the field sampling program validated
both assumptions. For all runs of the dynamic water quality model,
chloroform was treated as conservative (i.e., no chemical formation of
chloroform was assumed) and a time step of 30 minutes was used. The
resulting predicted time history of chloroform is plotted in Figure
3.5.15, along with field sampling results for three sampling stations.
In modeling TTHM, concentration was set at 60 ug/L at Keystone,
the average concentration observed during the sampling program.
Concentrations of TTHM (corresponding to the formation of
107
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479-
Feet
(MSL) 478 -
I 1-—1 1 1 I
1000
800 -
600 -_
400 -j
200 ~
GPM 0 -_
-200-
-400-3
•600 -m
-800-
-1000
Flow Into Lawn Ave. Tank
1 - 1 - 1 - 1 - 1
10 20
Time (Hours)
30
4C
GPM
20
Time (Hours)
Figure 3.5.14. Comparison of measured and simulated hydraulic conditions.
108
-------
400-
300-
1 ,200-
J >OH
At Mainland
, • 0
400
300-
:200-
1 0-
400-
S 30°
c •
I 200-
I
100-1
i i r I i
10 20 ,- 30
Time (Hours)
Field Measurements
s,^.— Model Results
40
At Hatfietd Packing Co.
1.0 20 30 40
Time (Hours)
At Lawn Ave. Tank
i... i , i ,., i I I
•10 20 ' 30 40
Time (Hours)
Figure 3.5.15. Comparison of modeled and measured hardness a selected
stations.
109
-------
bromoform) were assumed to be 10 ug/L at all wells. This value is
purely an assumption since water quality was not measured at the
wells during the sampling period. Additionally, historical
measurements of TTHM or bromoform were not available because only
the precursors of bromoform are present in the wells rather than
bromoform itself. Initial concentrations of TTHM of 34 ug/L at Lawn
Avenue Tank, and 5 ug/L at the elevated tank and Chestnut Street
standpipe were used based on the results of the field sampling
program. Modeling results for TTHM are plotted in Figure 3.5.16 for
three sampling stations along with the sampling results at those
locations. The spatial variation of TTHM, as predicted by the model,
are plotted in Figure 3.5.17 for hour 9 (9 hours after the start of the
simulation) and in Figure 3.5.18 for hour 24. Hour 9 corresponds to a
period of high demand when most wells are operating and the spatial
influence of Keystone water is at a minimum. Hour 24, at 7:00 a.m.,
corresponds to the end of a low demand/low well pumpage period; a
time at which the Keystone water has traveled to its greatest extent
through the system.
The hardness of the water sources for the North Penn system
varies considerably. During the sampling period average hardness for
the Keystone water was 318 mg/L. No measurements of hardness were
taken at the wells during the sampling period, but based on routine
sampling during the months previous to and following the sampling
period, average hardness for the wells varied from 179 to 413. The
results of the predicted hardness as compared to sampling
measurements are presented in Figure 3.5.15 for the three sampling
stations.
Examination of the results of the application of the dynamic water
quality model to the North Penn system as reflected in Figures 3.5.15
to 3.5.19 indicate both close agreement between predicted and
observed results and some anomalous behavior. For chloroform,
TTHM, and hardness the general levels of concentration compare very
favorably to the observed values at the three selected sampling
stations (Figures 3.5.16 and 3.5.17). In each case, there are some
differences in the timing of peak or minimum values. When the spatial
variation of predicted TTHM concentrations are compared to the
historical average TTHM level, the same general patterns are
apparent. Additionally, the predicted patterns bracket the pattern
corresponding to the long-term historical average; a result that would
be expected since the two selected times correspond to the extreme
spatial patterns during the sampling period.
There are several known factors which could contribute to the
variation between predicted and observed values. These factors are
summarized below:
110
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30
20-
I
10-
o
10 20 30
Time (Hours)
40
At Hatfield Packing Co.
10 20 30 40
Time (Hours)
10 20 30 40
Time (Hours)
Field Measurements
*s^— Model Results
Figure 3.5.16. Comparison of modeled and measured chloroform at selected
stations.
111
-------
50-
40
30~
20-
10-
I I
10
Field Measurements
•N^- Model Results
Time (Hours)
10
50-
I
10 20 30
Time (Hours)
40
I 40'
30-
O
20-
10-
At Lawn Ave. Tank
I I I I I I
10 20 30 40
Time (Hours)
Figure 3.5.17. Comparison of modeled and measured TTHM at selected stations.
112
-------
'60—
Figure 3.5.18. Simulation of TTHM values in North Penn distribution network
(Hour 9).
1. Observed temporal variations in the concentration of TTHM,
chloroform, and hardness at Keystone were not represented in the
simulation.
2. Field data on TTHM concentrations at the wells were not available
and thus assumed values were not based on observed values.
3. Field data on hardness in well water were not available during the
sampling period so that representative values based on sampling
at other times were used.
113
-------
Keystone
Figure 3.5.19. Simulation of TTHM values in North Penn distribution network
(Hour 24).
4. The nonconservative aspects of TTHM and chloroform were not
represented in the model application.
5. Flows from the hydraulic model were aggregated into periods of
various lengths thus losing some accuracy in representing high
frequency hydraulic occurrences in the model.
Dynamic Model and Flow Tracing
The dynamic model was combined with the flow tracing to predict
the movement of contaminants over time in the North Penn system.
Figure 3.5.13 illustrates that the contaminant can move back and
114
-------
forth in the pipes under varying demand and hydraulic scenarios. In
order to extend the contaminant propagation modeling capability, the
tracking model was coupled with the dynamic hydraulic scenarios to
predict contaminant movement.
To illustrate this effect, an initial influent of 100 units of
contaminant was assumed injected at the Keystone tie-in at time equal
to zero. Assuming time periods that were representative of a typical
day (Figures 3.5.20a, b, c, and 3.5.21a, b, c, d), the contaminant was
tracked in the North Penn system. Figures 3.5.20a, b, and c show the
dispersion of the contaminant at t = 300 min. As shown, the
contaminant is localized in the southeastern portion of the system. At t
= 540 min, the contaminant has spread to the central portion of the
system. By t = 960 min, the contaminant is beginning to disperse. At
times 1,440 min and 1,560 min, the contaminant is well dispersed and
is beginning to disappear. By t = 2,040 min (nearly a day and a
half),the contaminant still persists at very low levels, but is virtually
gone from the system.
To more clearly illustrate this effect, a time trace of contaminant
movement was calculated at three points in the system. These points
are shown in Figure 3.5.22. Figure 3.5.23 shows the predicted time
trace of the contaminant at points 3, 240, and 276. Point 3 shows the
contaminant moving past the monitoring point into the system. Point
240 is a point of maximum mixing in the system in which water tends
to move back and forth. Point 276 also illustrates the retention of
contaminant in the pipe as water moves back and forth past a given
point.
Discussion
Based on the results to date, steady-state predictive modeling
appears to be a reasonable first step to characterize the distribution of
water quality in multi-source systems. Although point prediction
capability is probably not accurate, and is likely highly sensitive to
hydraulic assumptions, general trends can be established and then
verified through field sampling.
Steady-state prediction might be better in systems that are less
dynamic in terms of operation, with fewer sources, than the NPWA
distribution system. Steady-state assumptions also might be more
consistent with a less dynamic system. In addition, the NPWA system
is somewhat "disjointed," due to the manner in which it was developed,
through connection of a number of separate systems. Thus a number of
portions of the distribution system are connected by a single pipe. Once
the hydraulic solution establishes flow direction, the system is
effectively "partitioned," at least in certain areas, into zones of uniform
concentration.
The steady-state algorithm as applied in this case did not take into
account the concentration of Keystone water resident in the tanks.
115
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Node with O10 based on injection of spike of C = 1000 at NP34 during period 1
End of Period 1
300 Min)
Lawn Ave. Tank
Souderton Zone
S6
NP21
2 mg Tank
/Offkfe"Standpipe
bfillcrest Zone
,L16
x Sampling Points
» Storage Tanks
• Wells
NP = North Penn
L=Lansdale
SL = Souderton
••••• Pressure Zone Boundaries
Node with C> 10
Based on Injection of
Spike of C -1000 at NP34
During Period 1
Keystone Tie-In
"Model
Figure 3.5.20a. Simulated contaminant dispersion in North Penn system.
According to the "late night" scenario (in which wells are off), at least
some Keystone water will enter the tanks, and then be discharged into
the distribution system as another "source" of Keystone water. This is
a more complex analysis, but, in the majority of the hydraulic
scenarios selected in this study, tanks were filling, rather than
discharging to the system.
A less tractable problem is the issue of residence time of water in
the pipes. The steady-state analysis fails to take into account water:
already in the pipes of the distribution system. For example, the late
night scenario results in Keystone water flowing throughout the
system, for all demands. In fact, much of the water in the pipe is well
116
-------
Lawn.Ave. Tank
Souderton Zone
End of Period 3
(T = 540 Min)
Hilltop Standpipe
"""*" NP30 !
315
NP26
Lansdale Low Zone
- Hatfield
n
s.
2 mg Tank
Office Stand
/ / Hillcrest Zone
•*• ' -
Mainland
Sampling Points
Storage Tanks : '•; '
Wells '•/'..:..
NP = North Penn
L = Lansdale ' ,
SL=Souderton
Pressure Zone Boundaries
4jHillcrest
L18'Tank
Node 5
NP34
Links"
Keystone Tie-In
, . „ Node 1
Figure 3.5.20b. Simulated contaminant dispersion in North Penn system.
water, arid the pipes act as a reservoir of this water, even though wells
are off. Obviously, blending takes place, as is seen at the Mainland
sampling site, rather than water being either fully well water or fully
Keystone water. The volume of water in the pipes of the distribution
system (as represented in the water supply simulation model, i.e.,
neglecting smaller pipes) was calculated to be 2.8 million gallons, or
over half of the average daily supply of 5 million gallons for the NPWA
distribution system. Thus, the reservoir effect is likely to be significant
and cannot be taken into account in traditional steady-state modeling.
117
-------
Lawn Ave. Tank
Souderton Zone
End of Period 3
(T = 960 Win)
Hilltop Standpipe
,2 mg Jank
/Offjde Standpipe
HillcrestZor
NP26
NP14
x Sampling Points
* Storage Tanks
• Wells
NP*5 North Penn
L=Lansdate
SL=« Souderton
— Pressure Zone Boundaries
Keystone Tie-In
Model
Figure 3.5.20C. Simulated contaminant dispersion in North Penn system.
This research includes extending the steady-state modeling
approach to the prediction of time of travel from one point in the
distribution system to another, and to calculating the percentage of
water that supplies a given point in the system from all other sources.
Both of these calculations have implications with regard to water
quality and propagation of contaminants within the system.
Development of a dynamic water quality model provides insight into
the actual operation of the system and more accurately models the
interaction of water quality and hydraulic behavior.
118
-------
End of Period 4
(T = 1440 Min)
Hilltop_ Standpipe'
Lawn Ave. Tank TsiP2VNP3°
Souderton Zone S4
NP21
19
__ Hillcres
i'.L18 Tank
NP14
x Sampling Points
• Storage Tanks
• Wells
NP = North Penn
L = Lansdale
SL = Souderton >
— Pressure Zone Boundaries
Keystone Tie-In
Node 1
Figure 3.5.21 a. Simulated contaminant dispersion in North Penn system.
This research clearly indicates the need to obtain more
representative monitoring results than are normally acquired from
distribution system sampling. As can be seen from Figure 3.5.6,
contaminant values can vary greatly over a relatively short time at a
given point. There are also no doubt weekly and yearly cycles which,
when combined with hydraulic and mixing variations, will have great
effect on the contaminant levels at a given point, in a distribution
system. To provide insight into these variations, several automated
samplers are being tested foV installation in the North Penn system.
Currently a rotary sampler fitted with an EPA-designed capping
119
-------
END OF PERIOD 5
(T= 1560 MINI
HiIltog,Standpipe '
Lawn Ave. Tank
Souderton Zone
NP26
NP14
_Lansdale Low ZoneTjJT
NP21
2 mg,Jank
, 'Offi6e Standpipe
/ Ijrfillcrest Zone
4 Hillcre
Tank
Sampling Points
Storage Tanks
Wells
NP = North Penn
L = Lansda!e
SL = Souderton
Pressure Zone Boundaries
Keystone Tie-In
Node 1
Figure 3.5.21 b. Simulated contaminant dispersion in North Penn system.
system is being field tested. This sampler will allow samples to be
taken at specified intervals for several days. The approach appears
promising.
Conclusions and Recommendations
Steady-state predictive modeling of water quality can provide
insight into overall water quality variations and patterns within a
distribution system, although "point prediction" of water quality is
less feasible. Interpretation of predictive modeling results must be
120
-------
End of Period 6
(T = 1800 Win)
Hilltop Standpipe
4 -' ~" '
Lawn Ave. Tank V;,,
Souderton Zone
NPi5;
NP26
Lansdale Low Zone""?5
L26
Hatfield
2 mg.Tank
/Office Standpipe
/ /' Hillcrest Zone
4_Hiilcres
Tank
Mainland NP33
Sampling Points
Storage Tanks
Wells
NP = North Penn
L = Lansdale
SL = Souderton
Pressure Zone Boundaries
Keystone Tie-In
Node 1
Figure 3.5.210. Simulated contaminant dispersion in North Penn system.
121
-------
End of Period 7
(T = 2040 Win)
Hilltop, Standpipe
Lawn Ave. Tank *j\jp2i_f NP30
Souderton Zone
NP21
Ml
•*- ,2 mg,Jank
/Office Standpipe
/ / Hillcrest Zone
i. i
NP26
Sampling Points
Storage Tanks
Wells
NP = North Penn
L=Lansdale
SL=Souderton
. Pressure Zone Boundaries
Keystone Tie-In
Node 1
Figure 3.5.21 d. Simulated contaminant dispersion in North Penn system.
made in light of an appreciation of the hydraulics of the system, in
particular, an understanding of the flow patterns and directions that
create the gradients of concentration. Quality modeling is based on
hydraulic modeling, and is thus highly sensitive to hydraulic modeling
assumptions and results. Field quality data are important in
developing, verifying, and understanding predictive models. Such
quality data should be available at time intervals sufficient to reflect
daily changes in system dynamics. Having ihe tools to predict time of
travel between points in a system and to estimate the quality of water
122
-------
North
Keystone
Figure 3.5.22. Sample tracking points in North Penn system.
provided to any point from any source will allow for realistic water
quality monitoring strategies.
Future research should include examining the sensitivity of the
steady-state models to changes in hydraulic assumptions. Further
algorithmic development should be devoted to additional development
and testing of dynamic models. This work should also be extended to a
study of bacterial contamination from multiple-source and single-
source systems with elevated tanks. The model might be helpful to
epidemiologists in analyzing exposure profiles for comparison of attack
rates.
123
-------
s
CM
j
st
_ o
> CO
»t
. o
• CO
CO
SOOOOOOOOOOOOOOOOOO
f-ajtn^'COCMr-ocncot-.coin^fcocMT-
Figure 3.5.23. Prediction of contaminants movement at various locations in North
Penn system.
124
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Acknowledgements
The author would like to acknowledge the assistance of Ms. Diane
Koutledge and Ms. Patricia Pierson in preparing this manuscript.
References
1. Clark, R. M. and Males, R. M. "Simulating cost and quality in
water distribution." J. Water Resour. Ping, and Mgmt ASCE
ll(4):454-466,1985. '
2. Chun D. G. and Selznick, H. L. "Computer modeling of
distribution system water quality." In: Computer Applications in
Water Resources, ASCE, New York, New York, 448-456, June
198o.
3. Gessler, T. and Walski, T. M. Water distribution system
optimization. TREL-85-11, Waterways Experiment Station US
Army Corps of Engineers, Vicksburg, Mississippi, October 1985.
4. Clark, R. M; and Males R. M. "Developing and applying the water
simulation model." J.AWWA.78(8):61-65,1986.
5. Males, R. M., Clark, R. M., Wehrman, P. J. and Gates, W. E
Algorithm for mixing problems in water systems. "Journal of
Hydr.Engrs., ASCE, 111(2):206-219,1985.
6. Males, R. M., Grayman, W. M., and Clark, R. M. "Modeling water
quality in distribution systems." J. Water Resour. Ping and
Mgmt., ASCE, 114(2): 197-209,1988.
7. Grayman, W. M., Clark, R. M., and Males, R. M. "Modeling
distribution system water quality: A dynamic approach." J Water
Resour. Ping. andMgmt., ASCE, 114(3):295-312,1988.
8.
i ,R' M" Grayman> W- M., Males, R. M., and Coyle, J. A
Modeling contaminant propagation in drinking water
distribution systems." Aqua, No. 3,137-151,1988.
125
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Treatment Plant Evaluation During a Waterborne
Outbreak
by: Gary S. Logsdon
Director, Water Process Research
Black & Veatch Engineers-Architects
1025 Alliance Road, Suite 101
Cincinnati, Ohio 45242
(513) 984-6630
[At the time this article was written, Dr. Logsdon was with
the Risk Reduction Engineering Laboratory
U.S. Environmental Protection Agency]
Introduction
Evaluation of a water treatment plant during a waterborne
disease outbreak represents a challenge different from many other
types of engineering work. During an outbreak a great sense of
urgency, or even crisis, can be expected. Disease outbreaks, if caused
by a pathogen in drinking water, are likely to involve public
notification and recommendations to boil water. News organizations
naturally have an interest in the outbreak, its possible causes, and the
remedies needed to solve the present problem and prevent future
problems. Into this environment comes the engineer selected to
evaluate the facility and present recommendations.
This paper has been prepared to serve as a guide to engineers who
are called upon to work in the difficult circumstances that are present
during a waterborne disease outbreak. When events are moving at a
hectic pace, referring to a document prepared when the author had
time to reflect on the nature of the work to be done during an
evaluation may be helpful to the engineer who is attempting to quickly
resolve complex issues on-site. A major portion of this paper has been
adapted from "Evaluating Treatment Plants for Particulate
Contaminant Removal."(l)
Getting started
The first task for the engineer is to get acquainted. This involves
meeting regulatory and public health officials, local government
officials, and water utility personnel at both the operating and the
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management levels. Finally, the engineer must acquaint himself or
herself with the treatment facilities. One useful approach to the latter
task is to tour the entire plant first to obtain an overview of the
situation, and then to go through it again for a detailed inspection.
During the initial phase of the investigation the engineer must convey
a spirit of cooperation and helpfulness. An outsider is dependent on the
good will and assistance of the local persons who are experiencing
difficulties, and who may be quite upset because of what is happening.
Tact, humility, genuine concern, and a sincere desire to help should go
far to gain acceptance for the engineer and support for the evaluation
job that needs to be done.
During an outbreak, the water utility staff have to produce water
and conduct business as usual, in addition to trying to provide
information to numerous regulatory officials, news persons, and the
general public. Additional information is also necessary for a
successful plant evaluation. To reduce the load on the staff, the
engineer can also use other sources of information. The water utility's
offices and laboratory often contain much, valuable information that
would aid the engineer in making a plant evaluation. Facility
blueprints and diagrams can be used for preliminary hydraulic
evaluations. Water quality records should be obtained. Chemical,
microbiological, and physical quality of treated water should be
reviewed and compared with corresponding data for raw water. How
much improvement in water quality is attained by treatment? How
often does raw water vary and what are the influences of raw water
quality changes on treated water quality? Factors such as low
temperatures in winter, spring runoff, summer and fall algal blooms,
and low flow conditions may influence finished water quality. The
unique aspects of each water source should be kept in mind as plant
evaluations are made.
Review of operating records also can be a useful way to gain an
understanding of the water utility's disinfection practice. Information
should be available on type of disinfectant used and history of
disinfectant doses and residuals. When combined with flow data and
information on disinfectant contact time, the residual data can be used
to estimate C-T values.
Records of disinfectant dosage and residual should be reviewed.
(Ozone is very reactive and residuals for this disinfectant remain in
drinking water for only a short time, so most of the following remarks
do not apply to ozone.) The quality of disinfectant added per day can be
estimated by multiplying the rate of flow by disinfectant concentration
at a point shortly downstream of its addition, but after good mixing.
This approximation can then be compared to the plant's records for the
amount of chemical fed per day. Disinfectant demand can influence
this comparison of dose vs. residual, but comparing measured chemical
usage vs. calculated usage should reveal gross problems in disinfectant
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chemical feed control or in the measurement of disinfectant
concentrations in water.
Records of residual measurement should be reviewed to determine
the frequency with which such measurements are made. If a
continuous analyzer is not used, how frequently are grab sample
measurements made? The adequacy of this frequency should be
considered, and it is somewhat related to the variability in raw water
quality. Disinfectant residual should be measured frequently enough
to ensure that a change in disinfectant demand has not produced an
inadequate residual.
Records should also be studied for indications that disinfectant
demand is determined. Sudden changes in disinfectant demand can
cause temporary depletion of the residual, and this might allow
penetration of the treatment barrier by pathogens.
Reviewing and developing data related to the water utility will
consume extra time at the beginning of a treatment plant
investigation, but the time invested in this effort can be expected to
yield a more productive and insightful evaluation.
Monitoring and Quality Control
Analytical methods and quality assurance deserve careful
attention. The methods used should be in accordance with regulatory
requirements. Measurements that are very important for clarification
plants include coagulant dose, residual coagulant, pH, and turbidity.
Disinfectant residual is a key measurement at all plants. A recently
published paper gives additional information on the measurement of
disinfection residuals (2). Quality assurance procedures should be
reviewed, and if necessary, the advice of a chemist familiar with
disinfection chemistry should be sought. This is especially true for
chlorine dioxide and ozone, both of which can present serious
analytical difficulties. The disinfection process is really not under the
control of treatment plant staff if they are not able to measure
concentrations of disinfectant in the water accurately.
Handling and analysis are important for turbidity samples. These
samples should be taken to the laboratory promptly when grab
samples are analyzed. The sample cell must be scrupulously clean
when it is inserted into the turbidimeter, and there should be no air
bubbles in the sample. This can be a problem when the water is cold
but it is vital to obtain accurate measurements. Turbidimeters should
be calibrated with secondary standards as often as instructions from
instrument manufacturers and experience indicate (whichever is
shorter). Calibration with formazin or some other approved primary
standard should be done as frequently as required by Standard
Methods (3) or the EPA Methods Manual. Most improper practices in
handling low turbidity water samples cause the turbidity
measurement to be higher than the actual value. Careful handling and
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measurement of grab samples may reveal that filtered water turbidity
is actually lower than it was thought to be when sufficient analytical
precautions were not taken.
When a filtration plant is inspected, the engineer should pay
careful attention to the sampling points for filtered water turbidity, to
sample handling, and to instrument calibration. To really be able to
control filter operation, the plant operator should be able to sample
each filter effluent independently, at a point where there has been no
mixing with water from any other filter. Although sampling at a point
of entry into the distribution system (e.g. clearwell effluent) is
required by the turbidity regulation, clearwell sampling reveals very
little about the specific condition of an individual filter, because of the
blending that occurs in the clearwell.
A review of plant records should show that adequate
documentation exists, both for routine monitoring and for the
measurements needed for quality control testing. Unless this is
available, the validity of the process monitoring data is questionable.
Plant Hydraulics and Flow Patterns
Hudson (4) stated that one of the two categories of deficiencies
causing many water treatment plants to fail to perform as well as
expected was hydraulic inadequacies, including flow distribution,
incorrect baffling, and a lack of consideration for mixing intensity
criteria. Because of its great importance, plant hydraulics should not
be overlooked.
The quality of filtered water can be affected by plant hydraulics.
The adverse influences of changes in rates of flow and flow patterns on
water quality can be very important. As flow rate is increased,
detention time decreases. Under some circumstances, shorter times for
flocculation or sedimentation can lead to poorer water quality.
Increased flow rates that result in higher filtration rates may have this
effect also, especially if the floe is weak.
Flow metering and measurement are important because
evaluating and controlling plant performance are difficult if flows are
unknown. In order for a plant to be operated properly the total flow
rate has to be known, on an instantaneous basis or by calculations
based on volumetric measurement. Is such equipment in place and
working? In addition, whenever flow is divided in a plant, some means
should be available for operators to understand how the flow is being
split.
Flocculators, settling basins, or filters operated in parallel should
have provisions for determining'flow. Unequal division of flow to two
identical settling basins could result in overloading one basin, with
poorly settled water as a result.
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Pressure filters operated in parallel can be operated in a variable,
declining rate mode, with the most clogged filter operating at the
slowest filtration rate; however, flow meters should be provided on
each filter so operators can determine the actual rate of filtration.
Again, if one or more filters is clogged and not carrying a fair share of
the load, other filters may be overloaded and operating at excessively
high rates. This can lead to turbidity breakthrough.
Even if improper division of flow among basins or filters is not a
problem, poor distribution of flow within a basin might be.
Flocculation basins should be operated in a plug flow mode so similar
mixing energy and time (GT) can be provided to all of the water. An
unbaffled flocculating basin resembles a continuously stirred reactor.
A properly baffled flocculation basin can have flow characteristics that
approach plug flow. Hudson (4), calculated that in a single
compartment, continuously stirred basin, 39 percent of the water will
pass through the basin in a time shorter than 50 percent of the
nominal residence time. In a series of five baffled, continuously stirred
basins, only 11 percent of the water will pass through the basin in a
time shorter than 50 percent of the nominal residence time.
Because of the importance of hydraulic patterns, short circuiting,
and detention times, it may be appropriate to evaluate flows by
performing tracer studies. Lippy (5) recommends this approach and
provides suggested procedures. Although dyes are very sensitive
tracers, their use in drinking water facilities may be inadvisable.
Other tracers are available, including lithium, chlorine, and fluoride.
Some caution should be applied if fluoride is used as a tracer upstream
of the filters. Standard Methods indicates that polyvalent aluminum
can complex with fluoride, the extent depending on pH and the relative
levels of fluoride and complexing species. In an extensive study of
water filtration at Duluth (6), pilot plant studies indicated the addition
of fluoride before filtration required an additional 2 to 4 mg/L of alum
to obtain the same level of turbidity that could be attained in the
filtered water when no fluoride was added. Chlorine can also be used as
a tracer, but chlorine demand and the effects of sunlight can influence
results. If chlorine demand is satisfied, sunlight is not a factor in closed
basins or during hours of darkness.
An excellent guide to evaluation of flow characteristics and
residence times is found in "Residence Times in Pretreatment",
Chapter 5 of Water Clarification Processes: Practical Design and
Evaluation (4). Hudson (4) recommended use of step doses, i.e. a sudden
increase of tracer fed on a continuous basis, rather than use of a slug
dose. The tracer concentration range that must be measured when a
step dose is used is much smaller than when a slug dose is used. This
usually makes tracer analysis much easier.
Even if hydraulics are satisfactory, problems related to flow can
occur. At water filtration plants, capacities for production and storage
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of finished water should be in balance with demand. That is, the
volume of storage should be adequate to equalize or moderate
differences between demand and production.
Process equipment that incorporates mixing, flocculation and
clarification should be operated with a minimum of rate changes.. An
example of this type of equipment is the upflow clarifier, which
establishes an equilibrium when the settling velocity of floe particles
in the sludge blanket equals the rise rate of the water in the basin.
Increasing water production in this type of clarifier causes higher
upward velocity of the water, and can result in washing out some of the
floe in the sludge blanket. That floe then goes on to the filter, where it
could cause shorter filter runs or perhaps pass through into the
finished water.
Water filters should be run at constant or gradually declining
rates, if possible. If storage is inadequate to provide for increases in
demand, then frequent rate changes may be imposed on filters. Some
changes will of course be necessary from time to time, but if the volume
of storage is small enough to require making several filtration rate
changes each date, the benefits of increasing this storage to provide
smoother plant operation ought to be seriously considered.
Chemical Feed Selection and Control
Deep bed, granular media filters that are operated at rates of 2
gallons per minute per square foot, or greater, will not work efficiently
if the raw water is not coagulated properly before it is applied to the
filters (7). In many cases, flocculation and sedimentation are also
necessary. This has to be understood clearly by the engineer who
evaluates the plant and by the operators who are responsible for
controlling coagulation and filtration. Shutting off coagulant
chemicals when raw water turbidity is lower than the 1 Nephelometric
Turbidity Unit (NTU) Maximum Contaminant Level is never a correct
opening procedure, although this mistake has been made numerous
times in the past.
Adding coagulant chemicals is essential, and maintaining proper
control of the coagulation reaction is very important. The efficacy of
inorganic coagulants, especially alum, depends on the pH of^he-water.
Chemical dose is important, both for inorganic coagulants and for
polymers. Inadequate doses result in inadequate particle
destabilization and coagulation. Overdoses of cationic polymers can
result in restabilization of particles. Application of the correct dose of
coagulant, on the other hand, results in destabilization of particles,
leading to effective flocculation, sedimentation, and filtration. A more
detailed explanation of these interactions is given in Chapter 6,
"Coagulation" in Coagulation and Filtration: Back to the Basics (8).
Review of this material is recommended.
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Close attention should be given to the procedures used to select
coagulant dose and pH at a filtration plant. Some utilities base
coagulation chemistry on past practice. This may be helpful if careful
records are kept and charts are prepared so changes in water
chemistry can be identified. Even so, actual dose selection should be
done by jar testing or other methods. The initial advice given on
coagulation when the plant was started shouldn't be assumed to be
valid to perpetuity.
Probably the most common approach to coagulant evaluation is jar
testing. For turbid waters, jar test results can be based on the turbidity
of the.settled water. For low turbidity waters, perhaps 5 to 10 NTU or
lower, changes in raw water turbidity may not be sufficient to clearly
indicate optimum chemical doses. Also, at direct filtration plants,
sedimentation is not practiced, so evaluating settled water is
inappropriate for dose selection. Wagner and Hudson (9) suggest the
water from jar tests can be filtered through Whatman No. 40 paper
after coagulation and flocculation. They report similar results for
filtered water turbidity using this procedure, for water from pilot
filters, and for water from full scale filters. Neuman (10) emphasized
the importance of performing jar tests with test water at the same
temperature as water in the plant. This is especially important when
cold water is being treated in the plant, because proper pH and doses
may be different when this water is warm. Griffith and Williams (11)
evaluated jar testing at Phoenix, Arizona, and reported that the mode
of addition of coagulant chemical influenced jar test results. Moffett
(12) reported higher doses of alum were needed to destabilize cdlloidal
suspensions in jar tests when coagulant was added on top of the water
rather than at the impeller. If a chemical is added by mixing-into the
water, rather than by pouring it on top of the water in the full scale
plant, this practice should be mimicked in jar testing. This can be done
by using funnels. The dose of coagulant chemical is added and then
followed by a rinse of test water in sufficient quantity to displace all
coagulant from the stem of the funnel. If rapid mix times are being
evaluated, or if the mixing time is short, chemicals should be added to
all jars simultaneously. During a plant evaluation, it is a good idea to
watch the plant staff perform a jar test. Also inquire about the
frequency of jar testing and the bases for deciding when to test, i.e.
seasonal changes, change in river flow, changes in turbidity, etc. This
information will provide the background needed to suggest
improvements to their testing program. Hudson and Wagner (13) have
published an excellent paper on performing jar tests and interpreting
the results.
Another approach to dose control is based on measurements of
electrpphoretic mobility. This is based on the magnitude of the
electrical charge on the surface of the colloids. If this charge is large, it
can cause the particles to repel each other and remain in suspension! If
it is small (near zero), particle collisions can occur and these particles
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will stick together and grow into floes that settle out, or stick on the
filter media.
Electrophoretic mobility can be determined by instruments that
measure zeta potential or streaming current potential. Zeta potential
measurements are based on the velocity of individual colloids or
groups of colloids. Zeta potential is usually determined on grab
samples. Streaming current potential is based on the average of the
electrophoretic mobility of all of the particles in the water sample.
Streaming current detectors analyze discrete samples, but can be set
up to do so repeatedly so they approximate continuous, on- line
analysis. The use of streaming current detectors to control coagulant
dose was evaluated by Dentel and Kingery (14). When electrophoretic
mobility techniques are used to adjust coagulant dose, the validity of
these approaches should be verified form time to time by performing a
series of jar tests or pilot filter studies with doses at, above, and below
the recommended concentration.
Some water utilities use pilot filters to determine appropriate
coagulant doses. Proprietary equipment used to filter coagulated water
after the rapid mixing step was described by Conley and Evers (15). In
these systems, turbidity is monitored continuously, and water is
filtered at a high rate. When the turbidity of the water produced by the
pilot filter is unsatisfactory, a change in coagulation chemistry is
needed. Research on a pilot filter concept for dose selection was
conducted by Kreissl et al. (16) about two decades ago. Kreissl and his
associates operated clean pilot filters at high rates, to simulate
conditions that would be encountered when a clogged filter was
operated at a normal rate. They were able to determine coagulant dose
with the pilot filters, and in addition, were able to obtain information
on floe strength and the ability of the system to resist turbidity
breakthrough at the end of a filter run. They reported that pilot filter
systems could be used to select optimum chemical dosage at filtration
plants that did not employ flocculation and sedimentation.
Tests employing pilot filters rather than the electrophoretic
mobility measurements, or jar tests, are appropriate for determining
floe strength and the proper dose for nonionic polymers. Visible
changes in floe formation sometimes can be seen in jar tests, but no
data on floe strength or head loss in filters can be obtained this way.
Nonionic polymers are often used to bridge smaller floe particles
together and form large floe that settles better, or to produce a tougher
floe that resists turbidity breakthrough during rate changes. Better
control of turbidity and Giardia cyst removal, even during filtration
rate increases, was attained (17) in direct filtration research, when
alum and 0.1 mg/L of nonionic polymer were used to condition the
water. In a later study Logsdon et al. (18) reported that a slightly
anionic high molecular weight polymer could strengthen alum floe and
assist retention of the floe and cysts in an 0.9 mm effective size
anthracite filter. In this case, polymer-conditioned alum floe filtered in
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0.9 mm media produced an effluent turbidity (0.10-0.16 NTU) similar
to turbidity produced by 0.42 mm sand filtering alum floe (0.08-0.16
NTU) in an earlier experiment. In both cases, filters were ripened and
not exhibiting turbidity breakthrough. Use of polymer can sometimes
permit production of high quality water in spite of less than optimum
facilities or operation.
After appropriate doses of chemicals are selected for coagulation, it
logically follows that the capability to feed the chemicals must exist.
Techniques for adding chemicals to raw water vary greatly, from those
that give poor results to those that provide efficient and effective
utilization of these materials. Several aspects must be considered.
These include flexibility in the locations and order for adding
chemicals, the actual means of adding and dispersing chemicals,
techniques for measuring feed rates and thus verifying the doses, and
the kinds of chemicals that are fed.
Flexibility should include the capability to add some chemicals at
more than one point in the treatment train. Points of addition may
include near the beginning of a raw water transmission main, just
before a rapid mixer, in a rapid mix tank, just before flocculation,
before filtration, after filtration as water flows to a clearwell, and into
backwash water. A sufficient number of feed points should be available
so that each chemical has the opportunity to mix completely with the
water and function as intended rather than react in a concentrated
form with some other chemical being added to the water. Hudson (4)
suggested the use of plastic water meters or plastic volumetric
cylinders to measure actual feed rates on a short term basis.
The actual mode of chemical addition is extremely important.
Inorganic coagulants and polymers should be diluted sufficiently to
readily dissolve and disperse in the raw water, but they shouldn't be
diluted so excessively that they become less effective. This aspect of
alum feeding was discussed by Griffith and Williams (11) who reported
dilution of liquid alum to a 1.5 percent solution gave them satisfactory
results. They also indicated that another investigator found alum
diluted to 0.3 percent was less effective. Manufacturers'
recommendations should be followed for polymer dilution.
Chemical pumping equipment and piping should be evaluated. A
commonly used feed pump is the diaphragm pump, which pumps in
pulses. If these pumps are used to add coagulant to raw water flowing
in a pipe, and they operate at 60 cycles per minute, with pumping
occurring only 0.5 second of each 1.0 second cycle, only half of the
water comes in initial contact with alum solution. If water in the pipe
is flowing at 6 feet per second, every other 3 foot segment has no alum,
while the other segments have twice the desired dose. Alum hydrolyzes
in a fraction of a second (19). Thus, some of the chemical reactions will
take place before the overdosed and underdosed water are mixed. The
pulsed flow pattern from a diaphragm pump can be converted to
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continuous flow by installation of a vertical pipe which is filled with
air and capped or by use of a pneumatic tank (an accumulator)
downstream from the pump. These devices should be used with
diaphragm pumps to obtain maximum efficiency with coagulant
chemicals.
Because of the rapidity with which coagulation occurs, the stream
of coagulant chemical must be dispersed throughout the entire
quantity of raw water in a nearly instantaneous fashion. Chemicals
added in a rapid mix chamber should be delivered close to the impeller.
Chemicals fed into a pipe should be fed with jets pointed upstream
against the flow. Multiple injection points across the area of flow, as
suggested by Forbes et al. (20), are preferred to a single injection point.
Another technique for adding chemicals in pipes is the use of orifice
plates to create turbulence. Commercially fabricated motionless
mixers (static mixers) are also available for in-line rapid mixing. The
least preferred way of adding coagulant chemicals is to simply dribble
or drop a steam of coagulant onto the surface of the raw water. This is
sometimes done in older water plants that employ a rising well for
rapid mixing, but improved results can often be obtained by providing
more rapid ways of mixing.
The plant evaluator should note the variety of chemicals being fed.
These can include inorganic coagulants, polymers, acid, lime, caustic
soda, soda ash, corrosion inhibitor, powdered activated carbon,
disinfectant, ammonia, and fluoride. The order of chemical addition
can be important. For guidance on this, reference works should be
checked, or jar tests should be performed. Certain chemicals interfere
with others. For example, powdered activated carbon added for taste
and odor control may decrease the chlorine residual, polyphosphate
added to sequester iron may reduce the efficacy of zinc as a corrosion
inhibitor for asbestos cement pipe, (21) and fluoride added before
filtration may complex with aluminum and necessitate use of more
alum to coagulate the water (6). The sequence used for chemical
addition at a specific plant is often based on past practice which has
been developed over the years on an ad hoc basis. Conducting jar tests
to verify whether this sequence actually gives the best results is
advisable.
At diatomaceous earth (DE) filtration plants, coagulant chemicals
generally are not used. During the DE filtration process, a small
amount of diatomaceous earth (the body feed) is added to the raw
water. The DE particles, and the particles in the raw water, are
filtered out on the surface of a cake of diatomaceous earth that covers
the filter leaves, or septa. Adding DE body feed to the raw water helps
to maintain the high porosity of this cake, and thus maintains the good
performance characteristics of the filter.
The dose required for body feed is dependent upon the
concentration and nature of the particles in the raw water. As raw
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water particulate matter increases in concentration, the body feed dose
is increased to maintain a similar ratio of particles in the filter cake.
Rigid particles have good filtration characteristics, whereas gelatinous
or compressible particles, such as alum floe or algae, tend to clog the
filter cake. The body feed dose for rigid solids may be as low a 1 mg/L of
DE per 1 mg/L of solids. For compressible solids, the ratio of DE to
solids can be as high as 10 mg/L of DE per 1 mg/L of solids (22).
The grade of DE (size distribution of diatomite particles) is also
important. Removal of particles generally occurs by straining, and a
smaller pore structure is needed for removal of bacteria than for
Giardia cysts. Finer grades of DE are needed for removal of smaller
sizes of particles, but the smaller pores result in a more rapid build-up
of head-loss. A trade-off exists between better effluent clarity produced
by finer grades of DE, and longer filter runs produced by coarser
grades.
Rapid Mixing
Moffett (12) called rapid mixing the most important step in the
water treatment process. Letterman et al. (23) evaluated rapid mixing
and showed that a short period of high intensity mixing was the
method of choice. Vrale and Jorden (24) evaluated rapid mixer
configurations and for alum coagulation, concluded that in-line mixers
(plug flow reactors) were superior, whereas backmix reactors were less
effective. Backmixing systems were better for lime softening, because
the wide distribution of residence times in these mixers provides some
crystallized CaCOs particles which can promote more rapid
precipitation.
Another commonly used approach to rapid mixing is addition of
chemicals at a hydraulic jump. If this method is used, the chemicals
should be distributed across the entire width of the flow because of the
rapidity with which coagulation reactions occur. In addition, these
chemicals should be added just upstream of the hydraulic roll because
this is the zone of higher velocity and lesser depth.
Many plants may have only one rapid mixer. If sequential
additions of chemicals gives superior jar test or pilot plant results,
adding more mixing capability may be advisable. Options include in-
line mixing in a raw water line before the rapid mix compartment, or
perhaps subdividing the original rapid mix compartment. Earlier
practice typically provided more residence time in rapid mix basins
than is needed for good mixing, so subdividing may be feasible. The
amount of energy applied in the mixing basin may influence the
quality of the water produced. However, it is not likely that frequent
variations in mixing energy will be used by operating personnel. In a
plant evaluation, the engineer should consider whether or not large
scale changes in mixing energy should be made, rather than try to fine
tune the system by factors of 5 to 10 percent.
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Flocculation
At filtration plants that employ coagulation, flocculation is the
first optional process. In-line direct filtration plants do not employ
flocculation. Other direct filtration plants use flocculation but omit
sedimentation. Conventional filtration plants use rapid mixing,
flocculation, sedimentation, and filtration. At a direct filtration plant,
flocculators are operated to produce a small, dense floe that will store
well in the filter, whereas at a conventional plant a floe that settles
well is desired. These differences in purpose should be considered in a
plant evaluation.
Because of the need to have some uniformity in flocculation time,
these basins should be well baffled so that plug flow can be
approximated. Research by Argaman and Kaufman (25) indicated that
for equivalent removal of particulate matter, a four-compartment
flocculator required less flocculation time and energy (GT) than a
single basin flocculator. They stated, "Systems with equal overall
residence times will perform better as the degree of
compartmentalization increases." Their work suggests that if
performance is poor and a single compartment flocculator is being
used, addition of baffles should be undertaken to provide multiple
compartments within the same space.
Floe formation and breakup and the basis for tapered energy input
are discussed in Chapter 3, "Flocculation" in Coagulation and
Filtration: Back to the Basics (26). A high energy input at the
beginning of flocculation promotes many collisions among the small
coagulated particles that have entered the flocculation basin. As they
travel through the basin, the floe particles grow. Larger particles need
less energy for transport, and excessive energy might even break up
the larger floes. Because flocculation is dependent on both energy
input and flocculation time, prevention of short circuiting is
important. With completely mixed flow or when short circuiting
occurs, a substantial portion of the coagulated water will pass through
the flocculation basin in a time less than the theoretical detention
time, and thus the GT input for that water will be less than desired.
Jar tests can be used to develop preliminary estimates of the
flocculation time and energy needed to produce acceptable results in
the full scale plant.
The time needed for flocculation at direct filtration plants should
be determined on a case by case basis. For example, in order to treat
cold water, 30 minutes of flocculation was employed at a direct
filtration plant in Springfield, Massachusetts (27). Greater or smaller
times may be appropriate in different circumstances.
One indication of the benefits provided by extra detention time
when cold water is treated is illustrated by the change in performance
observed for a pressure filtration package plant at Cayuga, N.Y. (28).
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This plant employed three pressure filtration units. The first tank
contained a coarse medium, the second was a multimedia filter, and
the third contained granular activated carbon. The first unit was used
to promote flocculation and remove the larger suspended solids. The
second filter was to reduce turbidity to a fraction of 1 NTU, and the
GAG unit was to remove taste-and-odor causing substances. During
times of moderate temperature, the units worked as intended, and the
first two filters removed 98 to 99 percent of the turbidity- causing
particulate matter. Filtered water was less than 0.20 NTU more than
50 percent of the time. During winter, however, when water
temperatures were below 5°C, turbidity removal by the coarse media
filter and the multi-media filter dropped to about seventy percent.
Fortunately the GAG filter did a good job of turbidity removal, and the
final filtered water turbidity remained low, but this situation produced
a higher solids load on the GAG than was desirable. The reason for this
performance may have been inadequate contact time from the point of
coagulant addition to the multi-media filtration step.
Cleasby et al. (29) compared direct in-line filtration with
coagulation-flocculation-filtration on water obtained from a gravel pit
near Ames, Iowa. In parallel experiments, they found that the filter
receiving flocculated water had a shorter initial improvement period
after backwashing, produced a lower turbidity, and had a lower rate of
head loss buildup than the in-line filter. However, the filter receiving
flocculated water often developed turbidity breakthrough before the
in-line filter did.
Although flocculation may be needed for treatment of cold water, it
can be appropriate in other circumstances too. When the Southern
Nevada Water System, serving the Las Vegas area, expanded its direct
filtration plant from 200 MGD to 400 MGD flocculation was added to
reduce alum floe carryover and powdered activated carbon (PAG)
breakthrough (30). The original plant was constructed to provide rapid
mixing and filtration (31). According to Monscvitz et al. (30), about
half of the aluminum added as alum in the flash mixers was being
carried over into the finished water during in-line filtration. Also,
when PAG was added to control tastes and odors, the filtration rate
had to be reduced from 5 to 2 gpm/sf to prevent breakthrough of the
PAG. Pilot plant studies showed that 15 to 20 minutes of flocculation
would reduce carryover of aluminum and prevent breakthrough of the
PAG.
A variety of questions should be considered when flocculation is
examined during a plant evaluation. If the plant doesn't employ
sedimentation, is flocculation needed? Considering the present state of
knowledge about flocculation, and the different results obtained at
various locations, this question probably should be resolved by a
program of pilot testing. If flocculation is used, is the baffling provided
adequate to obtain a narrow distribution of hydraulic detention times?
If short circuiting is not a problem, is the energy input sufficient to
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cause collisions between coagulated particles, but not great enough to
break up floes already formed?
Sedimentation
Sedimentation is a necessary treatment step when the raw water
contains large amounts of particulate matter or humic substances
(color), or if the coagulation that gives the desired finished water
quality results in the production of large volumes of filter clogging floe.
If a sedimentation basin is in use, but seems to be ineffective, some
questions are in order. One that is not obvious is whether the engineer
selected the correct solids separation process in the initial design.
When soft, highly colored waters are treated for color and THM
precursor removal, a light, fluffy, poorly settling floe is often formed.
When algae are present in raw water, floe particles containing algal
cells may be floated to the surface of a sedimentation basin by bubbles
of oxygen produced during photosynthesis. When floe is resistant to
settling, dissolved air flotation may be a more appropriate solids
separation step. The evaluator should analyze the reasons for floe
behavior and adjust the treatment process to match the situation.
At locations where the floe should settle, but it doesn't, the
engineer should look for problems in settling basin design or operation.
Settling theory is based upon an idealized concept which assumes the
velocity of water in the basin is distributed uniformly, and all of the
water progresses form the inlet end to the outlet end of the basin. The
minimum settling velocity for particle removal in the basin is
calculated by dividing the depth of the water by the transit time for
inlet to outlet.
Real world conditions, unfortunately, differ considerably from this
ideal situation. Short circuits in settling basins are a fact of life.
Among the causes for short circuiting are effects of wind, temperature
(and therefore density) differences, inlet design, and outlet design.
Finding remedies to poor settling basin behavior can require using a
variety of approaches.
Temperature differences and the resulting density currents may
be present if large diurnal temperature differences are occurring. Cold
nights, sunny days, and a shallow raw water source susceptible to
these changing conditions can produce rapid temperature changes in
the raw water and in the treatment plant. A series of temperature
measurements in the raw water and at various places in the treatment
plant, especially across the width and depth of the settling basins,
should reveal the extent of these temperature differences and indicate
whether this might be a problem.
Checking a settling basin for hydraulics and flow distribution
problems can be more involved. Tracer studies may be needed. Another
approach is visual observation. Hudson (4) published an aerial
photograph showing clouds of floe in a settling basin with a poorly
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designed inlet. Identification of these localized clouds of floe and close
observation of basin outlets will frequently reveal design deficiencies.
When viewing underwater objects, greater success generally is
attained by looking down from above than by looking across and down
into the water. Application of the "look down from above" concept may
necessitate viewing settling basins from the tallest nearby structure at
the waterworks, or more probably, viewing the basins from a small
airplane or helicopter.
After tracer tests and visual observations are made, some physical
adjustments may be necessary. If it is possible, some trial and error
modifications to baffle arrangements could be attempted before
permanent modifications are put in place. If baffle modifications are
not sufficient, or if a higher treatment rate is needed, addition of tube
settlers should be considered. Conley and Hansen (32) have discussed
the addition of tube settlers to existing basins, and have provided
guidance for design of these installations.
When settling is evaluated, the engineer should keep in mind
Walter Conley's paper, "Integration of the Clarification Process" (7).
The goal of treatment is to produce the highest quality filtered water,
not the best quality of settled water that can be attained. Thus,
improvements made to settling basins, while important, do not
constitute the end of the upgrading job. The filters must also be
evaluated.
Filtration
The final, and in some plants only, step for removal of partieulate
matter is filtration. Improperly designed, operated, or maintained
filters can contribute to poor water quality, even if pretreatment is
good. On the other hand, if pretreatment is wrong, or worse yet*
nonexistent, the best rapid rate filter is not going to completely
salvage the situation and produce high quality filtered water. Again,
the idea of integration of the clarification process is important.
Several aspects of filtration should be investigated. These include
filter bed design, filtration rate and the methods used for rate control,
filter washing and physical condition of the beds, hydraulics, and
water quality monitoring.
Filter bed design has varied through the 1900's. Beds of sand, often
with an effective size of about 6.4 or 0.5 mm, were commonly used for
about half of this century. Work by a number of researchers (7,33,34),
helped to bring multi-media filter beds to the attention of engineers
and water utility managers. Dual media (anthracite coal and sand)
beds were used to provide larger pore spaces for storage office within
the depth of the bed, rather than just near the surface, as occurs with
rapid sand filters. Even when operated at rates higher than
conventional rapid sand filter rates, dual media filters provide
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excellent filtered water turbidity because of the finer layer of sand
below the coal.
Both Conley and Pitman (33) and Robeck et al. (34) reported on the
use of polyelectrolyte to toughen the floe and control turbidity
breakthrough in the latter phase of the filter run. Conley and Pitman
observed polyelectrolytes could be used with sand filters, but the fine
sands that produced exceptionally clear water also developed high
head loss. Robeck et al. reported that a polyelectrolyte coagulant aid
helped achieve the optimum strength of floe, and that adequate floe
strength prevented the passage of coliform bacteria, virus, carbon, or
floe. Logsdon et al. (18) compared different filter media configurations
for turbidity and Giardia cyst removal. They also presented data on the
effects of chemical pretreatment on filter run length. Head loss
development occurred several times faster in the sand filter, compared
to dual media. Estimated times to 8 foot head loss at a 3 gpm/sf
filtration rate were 13 and 28 hours with sand filters for runs with
alum and high molecular weight, slightly anionic polymer vs. four
days with the dual media filters.
If hydraulics for filtration and backwashing permit, conversion of
sand filters to dual or tri-mixed media filters may enable a water
utility to operate filters at higher rates while maintaining or
improving upon effluent turbidity and lengthening filters runs. The
feasibility of making such a change should be carefully evaluated by
the engineer before modifications are begun. Surface wash should be
provided, if it was not used with the original rapid sand filter.
Dual and mixed media concepts were developed so filters would
have coarser grains at the top and finer grains at the bottom after
backwashing. When a bed of mixed particle sizes of uniform specific
gravity is backwashed, the resulting gradation is fine to coarse from
top to bottom. Use of appropriately sized filtering materials of different
specific gravities ranging from 1.4 for some anthracite media to 4.1 for
some garnet media has brought about the desired coarse to fine
gradation.
Design of multi-media beds must be approached with caution,
especially if conversion of rapid sand filters is contemplated. In this
situation, constraints on the backwashing rate may exist. In any case
when more than one type of filter media is used, calculations should be
made to verify the materials selected have similar fluidization
velocities. Cleasby (35) has suggested doing this by calculating the
fluidization velocity for the DIQ size and the Dgo size (10% by weight
smaller than DIQ and 90% by weight smaller than Dgo) grains for each
filtering material. Fluidization velocities for the DIQ grain size of all
materials should be similar. Fluidization velocities for the larger Dgo
sizes will be greater than the DIQ velocities, but they too should be
similar for all of the filtering materials. A small uniformity coefficient
will narrow the range in fluidization velocities for the coarse and fine
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grains of each material. The circumstance to avoid is having a multi-
media filter with one layer that fluidizes at a different backwash rate
from that needed by the other layer or layers. If this occurs the plant
operator may have to backwash at a rate not adequate to fully clean
the layer that is not so readily fluidized, to avoid washing out the
easily fluidized layer. Pilot plant studies can be used to demonstrate
performance of the design.
Because of the difficulty associated with sampling individual
materials in a multi-media filter bed that has been in use, the engineer
probably will have to rely on a check of filter media specifications or
conduct visual observations during backwashing to look for problems
in this area.
Filter rate is an important factor in determining water quality.
Rate increases are especially important, because sudden increases can
cause deterioration of filtered water quality. Cleasby et al. (36)
evaluated the effect of filtration rate changes on effluent quality and
reported that both the magnitude of the rate increase and the rapidity
of the increase were important. Operators need to understand the
concept that gradual increases are less detrimental to quality, and
they should manage filtration rate changes accordingly. Logsdon et al.
(17) reported than an abrupt rate increase caused higher levels of
turbidity and Giardia cysts when alum was used, but a stronger floe
consisting of alum and nonionic polymer resisted breakthrough during
a rate increase. Both of these studies provide strong support for
minimizing filtration rate increases and avoiding a stop-start
operating mode for filters.
Research on filtration rate increases was conducted at the Duluth
Filtration Plant (6). Typical coagulation practice involved use of 10 to
15 mg/L of alum, about 0.10 mg/L of nonionic polymer, and a pH of 6.8
to 7.3, depending on water temperature. Three rate change conditions
were evaluated: 1) starting a dirty filter after shutdown overnight by
going from 0 to 3.25 or 4.87 gpm/sf in 15 minutes; 2) going for 3.25 to
4.33 gpm/sf or from 4.87 to 6.49 gpm/sf in 60 seconds when one filter
was removed from operation for backwashing; and 3) starting a clean
filter and going from 0 to 3.25 or 4.87 gpm/sf. Both the dual media
filter and the mixed media filter were restarted when clogged (9.0 ft.
head loss) and about 2 x 106 amphibole fibers per liter (F/L) were
detected in the effluent. Slowly restarting dirty filters at lower head
loss conditions did not produce this effect. Typical filter effluent during
normal operation had amphibole fiber counts of 0.04 x 106 F/L or lower.
Filters should be operated continuously from the beginning of a
run until terminal head loss is reached, and then washed. Dirty filters
should not be restarted (37). Logsdon et al. (17) showed that Giardia
cysts previously stored in a filter were discharged during a turbidity
breakthrough, when no cysts were present in the influent water. In
filtration research in which precipitated iron was the particulate
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matter to be removed, Cleasby et al. (36) maintained a constant
influent iron concentration of 10 mg/L and observed peak effluent
concentrations as high as 44 to 135 mg/L under instantaneous rate
increases of 25% to 100%. Contaminants stored in a filter can be
discharged later if the filter is operated improperly.
When a filtration plant is evaluated, typical plant operations
should be observed. Pay close attention to filter start-up, rate changes,
and backwashing procedures. Are changes in flow made smoothly and
slowly, or rapidly and in jerky steps? The latter will be detrimental to
water quality. If a plant has conventional rate of flow controllers, their.
condition should be checked. Are they well maintained and
functioning smoothly, or neglected and erratic, or even unworkable?
Hudson (4) noted that under fixed conditions rate controllers may
cause surge amplitudes of 2 to 10 percent of head loss. He suggested
evaluating this problem by measuring the distance between extreme
levels in water piezometer tubes which occur within a one minute
period. These measurements should be repeated several times to
obtain representative values. Water piezometers are very sensitive
indicators of filter surges and they are economical to install. Their use
should be considered when filters are studied.
Other aspects of filter hydraulics are also important. Flow
measurements should be made for each filter so the operator knows the
actual rate of filtration. Has flow measurement capability been
provided for each filter? This is not always done. If a flow measurement
device exists, does it work, and when was the last time it was
calibrated by a reliable method, such as measuring the rate for
drawdown of water over the filter? In order for the plant operator to be
in control of the filtration process, he or she must know what is going
on.
If water quality is seriously degraded at the start of a new filter
run at least three remedies may be considered. The simplest approach
may be to start a new run very gradually, rather than abruptly. This
concept was evaluated by Amirtharajah (38). If the filtration rate is
increased uniformly from zero to the desired value over a period of 10
to 30 minutes, the effect of restarting after backwash may be
diminished. Another approach, used by Harris (39) at the Contra Costa
County Water District was to add nonionic polymer! to the backwash
water. He indicated turbidity of the initial water produced following
backwash was on the order of 0.10 NTU when the filter media was
preconditioned with polymer.
Chen (40) studied filter bed conditioning with polymer using the
U.S. Environmental Protection Agency's Drinking Water Research
Division Pilot Plant. When the facilities were operated in the direct
Magn£floc985-N. Mention of commercial products does not
constitute endorsement.
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filtration mode, both turbidity and the concentration of particles in the
7 to 12 um size range could be lowered somewhat at an 8.5 gpm/sf
filtration rate and even more at a 5.0 gpm/sf rate, but some quality
deterioration occurred at the beginning of the runs in nearly every
trial.
If these approaches do not work, another way to improve water
quality is to practice filter to waste. This may, however, require some
extensive physical modifications which could be expensive to
implement. Filter to waste may be impractical under some conditions,
even if the physical arrangement of the plant permits these
modifications. Cleasby et al. (29) reported that when direct in-line
filtration was practiced with a cationic polymer as the primary
coagulant, the initial turbidity improvement period lasted several
hours in some runs. Filter to waste could not be used for such a long
time. In cases like this, a better approach will be to change coagulation
practice so that the ripening or improvement period is drastically
shortened.
During a plant evaluation, the condition of the filter beds should
be determined by examining a filter that is about ready to be
backwashed. After the water is drawn down below the media surface,
the engineer should climb down into the filter for a close look. Is the
surface of the bed level, or do obvious hills and valleys exist? If so, this
indicates problems with bad flow distribution during backwashing or
filter operation. Does the filter surface contain mud balls or caked
mud? This indicates cleaning during backwashing may be inadequate.
After the filter has been washed, the filter should be drawn down
again so the inspection can be repeated. At this time the cleanliness of
the media can be evaluated. Filter media should be clean after
backwashing. Clean grains of sand, coal, or garnet will not be
noticeably cohesive, even when wet. If filter media can be squeezed by
and into a ball, dropped on to the filter bed from waist height, and
remain intact; or if filter media is spongy when squeezed into a ball it
may not be as clean as it should be to function properly.
When a filter is being backwashed the engineer should observe it
carefully, watching for sand boils at the beginning of the wash. Baylis,
Gullans, and Hudson (41) wrote that filter backwashing should begin
slowly and be increased with care. They felt that at least 30 seconds
should be allowed to bring the backwash flow to its full value. Serious
support gravel disturbances or disruption of false-bottom filter
underdrains can be produced by sudden application of the full flow of
backwash water.
Some form of washing assistance is necessary, because backwash
alone usually does not adequately clean filter media. U.S. practice
generally has been to use surface wash, although air-assisted
backwashing has been used successfully in some plants. Performance
of surface wash also should be observed. The objective of this
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inspection is to verify that the water flow from the washing device is
uniform and the arms are rotating smoothly, surface wash spray
nozzles can become clogged during use and should be inspected
routinely. Polymer floe tend to be more sticky than alum floe, so if
polymers are used to assist in conditioning the raw water, careful
attention to the surface wash is certainly appropriate.
When air scour is used, careful operation is essential. Introducing
air into the filter too abruptly can damage the filter bottom.
Simultaneous application of both air and wash water has the potential
to cause media to wash out of the filter bed. If wash water is applied to
carry away dirt removed from the filter by air scour, the washing
should be very gentle during the air scour step and for as long as air
remains in the bed. Dirt loosened by the scouring process can be
carried away at velocities lower than those needed for fluidization of
media. Fluidization, if practiced, should occur only after the air has
been removed from the filter bed.
A key aspect to attaining the best possible performance of filters is
adequate monitoring and control. Filtered water turbidity, at a plant
that practices effective coagulation, can give an indication of the
efficacy ofGiardia cyst removal (17,18,42); asbestos fiber removal (43),
and of particle removal in general, even though concentrations of cysts
or fibers are below the levels that can be detected by a turbidimeter.
Research results suggest that when filtered water turbidity is very
low, removal of cysts or fibers is effective, as is removal of light
scattering particles in general. One differing view of the value of
turbidity measurements has been presented by Brazos et al. (44) who
contended there was very little relationship between turbidity
reduction and removal of total bacterial cells as determined by direct
microscopic count.
Hudson (4) stated that much credit for improvement in water
quality is due to the development of reliable water quality monitoring
devices, including turbidimeters. He wrote:
"In a number of plants, filtered-water turbidity levels prior to
the initiation of turbidity monitoring were commonly held in the
range of 0.2-0.5 NTU. After the initiation of monitoring, operators
could observe episodes of quality deterioration and develop
techniques to prevent such episodes, gradually revising their
personal quality goals to new levels and commonly reducing the
filtered-water turbidity to 0.02-0.05 NTU, an order-of-magnitude
improvement. This process takes one to two years, but once having
become accustomed to the production of water quality at such
levels, the operators of these plants become intolerant of filtered
water with more than about 0.06 NTU."
"One of the axioms of water quality control is that, as the
clarity of water is improved by improved treatment, there is a
parallel reduction of color, taste and odor, bacteria and viruses,
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and often of iron, manganese and alumina levels. While few
consumers can detect turbidity at a level of 1 NTU except in
bathtubs or swimming pools, the use of much lower levels of
turbidity brings about a corollary improvement of other water
quality parameters. The turbidity measurement is quick and
convenient, and although it is used at levels lower than those of
consumer awareness, it provides a most useful, rapid means of
control of treatment processes."
A similar sentiment was recently expressed. James R. English (45)
wrote in a letter to the Editor AWWA's Mainstream that turbidity of
filtered water at the Sidney N. Peterson Water Treatment Plant
operated by the San Juan Suburban Water District in Roseville,
California averaged 0.04 NTU. He also stated, "Achieving the levels of
water quality we have has completely changed the self-image of each
plant operator. They begin to worry when turbidity levels reach 0.06
NTU, and when that happens, they typically find that a drift has
occurred in the chemical feed." This sounds very much like Hudson's
comment on operator attitudes. Plant operators should continue to
strive to find better, more cost-effective ways to treat water and
improve finished water quality.
Other evidence of the attainability of very low filtered water
turbidity was presented by Schleppenbach (6). The Duluth Filtration
Plant produced water with effluent turbidity consistently below 0.10
NTU for over two years, after the initial start-up and learning period
at the new plant. This occurred over all four seasons, and a range of
water temperatures from 1°C to about 12°C.
A key to success at Duluth, and an approach used by many
filtration plants, is the continuous monitoring of effluent turbidity.
Conley (7) stated, "Continuous monitoring of filter plant effluent with
sensitive turbidimeters is essential for intelligent management of the
plant." A similar opinion was held by Robeck et. al. (34) who wrote, "Of
course, monitoring for turbidity passage on a continuous, or half hour
interval, basis also allows the operator to know the status of the filters
and the influence of chemical dose changes."
"Intelligent management", and "knowing the status" are the key
words. When turbidity is monitored consistently, operators are able to
observe trends in turbidity, and changes can be acted upon soon after
they occur. The operator is not put in the position of knowing what the
turbidity is only once every two, four, or eight hours, as might be the
case when turbidity is measured only in grab samples taken to the
laboratory for analysis. Grab sample measurements may be needed for
compliance purposes, and they can be used as a means of adjusting the
calibration of continuous flow turbidimeters. However, there are many
benefits associated with continuous measurements. When
turbidimeters are installed on all filters, readout devices can be placed
in operational control panels so the operator has instantly available
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information to 'act upon. Chart recorders can be used to provide written
records which are useful for compliance purposes and periodic reviews
of the performance of the plant.
The diatomaceous earth filtration and slow sand filtration
processes differ in numerous ways from the coagulation-filtration
process which has been the subject of much of this paper. Coagulation
for pretreatment is not employed in either slow sand filtration or DE
filtration, and this difference is crucial. Many of the steps that are part
of the evaluation of a conventional plant or direct filtration plant
employing coagulation can be omitted. These include: coagulation dose
determination, coagulant feed, rapid mix, flocculation, and
sedimentation. However, in all filtration processes flow patterns,
hydraulics, rate changes, and cycles of on- off operation are very
important. These factors need to be considered in the evaluation of any
kind of filter. Other aspects of filtration are process-specific and relate
to slow sand or DE facilities. They are discussed in the paragraphs that
follow.
Slow sand filtration relies extensively on the development of a
biological population within the filter to accomplish removal of
particulate contaminants. Therefore applying disinfectant chemical to
raw water ahead of a slow sand filter can be detrimental to filtered
water quality. Because slow sand filters frequently treat raw water
that has been given no pretreatment, few options are available to the
operator of a slow sand filter plant that is not providing water of
satisfactory quality. These filters generally are designed to treat water
within a certain range of conditions. Those who attempt to use a slow
sand filter outside the established conditions are inviting problems.
Three important factors in successful operation are proper media
size, adequate media depth, and proper filtration rate. Proper media
design for a new plant is the design engineer's responsibility. However,
after a sufficient quantity of sand has been scraped away during the
cleaning process sand is again added. This process, called resanding, is
often done by the utility without the assistance of an engineer. Slow
sand filters need media with an effective size (e.s.) of 0.15 to 0.30 mm
(46); therefore, resanding with sand intended for a rapid sand filter ,
(0.45 to 0.55 mm e.s.) can cause deterioration of the effluent. An
engineer evaluating a slow sand filter should try to learn what size
sand is in use.
Scraping a filter repeatedly results in a progressively shallower
bed. Failure to resand can have very serious consequences. Visscher et
al. (46) suggest that the minimum depth of the sand bed should be 0.5
to 0.6 m. Scraping sand away and failing to replace it periodically will
eventually cause the bed to be too shallow to be effective. Finally, in an
attempt to obtain higher water production, the utility may operate the
filter at a rate which results in reduced efficiency for removal of
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turbidity and microorganisms. Visscher et al. recommend a range of
0.1 m/hr to 0.2 m/hr for slow sand filters.
If a waterborne disease outbreak has been caused by passage of
microorganisms through a sjow sand filter, the investigating engineer
should base his or her actions on the probability that something
related to the condition or operation of the filter is seriously wrong
Historically slow sand filters have provided very good protection
against the transmission of waterborne disease. If a slow sand filter is'
not performing effectively, the filter beds should be dewatered one at a
time, and the condition of the sand beds should be evaluated. The sarid
surface should be level (observe this as the water surface drops below
the sand) with no erosion of sand caused by influent water. The sand-
wall interface should be free of cracks where water could short circuit
the bed and avoid filtration. Core samples could be taken at a variety
of locations within the bed to provide data on sand size and depth to the
support gravel.
Because slow sand filtration is not widely used in the United
States, many water treatment engineers have little or no experience in
working with the process. Information on slow sand filtration has been
published in JAWWA from time to time since December, 1984, when a
number of papers on the topic appeared in a single issue. The
International Reference Centre's book on slow sand filtration (46) is
also an excellent reference work. Sloti Sand Filtration by Huisman
and Wood (47) published in 1974 is older but very helpful. This is
available from the World Health Organization and WHO publication
sources. Engineers without slow sand experience should find these
sources to be quite useful.
Diatomaceous earth filtration generally does not involve the use of
coagulant chemicals to pretreat the raw water. Gelatinous particulate
matter tends to blind the DE filter cake, so using alum or iron to treat
the raw water would cause shorter filter runs. The size of particulate
matter passed by the filter is determined by the grade of DE employed.
Fine potable water grades can be used to remove bacteria Coarse
potable water grades will remove Gidrdia cysts. The removal
mechanism for cysts is straining (48). For effective virus removal the
diatomaceous earth should be conditioned with a coagulant to promote
attachment of virus particles onto the DE.
Passage of microorganisms can be caused by using a grade of DE
which is too coarse to remove the organisms in question and by a
number of equipment and procedural problems. The filter leaves or
septa, must be kept very clean. Backwash at the end of a run should be
thorough Septa should^be inspected visually at periodic intervals.
With certain types^ of filters this can be easily accomplished, but
pressure filters with bolted pressure domes on both ends of the cylinder
can be very difficult to open and inspect. Filter septum integrity must
also be maintained. A hole in a septum can permit raw wateV to short
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circuit past the filter cake instead of flowing through it. A precoat
thickness of 0.1 Kg/m2 (0.2 pounds/ft2) is recommended for Giardia
cyst removal, and body feed always should be used (17). Any
interruption of a DE filter run should be followed by backwashing and
precoating the filter. Reuse of dirty diatomite could result in
penetration of the DE filter by microorganisms.
Diatomaceous earth filtration, like slow sand filtration, has not
been as widely employed as coagulation-filtration. Additional
information on DE filtration is available in AWWA Manual M30 (22)
and in Chapter 15, "Precoat Filtration," (49) in Water Treatment Plant
Design.
Disinfection
Disinfection is a key factor in the evaluation of a treatment plant.
Among the tasks that need to be accomplished are review of plant
records and monitoring procedures (already discussed), evaluation of
disinfection equipment, and inspection of physical processes.
The four chemical disinfectants most commonly used in the United
States are free chlorine, chloramine, chlorine dioxide and ozbne.
Although ozone was seldom used in past decades, interest in ozone is
rising. A number of new installations have been completed in recent
years, and more seem likely to be built. Ozone differs from other
chemical disinfectants in its lack of a long lasting residual and in its
high level of efficacy. Like chlorine dioxide, ozone must be generated
on site. .
Review of disinfection equipment is an important aspect of a plant
evaluation. If the disinfectant is purchased and fed, the feed facilities
should be adequate to provide the needed dose, and a sufficiently large
inventory should be on hand. If regulatory requirements are
established on the size of this inventory, they should be followed. If the
disinfectant is produced on-site, the production capacity of the
equipment should be large enough to satisfy the expected disinfectant
demand and maintain the desired residual, with allowances for
maintenance and repair of the system. On-site production is necessary
for ozone and chlorine dioxide, and is sometimes used for chlorine.
The dependability of the equipment is important because
disinfectant must function properly any time water is produced.
Rugged and reliable disinfection equipment is especially needed at
facilities which are remote or are staffed on a part-time basis rather
than around the clock. Redundancy of facilities and availability of
emergency power should be evaluated also. The type of monitoring
equipment and its reliability are important to ensure continuous
disinfection with an appropriate concentration of chemical. Because
emergencies can occur, disinfection facilities should be provided with
alarms and sensors to detect problems of inadequate residual, loss of
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feed, or other similar situations, such as an inadequate amount of
chlorine remaining in a storage tank (50).
Physical processes and facilities play an important role in
disinfection. Ideally, disinfectant should be added to water and
immediately mixed thoroughly. Following initial mixing, contact time
should be provided. The actual contact time is most easily defined and
is most uniform when plug flow conditions occur.
Disinfectants should be added at locations where they are
compatible with the entire process train and with other chemicals that
are being added at the same point. For example, adding powdered
activated carbon and chlorine at the same location is a wasteful use of
both chlorine and carbon. Adding chlorine to an iron-bearing well
water ahead of an aeration tower might be inappropriate if the
chlorine oxidized all of the iron and the aerator then served no useful
function. Adding chlorine in the plug flow conditions that prevail in
the middle of a settling basin would be counter productive, because the
chlorine would not mix with all of the water until it flowed out of the
basin. Sometimes in water treatment plant design the disinfection
process has been "added on" rather than specifically engineered. This
can be dangerous if all of the essential concepts have not been
considered. . .
When chlorine is added to water, it should be mixed quickly,
thoroughly, and uniformly throughout the water. According to Water
Treatment Plant Design (51), if the ammonia concentration in water
equals or exceeds about 10 percent of the chlorine dose, initial mixing
of chlorine is especially important. Failure to attain thorough and
rapid dispersal and mixing of chlorine can result in break point
chlorination where some portions of the water have substantially
higher than average concentration of chlorine and other portions have
opportunity for little or no reaction .with much lower than average or
minimal concentration of chlorine. In the presence of ammonia,
chlorine appears to be more effective during the first few seconds of
addition, hence the greater importance of mixing that is accomplished
very rapidly and with a minimum of backmixing. In-line mixers,
diffusers across pipes, and hydraulic jumps (51) would be appropriate,
but traditional stirred rapid mix basins with backmixing are not
desirable in this circumstance. :
At some-utilities, clearwells have been used to provide disinfectant
contact time. Contact time is variable in clearwells because they are
also used as flow equalization basins, storing water when production
exceeds demand and providing water when demand exceeds
production. Contact times approach a minimum during periods of high
flow when the volume of water in storage is low and the clearwell is
emptying faster than it is being filled. Even with adequate baffles to
attain plug flow, the contact time in a clearwell may not be adequate if
this structure is used to provide disinfectant C-T.
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C-T Determination
Use of the C-T concept (concentration of disinfectant (C)
multiplied by contact time (T) in minutes) is required for unfiltered
surface waters in the Surface Water Treatment Rule and is
recommended by EPA for plants that filter surface water. Contact time
is a function of the flow rate in a treatment plant, contact basin, or
pipeline. Accurately estimating this contact time is as important as
accurately measuring the disinfectant residual.
Contact time in pipes, basins and tanks is related to flow, volume,
and vessel shape. When plug flow is closely approximated, the actual
contact time closely approximates the theoretical detention time.
According to Water Treatment Principles and. Design (50) baffled
serpentine contact chambers having length to width ratios exceeding
200 give a close approximation of plug flow.
Basins having other configurations, such as clearwells,
sedimentation basins, and flocculation basins probably need to be
evaluated by tracer test to determine the residence time. Dead spots
and short circuiting can occur allowing portions of the water to flow
through these basins in considerably shorter times than the
theoretical detention time that would be calculated by dividing the
basin volume by the flow rate.
Lippy (52) presented an interesting, example of theoretical
detection time vs. actual contact time in a review of a giardiasis
outbreak in New Hampshire. The treatment plant had a 0.5 million
gallon storage facility plus a 0.04 million gallon pumping well that
followed the postchlorination injection point. Based on volume
displacement calculations the 0.54 million gallons should have
provided 7 hours of contact time. In actual practice, a substantial boost
in chlorine dosage was followed by a measurable increase in the
chlorine concentration of the water discharged from the pumping well
only 1-3/4 hours later. Contact time is not always what it appears to be.
Actual contact times measured by tracer studies should be used to
evaluate disinfection practice unless plug flow conditions are know to
exist.
After the contact time and disinfectant residual are determined,
the C-T. product can be calculated. C-T. values needed for
inactivation of microorganisms vary depending on water temperature.
The efficacy of free chlorine and chloramine is also dependent upon pH,
so both temperature arid pH data are needed to judge efficacy of the
C-T. product. Records of water quality measurements should be
reviewed to determine these parameters.
Summary
Well-operated water filtration plants and properly managed
disinfection processes should present effective barriers to the passage
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of microorganisms into drinking water, especially when both filtration
and disinfection are used. Occurrence of a waterborne outbreak is
evidence that severe problems have developed, and corrective action is
needed. This paper presents a discussion of measures that can be taken
by an engineer investigating a treatment plant during or after an
outbreak.
References
1. Logsdon, G. S., Evaluating treatment plants for particulate
contaminant removal, J. AWWA, 79:9:82- 92,1987.
Gordon, G., Cooper, W. J., Rice, R. G. and Pacey, G. E., Methods
of measuring disinfectant residuals, J. AWWA, SO'9-94-108
1988.
American Public Health Association, Standard Methods for the
Examination of Water and Waste water, 17th Ed APHA
Washington, D.C., 1989. ' ' '
Hudson, H. E., Jr. (1981) Water Clarification Processes:
Practical Design and Evaluation. Van Nostrand Reinhold
Company, New York.
Lippy, E. C., Engineering aspects of waterborne outbreak
investigation, In: G. F. Craun, (ed.), Waterborne Diseases in the
United States,
-------
14. Dentel, S. and Kingery, K. M., Use of streaming current
detectors in water treatment, J. AWWA. 81:3:85-94,1989.
15. Conley, W. R., and Evers, R. H., Coagulation control, J. AWWA,
60:2:165-174,1968.
16. Kreissl, J. F., Robeck, G. G., and Sommerville, G. A., Use of pilot
filters to predict optimum chemical feeds, J. AWWA, 60:3:299-
314,1968.
17. Logsdon, G. S., Symons, J. M., Hoye, R. L., and Arozarena, M.
M., Alternative filtration methods for removal of Giardia cysts
and cyst models, J. AWWA, 73:2:111-118,1981.
18. Logsdon, G. S., Thurman, V. C., Frindt, E. S., and Stoecker, J.
G., Evaluating sedimentation and various filter media for
removal of Giardia cysts, J. AWWA, 77:2:61-66,1985.
19. Hudson, H. E., Jr., and Wolfner, J. P., Design of mixing and
flocculating basins, J. AWWA, 59:10:1257-1267,1967.
20. Forbes, R. E., Nickerson, G. L., Hudson, H. E., Jr., and Wagner,
E. G., Upgrading water treatment plants: an alternative to new
construction, J. AWWA, 72:5:254-261,1980.
21. Weston Water Utility, Schofield, Wisconsin, Control of Asbestos
Fiber Loss from Asbestos-Cement Watermain, EPA-600/2-84-
014, U.S. Environmental Protection Agency, Cincinnati, Ohio,
1984.
22. Mclndoe, R. W., Logsdon, G. S., Ris, J. L. and Wirsig, A., Precoat
Filtration, AWWA Manual M 30, American Water Works
Association, Denver, Colorado, 1988.
23. Letterman, R. D., Quon, J. E., and Gemmell, R. S., Influence of
rapid mix parameters on flocculation, J. AWWA, 05:11:716-722,
1973.
24. Vrale, L., and Jorden, R. M., Rapid mixing in water treatment,
J. AWWA, 63:1:52-58,1971.
25. Argaman, Y., and Kaufman, W. J., Turbulence and flocculation,
J. of the Sanitary Engineering Division, ASCE, 96:2:223-241,
1970.
26. Letterman, R. D., Flocculation, In: Coagulation and Filtration:
Back to the Basics, AWWA Seminar Proceedings, No. 20155,45-
47,1981.
27. Sweeney, G. E., and Prendiville, P. W., Direct filtration: an
economic answer to a city's water needs, J. AWWA, 66:2:65-71,
1974.
28. MacNeill, J. S., Jr., and MacNeill, A., Feasibility Study of
Alternative Technology for Small Community Water Supply,
154
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29.
30.
31.
32.
33.
34.
36.
37.
38.
39.
40.
EPA-600/2-84-191, U. S. Environmental Protection Agency,
Cincinnati, Ohio, 1985.
Cleasby, J. L., Hilmoe, D. J., Dimitracopoulos, C. J., and Diaz-
Bossio, L. M., Effective Filtration Methods for Small Water
Supplies, EPA 600/2-84-088, U.S. Environmental Protection
Agency, Cincinnati, Ohio, 1984.
Monscvitz, J. T., Rexing, D. J., Williams, R. G., and Heckler, J.,
Some practical experience in direct filtration, J. AWWA
70:10:584-588,1978.
Spink, C. M., and Monscvitz, J. T., Design and operation of a
200-MGD direct filtration facility, J. AWWA 66-2-127-132
1974.
Conley, W. E., and Hansen, S. P., Advanced Techniques for
Suspended Solids Removal, In: R. L. Sanks, (ed.), Water
Treatment Plant Design for the Practicing Engineer, Ann Arbor
Science Publishers, Ann Arbor, Michigan, 1978.
Conley, W. R., and Pitman, R. W., Test program for filter
evaluation at Hanford, J. AWWA, 52:2:205- 214,1960.
Robeck, G. G., Dostal, K. A., and Woodward, R. L., Studies of
modifications in water filtration, J. AWWA, 56:2:198-213,1964.
35. Cleasby, J. L., Unconventional Filtration Rates, Media, and
Backwashing Techniques, In: E. A. Glysson, D. E. Swan, and E.
J. Way, (eds.), Innovations in the Water and Wastewater Fields,
Butterworth Publishers, Boston, Massachusetts, 1985.
Cleasby, J. L., Williamson, M. M., and Baumann, E. R., Effect of
filtration rate changes on quality, J. AWWA> 55:7:869-880
1963.
Logsdon, G. S., Mason, L. and Stanley, J. B., Jr., Trouble-
shooting an existing treatment plant, In: Filtration: Meeting
New Standards, AWWA Seminar Proceedings No 20028 109-
125,1988.
Bucklin, K., Amirtharajah, A., and Cranston, K. O., The
Characteristics of Initial Effluent Quality and Its Implications
for the Filter to Waste Procedure, AWWA Research Foundation
Research Report, AWWARF, Denver, Colorado, 1988.
Harris, W. L., High-rate filter efficiency, J. AWWA, 62:8:515-
Chen, Cheng-Tyng, Filter Preconditioning to Reduce Initial
Degradation in Effluent Water Quality, Master of Science
Thesis submitted to University of Cincinnati, Cincinnati Ohio
1986.
155
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41. Baylis, J. R., Gullans, O., and Hudson, H. E., Jr., Filtration, In:
Water Quality and Treatment, Third Edition, American Water
Works Association, 1971.
42. Al-Ani, M. Y., Hendricks, D. W., Logsdon, G. S., and Hibler, C.
P., Removing Giardia cysts from low turbidity waters by rapid
rate filtration, J. AWWA, 78:5:66-73,1986.
43. Logsdon, G. S., Symons, J. M., and Sorg, T. J., Monitoring water
filters for asbestos removal, J. of the Environmental Engineering
Division, ASCE, 107:6:1297-1315,1981.
44. Brazos, B. J., O'Connor, J. T., and Lenau, C. W., Seasonal Effect
of Total Bacterial Removals in a Rapid Sand Filtration Plant,
In: Proceedings 1986 AWWA Water Quality Technology
Conference, 795-833,1986.
45. English, J. R., Meeting turbidity standards, Letter to Editor of
AWWA Mainstream, 31:1:2,1987.
46. Visscher, J. T., Paramasivam, R., Raman, A., and Heijnen, H.
A., Slow Sand Filtration for Community Water Supply,
Technical Paper 24, International Reference Centre for
Community Water Supply and Sanitation, The Hague, The
Netherlands, 1987.
47. Huisman, L. and Wood, W.E., Slow Sand Filtration, World
Health Organization, Geneva Switzerland, 1974.
48. Walton, H.G., Diatomite filtration: why it removes Giardia from
water, In: P.M. Wallis and B.R. Hammond, (eds.), Advances in
Giardia Research, The University of Calgary Press, Calgary,
Alberta, 113-116,1988.
49. Baumann, E. R., Precoat Filtration, In: R. L. Sanks, (ed.), Water
Treatment Plant Design, Ann Arbor Science Publishers, Inc.,
313-370,1978.
50. James M. Montgomery Consulting Engineers, Inc., Water
Treatment Principles & Design, John Wiley & Sons, New York,
N.Y., 1985.
51. American Society of Civil Engineers and American Water
Works Association, Water Treatment Plant Design, 2nd Ed.,
McGraw-Hill, New York, N.Y., 1990.
52. Lippy, E. C., Tracing a giardiasis outbreak at Berlin, New
Hampshire, J. AWWA, 70:9:512-520, 1978. Magnifloc 985-N.
Mention of commercial products does not constitute
endorsement.
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IV. Engineering and Water Quality Concerns
Surface Water Source Protection
by: Joseph L. Glicker, P.E.
Water Quality Director
Portland, Oregon Water Bureau
1120 SW 5th Avenue
Portland, OR 97204
(503) 796-7471
Maintaining the best possible water quality is the goal of any
surface water source protection program. This chapter outlines the
process by which a surface water source protection program can be
developed, the most common threats to water quality in surface
supplies, and the general means used to control those threats. It also
describes some of the upcoming problems and needs in the field of
watershed protection. The City of Portland, Oregon's, Bull Run
watershed is used to illustrate application of some of these concepts.
Why Watershed Protection?
Providing high quality water at the consumer's tap is both a desire
and a legal requirement for water suppliers in the United States. In
meeting this need and desire, a water utility can rely on only two
things - the initial quality of its raw water and the treatment and
control processes that are applied to that water as the water moves
towards the tap.
While water treatment processes can significantly reduce the
amount of contaminants present in a raw water, the costs and the
potential risks from residual amounts of contaminants left after
treatment may be too high. This is particularly true as the number of
contaminants of concern increases and the level of risk that the public
is willing to accept decreases. Some water systems may require
additional steps in the treatment train, such as aeration or granular
.activated carbon (GAG) filtration, at great increases in treatment
costs. The efficacy of these treatment processes is usually measured by
the percentage of contamination in the raw water they are capable of
removing and not necessarily by the absolute contaminant level they
attain. Thus, even with the addition of these treatment steps, the risks
of chronic or acute disease from the contaminants remaining in the
raw water may still be greater than the consumer is willing to accept.
Allowing degradation of a water source because of the presence of, or
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the ability to construct, a water treatment plant, therefore, will not
maximize protection of public health.
A watershed protection program is an important element of the
multiple barrier approach to providing high quality water. Watershed
protection programs minimize the need for more extensive treatment
processes and therefore, minimize the resulting public health risks
associated with the existing barriers. Such programs are useful to all
water systems regardless of the status or nature of their treatment
processes.
Developing a Watershed Protection Program
The first step in developing an effective watershed protection
program is to be thoroughly familiar with the watershed and the
problems that it faces. The water supplier should determine watershed
boundaries, physical features, land uses, hydrology, geology, and their
relationship to the water supply facilities. Most importantly, the
sources of contamination should be identified and their impact on
water quality within the watershed studied. Formal sanitary surveys
are a useful tool in this process.
Before a watershed protection program can be developed, the basic
goals, principles, and use patterns for the watershed must be
identified. These goals will greatly impact the choice of control
strategies and protection measures. Examples of types of goals for
different watershed protection programs include:
1. Preventing any human-caused situations, such as a hazardous
material spill or an aerial pesticide spray program, from
contaminating the supply.
2. Mitigating the effects of any natural disasters (flood, fire,
windstorms) that may occur.
3. Providing a specific water quality to minimize treatment processes
(for example, to be able to use the water with only disinfection or
with only conventional treatment but without GAG).
Once the aims of the watershed protection program have been
identified, the watershed has been characterized, and the sources of
contamination inventoried and studied, the water utility can begin
developing specific means of achieving the goals. These means may
include physical facilities such as stormwater sediment traps or
containment facilities around hazardous material storage sites;
operational programs such as inspecting septic tank designs; and
management controls such as lot size restrictions or buffer setbacks
from streams and reservoirs.
The final step in developing a watershed protection program is
monitoring and evaluating the chosen control measures to determine if
the identified objectives are being achieved. Any deficiencies noted by
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the monitoring and evaluation process should then be corrected and
improved through ongoing upgrading of the watershed control
program.
Land Use Impacts on Water Quality
When using a process like the one outlined above, the water utility
will likely find that some of the water quality and land use concerns
found across the country will apply. A national survey of surface water
utilities conducted in the early 1980s (1) found that agricultural
cropland was the primary rural source of water quality problems,
followed by forestry activities, feedlots, and large dairy operations and
mining. The most significant urban sources of contamination were
stormwater runoff from urban and industrial areas and septic tanks.
Other sources of contamination include grazing; recreation; municipal
and industrial point source discharges; commercial and residential
establishments and communities; and secondary uses of water supply
lakes, reservoirs, and intake streams for flood control, hydropower
generation, recreation, wildlife habitat, and irrigation.
These activities can impact a number of water quality parameters.
The National Urban Runoff Program (NURP) concluded that heavy
metals (primarily copper, lead, and zinc), coliform bacteria, suspended
solids, and nutrients (nitrogen and phosphorous) were the variables
most influenced by urban runoff (2). Forest management activities
(road construction, logging, slash burning) can increase soil erosion
and thus sediment and turbidity (3), nitrate concentrations (4), and
color (5). Agricultural land runoff can increase sediment, turbidity,
dissolved solids, nutrients, bacteria, pathogenic organisms including
cryptosporidium from feedlots and grazing areas, and herbicides and
pesticides (6). Acid rain can result in elevated levels of mercury,
aluminum, cadmium, lead, asbestos, and nitrates (7), particularly in
small surface streams. While recreational uses of remote mountain
streams increase the likelihood of Giardia contamination, cysts have
been found even in very low use areas (8,9). Table 4.1.1 is a summary
matrix of typical watershed land uses and the water quality variables
they can affect, which was developed based on these and other
references.
Typical Watershed Programs
Influence over these land use activities is highly dependent on the
degree and method of control available to water utilities. Direct
ownership of the land provides the most direct control over land use
activities, but other avenues are available when the watershed is only
partially owned, or not owned, by the utility. Written agreements with
land owners, zoning ordinances, and various local, state, and federal
planning and environmental laws and regulations all form the basis
for regulating land use activities that may harm water quality. Buffer
setbacks from-streams and reservoirs, land use prohibitions, entry
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Table 4.1.1. Land Use-Pollutant Analysis Matrix
Land Use
Concern or
Management Tur- Suspended
Problem bidity Sediment pH
Nitro- Phos- Giar- Crypto- Virus-
Algae gen phorous 'dia sporidium es
Cropland x
Runoff
Dairy Feedlots x
Grazing x
Recreation
Timber x
Management
Road x
Construction
Mining x
Industrial x
Discharge
Sewage x
Discharge
Sepb'c Tanks
Urbanization x
Hazardous
Waste
Disposal
Hazardous
Waste
Transport
Acid Rain
X X X X X X ' X
X X X X X X -X
x x x x x x ' , x
X X ' X
X XXX
x xxx
XX
X X X X X •"•-;.-.
X XXX X X XX
X X X X XXX
X X X X X X X X
X
(Continued)
controls, large lot sizing, limitations on allowed impervious surface
area on a site, utility approvals of facility designs, sanitary
regulations, land acquisitions and exchanges, hazardous materials
transportation restrictions, and special storm- and wastewater
treatment facilities are all measures that can be taken in individual
circumstances to control potential problems. -
The literature describes examples of various watershed protection
programs developed by water utilities. Burby et al. 'described an
example of "downzoning," the use of large minimum lot sizes
(anywhere from 1 to 10 acres depending on soils, slopes, proximity to
streams, and other local conditions) to control development pressure,
in Newark, New Jersey's watershed (1). The Scituate Watershed Study
for Providence, Rhode Island, discussed methods for reducing risk from
hazardous materials spills and other road runoff (id): Portland,
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Table 4.1.1. (Continued)
Land Use
Concern or
Management
Problem
Cropland
Runoff
Dairy Feedlots
Grazing
Recreation
Timber
Management
Road
Construction
Mining
Industrial
Discharge
Sewage
Discharge
Septic Tanks
Urbanization
Hazardous
Waste
Disposal
Hazardous
Waste
Transport
Acid Rain
Total Fecal other
Coli- Coli- Pesti- Synthetic Heavy Manga-
form form THMs cides VOCs Organics Metals Iron nese
x x x x x xx
XXX
XXX
x x
X X X X XX
X XX
XX X
xxx x xx x
x x x x x x xx x
x x x x x
xxxxx x xx x
XXX X
x x x x
XX X
Maine's use of septic system inspection was described by Grady (11)
Controlling forest management activities in the Northwest was
described for Seattle, Washington (12), and Portland, Oregon (13) A
recently completed study funded by the American Water Works
Association Research Foundation (AWWARF) documents those
watershed control practices that have been found most effective across
the country.
Monitoring and evaluating the effectiveness of the control
measures can occur on several levels. Utilities can conduct water
quality monitoring programs for parameters of concern in streams,
reservoirs, and local potential contamination sources. Models for
evaluating control options have been developed (15) and can be
adjusted over time based on the observed results. The utility can also
perform onsite inspections for conformance with any restrictions and
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can implement research programs to upgrade information on which
decisions are based. The results of these activities must be periodically
compared to the goals of the watershed protection program and to the
list of control measures being used to determine whether the program
is performing as desired and how it can be improved over time.
Directions in Watershed Protection Programs
The promulgation and implementation of EPA's Surface Water
Treatment Rule, along with other environmental legislation, has and
will have a significant impact on watershed management programs.
Many watershed programs are being pushed from two somewhat
contradictory directions; at the same time that watersheds feel
pressures for increased use and activity, the requirements of
legislation, regulation, and public expectations lead to a need for
greater control and oversight of those activities. While the public is
generally aware of the contamination potential from domestic and
industrial waste point discharge sources, they are less aware of the
subtle effects that increased urbanization, demands for recreational
opportunities, and other lifestyle related activities can have on water
quality. For example, development of homes in rural areas may result
in increased contamination from sewage discharges if sewers are not
available, septic tanks are improperly installed, or the capacity of the
soil for septic systems is exceeded.
These problems raise difficult questions of equity concerning the
distribution of costs that result from the need to control these activities
and to clean up or provide treatment if control measures fail. A
community may be forced to install expensive new water treatment
equipment if watershed degradation occurs because of some
uncontrolled land use, while the individual responsible for the use may
not have to bear any cost. Conversely, a community may wish to
impose significant expense on a land user to control a contamination
source, when the land owner receives no benefit and may not even.
have had to worry about the contaminant if the community was not
using the area as a watershed. As an example, a developer may be
restricted to 5-acre lot size and be required to install stormwater
retention ponds for sediment control in a watershed subdivision, while
getting water supply from an entirely different water source.
These issues are also complicated by uncoordinated and conflicting
environmental legislation and regulation at both the state and federal
levels. Those with responsibilities for drinking water programs may
not be aware of actions being taken by other agencies which will affect
the land use and water quality that a watershed may produce. The
reverse is also true. The following is one classic example. In the
Surface Water Treatment Rule proposed in November 1987, EPA
requires that suppliers of unfiltered water must have ownership or
written agreements with landowners in the watershed. In a regulation
proposed by the U.S. Forest Service in September 1987 concerning
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management of municipal watersheds, any new such formal
agreements were specifically prohibited. Another example was a
recent effort by the State of New York Department of Environmental
Conservation to eliminate chlorination of wastewater treatment plant
effluent as it is discharged into streams within the watershed of the
City of New York, at the same time that the State Health Department
was asking the city to provide stricter controls on sources of
contamination in the watershed. To fully protect the nation's water
resources, greater coordination will be needed between agencies
responsible for Clean Water Act implementation, Safe Drinking Water
Act implementation, ground-water protection measures, and land use
planning with agencies responsible for watershed protection.
The role of the public in watershed issues is also increasing.
Communities are no longer as willing to leave decisions about
watershed management to government officials and technical experts.
The public increasingly wants a say in the decision-making process
through advisory committees, environmental impact statements
public hearings, and discussions with staff.
Portland's Watershed Protection Program
The watershed protection program for the Bull Run watershed
which serves as the primary water supply for Portland, Oregon, is a
good example of the implementation of watershed protection issues
and processes. This watershed is located about 25 miles east of
Portland on the western slopes of the Cascade mountains. The physical
drainage covers about 68,000 acres. About 95% of this land is owned by
the federal government and administered along with a 30 000-acre
buffer by the U.S. Forest Service as the Bull Run Management Unit
The watershed is dominated by Douglas Fir-Western Hemlock forests
and ranges in elevation from 800 to 4,500 feet. Two manmade
reservoirs and an enhanced natural lake serve as source water storage
facilities for the City of Portland. Raw water turbidity averages about
0.4 NTU, with a typical range of 0.2 to just over 1 NTU. Monthly mean
total cohform levels are below 15 plaque-forming units (PFU)/100 mL
and 90th percentile levels are below 35 PFU/100 mL. Corresponding
values for monthly fecal coliforms are 2 and 5 PFU/100 mL
Conductivity averages about 20 micromhos/cm, pH ranges from 6 8 to
7.4 units, and temperature from 0.5 to 15°C. The only treatment
provided for the water is disinfection with chlorine and ammonia.
Management of the watershed is governed by special"federal
legislation (PL 95-200, 1977), which makes continued production of
pure clear raw potable water" the primary management goal and
allows secondary uses compatible with that goal. The basic watershed
management program is described in a Final'Environmental
Statement (FES) issued by the Forest Service for the watershed (16)
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This FES was developed through a public process in which the
Portland Water Bureau participated heavily.
The FES prohibits agricultural, recreational, and residential uses
in the watershed. It allows timber harvesting where needed to protect
water quality or where found to have no impact on it, and it prohibits
pesticide use within the watershed boundaries. All road access points
are gated and locks and keys changed every year. An Administrative
and Operations Plan (17) has been developed to implement procedures
and regulations on day-to-day activities such as road maintenance,
reservoir operations, spill response, sanitation requirements for
workers and contractors when in the watershed, and fire protection.
All specific activities or projects that are not covered by these broader
documents or that are undertaken to implement the general directives
they provide are also assessed in an environmental planning process.
As an example, while the FES generally calls for removing timber
downed by windstorms due to the fire hazard it creates, the Forest
Service also completed a separate Environmental Impact Statement
(18) to specifically determine which acres were to be harvested after a
severe 1983 storm. ,
In order to monitor management program performance, water
quality standards have been developed at key stream locations in the
watershed (19). These standards, based on historical water quality
data, are used to identify and correct any degradation of water quality
that may result from any management action. A network of about 2d
longer term monitoring stations is supplemented by shorter term
project monitoring stations centered around specific activities, to help
interpret any changes in water quality detected by the standards.
Physical and biological parameters such as turbidity, pH, conductivity,
sediment, total and fecal coliforms, and HPC are monitored weekly at
these sites. Nutrients such as nitrogen and phosphorous are examined
every other week. Other parameters, such as chlorophyll a, metals,
and pesticides-are tested on schedules ranging from weekly to yearly
depending on the location and parameter. Special studies, such as a
several-year effort to document raw water Giardia cyst levels, are also
conducted.
In addition to water monitoring, onsite field inspection of all
approved activities takes place daily to ensure conformance to
environmental plans. Natural resource inventories are conducted both
before and after activities such as logging to ensure that the activity
did in fact accomplish its intended results. Both water monitoring and
field inspections provide feedback to the planning process to be used in
improving future projects; reports describing these results are
prepared and distributed to the public yearly.
Public involvement in the management program is heavy. A
citizen advisory committee has been established and meets monthly to
provide guidance to both the Forest Service and the City of Portland, in
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reviewing watershed management plans and policies. Special open
public meetings are held to discuss particular management issues of
concern. Watershed tours are arranged for interested parties. The
Portland Water Bureau prepares and distributes public information
summaries and materials on water quality and watershed
management issues. It also works closely with local media to ensure
that information on activities is distributed throughout the
community. .
The major issues within the Bull Run watershed are related: the
impacts of logging activities on water quality and the .long-term
relationship between human watershed management activities and
natural catastrophes such as wind, pire, and flood. The watershed
program must balance the risks between acting and failing to act. It
must look at whether conducting "management" such as logging will
improve or harm the watershed in both the short and long term and
whether short-and long-term risks are worth the benefits.
In order to better answer these questions, research, studies, and
operational improvements are continually being made in the
watershed program. Recently, experimental remote turbidity
monitoring stations have been installed, a special study on sediment
sources and transport in the watershed was conducted, an independent
peer review of the watershed monitoring program was commissioned,
and a study was conducted to quantify the levels ofGiardia cysts at the
raw water intake.
The Portland Water Bureau works closely with the County Health
Department to track giardiasis in the community (giardiasis has been
a reportable disease in Oregon since 1982). A study was recently
completed comparing the observed risks of getting giardiasis in
Portland with those in a nearby similar filtered water community (20).
No-evidence of waterborne transmission of giardiasis in Portland's
unfiltered supply was detectable from the epidemiological data. '
Being responsive to the public concerns about the watershed
includes continually assessing the public health impact of the
watershed and water treatment programs. The results from these and
similar activities will be used to ensure that the water quality
produced by the watershed continues to be the best possible and
continues to meet all requirements of use as an unfiltered, disinfected
supply.
Conclusions
An effective watershed management program depends upon a
conscious decision-making process. This process involves
understanding and characterizing the watershed, identifying the goals
of watershed management and the means of achieving those goals, and
monitoring the effectiveness of protection measures to provide
feedback for improving the watershed program. A process such as this
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is likely to identify the land uses and activities which have been found
to impact water quality and the measures found effective at controlling
them elsewhere. Portland's watershed protection program for the Bull
Run watershed is an example of a successful program that meets its
stated goal of providing "pure, clear, raw, potable water."
Watersheds across the country are facing increased pressure from
urbanization, demand for recreational opportunities, and other access
to the resources they contain. Combined with stricter federal and state
drinking water regulations such as the Surface Water Treatment Rule
and other environmental regulations, these pressures make the need
for better watershed management programs critical. As the number of
contaminants a water supplier must be concerned about increases, the
risks the public is willing to accept decrease, and the cost and
complexity of advanced treatment processes required for these
contaminants increase, watershed protection becomes more essential
for all water suppliers. In order to meet these challenges, federal and
state agencies with responsibilities for activities that can impact
watersheds must develop greater coordination, and must encourage
more effective public involvement.
References
1. Burby, R.J., Kaiser, E.J., Miller, T.L., and Moreau, D.H.
Drinking Water Supplies Protection through Watershed
Management. Ann Arbor Science Publishers, Ann Arbor,
Michigan, 1983.
2. U.S. Environmental Protection Agency. Results of the
Nationwide Urban Runoff Program, Volume I - Final Report.
Washington, D. C., 1983.
3. Beschta, R.L. Long-term patterns of sediment production
following road construction and logging in the Oregon coast
range. Water Resources Research. 14(6): 1011,1978.
4. Pierce, R.S., Martin, C.W., Reeves, G.F., Likens, G.E., and
Borman, F.H. Nutrient loss from clearcutting in New Hampshire.
In: Proceedings of a Symposium on Watersheds in Transition,
American Water Resources Assoc., 1972.
5. Taylor, R.L., Adams, P.W., Nelson, P.O., and Seidler, R.J. Effect
of Hardwood Leaf Litter on Water Quality and Treatment in a
Western Oregon Municipal Watershed. Oregon State University
Water Resources Research Institute, WRRI-82,1983.
6. Christensen, L.A. Water Quality: a Multi-disciplinary
Perspective, in Problems and Potentials for Agricultural
Communities. American Water Resources Association. 1984.
7. Quinn, S.O. and Bloomfield, N. (eds.) Proceedings of a Workshop
on Acidic Deposition, Trace Contaminants and their Indirect
166
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Human Health Effects: Research Needs. EPA Environmental
Research Laboratory, Corvallis, Oregon, 1985.
8. Suk, T.J., Sorenson, S.K. and Dileanis, P.O. The relation between
human presence and occurrence of Giardia cysts in streams in the
Sierra Nevada, California. J. Freshwater Ecology. 4(1):71,1987.
9. Ongerth, H. J. Public health viewpoint, AWWA Joint Discussion:
Watershed Management and Reservoir Use. J. AWWA. 56(2) 149,
1964.
10. Joubert, L.B. Watershed protection strategies for the Scituate
reservoir. J. New England Water Works Assoc. 99(2):136,1985.
11. Grady, Robert P. Source protection of a multipurpose lake: a
utility's perspective. In: R.B. Pojasek (ed.). Drinking Water
Enhancement through Source Protection. Ann Arbor Science
Publishers, Ann Arbor, Michigan, 1977.
12. Monahan, J.E. and Courchene, J.E. Seattle's watershed
protection and monitoring program. In: 1986 AWWA Water
Quality Technology Conference Proceedings, Portland, Oregon,
1977.
13. Robbins, R. Development and application of water quality
standards for the Bull Run watershed. In: Proceedings of the
Water Quality Technology Conference - 14. AWWA, November
1986.
14. Robbins, R.W., Glocker, J.L., Bloem, D.M., and Niss, B.M.
Effective Watershed Management. American Water Works
Association Research Foundation (in Press).
15. Donigian, A.S. and Rao, P.S. Selection, application, and
validation of environmental models. International Symposium on
Water Quality Modeling of Agricultural Nonpoint Sources,
Logan, Utah, 1988.
16. U.S. Forest Service. Bull Run Planning Unit Final
Environmental Statement. Mt. Hood National Forest, 1979.
17. U.S. Forest Service. Bull Run Watershed Administration and
Operation Plan. Mt. Hood National Forest, 1984.
18. U.S. Forest Service. Bull Run Slowdown Final Environmental
Impact Statement. Mt. Hood National Forest, 1987.
19. U.S. Forest Service. Water Quality Standards for Bull Run
Watershed Management Unit. Mt. Hood National Forest, 1987.
20. Glicker, J. and Edwards, R. The risk of endemic giardiasis from
an unfiltered protected water source. In: Proceedings of the
Water Quality Technology Conference -15. AWWA, 1987.
167
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Principles of Water Filtration
by: Gary S. Logsdon
Director, Water Process Research
Black & Veatch Engineers-Architects
1025 Alliance Road, Suite 101
Cincinnati, Ohio 45242
(513) 984-6630
[At the time this article was written, Dr. Logsdon was with
the Risk Reduction Engineering Laboratory,
U.S. Environmental Protection Agency]
Introduction
Three distinctly different filtration processes are generally used by
water utilities in the United States - slow sand filtration, diatoma-
ceous earth filtration, and coagulation followed by rapid rate filtration.
An understanding of the similarities and differences of each of the
three processes would be enhanced by knowledge of principles related
to colloid stability and particle removal in a packed bed. This article,
therefore, presents a brief review of filtration principles, followed by a
discussion of the capabilities of the three kinds of filtration processes.
Colloid Stability and Coagulation
Many of the particles that need to be removed by filtration are in
the colloidal size range 0.001 to 0.1 um (1). Colloidal particles possess a
surface electrical charge, typically a negative charge for the colloids of
concern in water filtration. Information on the origin and nature of
such charges is given by Sawyer and McCarty (1). Because of their
surface charge, colloids tend to remain in suspension and are said to be
stable. Positively charged inorganic coagulants or polymers are used to
overcome the effects of the negative surface charges of colloids,
destabilizing them, so that they will agglomerate and slowly settle to
the bottom of sedimentation basins or stick to grains of filtering
material in the filtration process. It is absolutely essential that colloids
be destabilized if rapid rate (rapid sand or multi-media) filtration is
used, because stable (uncoagulated) colloidal suspensions cannot be
filtered effectively by these filters.
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Use of the correct dose of coagulant is important. Dose control can
be accomplished in a variety of ways. One traditional approach is jar
testing. For waters of perhaps 10 NTU or higher, jar testing combined
with continuous monitoring of the turbidity of the filtered water at
individual filters is an approach frequently used. If raw water quality
can change rapidly or the raw water turbidity is low (below 10 NTU),
jar tests may not be very effective, because of the time required for
testing or the smaller differences in raw and settled water turbidities.
In such instances, coagulant dose control by zeta potential
instrumentation, a streaming current detector, or a pilot filter may be
appropriate. Wagner and Hudson suggested that filtration using
Whatman No. 40 paper could give information on the treatment levels
that produce acceptable water quality (2). The chemical dosing
requirements for successful treatment of a raw water are not constant.
It is essential that testing be done periodically to determine the
optimum conditions for coagulation; this is often not done, especially at
small plants.
Particle Removal in a Packed Bed
Fundamentals of granular media filtration have been studied by
numerous researchers in recent decades. A review of much of this work
was recently published in the Journal of the American Water Works
Association (3). An excellent description of principles of particle
removal by individual collector grains and by a packed bed of collector
grains (a filter bed) was given in Water Treatment Principles and
Design (4). The following discussion of particle removal in a packed bed
is based on this information.
Removal of particles by three different mechanisms is shown in
Figure 4.2.1, which depicts a single collector grain. The four solid lines
labeled A, B, C, and D represent streamlines in a laminar flow
condition. Unless acted upon by outside forces, small particles will
follow the streamlines. The small particle (less than 1 um) is acted
upon by water molecules and moves in a random fashion (Brownian
movement). Brownian movement can cause the small particle to
deviate from the streamline and may even cause it to collide with the
collector grain. The equation for particle removal efficacy by Brownian
movement (FIB) is given in Figure 4.2.2. The particle in streamline B is
removed by sedimentation. Because it is more dense than water, this
particle's motion is influenced by gravity as the streamline curves
around the collector grain. Sedimentation causes the particle to
deviate from the streamline. The equation for efficiency of removal by
sedimentation (%) is also given in Figure 4.2.2. In streamline C, a
large particle moves along the streamline. It is sufficiently large that
it touches the collector grain and is removed by interception. Figure
4.2.2 gives the equation for interception efficiency (nj) as well. The
overall efficiency of a single collector is the sum of the efficiencies for
the various removal mechanisms (Figure 4.2.3).
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C D
Sedimentation, S
Brownian
Transport, B
Interception,
Figure 4.2.1. Transport mechanisms for particle removal.
Adapted from James M. Montgomery Consulting Engineers, Inc. Wafer Treatment
Principles and Design. John Wiley & Sons, New York, NY, 1985.
A filter bed consists of many collectors stacked on top of one
another, as well as side by side. Figure 4.2.4 is a representation of a
gravity filter. The equation for particle removal by a filter bed is given
in Figure 4.2.3. This equation includes overall collector efficiency (q)
and several other factors. Those most easily modified by the filter
designer are dm, the filter media diameter, and L, the depth of the
filter bed. Particle removal efficiency increases as L increases and dm
decreases. The factor most easily modified by the operator is a, the
171
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n=0.9[kT/lldpdmV0]2'3
Brownian
Interception n, = 1-5 (d pd m)"
Sedimentation n« • [(Pp • Pw) 9fl
Definition of Terms
K
T
dp
» Boltzman Constant Vo = Approach Velocity
» Absolute Temperature pp '= Density of Particle
- Dynamic Viscosity pw - Density of Water
- Diameter of Particle g ' = Acceleration of Gravity
dm = Diameter of Media
Figure 4.2.2. Single collector efficiency equations.
Adapted from James M. Montgomery Consulting Engineers, Inc. Water Treatment
Principles and Design. John Wiley & Sons, New York, NY, 1985.
Collector Efficiency
n • la + ni + nf
Filter Efficiency
exp
Definition of Terms
V » Shape Factor
e0 - Porosity of Clean Bed
L - Depth of Filter Bed
a = Attachment Efficiency
Figure 4.2.3. Overall collector efficiency and filter efficiency.
Adapted from James M. Montgomery Consulting Engineers, Inc. Water Treatment
Principles and Design. John Wiley & Sons, New York, NY, 1985.
particle attachment efficiency. For stable colloids, a would approach
zero, indicating that no colloids that touch the collector grain would
stick. For destabilized colloids, if 100 percent of the particles that touch
the collector stick to it, a = 1.0. Coagulation should be optimized, so
that a can approach 1.0. Particle size also influences removal
efficiency. Particles in the 1 um size range tend to be removed least
efficiently.
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Filtration Rate
Influent Cone.
AH (Total Available Head)
Effluent Cone.
,, . , . Flow Control v-,.
Figure 4.2.4. Gravity filtration through granular media.
Removal of very large agglomerations of particles is accomplished
with yet another method. For any packed bed of collector grains, there
exists a limit on the size of particle that can pass through the pores.
Particles that are too large to pass through a packed bed are removed
by the mechanism of straining. Very large floe particles are removed in
this way in sand filters. Giardia cysts are removed by straining in the
diatomaceous earth filtration process. In slow sand filtration and in
rapid rate filtration, Giardia removal occurs by the mechanisms
described above for particle removal in a packed bed. In slow sand
filters, larger organisms may prey upon Giardia cysts for a food source
and accomplish removal in this fashion also.
A summary of factors influencing particle removal efficiency is
given in Tables 1 and 2. Attachment efficiency o plays a critical role in
particle removal. Control of coagulation chemistry to maximize a. is
essential for effective operation of coagulation-filtration plants.
Particle density, pp, and concentration of particles in raw or influent
water, C0, can be modified to a limited extent. Flocculation energy can
influence pp, as more intense flocculation can promote formation of
denser floes. Effective sedimentation can reduce particle concentration
by perhaps one order of magnitude, and should thus result in a reduced
concentration of particles in the filter effluent. Filtration rate is a
function of both design and operation. Higher filtration rates reduce
particle removal by Brownian movement and sedimentation according
to the equations in Figure 4-2.2. In addition, the larger shear forces
associated with higher filtration rates would tend to remove a larger
fraction of particles that did touch and attach to collector grains,
including those removed by interception. Decreasing the filter media
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size creates more surfaces for attachment and places more collectors in
the same bed depth, increasing particle removal efficiency. Increasing
filter bed depth increases the number of collectors a particle must
successfully pass in order to reach the effluent. Thus deeper beds are
more effective for particle removal, all other factors being equal.
Table 4.2.1. Factors Influencing Particle Removal Efficiency
Parameter
Attachment Efficiency, a
(0.05 - 1.0)
Particle Density, pp
(1.02 - 2.5 g/cm3)
Influent Particle Concentration, C0
(<1 -100mg/L)
Change in
Parameter
t
t
t
Change in Effluent
Concentration
i
i
t
Adapted from R.D. Letterman, private communication, 1988.
Table 4.2.2. Design Factors Influencing Particle Removal Efficiency
Parameter
Filtration Rate, V0
(0.04 - 1.0 gpm/ft2)
Medium Grain Diameter, dm
(0.3 - 3.0 mm)
Bed Depth, L
(1 - 8 ft)
Change in
Parameter
T
t
t
Change in Effluent
Concentration
T
t
1
Adapted from R.D. Letterman, private communication, 1988.
Filter run length is also an important concern. Efficient plant
operation requires runs of sufficient length so that most of the water
produced can be used for the needs of the community. When runs are
very short, a substantial portion of treated water has to be used to
backwash filters. This is inefficient and wasteful of both water and
plant labor.
Wetter Treatment Principles and Design lists factors that influence
filter run length (Table 4.2.3) (4). Increasing filter media diameter
lengthens the time to maximum head loss by enabling the filter to
store large floe particles. Large media grains are less efficient as
collectors, however, so turbidity breakthrough could occur faster with
larger media. Because deeper filter beds are more effective particle
collectors, they lengthen the time to turbidity breakthrough, but
deeper beds also have higher clean bed head loss and thus reach
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maximum head loss sooner. Higher filtration rates push particles
through and out of filter beds faster and cause higher head loss. Both
effects reduce run length. Higher influent particle concentration raises
the effluent concentration, causing the filter to clog sooner. Stronger
floe adheres in the pore spaces of the filter bed more effectively,
thereby increasing time to turbidity breakthrough. On the other hand,
stronger floe can increase head loss or even "blind" a filter. Higher
porosity in the filter bed provides passages for floe particles through
the bed and decreases time to turbidity breakthrough. Greater
porosity, by providing more floe storage volume, increases time to
maximum head loss. Denser deposits of floe in the filter bed result in a
slower decrease in the porosity of the bed as floe is removed. This
hastens turbidity breakthrough but permits storage of more floe, thus
reducing the rate of head loss buildup. Table 4.2.3 shows that
numerous conflicting circumstances must be dealt with in both the
design and operation of filters. Balancing of various factors and
achieving suitable tradeoffs are necessary for successful design and
operation of water filters.
Table 4.2.3. Design Factors Influencing Particle Removal Efficiency
Effect of Parameter Increase
Parameter Increased
Time to Turbidity
Breakthrough
Time to Maximum
Head Loss
dm
L
Q/A (v0)
C0
Floe Strength
Porosity (e)
Deposit Density
1
t
i
1
t
i
i
t
i
1
1
i
t
t
Adapted from James M. Montgomery Consulting Engineers, Inc. Water
Treatment Principles and Design. John Wiley & Sons, New York, NY, 1985.
Regardless of the type of process used, filtration tends to be
performed on a batch rather than a continuous basis. All filters, if
operated for a sufficiently long time, will remove enough particulate
matter from the influent water to change the hydraulic characteristics
of the filter. As more and more particles are stored in the filter,
progressive clogging occurs. This clogging can eventually cause the
head loss across the filter to increase to a maximum operating value. A
clogged, or dirty, filter must be removed from service, cleaned, and
prepared for service again. Various filtration processes differ in the
frequency and nature of the cleaning step. This is explained in the
following sections.
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Slow Sand Filtration: Principles and Capabilities
Slow sand filtration originated in Great Britain in the 1800s. In
this process, uncoagulated water is applied to a sand bed about 1 meter
deep at a filtration rate (approach velocity) of about 0.1 meter/hour
(m/hr.). Removal of microorganisms is aided by biological processes
that occur on and in the filter bed, where an ecosystem becomes
established with extended use. On the top of the media, a biologically
active scum layer (schmutzdecke) builds up and assists in filtration. As
the water enters the schmutzdecke, biological action breaks down
some organic matter, and inert suspended particles may be physically
strained out of the water. The water then enters the top layer of sand
where more physical straining and biological action occur and
attachment of particles onto the sand grain surfaces takes place. Also
some sedimentation may occur in the pores between the media grains
where velocity is sufficiently slow. In a slow sand filter, the efficiency
of particle attachment (a) may be enhanced by production of natural
polymers by the biota living in the schmutzdecke and in the filter bed.
Slow sand filter maintenance generally is not complicated or time
consuming, and can be done in times as short as 1 hour at small slow
sand filtration plants. Filter cleaning, however, is somewhat involved.
When the depth of the schmutzdecke layer increases, clogging the top
sand layer and causing the head loss through the filter, to reach a
predetermined level, the filter must be drained and the top layer of
sand removed. This period of scraping is the only time during normal
operations in which additional manpower may be required to operate
the filter. The amount of time between scrapings will depend on how
much material is filtered out of the water. Thus high turbidity levels
would shorten the cycle. Slow sand filter runs can be as short as 1 or 2
weeks when muddy or algae-laden waters are treated. The runs can
last for several months when very clear waters are filtered.
Slow sand filters are uncomplicated and easy to operate, but they
can be used successfully only with a good quality of raw water
(turbidity usually less than 10 NTU and no undesirable inorganic or
organic chemicals present). In comparison to other filtration processes,
they also require large land areas per volume of water treated, and
thus would probably not be used by large water utilities to the United
States. Slow sand filtration is a very effective treatment process for
control of microorganisms, however, when the raw water quality is
appropriate.
Slow sand filters have been shown capable of removing 99 to 99.99
percent of the raw Giardia cysts in water (5,6,7). Using pilot filters
Bellamy et al. found that cyst removal did not deteriorate after filter
scraping (5). Pyper observed that at 7.5'C to 21SC, cyst removal was
99.98 percent to 99.99 percent. At 0.5'C to 0.75"C, removal ranged from
99.36 percent to 99.91 percent; however, at 0.5"C, cyst removal
deteriorated to 93.7 percent when both Giardia cysts and primary
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unchlorinated sewage effluent were added to the raw water
simultaneously. In this situation, the loading of organisms in the
influent water may have been greater than the established biological
population of the slow sand filter could cope with.
Schuler et al. studied the removal of Cryptosporidium oocysts and
found that a pilot-scale slow sand filter was at least as effective for
removal of Cryptosporidium oocysts as for Giardia cysts (8). Removal
of Cryptosporidium was 99.98 percent or greater.
Total coliform removal was found to be adversely influenced by
increases in filtration rate from 0.04 to 0.4 m/hr (5), by decreases in
filter bed depth from 0.97 to 0.48 m (6), by increases in sand size from
0.13 mm to 0.61 mm and by decreases in temperature from 17°C to 2°C
(6). Of these parameters, the 0.61-mm sand size would be greater than
sizes typically used in slow sand filters and might have accentuated
the adverse impact of that variable. The use of 0.61-mm sand resulted
in average total coliform removal of 96 percent versus 99.4 percent for
0.13-mm sand. Temperature decreases from 17°C to 5°C or 2°C resulted
in deterioration in coliform removal from the 99 percent level to about
90 percent for the colder waters. Cleasby et al. found that total coliform
removal was lower during the first 2 days after scraping than during
the remainder of the run (9). In some instances, differences in the two
time periods were slight, but five of nine runs exhibited coliform
removals ranging from 82 to 95 percent during the first 2 days. During
the remainder of the runs, removals ranged from 97 to 100 percent.
Virus removal has been reported to be influenced by both
temperature and filtration rate (10). At 5 to 8°C, poliovirus removal
was 98.25 percent at a filtration rate of 0.5 m/hr, but was 99.68 percent
at a rate of 0.2 m/hr . At 16 to 18°C, poliovirus removal was higher,
ranging from 99.865 percent at 0.4 m/hr. to 99.975 percent at 0.2 m/hr.
These encouraging results suggest that slow, sand filtration would be
effective against a broad spectrum of microbiological contaminants.
Diatomaceous Earth Filtration: Principles and Capabilities
During World War II, an intense effort was made to develop a
filtration process that would achieve practically complete removal of
E. histolytica cysts. The result was the application of the diatomaceous
earth (DE) filtration process to potable water treatment. In DE
filtration, a thin coating (3 to 5 mm) of diatomaceous earth is placed on
a filter septum by recirculating a slurry of DE through the filter until
essentially all of the DE is on the filter septum. After the filter cake is
established in the precoating process, raw water that has been dosed
with a small amount of DE is passed through the filter. Particles are
removed, and they accumulate on the filter cake. Because DE (the body
feed) is added to the raw water, the good hydraulic characteristics of
the filter cake are maintained, and long filter runs can be attained.
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When a run is terminated, the filter cake is removed, disposed of, and
fresh DE is used to recoat the clean septum.
In DE filtration, mechanisms of particle removal depend on
particle size. Walton presented information, including photomicro-
graphs, that showed that DE filters remove Giardia cysts by a
straining or screening action (11). Because of the size of these cysts,
their passage through the DE filter cake is physically blocked. On the
other hand, removal of bacteria could occur by straining when very
fine grades of DE are used, or it could occur by means of surface
attachment when coarse DE grades are coated with positively charged
aluminum or iron precipitates or cationic polymers. Virus removal
would occur primarily by surface attachment, regardless of the grade
of DE being used, as long as the viruses were not attached to larger
particles in the raw water.
Diatomaceous earth filters have been studied for removal of a
variety of contaminants and have been shown to attain excellent
removal of Giardia cysts over a broad range of operating conditions.
Cyst removal exceeding 99 percent, and often 99.9 percent, were
reported by Lange et al. (12) for filtration rates of 2.4 to 9.6 m/hr, for
temperatures from 3.5 to 15°C, and for four different grades of
diatomaceous earth (Celite 545*, Celite 535*, Celite 503*, and Hyflo
Super-Gel*). Logsdon et al. reported that when sufficient DE precoat
and body feed were used, removal of 9um radioactive beads was nearly
always 99.9 percent or higher (13). Use of a precoat of at least 1.0 kg/m2
was shown to be appropriate for obtaining most effective removal of
the 9um particles. They also reported that 11 filter runs were made
with G. muris cysts at filtration rates of 2.2 to 3.5 m/hr, with Celite
535* precoat and body feed. Cyst removal exceeded 99.0 percent in all
runs, and exceeded 99.9 percent in five of the runs. DeWalle et al.
reported on four.DE filter runs conducted for Giardia cyst removal
(14). Cyst removal exceeded 99 percent in each of the four runs. The
overall results of all research for Giardia cyst removal indicate that
DE filtration is very effective for controlling Giardia cysts. Factors
important to continued effective performance are using adequate
precoat and body feed and keeping the septum very clean (good
cleaning at the end of each run).
Schuler et al. reported on an evaluation of DE filtration for
removal of both Giardia cysts and Cryptosporidium oocysts (8). In four
of five runs, no Giardia cysts were recovered in the filtered water.
Cryptosporidium results were not quite as good, but removal exceeded
99.99 percent in all runs. Removal of total coliform bacteria by DE
filtration was studied extensively at Colorado State University by
Lange et o/.(12). Coliform removals were strongly influenced by the
grade of diatomaceous earth used. Coarser grades attained removals
ranging from 30 to 50 percent for Celite 545*, 50 to 70 percent for
Celite 503*, and 92 to 96 percent for Celite 512*. Total coliform
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removal with Super-Cela
percent.
was 99.92 percent to greater than 99.98
Brown et al. reported that a high percentage of removal could be
attained for poliovirus when coated DE filter aid was used or when
cationic polymer was added to the raw water (15). In one 12-hour filter
run, diatomaceous earth coated with 1 mg of cationic polymer per gram
of DE produced filter water in which no viruses were recovered from 11
samples (removal > 99.95 percent). In one instance, in which 1 of 12
samples was positive, virus removal was 99 percent. In a 12-hour run
in which uncoated DE was used and 0.14 mg/L of cationic polymer was
added to the raw water, no viruses were recovered from any of the 12
samples analyzed.
Work was done at Colorado State University to improve the
capabilities of DE filtration. In order to alter the surface properties of
diatomaceous earth, aluminum hydroxide was precipitated to the
surface of a DE slurry. With 0.05 grams of alum per gram of Celite
545®, total coliform removal was 99.86 percent, as compared to 30 to 50
percent removal for uncoated Celite 545®. For the same grade of DE,
turbidity removal was 98 percent for coated DE versus less than 20
percent for uncoated DE (12). These results show that the straining
mechanism of removal can be augmented by a surface attachment
removal mechanism if DE is given an electropositive coating.
Diatomaceous earth filtration, like slow sand filtration, is most
appropriate for use with high quality raw water. Limits on the quality
of raw water that would be appropriate for DE filtration are not easy to
set. The process removes particulate matter by trapping it within the
filter cake. As the concentration of particulate matter in raw water
increases, the load applied to the filter cake increases. To maintain
high permeability of the filter cake and good head loss characteristics,
body feed diatomaceous earth is added to the raw water. A rule of
thumb is that higher raw water particle concentrations require more
body feed, if the nature of the particles does not change. The nature,
and especially the compressibility, of the particles being removed is
quite important. Rigid turbidity-causing particles, such as very fine
sand, would not block or blind the filter cake, but compressible
particles, such as algae, coagulation floe, precipitated iron, or
biological matter could. Pilot filtration studies are advisable if the
water in question is not already being treated by DE filtration. Such
studies would establish the appropriate grade of DE to ensure the
desired effluent turbidity, the amount of body feed to add under
conditions of the test runs, and the approximate length of filter run to
expect.
Coagulation-Filtration: Principles and Capabilities
Near the turn of the century, engineers in the United States began
to recommend the use of rapid sand filters for treatment of muddy
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surface waters like those found in the Ohio River and Mississippi River
valleys. These filters treat water that has been conditioned by
coagulation, flocculation, and sedimentation to remove most of the
particulate matter; this results in longer filter runs. Rapid sand filters
typically were operated at rates of 5 m/hr in the first half of this
century. Since about 1960, use of dual media (coal and sand) or mixed
media (coal, sand, and garnet or ilmenite) has permitted operation of
rapid rate filters at rates as high as 10 to 24 m/hr. For clear waters (10
NTU or lower) the sedimentation and sometimes flocculation processes
may be omitted, and the process is known as direct filtration. In direct
filtration, all removal of particulate matter occurs within the filter.
Effective coagulation is essential for successful operation of rapid
rate filters, with or without sedimentation, because surface
attachment is the mechanism by which particle removal occurs. The
principles of colloid destabilization and attachment onto collector
grains described earlier in this paper are especially important for
coagulation-filtration. Unless the coagulation step is properly
managed, this process cannot attain the high degree of particle
removal of which it is capable. When the raw water is not coagulated
at all, or is not coagulated properly, coagulation-filtration simply
cannot attain maximum particle removal on a consistent basis.
After a rapid rate filter has been in service for a period of time,
filtration is stopped and the filter bed is cleaned by backwashing.
Typically surface wash is also used to supplement the cleaning action
of the backwash water. Some newer installations employ an air scour
step to enhance the cleaning of media. Backwashing needs to be done
carefully so that the filter bed is thoroughly cleaned but not unduly
disturbed by the backwashing procedure. Careless handling of water
or air during the backwashing could even result in damage to the filter
bed and the media support system.
Most U.S. coagulation-filtration research for Giardia cyst removal
has focused on the coagulation-filtration (in-line) or coagulation-
flocculation- filtration (direct filtration) variations of the process,
because waterborne giardiasis outbreaks tended to be observed in
regions of the country that had low turbidity waters, which were
thought to be suitable for such treatment. Research by Logsdon et al,
(13), DeWalle et al. (14), and Al-Ani et al. (16) involved coagulation
with alum, or alum plus a polymer; filtration through sand or dual
media at 5 to 14 meters/hr; and temperatures ranging from 3 to 20°C.
Later research (17) was conducted on conventional treatment, with
alum or alum and polymer, dual media, and three monomedia types
(sand, anthracite, GAG); filtration at 7 m/hr; and at room temperature
(about 25°C).
Results of the three cited direct filtration studies indicate that
Giardia cyst removal can exceed 99.0 percent or even 99.9 percent
when the raw water is coagulated properly and filtered. Results of
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Logsdon et al (1981) and DeWalle et al. (1984) indicated that with
proper pretreatment, cyst removal exceeded 99.0 percent when filtered
water turbidity was below 0.30 NTU. Al-Ani et al showed that cyst
removal of 99 percent or more was likely to occur if turbidity removal
was 70 percent or more, when raw waters in the 0.2 to 1 NTU range
were treated (16). This would produce filtered waters in the 0.06 to
0,30 NTU range.
.All of the above researchers showed that dependable cyst removal
results can be attained if a clear water (about 1 NTU) is filtered
without being properly coagulated. Use of no coagulant, or of an
improper dose, resulted in erratic cyst removal results. In addition,
DeWalle et al. showed that for alum coagulation, proper pH is
necessary when testing soft, low alkalinity water (14). They observed
effective treatment at pH 5.6 and 6.2, but at pH 6.8 with alum
coagulation, cyst removal was reduced from 99 to 95 percent.
The coagulation-filtration process can remove a variety of
contaminants. Robeck etal. showed that direct filtration could remove
90 percent to 99 percent of viruses, while conventional treatment
removals consistently were 99 percent (18). McCormick and King
stated that coliform removal by direct filtration was practically 100
percent when filtered water turbidity was 0.10 NTU or less (19).
Coagulation and filtration can be used to treat a very wide range of
raw water quality. Turbidities in the range of hundreds of NTU can be
treated when sedimentation is used. Water utilities along the Missouri
River sometimes employ plain sedimentation or chemically assisted
sedimentation ahead of a conventional treatment train. Such plants
can handle raw water of 1,000 NTU or more.
An important consideration at all plants employing coagulation is
that the level of operating skill needed is substantial. In order to
effectively and efficiently control the coagulation-filtration process
and attain low filtered water turbidity, operators need to understand
•the chemical aspects of coagulation, regardless of plant size. Large and
medium-sized plants are able to hire and keep trained operators who
can effectively operate coagulation-filtration plants. Small plants,
however, may not have the resources to hire or train operators who
have a solid understanding of coagulation. This can lead to problems of
poor treated water quality, if operators are unable to adjust treatment
when raw water quality changes.
Summary
1. The mechanisms by which particles are removed in packed beds
are understood to an extent sufficient for developing general
. concepts of filter design and operation.
2.
Each of the three filtration processes reviewed is different, and no
single process is ideal in every circumstance.
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3. As process complexity increases, from slow sand filtration to DE
filtration, to coagulation-filtration, the skill level needed for
effective operation increases, and producing high quality filtered
water increasingly becomes dependent on operator skill and
ability.
4. When properly designed and operated, the filtration processes
reviewed can substantially reduce the concentration of
microorganisms in drinking water.
References
1. Sawyer, C.N. and McCarty, P.L. Chemistry for Environmental
Engineering, Third Edition. McGraw-Hill, New York, N.Y.
1978.
2 Wagner, E.G. and Hudson, H.E., Jr. Low-dosage high-rate direct
filtration. J. AWWA, 74:5:256-261,1982.
3. Amirtharajah, A. Some theoretical and conceptual views of
filtration. J. AWWA, 80:12:36-46,1988.
4. James M. Montgomery Consulting Engineers, Inc. Water
Treatment Principles and Design. John Wiley & Sons, New
York, N.Y., 1985.
5. Bellamy, W.D., Hendricks, D.W., and Logsdon, G.S. Slow sand
filtration: influences of selected process variables. J. AWWA,
77:12:62-66,1985.
6. Bellamy, W.D., Silverman, G.P., and Hendricks, D.W. Filtration
of Giardia cysts and other substances: Volume 2. Slow Sand
Filtration. Report No. EPA 600/2-85/026, U.S. Environmental
Protection Agency, Cincinnati, Ohio, 1985.
7. Pyper, G.R. Slow sand filter and package treatment plant
evaluation: operating costs and removal of bacteria, Giardia,
and trihalomethanes. Report No. EPA/600/2-85/052, U.S.
Environmental Protection Agency, Cincinnati, Ohio, 1985.
8. Schuler, P.P., Ghosh, M.M. and Boutros, S.N. Comparing the
removal of Giardia and Cryptosporidium using slow sand and
diatomaceous earth filtration. In: Proceedings 1988 AWWA
Annual Conference, pp. 789-805,1988.
9. Cleasby, J.-L., Hilmoe, D.J., and Dimitracopoulos, C.J. Slow
sand and direct in-line filtration of a surface water. J. AWWA,
76:12:44-55,1984.
10. Poynter, S.F.B. and Slade, J.W. The removal of viruses by slow
sand filtration. Prog, in Water Tech., 9:1:75-88,1977.
11. Walton, H.W. Diatomite filtration: how it removes Giardia from
water. In: Wallis, P.M. and Hammond, B.R. (eds.), Advances in
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Giardia Research, Univ. of Calgary Press, Calgary, Alberta, pp.
113-117,1988.
12. Lange, K.P., Bellamy, W.D., Hendricks, D.W., and Logsdon,
G.S. Diatomaceous earth filtration of Giardia cysts and other
substances. J. AWWA, 78:1:76-84,1986.
13. Logsdon, G.S., Symons, J.M., Hoye, R.L., Jr., and Arozarena,
M.M. Alternative filtration methods for removal of Giardia
cysts and cyst models. J. AWWA, 73:2:111-118,1981.
14. DeWalle, F.B., Engeset, J., and Lawrence, W. Removal of
Giardia lamblia cysts by drinking water treatment plants.
' Report No. EPA-600/2-84-069, U.S. Environmental Protection
Agency, Cincinnati, Ohio, 1984.
15. Brown, T.S., Malina, J.F., Jr. and Moore, B.D. Virus removal by
diatomaceous earth filtration - Part 2. J. AWWA, 66:12:735-738
1974.
16. Al-Ani, M.Y., Hendricks, D.W., Logsdon, G.S., and Hibler, C.P.
Removing Giardia cysts from low turbidity waters by rapid rate
filtration. J. AWWA, 78:5:66-73,1986.
17. Logsdon, G.S., Thurman, V.C., Frindt, E.S., and Stoecker, J.G.
Evaluating sedimentation and various filter media for removal
of Giardiacysts. J. AWWA, 77:2:61-66; 1985.
18. Robeck, G.G., Clarke, N.A., and Dostal, K.A. Effectiveness of
water treatment process in virus removal.
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Principles of Drinking Water Disinfection for
Pathogen Control
by: John C. Hoff
Risk Reduction Engineering Laboratory
Drinking Water Research Division
U.S. Environmental Protection Agency
26 W. Martin Luther King Dr.
Cincinnati, Ohio 45268
(513) 569-7331
Introduction
The 1986 Amendments to the Safe Drinking Water Act mandate
that EPA establish disinfection requirements to ensure the control of
waterborne pathogens by drinking water treatment. The requirements
being developed constitute a major change in disinfection practice in
the United States and are the result of application of knowledge gained
over a period of more than 75 years. The purpose of this paper is to
provide some insight into the basis for these changes and the principles
of sound disinfection practices.
Historical Aspects
The introduction of water filtration and use of chlorine for
disinfection around the turn of the century resulted in dramatic
decreases in waterborne disease in the United States (1,2). Continuous
application of chlorine for drinking water disinfection was first used in
the United States in 1908 (3). In the same year, Chick noted the
analogy between rates of inactivation of bacteria by chlorine and
chemical reaction kinetics, which she incorporated into an equation
now known as Chick's Law (4). Watson analyzed Chick's data in terms
of the relationship of disinfectant concentration (C) and contact time
(t) to killing efficiency, developing an equation now known as Watson's
Law (5), which in simplified form constitutes the C-t concept (6). The
C-t concept provides a major basis for the disinfection regulations
currently being developed.
Subsequent developments in knowledge of chlorine chemistry led
to the use of combined chlorine rather than free chlorine because of the
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stability of the chlorine residual. Studies on comparative biocidal
efficiency, however, led to a reemphasis on free chlorine use because of
its greater killing ability. This was reinforced by the results of studies
in the 1950s on inactivation of enteric viruses, which were shown to be
extremely resistant to chloramines. The recognition of giardiasis as a
major cause of waterborne disease outbreaks in the 1970s provided
additional emphasis on the need for more stringent disinfection
requirements, because of its resistance to chlorine at the then
normally applied dosage and contact times. Concurrently, the
recognition of possible long-term health effects from chlorine by-
products led to active consideration of other chemical disinfectants as
alternatives to chlorine.
Relationships of Pathogen Characterises to Disinfection
Efficiency
The pathogens that must be controlled by drinking water
disinfection (bacteria, viruses, protozoans) comprise a diverse group
with regard to occurrence, size, mode of existence, and resistance to
drinking water disinfectants. The primary source of almost all of the
important drinking waterborne pathogens is the intestinal tract of
animals. The notable exception is Legionella pneumophila, a
respiratory pathogen that multiplies, apparently in association with
amoeba, in warm water environments, including drinking water
distribution systems. Because of its life cycle, this pathogen must be
controlled by means other than disinfection during water treatment.
With the exception of Legionella pneumophila, there is little or no
evidence that any of the important drinking water pathogens multiply
significantly in the water environment outside an animal host. Other
characteristics that relate to control of these pathogens by drinking
water disinfection include host specificity, infectious dose, innate
disinfectant resistance, and size and its relationship to the effects of
turbidity on disinfection efficiency.
Host Specificity
Bacterial enteric pathogens normally multiply in the intestinal
tract of humans and other animals. Some show a degree of host
specificity but many can infect a broad range of hosts (e.g., Salmonella,
Campylobacter). In contrast, the enteric virus pathogens show a high
degree of host specificity and there is no evidence of waterborne
outbreaks caused by viruses from other than human sources.
Consequently, water systems using source waters from protected
watersheds not subjected to wastes from human sources are not
required to orient their disinfection processes toward control of
viruses. This becomes an important consideration when certain
disinfectants, notably chloramines, are used because of the extremely
high resistance of viruses to this disinfectant.
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With regard to waterborne protozoan pathogens, host specificity is
not yet clear. For Giardia lamblia, the waterborne protozoan pathogen
currently of most concern, problems of lack of species definition,
apparent strain-specific cross transmission characteristics, and
technical problems with many of the cross-transmission studies, make
it difficult to assess host specificity (7,8). Several waterborne
outbreaks do appear to have resulted from source water contamination
by animals other than humans (9). For Cryptosporidium, a protozoan
pathogen that is currently gaining significance as a recognized
waterborne pathogen, cross-species transmission seems to occur
readily (10).
Infectious dose
The infectious dose for most enteric bacterial pathogens tested is
very high (10* to 107 organisms), but for at least one Shigella strain,
the IDso was shown to be much lower (<100 organisms)(ll). The
infectious dose for enteric viruses also seems to vary. ID^QS for
poliovirus fed to humans varied from < 10 plaque forming units (PFU)
to >1 X 104 PFU in a number of published studies (11,12) depending
on host age, virus isolate used, and other factors. Infectious dose data
for viruses implicated as etiologic agents in waterborne disease
outbreaks (Norwalk, Hepatitis A, rota virus, etc.) are not available.
Similarly, for G. lamblia, results of infectious dose studies are not
consistent. Studies by Rendtorff (13) and Hibler et al. (14) indicate
ID50s of < 10 cysts in most cases, while Visvesvara et al. (15) reported
ID50s of 100 to 1,000 cysts and higher. Host specificity probably
influenced the results of these studies.
Overall, it would appear that the infectious dose for waterborne
pathogens, particularly viral agents and Giardia cysts, can be very low
and therefore removal and/or inactivation of these pathogens must be
conducted so as to be highly effective.
Innate resistance to disinfectants
Different types of waterborne pathogens show an overall similar
pattern of relative resistance to chlorine, the most commonly used
disinfectant, and to the alternative disinfectants currently considered
as viable alternatives (chlorine dioxide, ozone, and chloramine). The
degree of difference in resistance and, in some cases, the order of
resistance vary. In general, the bacterial pathogens are the most
sensitive overall, with the viral agents more resistant and the
protozoans much more resistant than either of the other two groups
(Table 4.3.1). Note that E. coli, representing enteric bacterial
pathogens, is as sensitive as or, in most cases, more sensitive than the
other agents. The viral agents show differences in their resistance to
specific disinfectants and are extremely resistant to chloramine.
Giardia cysts overall are more resistant to all disinfectants except
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chloramine. Chlorine dioxide and ozone are much more effective for
cyst inactivation than chlorine or chloramine.
Table 4.3.1. Summary of C-t Value Ranges for 99 Percent
Inactivation of Various Microorganisms by
Disinfectants at 5°C Disinfectant
Free Preformed Chlorine
Chlorine Chloramine Dioxide Ozone pH
Microorganism pH 6 to 7 pH 8 to 9 pH 6 to 7 6 to 7
£ CO// 0.034-0.05 95-180 0.4-0.75 0.02
Polio 1 1.1-2.5 768-3,740 0.2-6.7 0.1-0.2
Rotavirus 0.01-0.05 3,806-6,476 0.2-2.1 0.006-0.06
Phagef2 0.08-0.18 -
G. lamblia cysts 47 - > 150 - - 0.5-0.6
G.muris 30-630 1,400 7.2-18.5 1.8-2.0
Adapted from Hoff (6).
Because of their high degree of resistance, Giardia cysts are the
"target pathogen" in most cases in applying C-t values, as proposed
under the regulations being developed (16). In some instances,
however, viral pathogens are the target. More detailed discussions of
the specific application of C-t values for the proposed regulation are
given elsewhere in this volume (17,18).
Size
The different pathogens vary in size by more than two orders of
magnitude. The viral agents range from 0.02 to 0.07 m in diameter, the
enteric bacteria are on the order of 0.3 by 1 m, and Giardia cysts are
about 10 to 15 m in diameter. These differences have important
consequences with regard to their removal by some filtration processes
(19) and also influence their relationships to other particulate matter
including adsorption phenomena and inclusion within larger particles.
These associations have important implications for disinfection
effectiveness because of the possible prevention of contact between the
disinfectant and the microorganism. Such protective effects have been
documented by a number of investigations (20, 21, 22, 23). The high
degree of protection provided coliforms in wastewater effluent solids
provides indirect evidence that viruses would be similarly protected
because they are much smaller than bacteria. No direct evidence of
Giardia cyst protection by this mechanism is yet available. Because
they are about 10-fold larger than bacteria, this type of protection may
be of less significance for Giardia cysts.
Such protection underscores the need for removal of potentially
protective particles prior to disinfection. C-t values have been
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developed using purified preparations of microorganisms and this
should be kept in mind in applications to real-life situations. The
protective effects of organic particulate matter would render these
C-t values inadequate for effective disinfection unless the particulate
matter was removed.
Water characteristics
In addition to the factors described above relating to the types and
sources of pathogens present in the water, two other factors, water
temperature and pH, have important effects on disinfection efficiency.
Temperature effects are predictable, with inactivation rates increasing
at higher water temperatures. The available data indicate that
inactivation rates increase two- to threefold for every 10°C increase in
temperature (6).
The effects of pH are disinfectant specific. In general, within the
pH range encountered in drinking water treatment, both free chlorine
and chloramine become less effective as pH increases, chlorine dioxide
becomes more effective as pH increases, and ozone effectiveness is
relatively unchanged with changes in pH.
Little can be done to increase water temperature above ambient
conditions to enhance disinfection efficiency but water temperature
must be considered in evaluating disinfection requirements. While
water pH can be easily changed, corrosion control rather than
enhanced disinfection efficiency has usually been the primary
consideration in making such changes. Corrosion control generally
results in higher pH values and has often worked to the detriment of
disinfection efficiency, when chlorine or chloramine is used for
disinfection.
Mixing
Obviously, thorough mixing of the disinfectant with the water
being treated is necessary for effective disinfection. Although this is
widely recognized, the need for effective mixing does not seem to
receive the attention it deserves. For instance in the AWWA handbook
(1), effective mixing is discussed only as it relates to coagulation and
sedimentation, not as it relates to disinfection.
Summary
The pathogens that must be inactivated by drinking water
disinfection comprise a diverse group of microorganisms (bacteria,
viruses, protozoans) with regard to occurrence, size, mode of existence,
and resistance to disinfectants. Of the many chemical and physical
disinfecting agents available, only a few have been widely applied for
large-scale drinking water treatment in the United States or other
countries. These include free and combined chlorine, chlorine dioxide,
and ozone. The kinetic nature of microorganism inactivation by
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disinfectants was described many years ago and much information has
been developed on the comparative resistance of microorganisms and
the comparative effectiveness of disinfectants. Only recently have
efforts been made to apply this information to the development of
scientifically based drinking water disinfection requirements. This
approach is based on use of disinfectant concentrations (C) and contact
time (t) required for the inactivation of target pathogens under various
conditions of water pH and temperature. While the effects of
temperature increases are consistent with all disinfectants and
pathogens, i.e., inactivation rates increase as temperature increases,
pH effects vary depending on the disinfectant and target pathogen.
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References
1. American Water Works Association. Water Quality and
Treatment, McGraw- Hill, New York, New York, 1971.
2. Craun, G.F, Surface water supplies and health. J. AWWA
80(2):40-52,1988.
3. Craun, G.F. Statistics of waterborne outbreaks in the U.S.
(1920-1980). In: Waterborne Diseases in the United States. CRC
Press, Inc., Boca Raton, Florida, pp. 3-10.1986.
4. Chick, H. An investigation of the laws of disinfection. J. Hyg
8:92-158,1908.
5. Watson, H.E. A note on the variation of the rate of disinfection
with change in the concentration of disinfectant. J. Hyg. 8-536-
542,1908.
6. Hoff, J.C. Inactivation of microbial agents by chemical
disinfectants. EPA/600/2-86/067 U.S. Environmental Protection
Agency, Cincinnati, Ohio, 1986.
7. Woo, P.K. Evidence for animal reservoirs and transmission of
Giardia infection between animal species. In: Erlandsen, S.L.
and E.A. Meyer (eds.), Giardia and Giardiasis. Plenum Press,
New York, New York, pp. 379-400,1984.
8. Bemrick, W.J. Some perspectives on the transmission of
giardiasis. In: Erlandsen, S.L., and E.A. Meyer (eds.) Giardia
and Giardiasis, Plenum Press, New York, New York pp 379-
400,1984.
9, Craun, G.F. and W. Jakubowski. Studies of waterborne
giardiasis outbreaks and monitoring methods. In: Proc. Intnl.
Symp. on Water- Related Health Issues. Am. Water Resource
Assoc., Bethesda, Mary land, pp. 167-174, 1986.
10. Crawford, F.G. and S.H. Vermund. Human cryptosporidiosis.
CRC Critical Rev. Microbiol. 16:113-159,1988.
11. National Academy of Sciences. Microbiology of drinking water.
In: Drinking Water and Health, Vol. 1. National Academy of
Sciences, Washington, D.C., pp. 63-134,1977.
12. Ward, R.L. and E.W. Akin. Minimum infective dose of animal
viruses. CRC Critical Rev. Env. Control. 14:297-310,1984.
13.
14.
Rendtorff, R.C. The experimental transmission of human
intestinal protozoan parasites. II. Giardia lamblia cysts given in
capsules. Am. J. Hyg. 59:209-220,1954.
Hibler, C.P., C.M. Hancock, L.M. Perger, J.G. Wegrzyn, and
K.D. Swabby. Inactivation of Giardia cysts with chlorine at
0.5°C. Water Treatment and Operations. American Water Works
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Association Research Foundation, Denver, Colorado, 1987.
15. Visvesvara, G.S., J.W. Dickerson, and G.R. Healy. Variable
infectivity of Giardia lamblia cysts for Mongolian gerbils
(Meriones unguiculatus). J. Clin. Microbiol. 26:837-841,1988.
16. U.S. EPA. National primary drinking water regulations. Fed.
Reg. 52(212):42178-41222,1987.
17. Clark, R.L. Determination of C-T values. In: G.F. Craun (ed.)
Methods for the Investigation and Prevention of Waterborne
Disease Outbreaks. Report No. EPA/600/l-90/005a, U.S.
Environmental Protection Agency, Cincinnati OH, 1990. pp.
193-206.
18. Berger, P.S. and Regli, S. The Safe Drinking Water Act and the
regulation of microorganisms in drinking water. In: G.F. Craun
(ed.) Methods for the Investigation and Prevention of
Waterborne Disease Outbreaks. Report No. EPA/600/l-90/005a,
U.S. Environmental Protection Agency, Cincinnati OH, 1990.
pp. 23-28. .
19. Logsdon, G.S. Principles of water filtration. In: G.F. Craun (ed.)
Methods for the Investigation and Prevention of Waterborne
Disease Outbreaks. Report No. EPA/600/l-90/005a, U.S.
Environmental Protection Agency, Cincinnati OH, 1990. pp.
169-183.
20. Hoff, J.C. The relationship of turbidity to disinfection of potable
water. In: C.W. Hendricks (ed.) Evaluation of the Microbiology
Standards for Drinking Water. Report No. EPA-570/9-78-OOC,
U.S. Environmental Protection Agency, Washington, D.C.,
1978. pp. 103-117.
21. LeChevallier, M.W., T.M. Evans, and R.J. Seidler. Effects of
turbidity on chlorination efficiency and bacterial persistence in
drinking water. Appl. Env. Microbiol. 42-.159-167,1981.
22. Hejkal, T.W., P.M. Wellings, P.A. LaRock and A.L: Lewis.
Survival of poliovirus within organic solids during chlorination.
Appl. Environ. Microbiol. 38:114-118,1979.
23. Berman, D., E.W. Rice and J.C. Hoff. Inactivation of particle-
associated coliforms by chlorine and monochloramine. Appl.
Environ. Microbiol. 54:507-512,1988.
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Determination of C-t Values
by: Robert M. Clark, Director
Drinking Water Research Division
Risk Reduction Engineering Laboratory
U.S. Environmental Protection Agency
Cincinnati, Ohio 45268
(513) 569-7201
Introduction
Amendments to the Safe Drinking Water Act (PL93-523) highlight
the continuing problem of waterborne disease and mandate EPA to
promulgate (a) criteria by which filtration will be required for surface
water supplies and (b) disinfection requirements for all water supplies
in the United States. EPA's Office of Drinking Water is proposing to
use the C-t (Concentration times Time - disinfectant residual
concentration in mg/L multiplied by the disinfectant concentration
contact time in minutes) concept for determining the inactivation of
Giardia lamblia, one of the most resistant pathogens, likely to be
present in surface waters.
Among the known causes of waterborne outbreaks, Giardia is the
most frequently identified etiologic agent and is responsible for more
cases of waterborne illness than any other agent (1). The Office of
Drinking Water is developing criteria under which utilities using
surface water would be required to meet source water quality
conditions, maintain a protected watershed, and achieve C-t values
which provide a 99.9 percent inactivation of Giardia lamblia cysts, in
order to avoid filtration. If, for example, a utility in addition to meeting
other requirements, can demonstrate that through effective
disinfection, manifested by a sufficient C-t value, it can reduce
Giardia levels by 99.9 percent, then it would not be required to filter.
In this chapter a mathematical model is developed based on the
C-t concept, for inactivation of G. lamblia cysts by free chlorine. The
model is first applied to inactivation data from animal infectivity
studies. A procedure is then developed to select the best combination of
data sets available assuming that the animal infectivity data are
included in each combination. The model is then applied to the
combined data set to calculate the model parameters. A regulatory
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strategy is proposed for applying this model for the determination of
C-t values under the surface water treatment rule (SWTR).
The C-t Concept
The C-t concept in current use is an empirical equation stemming
from the early work of Watson and is expressed as (2):
K = Cnt
where
K
(1)
= constant for a specific microorganism exposed under specific
conditions
C = disinfectant concentration
n = constant, also called the "coefficient of dilution"
t = the contact time required for a fixed percent inactivation
It is based on the van't Hoff equation used for determining the nature
of chemical reactions in which the value n determines the order of the
chemical reaction (3,4).
The application of this equation to disinfection studies requires
multiple experiments where the effectiveness of several variables,
such as pH, temperatures, and the disinfectant concentration are
examined to determine how they affect the inactivation of microbial
pathogens. The disinfection concentration (C) and time (t) necessary to
attain a specific degree of inactivation (e.g., 99 percent) are plotted on
double logarithmic paper. Such a plot results in a straight line with
slopes (5,6). Figure 4.4.1 illustrates data plotted in this manner and
also the significance of the value of n in extrapolation of disinfection
data (7). When n equals 1, the C-t value remains constant regardless
of the disinfectant concentration used, i.e., disinfectant concentration
and exposure time are of equal importance in determining the
inactivation rate, or the C-t product, K. If n is greater than 1,
disinfectant concentration is the dominant factor in determining the
inactivation rate while if n is less than 1, exposure time is more
important than disinfectant concentration. Thus, the value of n is very
important in determining the degree to which extrapolation of data
from disinfection experiments is valid. In addition, Morris pointed out
that the evaluation of n is valid only if the original experimental data
follow Chick's Law (i.e., the rate of organism distraction is directly
proportional to the number of living organisms remaining at any
specified time), which is normally not the case (8).
Factors Affecting C-t
The destruction of pathogens by chlorination is dependent on a
number of factors, including water temperature, pH, disinfectant
contact time, degree' of mixing, presence of interfering substances
(which may be related to turbidity), and concentrations of chlorine
194
-------
o
o
o
F
11111 I I f
rrn—i m n i
LLLL
i i
ii i i i i
''III I
I I I I 1
"I'M
0 =5,
T- O)
Figure 4.4.1. Effect of n value on C-t values at different disinfectant
concentrations (C-t values given in parentheses).
195
-------
available (7). The pH, especially, has a significant effect on
inactivation efficiency because it determines the species of chlorine
found in solution.
The impact of temperature on disinfection efficiency is also
significant. For example, Clarke determined that in order to maintain
the same level of virus destruction by chlorine, contact time must be
increased two to three times when the temperature is lowered 10°C (9).
Disinfection by chlorination can inactivate Giardia cysts, but only
under rigorous conditions. Most recently, Hoff et al. concluded that
these cysts are among the most resistant pathogens known, and that
inactivation by disinfection at low temperatures is especially difficult
(10).
In Vitro Excystation
Excystation, the process by which the trophozoite breaks out of the
cyst wall, is considered an acceptable criterion for determining the
effects of chlorination on the viability of Giardia cysts (10). Using in
vitro excystation, Jarroll et al. have shown that 99.8 percent of G.
lamblia cysts can be killed by exposure to 2.5 mg/L of chlorine for 10
minutes at 15°C at pH 6, or after 60 minutes at pH 7 or 8 (11). At 5°C,
exposure to 2 mg/L of chlorine killed 99.8 percent of all cysts at pH 6
and 7 after 60 minutes (11). While it required 8 mg/L to kill the same
percentage of cysts at pH 6 and 7 after 10 minutes, it required 8 mg/L
to inactivate cysts to the same level at pH 8 after 30 minutes.
Inactivation rates decreased at lower temperatures and at higher pHs,
as indicated by the higher C-t values. Figures 4.4.2 and 4.4.3
summarize the data developed by Jarroll et al. It should be noted that
the nature of the excystation method limits the ability to' measure
percent survival at high inactivation levels. The assay involves
microscopic observation of the cysts. Therefore, to detect 1 viable cyst
in 1,000 (99.9 percent inactivation), several thousand cysts must be
observed to count enough viable cysts for statistical confidence at this
level. Since this has not been done, no data for achieving 99.9 percent
or higher inactivation levels are available from studies involving
excystation procedures.
Animal Infectivity Data
Quantification of the combined effects of pH, temperature, and
disinfectant concentrations require special techniques that take into
account the interaction of these variables so they can be described by a
single value. In this section, cyst inactivation data from animal
infectivity studies conducted by Hibler et al. are described (12). The
Hibler data are unique in that, unlike data from excystation studies,
they indicate the disinfectant conditions necessary to achieve greater
than 99.9 percent inactivation of G. lamblia cysts. These data could be
combined with excystation data for G. lamblia by Rice et al. (13),
Jarroll et al. (11), and Rubin et al. (14) to show the combined effects of
196
-------
Fefcent'Cyst
Survival
100,
pH6
pH7
Chlorine Concentrations
0 1 mg/l
2 mg/l
n 4 mg/l
• 8 mg/l
pH8
10
0.2-010 30 60 0 10 30 60 010 30
60
Contact Time (minutes)
Figure 4.4.2. Inactivation of G. lamblia cysts by free residual chlorine at 5°C.
chlorine concentration, pH, and temperature on different levels of
inactivatioh of G. lamblia cysts.
Hibler acquired G. lamblia isolates from several human sources
and maintained them by passage in Mongolian gerbils (12). Cysts
obtained from these animals were used to develop C-t values for
99.99 percent inactivation of G. lamblia cysts with chlorine at
temperatures of 0.5,2.5, and 5.0°C and at pH values of 6,7 and 8
197
-------
Chlorine Concentrations
Percent Cyst
Survival
100
a 3.0 mg/l
e 2.5 mg/l
pH6
pH7
pH8
10 _
0.2
0 10 30 50 0 10 30
Contact Time (minutes)
60 0 10
Figure 4.4.3. Inactivation of G. lamblia cysts by free residual chlorine at 15"C.
In these experiments, clean G. lamblia cysts at a concentration of
1.02 x 103 cysts/mL were exposed to selected chlorine concentrations at
appropriate pH and temperature. At specified time intervals for each
temperature and pH condition, chlorine activity was stopped by the
addition of sodium thiosulfate. The treated cyst suspension was
centrifuged, the supernatant poured off, and the cysts resuspended in a
small volume of buffer. Each of five gerbils, per test run, was fed 5 x
104 of the concentrated chlorine exposed cysts. Infectivity studies with
unchlorinated cysts showed that approximately five cysts usually
198
-------
constituted an infective dose. Table 4.4.1 shows a distribution of the
number of animals infected by chlorine exposed cysts.
Table 4.4.1. Distribution of animals infected by chlorine exposed
cysts.
Number of Infected Animals
PH
6
6
6
7
7
- 7
8
8
8
Temp
0.5
2.5
5
0.5
2.5
5
0.5
2.5
5
0
58
54
23
35
62
61
36
68
45
1 ,
15
7
10
7
6
7
10
12
9
2
5
4
6
5
4
4
8
4
3
3
3
3
5
1
4
4
3'
3
2
4
2
1
5
1
0
0
1
2
1
5
5
7
15
25
4
12
2
6
6
In order to analyze these data the following assumptions were
made. If all five animals were infected, then the C-t of the test run
produced less than 99.99 percent inactivation since cyst exposure
levels would have been reduced from 5 x 104 to less than 5 per animal.
If no animals were infected, then the C-t had produced greater than
99.99 percent inactivation, since cyst exposure levels would have been
reduced from 5 x 104 to less than 5 per animal. In the test runs where
one to four animals were infected, cyst exposure levels would
theoretically have been reduced from 5 x 104 to both 5 or greater cysts
(those infected) and to less than 5 cysts (those not infected) per animal.
Since in most of the test runs where one to four animals were infected
the largest number of infected animals was only one, it was considered
reasonable to assume all data where one to four animals were infected
represented 99.99 percent inactivation. The C-t of the test run
produced less than 99.99 percent inactivation.
The limitation of this experiment is that it is only appropriate to
assign a specific level of inactivation (i.e., 99.99 percent) to the case of
one to four animals infected.
Model Development
Statistical analysis was performed on the infectivity and
excystation data sets to determine the effects of inactivation level;
temperature, pH, and concentration of disinfectant on time to
inactivation (15). A multiplicative model was selected to best represent
the chemical reactions during the inactivation process:
199
-------
(2)
where
t = time to inactivation in minutes
I = inactivation level
C = concentration of disinfectant in mg/L
pH = pH at which experiment was conducted
temp = water temperature at which experiment was
conducted
R, a, b, c, d = model parameters
A log transformation of equation 2 yields: LOGio(t) = LOGio(R) +
(a)LOGio(I) + bLOGio(C) + cLOGio(pH) + dLOGio (temp) (3)
where LOGio is logarithm to the base 10.
Equation 2 was applied to each of the three data sets individually
and in various combinations (Hibler et al. (12), Jarroll et al. (11), Rice
et al. (13), and Rubin et al. (14). The Hibler et al. data were assumed to
be one element in all of the combinations studied (12).
In order to arrive at the optimal combination of these data sets,
equation 2 was modified to provide an alternative equation designed to
study the various combinations of data as follows:
t = R la Cb pHc tempd Ze
(4)
where
Z = indicator random variable
e = exponent ofZ
The indicator random variable was used to examine data set
compatibility and to move the regression intercept of slope to
compensate for data set difference. Significance of the indicator
random variable (Z) would support the hypothesis of different
regression surfaces, i.e., incompatibility of the data sets chosen. The
indicator random variable was created in such a way as to always
differentiate between the Hibler data and other data sets considered
(11,12,13,14). Table 4.4.2 contains the data set combinations and
regression diagnostics.
As shown by Table 4.4.2, the indicator random variable combining
the Hibler et al. and Jarroll et al. data bases was not significant. All
other data bases considered had a significant indicator random
variable at the 0.05 level of significance. A formal test for differences of
intercept and/or slope between the Hibler et al. and Jarroll et al. data
sets was conducted using the BMDP PIR procedure in the BMDP
statistical programming language. Test results indicated no difference
between the data sets. Thus, statistical analysis supports the choice of
the Hibler et al. and Jarroll et al. data as the data base for Giardia cyst
modeling procedures.
200
-------
Table 4.4.2. Regression Diagnostics for Data Set Combinations
Data Sets Considered R-Square Variables
Plots
Hibler, Rice, Jarroll, Rubin
Hibler, Rice, Jarroll, Rubin, Z
Hibler, Rice, Rubin
Hibler, Rice, Rubin, Z
Hibler, Jarroll, Rubin
Hibler, Jarroll, Rubin, Z
' , :
Hibler, Rice, Jarroll
Hibler, Rice, Jarroll, Z
Hibler, Rubin
Hibler, Z
Hibler, Rice
Hibler, Rice, Z
Hibler. Jarroll
Hibler, Jarroll, Z
0.6801
0.7316
0.6649
0.7899
0.6424
0.6879
0.8619
0.865
0.6483
0.7593
0.8548
0.8678
0.8452
0.8459
intercept, temp
not significant
intercept, temp
not significant
intercept, temp
not significant
intercept
not significant
intercept, temp
not significant
intercept, temp
not significant
all variables
significant
all variables
significant
temp
not significant
intercept
not significant
all variables
significant
all variables
significant
all variables
significant
Z not significant
non normal data
non constant var
non normal data
non constant var
non normal data
non constant var
non normal data
non constant var
non normal data
non constant var
non normal data
non constant var
non normal data
non constant var
non normal data
non constant var
non normal data
non constant var
non normal data
constant var
non normal data
constant var
non normal data
constant var
non normal data
constant var
non normal data
constant var
The resulting regression equation is:
: t = 0.121-0.27 e-0,8ipH2.54temp-0.15
Equation 5 multiplied by C yields
C-t = 0.121-0.27 C0.19 PH2.54 temp-0.15
(5)
(6)
201
-------
which is the C-t equation utilized. It can be shown that equation 6 is
equivalent to the Watson equation (Appendix A). The confidence
intervals for parameter estimation of equation 6 are:
R: (0.0384, 0.4096)
a: (-0.2321,-0.3031)
l+b:( 0.0792, 0.2977)
c:( 1.9756, 3.1117)
d: (-0.0724, -0.2192)
These confidence intervals were calculated using the Bonferroni
method (16).
Regulatory Application
There are many uncertainties regarding the various data sets that
might be utilized for calculating C-t values. The random variable
analysis shows the statistical incompatibility among these data sets.
More work needs to be done to define the impact of strain variation,
and in vivo versus in vitro techniques on C-t values. To provide
conservative estimates for C-t values, the authors suggest the
approach illustrated in Figure 4.4.4.
In Figure 4.4.4, the 99 percent confidence interval at the 4 log
inactivation level is calculated. First order kinetics are then assumed
so that the inactivation "line" goes through 1 at C-t = 0 and a C-t
value equal to the upper 99 percent confidence interval at 4 logs of
inactivation (Appendix B). As demonstrated, the inactivation line
bounds higher C-t values than all of the mean C-t values from
equation 6 as well as all of the Jarroll et al. data points (at inactivation
levels of 0.1 and 0.015) and the Hibler et al. data points (at an
inactivation level of 0.0001). Conservative C-t values, for a specified
level of inactivation, can be obtained from the inactivation line
prescribed by the disinfection conditions. For the example indicated in
Figure 4.4.4, the appropriate C-t for achieving 99.9 percent
inactivation would be 160. This approach (assumption of first order
kinetics) also provides a conservative basis for establishing credits for
a sequential disinfection step, e.g., the C-t values for 90 or 99 percent
inactivation.
Summary and Conclusions
Amendments 'to the Safe Drinking Water Act clearly require all
surface water suppliers in the United States to filter and/or disinfect to
protect the health of their customers. G. lamblia has been identified as
one of the leading causes of waterborne disease outbreaks in the
United States. G. lamblia cysts are also one of the most resistant
organisms to disinfection by free chlorine. EPA's Office of Drinking
Water has adopted the C-t concept to quantify the inactivation of G.
lamblia cysts by disinfection. If a utility can assure that a large enough
C-t can be maintained to ensure adequate disinfection, then,
202
-------
Inactivation Level
1.0000
0.1000
0.0100
0.0010
0.0001
0.0000
Ct-PRED.
Actual Ct
99% Conf. Interval
Suggested SWTR
CTs
I
I
I
l_
I
I
I
0 20 40 60 80 100 120 '140 160 180 200 220 240 260 280
. , Gt Values
Figure 4.4.4. Ninety-nine percent confidence levels using Hibler-Jarroll equation
for chlorine = 2 mg/l; pH = 7; temperature = 5°C.
depending upon site specific factors, installation of filtration may not
be required. Similarly, the C-t concept can be applied to filtered
systems for determining appropriate levels of protection.
The equation developed in this chapter can be used to predict C-t
values for inactivation of G. lamblia by free chlorine based on the
interaction of disinfectant concentration, temperature, and
inactivation level. The parameters for this equation have been derived
from a set of animal infectivity data (Hibler et al.) (12) and excystation
data (Jarroll et al.) (11). The equation can be used to predict C-t
values for achieving 0.5 to 4 logs of inactivation and, within
temperature ranges of 0.5-5°C, chlorine concentration ranges up to 4
mg/L. While the model was not based on pH values above 8, the model
203
-------
is still considered applicable to pH levels of 9 for reasons discussed
elsewhere (17). The equation shows the effect of disproportionate
increases of C-t versus inactivation levels. Using 99 percent
confidence intervals at the 4 log inactivation levels and applying first
order kinetics to these endpoints, a conservative regulatory strategy
for defining C-t at various levels of inactivation has been proposed.
This approach represents an alternative to the regulatory strategy
previously proposed (17).
References
1. Craun, G. F. Surface water supplies and health. J. AWWA.
80(2):40-56. February 1988.
2. Watson, H. E. A note on the variation of the rate of disinfection
with change in the concentration of the disinfectant. J. Hyg. 8:536-
592,1908.
3. Berg, G., Chang, S. L., and Harris, E. K. Devitalization of micro-
organisms by iodine 1. Dynamics of the devitalization of
enteroviruses by elemental iodine. Virol. 22:469-481,1964.
4. Fair, G. M., Geyer, J. C. and Okun, D. A. Water and Wastewater
Engineering. Vol. 2 Water purification and wastewater treatment
and disposal. John Wiley and Sons, Inc., New York, New York,
1968.
5. Fair, G. M., Morris, J. C., and Chang, S. L. The dynamics of water
chlorination. J. NewEng. Water Works Assoc. 61:285-301,1947.
6. Fair, G. M., Morris, J. C., Chang, S. L., I. Weil, and Burden, R. P.
The behavior of chlorine as a water disinfectant. J. AWWA.
40:1051-1061,1948.
7. Hoff, J. C. Inactivation of microbial agents by chemical
disinfectants. EPA/600/2-86-067, U.S. Environmental Protection
Agency, 1986.
8. Morris, J. C. Disinfectant chemistry and biocidal activities. In:
Proceedings of the National Speciality Conference on Disinfection,
American Society of Civil Engineers, New York, New York, 1970.
9. Clark, N. A., Berg, G., Kabler, P. W. and Chang, L. L. Human
enteric viruses in water: Source, survival, and removability.
Presented at International Conference on Water Pollution
Research, Landar, September 1962.
10. Hoff, J. C., Rice, E. W., and Schaefer, III, F. W. Disinfection and
the control of waterborne giardiasis. In: Proceedings of the 1984
Specialty Conference, Environmental Engineering Division,
ASCE, June 1984.
204
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11. Jarroll,,E. L., Bingham, A. K., and Meyer, S. A. Effect of chlorine
on Giardia lamblia cyst viability. Appl. Env. Microbiol., Vol. 41,
pp. 483-487, February 1981.
12/Hibler, C. P., Hancock, C. M., Perger, L. M., Wegrzn, J. G., and
Swabby, K. D. Inactivation of Giardia cysts with chlorine at 0.5°C
to 5:0°C. American Water Works Association Research
Foundation, 6666 West Quincy, Denver, Colorado.
13. Rice, E. W., Hoff, J. C., and Schaeffer, III, F. W. Inactivation of
Giardia cysts by chlorine. In: Applied and Environmental
Microbiology, Vol. 43, pp. 250-251, January 1982.
14. Rubin, A. Internal report of progress, through June 1, 1988. EPA
Project CR812238,1988.
15. Clark, R. M., Read, E. J., and Hoff, J. C. Inactivation of Giardia
lamblia by chlorine: A mathematical and statistical analysis.
Accepted for publication by the J. Env. Eng.
16. Neter, J. and Wasserman, W. Applied Linear Statistical Models.
Irwin: Hpmewood, Illinois, 1974.
17. Regli, S. EPA disinfection regulations. In: Seminar Proceedings
Assurance of Adequate Disinfection or C-t or not C-t, American
Water Works Association Annual Meeting 1987, pp. 1-7.
Appendix A
Equation 6 can be shown to be equivalent to equation 1 by dividing
equation 6 by Cb which yields:
C-bt = RIapHctenipd
Assuming a constant pH = pH,
temp = temp and 1 = 1 yields
K = RlapHbtempcId
therefore
where
; -b = n in equation 1 .
(A-l)
(A-2)
(A-3)
205
-------
Appendix B
A general relationship that relates C-t values at different
inactivation levels is:
ln(Nj/N0) = _Kj
ln(Nj/N0) ~ Kj
(B-l)
where
N{ = the number of organism left at time t,
NJ = the number of organisms left at time tj.
Table 4.4.B-1 summarizes the multiplication factors to be applied,
assuming first order kinetics, to convert a value of KI to an
equivalent value of Kj.
Table 4.4.B-1. Multiplication Factors to Convert C-t Values from one
Inactivation Level K, to Inactivation Level Kj
From Inactivation
Level i
90
99
99.9
99.9
Level j 90
-
1/2
1/3
1/4
rviuuipii
.99
2.01
-
2/3
1/2
er ior w
99.9
3.0
1 1/2
-
3/4
99.9
4.0
2.0
1 1/3
-
1 Multiplier for Ki, e.g., to convert 90 percent inactivation to 99 percent
inactivation, multiply by a factor of 2.
206
-------
Distribution Systems: Treated Water Quality Versus
Conform Noncompliance Problems
by: Donald J. Reasoner, Research Microbiologist
Edwin E. Geldreich, Senior Research Microbiologist
Drinking Water Research Division
Risk Reduction Engineering Laboratory
U.S. Environmental Protection Agency
Cincinnati, Ohio 45268
(513)569-7234
Introduction
The maintenance of water quality during distribution is a concern
for every water utility, regardless of geographic location and popula-
tion size served. Factors that adversely affect water quality, or the
determination of water quality, must be considered with respect to the
utility's ability to meet the requirements of the Primary Drinking
Water Standards and alleviate the health risk to consumers.
Water quality is dynamic; it will change as the water progresses
from one point to another in distribution. To prevent adverse change,
the factors that cause degradation in water quality must be understood
and appropriate steps taken to prevent degradation. Some of these
factors may not be reasonably controlled, but it is imperative that their
impact on water quality be assessed.
With respect to bacteriological quality, it is necessary to recognize
that the distribution system is an ecosystem that responds to physical
and chemical changes in the water passing through it. Our knowledge
about those changes and their effects on the bacteria in the system is
rudimentary. In general, control of bacterial populations m
distribution systems has been accomplished primarily by increasing
the disinfectant residual. However, it has become increasingly appar-
ent over the last few years that this is not always effective; there are
numerous instances where coliform bacteria have become established
in the distribution system and show up intermittently, or in some cases
pose a fairly persistent problem (1,2,3,4,5).
Distribution system problems caused 16 percent of waterborne
outbreaks and illnesses during the period 1971-1985 (6). However,
these figures represent only reported waterborne outbreaks; there may
have been other similar outbreaks that went undetected and there
207
-------
have been many distribution problems that have not been
epidemiologically investigated.
The occurrence of coliform bacteria in treated distribution system
water raises a warning flag that a problem exists that needs to be
addressed immediately. In the past, it was common to attribute
coliform positive samples to improper sample collection or laboratory
error, particularly when check samples turned out to be coliform free.
In recent years, however, there has been sufficient documentation of
intermittent and/or persistent coliform occurrences in treated distribu-
tion water systems to cause a change in attitude toward this problem.
The emphasis now is on reevaluating what actually is occurringj and
finding ways to control the bacterial levels in distribution systems.
Table 4.5.1 presents a summary of documented coliform occurrence
problems in public water supplies in the United States and Canada,
providing a perspective on this problem. These data were gathered
from published reports and from personal communications (E.E.G)
with state and U.S. EPA Regional personnel. Again, these are the
known and documented cases; how many similar occurrences have
gone either undetected or ignored is unknown.
There are two major concerns about the presence of coliforms in
distribution systems: 1) When a coliform occurrence problem arises,
does it signify an increased health risk? and 2) If the coliform bacteria
have colonized the system and there is no increased health risk, of
what value is the continued reliance on the coliform group of bacteria
as a sanitary indicator? The documented problems in Table 1 have not
been associated with increased infectious disease risk based on
existing surveillance systems. However, specific epidemiologic studies
to determine this risk were not conducted. On the other hand,
waterborne outbreaks have resulted in instances when coliforms were
found in the distribution system. How can coliform occurrences
associated with unsafe water quality be effectively differentiated from
those associated with safe water quality? At present, there has been no
research conducted to effectively answer the latter question and we
must treat all coliform occurrence problems as indicative of a potential
health problem in treatment or distribution, or both.
The geographical distribution of systems with coliform problems is
shown in Figure 4.5.1. As indicated, the problems have occurred
primarily in the eastern half of the United States. This may be due to
the age of the distribution systems involved and to the source water.
Origins of Microbial Flora in Drinking Water
The origins of the microbial flora in drinking water include source
water, treatment process exposures, formation of biofilms in the
distribution system, and post treatment contamination caused by
events such as main breaks and repairs, and cross-connections (Table
4.5.2). Source water and treatment process contributions are due,
208
-------
Table 4.5.1. Coliform Occurrences in Public Water Supply Distribution Systems
Location Population^ Reference
United States:
Nassau County, NY
(Fairfax Water Authority)
San Francisco, CA
Milwaukee, Wl
(summer fair line)
New Haven, CT
Worchester, MA
Fairfax, VA
Rochester, NY
Akron, OH
Lexington, KY
Grand Rapids, Ml
Flint, Ml
Ann Arbor, Ml
Springfield, IL
Miami Beach, FL
Muncie, IN
Salem-Beveriy, MA
Bethlehem, PA
Monmouth, NJ
Terre Haute, IN
Florissant, MO
Asheville, NC
Wilmette, IL
N. Andover, MA
Bennington, VT
Brockport, NY
Seymour, IN
Lakewood, NY
Kennebunk, ME
Newfields, NH
Eden Isles, LA
Total
2,600,000
678,974
964,988
761,337
646,352
598,601
241,741
237,177
204,165
181,843
159,611
107,316
99,637
96,298
95,000
75,875
70,419
68,000
61,125
55,372
53,281
28,229
16,284
16,000
9,776
4,600
3,941
3,294
700
500
8,120,652
_b
Tracy et at. (7)
Milwaukee Journal (8)
Centers for Disease Control (9)
_b
The Fairfax Journal (1 0)
Rochester Democrat and Chronicle (1 1 )
Akron Beacon Journal (12)
_b
Wierenga (3)
_b
_b
Hudson et al. (2)
AWWA Mainstream (13)
Earnhardt (14)
McFeters etal. (15)
_b
_b
_b
St. Louis Post-Dispatch (16)
O'Brien and Gere (1 7)
_b
_b
McFeters et al. (15)
Clark (18)
Lowther and Moser (1 )
Lakewood Post-Journal
McFeters et al. (15)
_b
Times-Picayune (20)
(continued)
209
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Table 4.5.1. (Continued)
Location
Canada:
Edmonton, Alberta
Vancouver, British Columbia
Halifax, Nova Scotia
Total
Population3
461,361
410,188
117,882
989,431
Reference
_b
_b
Martin et al. (5)
a Population based on 1980 census, or on population given in reference.
b Where references are not given, information was collected by E.E. Geldreich through
personal communications with State or U.S. EPA Regional Water Supply representatives.
respectively, to survival of source water bacteria that pass through the
treatment processes and bacteria from biofilm that sloughs from rapid
sand and carbon filters. Turbidity particulates, including carbon fines
from granular activated carbon filter beds and floe, may have bacteria
associated with them that are protected from the disinfectant. Water
supply treatment was not intended to produce sterile water but to
remove organisms of risk to public health. Coliform bacteria were
chosen as indicators of pathogen presence because of their longer
survival in the water environment and their presence may indicate
deficiencies in water treatment.
Water Pipe Environment
Once they pass through the treatment barriers, some of the bacter-
ia find conditions in which they can survive, adapt, and grow, thus
supporting the idea of the distribution system as an ecosystem or
natural habitat.
The distribution system becomes a repository for particulates that
include carbon fines, floe particles, soil particles, and biological
materials such as algae, bacteria, protozoa, etc. In addition, nutrients
dissolved in the water are available for bacterial growth, pipe walls
and sediments provide sorption sites for bacteria and nutrients, water
pH conditions are very suitable for bacterial growth, and the water
temperature may be conducive to bacterial growth, particularly if it is
above 10°C. Finally, flow conditions may favor bacterial growth
particularly those areas of slow or no flow (dead-ends) in the
distribution network. In fact, all conditions in the distribution system
except the presence of a disinfectant residual may contribute to the
growth of numerous heterotrophic bacteria.
Biofilms in Distribution Systems
Once in the distribution system environment, many bacteria reach
the pipe walls and sediments where they find conditions suitable for
survival and possibly growth. Bacteria may attach to the surfaces of
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Table 4.5.2. Sources of Microorganisms in Distribution Water
Source
Wood
Filter sand; filter effluent
piping; filter bed
Microorganism(s)
Klebsiella
C. freundii, E. coli,
Reference
Seidleretal. (21)
Wierenga (3)
Pipe tubercles, floe,
sediment, pipe biofilm
Pipe tubercles, pipe
biofilm
Treatment plant
Biofilm
GAG, sand, anthracite
filter beds
£ coli, K. pneumoniae
K. oxytoca, E. cloacae,
E. agglomerans
K. pneumoniae, K. oxytoca
E. agglomerans, E. cloacae
E. coli, C. freundii
K. pneumoniae, E. cloacae
Enterobacter sp.
K. pneumoniae, K. ozaenae,
K. rhinoscleromatis, E. coli,
E. cloacae, E. aerogenes,
E. agglomerans, C. freundii
K. oxytoca, K. pneumoniae,
E. coli, E. agglomerans,
E. cloacae, E. aerogenes,
E. sakazaki, C. freundii,
Serratia sp.
LeChevallier et al. (4)
Lowther and Moser
0)
Hudson et al. (2)
Reilly and Kippin (22)
Camper et al. (23,24)
Invertebrates
Nematodes
Open finished water
reservoir
Treatment breakthrough,
pipe break
Aerobacter aerogenes
(Enterobacter aerogenes)
Total conforms
E. aerogenes, E.
agglomerans,
K. pneumoniae, K. oxytoca
Tracy et al. (8)
Levy et al. (25)
Chang et al. (26)
Silverman et al. (27) ,
McFeters et al. (15)
pipes or particles by means of glycocalyx, which is composed of complex
mucopolysaccharide material. The organisms may also respond to the
low nutrient conditions by forming a capsule, which also provides some
protection from disinfection. Once attached to the pipe or sediment
surfaces, the organisms benefit by the presence of other organisms and
their capsular arid glycocalyx materials, and possibly by symbiotic
interactions in which metabolites released by other organisms may be
used as nutrients and vice versa. Biofilms develop in all aquatic
ecosystems and water distribution systems are no exception (28,29).
The interior of a pipe that has become corroded and tuberculated,
or that has developed a porous coating of inorganic minerals provides
tremendous surface area for bacterial colonization (30,31,32,33). It is
important to emphasize that no water system is free from biofilm; what
is most important is the degree of development of the biofilm. The
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development of biofilms that include coliform colonization is believed
to require relatively high levels of assimilable organic carbon (AOC) in
order for the coliforms to multiply. Conditions that favor the release of
coliform-containing biofilm from the pipe or sediment surfaces
contribute to the occurrence of coliforms in water samples taken for
analysis. Most coliform occurrence problems that have been
investigated have involved species of Klebsiella, Enterobacter, or
Citrobacter. Occasionally, Escherichia coli has beeri found, but the
infrequency of its occurrence indicates that this organism generally
does not colonize and grow in the biofilm. Rather, it remains an
indicator of recent sewage or fecal contamination.
Distribution system coliform problems have involved, or been
compounded by, such things as improper operation of filters and
inadequate or interrupted disinfection, as well as problems with
system maintenance. Dead-end and slow-flow areas of distribution
systems contribute highly to bacterial growth due primarily to the loss
of disinfectant residual and its inhibitory effect on bacterial growth.
For example, when sufficient nutrients are available in a standpipe or
storage reservoir that is not used for some period of time, coliform
bacteria may grow and then be dispersed when water is moved into the
distribution system. A coliform problem in. Florrisant, Missouri, was
due to water that had been held for several months in a standby
reservoir. In another case, residual food grade antifreeze used to
protect a water line to a festival site from freezing during the winter
non-use period provided nutrients for coliform regrowth the following
summer. The problem was cleared up by continuous line-flushing in
the spring until a low HPC was achieved and no coliforms were
detected in 100-mL samples; booster chlorination was applied during
the summer-use period to maintain a chlorine residual.
Water main repairs and new main construction may result in
coliform problems, usually because of inadequate flushing and
disinfection. Construction materials, such as wood, left in newly
constructed lines, have been implicated in coliform problems. In
Halifax, Nova Scotia, Klebsiella pneumoniae grew in association with
wooden material left from construction (5). To resolve the problem,
lime was added to adjust the pH to 9.1, which either inactivated the
organisms, or resulted in the formation of a carbonate scale that
trapped the organisms so that they could not slough off into the water.
The hydraulic effects of high-flow rates and high pressure can also
contribute to coliform problems, shearing biofilm organisms from the
pipe or sediment surfaces. High pressures can cause breaks in pipe
when pressure fluctuates suddenly due to rapid changes in demand.
Temporary flow reversals caused by sudden heavy demands in a
portion of the system can stir up and resuspend sediments, or cause
biofilms to shear or break loose. ;
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As indicated earlier, many of the systems that have identified a
cblifof m noncompliance problem are east of the Mississippi River and
north of the Ohio River (Figure 4.5.1). Characteristics shared by water
systems in this area include use of surface source waters, a significant
percentage of iron pipe in the system having been in service for more
than 75 years, and tuberculations in the iron pipe. Additionally,
systems with a problem of bacterial regrowth or aftergrowth
frequently lack an adequate disinfectant residual, or are unable to
maintain an adequate residual throughout the distribution system.
The microbial growth problem becomes worse during the warmer
period of the year, generally summer to late summer and even late fall
in many areas of the United States. Bioadsorption of nutrients during
cold .water periods may make more nutrients available for bacterial
growth during warm water periods.
Figure 4.5.1. Distribution of U.S. public water supplies with coliform occurrences
(numbers = occurrences).
The nutrient concentrations in the bulk water needed to support
coliform regrowth are probably greater than 50 to 60 ug/L measured by
an assimilable organic carbon bioassay (34). Nutrient levels needed for
the growth of noricbliforms are probably less than this, although van
der Kooij has indicated that general bacterial growth in distribution
water may be limited at AOC levels below 10 to 15 ug acetate carbon
equivalents/L (34). The magnitude of the actual nutrient
concentrations organisms encounter at the interface between the
213
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water and the pipe or particle surfaces where they reside is currently
unknown.
Coliform Case Study - Multiple Factors
Because of the difficulties many utilities have experienced in
solving persistent coliform problems, it is evident that a variety of
factors are involved and that simple solutions, such as boosting the
chlorine residual, are either not readily available or not satisfactory.
Differing circumstances at each utility have required different
remedies; in all cases, multiple factors have affected both the coliform
persistence and the remedy. Some utilities have not been able to
eliminate the coliform problem entirely and are faced with being out of
compliance with the coliform Maximum Contaminant Level (MCL)
during some portions of the year, or being close to being out of
compliance much of the time, both particularly during warm water
months.
In a recently published study of a persistent coliform problem in
New Jersey, coliforms were first isolated in June 1984 and have
persisted at varying levels since that time (35). The water received
conventional treatment (pretreatment, flocculation, clarification,
filtration, and postdisinfection). In studying this problem, investiga-
tors concluded that the treatment plant was not the main source of
coliforms since the plant met the coliform standard. However,
coliforms were found in 193 of 500 (38.6 percent) of the distribution
samples collected between May and August 1986. Coliform numbers
increased between the treatment plant (0.03/100 mL) and the first
distribution site (0.64/100 mL), and then decreased (0.20/100 mL) as
the water moved through the study area. Not only did coliform
numbers increase after treatment, but the species diversity also
increased. Coliforms found included E. agglomerans (44.2 percent) at
all locations, E. cloacae (24.4 percent) at all locations, K. oxytoca
(predominant in past episodes), K. pneumoniae, E. coli (6 percent), and
others. Noncoliform bacteria (HPC) levels often exceeded 106 CFU/mL
at the end of branch lines and included Flavobacterium and
Pseudomonas vesicularis. AOC levels in this study decreased from
about 130 pg/L in the treatment plant effluent to 56 ug/L at a
distribution sample site, a total decrease of 67 percent. Laboratory
studies showed that E. coli grew almost fourfold in dechlorinated
treatment plant effluent, but essentially did not grow in water from
the last distribution site with an AOC of about 56 ug/L.
Coliforms were found in pipe sediments at about 0.5 CFU/g
sediment and in tubercules released by the cleaning operation
(pigging) at >160 CFU/g tubercule. No coliforms were found in a
sample taken from a 20 cm2 area of cement pipe before cleaning, but
the sampling effort may not have been intensive enough to detect
coliforms. Ridgway and Olson have shown that the occurrence of
bacterial microcolonies on pipe walls is sparse and randomly
214
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distributed (patchy) (33); coliform occurrence within the biofllm is
likely to be patchy also.
Coliform bacteria were present in a variety of samples from this
system, sometimes at high densities such as in the tubercle material.
Although the water leaving the treatment plant contained only 0.03
coliforms/100 mL (30 times lower than the standard), fluctuations in
various treatment parameters such as pH and chlorine indicated the
need for tighter control on some of the plant processes.
Simple calculations indicate that at a treatment effluent quality of
0.03 coliforms/100 mL and a mean production of 32.7 MGD (range of
23.3 to 43iO MGD), the mean input into the distribution system was 2.2
x 107 coliforms per day (range of 1.5 x 107 to 2.8 x 107 coliforms per
day). Thus, a continuous seeding of the distribution system was
occurring. In addition, although the average free chlorine residual of
the water was 1.1 mg/L (range of 0.1 to 3.0.mg/L), simple laboratory
experiments with biofilm coliforms indicated only about 50 percent
inactivation of coliforms after 1 hour of exposure to 1.1-mg/L free
chlorine residual.
Comparison of the mean coliform level leaving the treatment plant
(0.03/100 mL) with coliform levels detected in the distribution water
by the standard coliform procedure using m-Endo medium (0.33/100
mL) indicated an 11-fold increase in the coliform concentration of the
distribution system water. Coliform recovery by the stressed coliform
procedure using m-T7 medium (0.41/100 mL) showed a 13-fold increase
in coliform concentration in the distribution water; evidence that
viable injured coliforms were present. Thus, not only was there a
continuous seeding of the system with coliforms, but the disinfectant
residual was not effective in significantly reducing the coliforms in the
system. A biofilm reservoir of coliforms existed in the sediment,
tubercles, and probably the pipe walls in some areas of the system.
The combination of factors in this scenario may represent
situations of persistent or intermittent coliform occurrence in other
distribution systems that were hot as well studied and documented. A
variety of such coliform problems need to be thoroughly studied and
analyzed to develop better information on the critical factors involved,
as well as to formulate the appropriate corrective actions to eliminate
the problem. The need for action to correct problems of coliform
occurrence is evidenced by the apparently increasing number of
reports of intermittent and persistent coliform outbreaks in municipal
water treatment and distribution systems.
Action Plan for Correction of Coliform Problem ,
The first task in correcting a coliform problem is determining
whether the coliform occurrences represent new contamination or the
recurrence of coliforms that have colonized the distribution system.
New contamination indicates a penetration of the treatment barrier
215
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and may represent a risk of a waterborne outbreak. Additional
coliform monitoring of the plant effluent may be instructive, including
the examination of larger sample sizes, i.e., 500 mL to 1 L, or composite
samples collected over a 24-hour period. Because penetration of the
treatment barrier by coliforms may occur along with particulate
materials, increased monitoring of the turbidity level of the treated
water entering the distribution system may also be helpful. For
systems that use disinfection as the only treatment barrier, storm
events and increased runoff from the watershed signal a need to be
more vigilant of the disinfection process and ensure the disinfectant
residual is adequate to compensate for any increased chlorine demand
in the water. A thorough evaluation of the treatment plant operation
and correction of any deficiencies, no matter how trivial they may
appear, is also needed to protect against transmission of possible
pathogens and aid in determining whether the coliform problem is new
contamination or possible colonization.
Other microbial determinations may yield additional .useful
information to evaluate health significance. HPC measurements will
add data on regrowth potential. Other indicator organisms such as
Ctostridium perfringens may be useful for filtered systems; its
presence indicates .poorly treated contaminated water since C.
perfringens does not grow in a pipe network.
Coliform occurrences due to treatment plant problems often
appear to be widespread in the distribution system and frequently can
be brought under control rapidly by correcting the treatment failure.
In addition to checking the treatment plant operations, operators
should thoroughly evaluate the distribution system integrity and
events that impact distribution system operation. Coliform
occurrences due to distribution system defects or operational problems
are often characterized by positive coliform samples only in the area
downstream from the defect. If due to a low pressure or reversed flow
situation, the coliform occurrences may be scattered and have no
specific pattern. A cross-contamination control program helps to
reduce potential contamination problems due to this source.
Knowledge of events that caused high water demand, such as fire
fighting, can be helpful in determining the factors that may be
involved in a coliform outbreak.
Regardless of whether treatment plant operation or a distribution
system problem is the source of coliforms, detection of Escherichia coli
or any other fecal coliform organism is cause for alerting the public to
boil their drinking water until the problem has been rectified and
coliforms can no longer be detected. .
If efforts to isolate the coliform source are unsuccessful, with
coliforms continuing to appear in a random fashion throughout all or
parts of the distribution system, and various efforts to eliminate the
coliform have failed, it is possible that the coliform colonized the
216
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biofilm on the walls of the distribution pipes. In such situations,
•Klebsiella, Enterobacter, or occasionally Citrobacter species
predominate, even in the presence of a free chlorine residual ranging
from 0.1 to 2.0 mg/L. These coliform occurrences may reflect either the
existence of a biofilm growth during warm water periods or the release
of biofilm as a result of an action such as a change in water pH to
reduce a corrosion problem.
• Each distribution system is unique and corrective measures for
suppressing coliform occurrences will vary both in kind and in the
degree of response. In all situations, however, an aggressive flushing
program applied to all sections of the distribution system is
recommended. This will clear the system of static water that may
contain high densities of bacteria and colloidal particulates, which
exert disinfectant demand and may reestablish a free chlorine residual
if the disinfection process does not involve chloramines. There may be
a period of 24 to 48 hours following the flushing when water quality
temporarily deteriorates and the water may have increased coliform
levels, as well as increased color, taste, and odor. Notifying the public
in advance of the flushing program and asking them to run the water
to waste at their taps will help restore the clean water appearance and
possibly reduce the number of telephone complaints. Some areas of the
distribution system may need more aggressive cleaning, such as
forcing of plastic plugs through the pipe (pigging) or mechanical
scraping to remove tuberculations and accumulated sediments.
In addition to flushing, it is important to establish a disinfectant
residual throughout the distribution system to control the biofilm.
Where free chlorine is used, it may be necessary to use 5 mg/L or more
during the warm water period, and coliform levels may undergo a slow
reduction rather than an immediate drop to nondetectable levels.
Systems that use chloramines may find it desirable to change to an
alternative disinfectant (free chlorine, chlorine dioxide) to control the
heterotrophic bacterial population and better disinfectant penetration
of the biofilm. In other systems, changing to the use of chloramines
may result in better coliform control because of rate limiting transport
of the disinfectant into the biofilm (34).
A pH adjustment may be helpful in bringing a coliform problem
under control. In at least one case, adjusting the pH to 8.5 and adding
linie was apparently successful in producing a less porous calcium
carbonate scale, resulting in less favorable conditions for bacterial
colonization. In another case, elevation of the pH'to 9.1 was effective in
reducing Klebsiella occurrence, possibly due to the destruction of
polysaccharide capsular and biofilm material (5). Lowering the pH
below 8.3 for corrosion control management has been implicated in
several coliform problems as a factor in creating porous sediments and
scale and apparently releasing embedded viable coliforms.
217
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Since coliform colonization in the distribution system does not
signify fecal contamination, the need to notify the public of coliform
noncompliance requires a slightly different approach, with two action
levels.
The first level of action is to form a task group to evaluate the
problem and examine the laboratory results to determine if the
occurrences are a result of coliform release from the biofilm in the
network, and therefore of little apparent public health threat. The task
group should be composed of utility, state, and federal water
authorities and should jointly recommend initiation of corrective
actions. It should meet at frequent intervals (weekly or daily, if
necessary) to review progress by the water plant operations in
correcting the deficiencies. The public should also be informed that
water treatment adjustments are in progress to improve water quality.
Local hospitals and medical clinics may also be alerted so they can
monitor for any evidence of waterborne disease as a precautionary
measure.
The second level of action is to issue a boil water order
immediately if any of the following occur:
• Illness that may indicate a waterborne outbreak
• Failure to follow-up on task group recommendations
• Breakdown in disinfection treatment
• Detection of E. coli or fecal coliform bacteria in the water
distribution system
Frequent boil water orders, however, may cause either public over-
reaction or apathy. One concern is that customers may seek water
supply alternatives (bottled water, point-of-use treatment), some of
which may be of less desirable quality than public water supplies. The
overall objective is to formulate an action plan that responds to the
problem and aims at correcting it, rather than driving the monthly
coliform average into compliance by increased sample analyses, thus
creating a false sense of compliance.
Summary
The overall quality of treated drinking water is subject to
deterioration during distribution. Factors that affect the rate and
degree of deterioration include source water characteristics and
quality, type of treatment and treatment effectiveness, disinfectant
residual, temperature, pH, flow rate, residence time, organic carbon
available for microbial growth, the numbers and types of
microorganisms present, and distribution system age and condition.
The bacterial load of the distribution water is significantly influenced
by several of these factors.
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The occurrence of cqliform bacteria, used as indicators of
treatment effectiveness and sanitary quality, in treated distribution
water causes concern because of the possibility that disease-causing
organisms may also be present. Coliform occurrences may cause the
utility to be out-of-compliance with the coliform MCL of the Primary
Drinking Water Standards, even though the coliform organisms may
pose no direct health risk. Thus, conditions that favor coliform
persistence or growth within a distribution system impact our ability
to discriminate between situations of real and apparent adverse health
risk.
During the past few years, documentation of intermittent and/or
persistent coliform problems in a number of water distribution
systems, primarily in the eastern half of the. United States, has
stimulated interest and concern about this problem. This information
suggests that there are conditions under which coliforms survive, or
may actively grow, in biofilms that develop on the pipe surfaces and
sediments in the distribution system. Continuing research into the
underlying causes of coliform persistence/colonization in distribution
systems is needed. Until our knowledge of these conditions becomes
more complete, we must rely on educated guesses to formulate
practical approaches to remediation and control of coliform
noncompliance problems.
References
1. Lowther, E.D. and Moser, R.H. Detecting and eliminating
coliform growth. In: Technology Conference Proceedings, WQTC-
12, 1984, Advances in Water Analysis and Treatment. American
Water Works Association, Denver, Colorado, 1985. pp. 323-336.
2. Hudson, L.D., Hankins, J.W., and Battaglia, M. Coliforms in a
water distribution system: a remedial approach J AWWA
75(11):564-568,1983. '
Wierehga, J.T. Recovery of coliforms in the presence of a free
chlorine residual. J. AWWA. 77(11) 83-88,1985.
LeChevallier, M.W., Babcock, T.M., and Lee, R.G. Examination
and characterization of distribution system biofilms Appl
Environ. Microbiol. 53:2714-2724,1987.
Martin, R.S., Gates, W.H., Tobin, R.S., Grantham, D., Sumarah
R., Wolfe, P., and Forestall, P. Factors affecting coliform bacteria
growth in distribution systems. J. AWWA. 74(l):34-37,1982.
Craun, G. Surface water supplies and health. J, AWWA. 80(1) 40-
52,1988.
7. Tracy, H.W., Camarena, V.M., and Wing, F. Coliform persistence
in highly chlorinated waters. J. AWWA. 58:1151-1159,1966.
3.
4.
5.
6.
219
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8. Milwaukee Journal. Summerfest: don't drink the water.
Milwaukee, WI. July 3,1985.
9. Centers for Disease Control. Detection of elevated le'vels of
coliform bacteria in a public water supply - Connecticut.
Morbidity and Mortality Weekly Report. 34(10): 142-144. March
15,1985.
10. Fairfax Journal, The. Consumer group critical of Alexandria
Tapwater. Fairfax, Virginia. May 8,1986.
11. Rochester Democrat and Chronicle. A puzzling bacteria invasion
in water supply. Rochester, NY. April 20,1986.
12. Akron Beacon Journal. Tainted water found in two areas. Akron,
Ohio. August 10,1983.
13. American Water Works Association. Health Department issues
boil water order in Miami Beach. AWWA Mainstream. 27(5):1,5,
1983.
14. Earnhardt, K.B., Jr. Chlorine-resistant coliform: the Muncie,
Indiana experience. In: Technology Conference Proceedings,
WQTC-8, 1980, Advances in Laboratory Techniques for Quality
Control, American Water Works Association, Denver, Colorado.
pp. 371-376,1981.
15. McFeters, G.A., Kippin, J.S., and LeChevallier, M.W. Injured
coliforms in drinking water. Appl. Environ. Microbiol. 51:1-5,
1986.
16. St. Louis Post-Dispatch. Florissant hard-boil spurs soft drink
sales. St. Louis, MO. March 6,1984.
17. O'Brien and Gere, Consulting Engineers. Report on water quality
study of Asheville, North Carolina. O'Brien and Gere, Consulting
Engineers, Charlotte, NC. June, 1971.
18. Clark, T.F. Chlorine tolerant bacteria in a water distribution
system. Public Works, pp. 65-67,1984.
19. Lakewood Post-Journal. Expect more turbidity as water system
fixed. Lakewood, NY. July 13,1982.
20. Times-Picayune. Water supply of Eden Isles cited by state. Eden
Isles, LA. October 28,1983.
21. Seidler, R.J., Morrow, J.E., and Bagley, S.T. Klebsiella in
drinking water emanating from redwood tanks. Appl. Environ.
Microbiol. 33:893-905,1977.
22. Reilly, J.K. and Kippin, J.S. Relationship of bacterial counts with
turbidity and free chlorine in two distribution systems. J.
AWWA. 75(6):309-312,1983.
220
-------
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
Camper, A.K., LeChevallier, M.W., Broadaway, S.C., and
McFeters, G.A. Growth and persistence of pathogens on granular
activated carbon filters. Appl. Environ. Microbiol. 50(6): 1378-
1382,1985.
Camper, A.K., Broadaway, S.C., LeChevallier, M.W., and
McFeters, G.A. Operational variables and the release of colonized
granular activated carbon particles in drinking water. J. AWWA
79(5):70-74,1987.
Levy, R.V., Hart, F.L., and Cheetham, R.D. Occurrence and
public health significance of invertebrates. J. AWWA 78(9V105-
110,1986.
Chang, S.L., Berg, G., Clarke, N.A., and Kabler, P.W. Survival
and protection against chlorination of human enteric pathogens
in free-living nematodes isolated from water supplies Jour Troo
Med.Hyg. 9:136-142,1960.
Silverman, G.S., Nagy, L.A., and Olson, B.H. Variations in
particulate matter, algae, and bacteria in an uncovered finished
drinking water reservoir. J. AWWA. 75(4):191-195,1983.
;Nagy, L.A., Kelly, A.J., Thun, M.A., and Olson, B.H. Biofilm
composition, formation and control in the Los Angeles aquaduct
system. In: Technology Conference Proceedings, WQTC-10,1982,
The Laboratory's Role in Water Quality, American Water Works
Association, Denver, Colorado, pp. 141-160,1983.
Donlan, R.M. and Pipes, W.O. Pipewall biofilm in drinking water
mains. In: Technology. Conference Proceedings, WQTC-14, 1986,
Advances in Water Analysis and Treatment, American Water
Works Association, Denver, Colorado, pp. 637-660,1987.
Tuovinen, O.H,, Button, K.S., Vuorinen, A., Carlson, L., Mair,
D.M., and Yut, L.A. Bacterial chemical and mineralogical
characteristics of tubercles in distribution pipelines J AWWA
72(11):626-635,1980.
Allen, M.J. and Geldreich, E.E. Distribution line sediments and
bacterial regrowth. In: Technology Conference Proceedings
WQTC-5- 1977, Water Quality in the Distribution System,'
American Water Works Association, Denver, Colorado, 1978.
Allen, M.J., Taylor, R.H., and Geldreich, E.E. The occurrence of
microorganisms in water main encrustations. J. AWWA, 72:616-
1980.
Ridgway, H.F. and Olson, B.H. Scanning electron microscopy
evidence for bacterial colonization of drinking water distribution
systems. Appl. Environ. Microbiol. 41(l):274-287,1981
221
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34. Van der Kooij, D., Visser, A., and Hijnen, W.A.M. Determining
the concentration of easily assimilable organic carbon in
drinking water. J. AWWA. 74(10):54O-545,1982.
35. LeChevallier, M.W., Lee, R.G., and Moser, R. Disinfection of
bacterial biofilms. American Water Works Service Company,
Report Authorization 270, Formula 40-00. Belleville, IL, 1987.
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V. Sampling and Analytical Methods
Environmental Sampling for Waterborne Pathogens:
Overview of Methods, Application Limitations and
Data Interpretation
by:Joan B. Rose
Department of Environmental
and Occupational Health
College of Public Health
University of South Florida
Tampa, FL 33612
(813) 974-3623
Introduction
Two key events that trigger the development of detection methods
for pathogenic microorganisms in water are the recognition of an agent
as a human pathogen and the waterborne transmission of the agent.
Although Cryptosporidium was first described in 1907 (1) and
Giardia, in 1859 (2), the recognition of waterborne cryptosporidiosis
and giardiasis in 1985 and 1965 came only after they had formally
been established as human pathogens in 1980 and 1960, respectively.
Similarly, Yersinia enterocolitica was described in 1923 and recognized
as a pathogen in 1963, but the first waterborne outbreak was not
documented in the United States until 1980 (3). Campylobacter was
initially identified and described as a human pathogen in 1971, and by
1978 a waterborne outbreak had been identified (Table 5.1.1).
Many of the enteric viruses have long been recognized as causing
waterborne disease, and methods have been developed and used for
their detection in wastewater since 1945 and in drinking water, since
1960 (5). In 1913, Sawyer first identified the polio virus in human
stools (6), and during the 1940s much debate ensued regarding the
potential for waterborne transmission of this and related
enteroviruses, such as echoviruses and coxsackieviruses. There was
convincing evidence that the hepatitis A virus (HAV) was transmitted
through water, but methods for its detection in the environment were
not developed until the late 1970s. The rotavirus and Norwalk virus
were identified in 1973 in human stools and in 1968 during an
outbreak, respectively (7). Methods for rotavirus detection in the
environment were under development by 1978 (8), concurrently with
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Table 5.1.1. Events Leading to the Recognition of Waterborne Disease for
Selected Pathogens
Waterborne outbreaks
Recognized as
Microorganism
First Total
First described human pathogen documented number3
Cryptosporidium
Giardia
Campylobacter
Yersinia enterocolitica
1907
1859
1971
1923
1980
1960
1971
1960
1985
1965
1978
1980
2b
95
11
, 2
* In the United States up to 1985 (4).
b In the United States up to 1987.
the first report of a suspected waterborne outbreak (9). Methods for
cultivating the Norwalk virus in the laboratory are not currently
available. In addition, there are over 100 different types of enteric
viruses that may be present in wastewater such as adenoviruses and
caliciviruses that are not well studied.
The causes of most diarrhea in the clinical laboratory are not
identified (10). Interestingly, identifiable agents in waterborne
outbreaks follow a similar pattern.
A number of reviews have compiled data on outbreaks in Scotland,
Sweden, the United Kingdom, and the United States (Table 5.1.2
[3,11-13]). The reports covered as few as 9 years in Sweden to as many
as 65 years in the United States. The total number of outbreaks
divided by the years of review gave a range of 0.7 to 3.5 outbreaks per
ye'ar in Europe, while the United States averaged 24 outbreaks per
year. In 23 to 59 percent of the outbreaks no causative agent was
identified, perhaps because the causative agent had not yet been
recognized as a human pathogen: Such was the case with
Cryptosporidium prior to 1980. Another problem may be inadequate
investigation of the outbreak due to inappropriate sampling or
inadequate analysis.
Table 5.1.2. Reported Outbreaks of Waterborne Disease up to 1987 (2,11-13)
, . Outbreaks " Unknown
Location years of Reporting Total Parasites Viruses Bacteria Etiology
Scotland 1955-1987 57 4 '4 4 16
Sweden
United Kingdom
United States
1975-1984
1937-1986
1920-1985
32
34
1587
1
,3
96
2
,a
97
11 ,
14
622 ,
19
8
699
' The eight unknown were classified as possibly viral.
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Bennett et al. (14) in a recent review of disease in the United States
summarized the data for 1985. They collected data on the total
incidence of enteric infections in the United States for the year 1985
and estimated the number of cases acquired through water as a
proportion of overall morbidity. They estimated 940,000 cases of
waterborne disease with 95 percent due to Campylobacter, Norwalk
virus, enteric Escherichia coli, Giardia, and Salmonella (Table 5.1.3).
For comparison only 1,561 cases of the disease occurred in the 16
documented waterborne outbreaks in the same year (15).
Table 5.1.3. Estimated Cases of Waterborne Disease in the United
States for 1985
Enteric Infections
Felt to be
Microorganism
Campylobacter
E. coli
Salmonella
Giardia
Norwalk virus
No. of Enteric
Infections from
all Cases (14)
3,100,000
200,000
2,000,000
120,000
6,000,000
i ransmittea oy
Water (14)
Percent
15
75
3
60
5
Cases
315,000
150,000
60,000
72,000
300,000
Cases Reported from
Waterborne
Outbreaks (15)
169
a
60
741
a
a None reported in 1985.
Current documentation of outbreaks does not begin to identify the
impact of waterborne disease in the United States. It is essential that
methods are developed and utilized for evaluating pathogen
occurrence in water as well as in humans. Such methods will provide
valuable information on potential risks of waterborne disease and the
occurrence of various waterborne agents, particularly during
catastrophic conditions (floods, treatment failures, contamination'
events, and outbreaks), which can then be used to implement
strategies to protect against waterborne disease transmission.
Overview of Methods to Recover Pathogens from Water
Filtration procedures have been developed to recover and
concentrate viruses and parasites from large volumes of water (100 to
1,000 L) and bacteria from smaller volumes (1 L). These procedures are
routinely followed by microscopic procedures, tissue culture
techniques, and bacteriological culture methods for the detection of the
microorganisms. Finally, more sophisticated techniques (immuno-
logical, biochemical) may be needed to specifically identify a pathogen.
Early attempts in the 1940s to recover viruses from water samples
involved the use of gauze pads; it was not until between 1976 and 1978
that electronegative and electropositive filters were designed
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specifically to recover enteric viruses from water (16). With a field
method and tissue culture techniques established for the enteroviruses
(polioviruses, coxsackieviruses, and echoviruses), documentation of
published reports on viral contamination in drinking water
dramatically increased (5). Specialized techniques for the detection of
rotavirus and HAV in cell culture were developed, and the first
updated isolations of these viruses from drinking water soon followed
between 1982 and 1984 (17-19).
The methods to recover enteric protozoa began with Giardia
during the first waterborne outbreak in 1965 (20). However the yarn-
wound filters which could be used readily to sample large volumes of
water were not introduced until the mid 1970s (21). Sauch, in 1985,
made another significant contribution when she developed a technique
that utilized a specific antibody to the Giardia cyst on membrane
filters for detection with fluorescence microscopy (22). By 1985 a
similar method had been developed for detecting Cryptosporidium
oocysts in water (23,24).
Culture methods developed for bacteria in the clinical laboratory,
using selective and differential media for growth and identification,
were used similarly for environmental samples. The techniques for
recovering these organisms from water were developed by individual
groups of investigators during specialized surveys (5,25,26). There has
been no single approach or strategy to evaluate or to optimize recovery
efficiencies for the bacteria (including filter type, sample volumes,
resuscitation procedures). Generally, the 100-mL sample volume used
for coliform analysis was deemed not to be sufficient; 1,000 mL
appeared more appropriate and could be collected with various types of
filtration systems (membrane filters, depth filters). Recent
developments have utilized naldixic acid and acridine orange to
microscopically enumerate, by an epifluorescence procedure, bacteria
in water samples which may be viable but nonculturable (27).
Current Status, Limitations, and Future of Methods for Detecting
Microorganisms in Water
The detection methods for viruses, parasites, and bacteria in water
samples are summarized in Table 5.1.4. Two major problems are
common to all the methods. The first deals with recovery efficiencies,
which may range from 1 percent to 70 percent, depending on the water
conditions and quality. Virus and parasite recovery may be good from
waters of drinking water quality; however, disinfection of the water
may lead to greatly underestimating the stressed bacteria that are no
longer cultivatible but still capable of initiating infections (28). The
second problem is that the time required to process the sample and
obtain the results could range from 2 to 80 days.
Within each group of microorganisms there are also unique
problems. Although the enteric viruses often are referred to
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Table 5.1.4. Routine Methods for Recovery and Detection of Viruses, Parasites,
and Bacteria in Water
Sample
Microorganism Filter Type Recovery Processing Detection
Viruses
Parasites
Bacteria
Electronegative
or electro-
positive car-
tridge filters
Yarn-wound
cartridge filters
Membrane
filters
Eluted with beef
extract
Washed from
filter
Placed in
enrichment
media
Organic
precipitation
Centrifugation
density
gradients
Selective media
Tissue culture
Microscopic
immuno-
fluorescence
Biochemical
testing,
serotyping
collectively, they form a very diverse group of microorganisms. There
are over 100 types of viruses from humans that could be found in
sewage; however, methods have been developed and are generally
utilized to recover and identify only a few of the enteroviruses. Tissue
culture systems are not available for the Norwalk-like viruses or for
many of the adenoviruses, and are not routinely used for HAV or the
rotaviruses. Therefore, little is known about the occurrence of these
important viral agents and their potential for causing waterborne
disease.
Cryptosporidium and Giardia can be specifically identified using
monoclonal antibodies. However, the viability of the oocysts and cysts
is unknown in environmental samples. This lack of knowledge leads to
ambiguity in interpreting their health significance, particularly when
the organisms are identified in treated waters. (See Table 5.1.5 for a
summary of methods limitations.)
Table 5.1.5. Limitations of the Methods for Viruses,
Parasites, and Bacteria
Group of
Microorganism Major Limitation
Viruses Methods are not available for many
important viruses (Norwalk).
Parasites Viability of oocysts and cysts is unknown.
Bacteria Methods are not quantitative.
Common to all Recoveries can be poor.
Quantitative results are normally obtained for viruses and
parasites, which are particularly beneficial when evaluating the
potential health risks. Qualitative data are most often reported for the
bacteria if a preenrichment step is utilized. A most probable number
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approach can also be used to estimate bacterial concentrations, but it
requires a tremendous amount of work. Regardless of approach,
without a preenrichment step, the bacteria may not be detected or may
be greatly underestimated. :. ' • "
Methods development is generally a slow process, but new
technology is being evaluated for detecting waterborne pathogens.
Approaches using molecular biology, such as gene probes, have been
utilized in the diagnosis of disease and are gradually being tested.for
environmental samples (29-31). Gene probes are pieces of nucleic acids
(DNA or RNA) which can bind to specific microorganisms and be
detected with radioactive or enzyme-linked markers. Gene «probe
methods are rapid and specific and could be used for pathogens such as
Norwalk virus. Although it will take many years to, develop and define
the limits of appropriate methods for environmental samples, gene
probes will be part of the future of available diagnostic tests.
Investigations of Waterborne Outbreaks
In most waterborne outbreak investigations, the etiological agent
is not recovered from the suspected water source/Often environmental
microbiologists are not a part of the investigative team; appropriate
equipment is not available; or samples are collected too late in the
outbreak, missing the contamination event. The few investigations
that have reported detecting the suspected pathogen in the water have
contributed greatly to the documentation and evaluation of the
outbreak.
During an outbreak of gastroenteritis in Georgetown, Texas
(population 13,000), virus samples were collected (17). The outbreak
began June 1, cases peaked June 13, and samples were collected from
the sewage and ground water supply on June 19. The epidemiological
investigation reported a 79 percent attack rate, but no pathogen was
identified in the fecal specimens. However, serological data
demonstrated a recent exposure to the coxsackie B3 virus in five of
seven individuals. Drinking, water samples were positive for coxsackie
B3 virus, coxsackie B2 virus, and HAV (Table 5.1.6).
Table 5.1.6. Viral Contamination of the Ground Water in the
Georgetown Outbreak
No. of Positive -. -
Samples/No, of •
Samples Collected Type of Sample Viruses Average pfu/100 l_a
5/5
2/3
1/3
Sewage CBS, CB2, HAV
4,580
Well
Tap water
Rotavirus
CB3, CB2, HAV
CBS
5.3
0.33
« Plague-forming units for coxsackieviruses (CB2, CB3).
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, Although the outbreak had officially ended June 23, by July 15, 29
cases of hepatitis were reported. Unfortunately, because the methods
for detecting HAV from water required weeks to obtain results,
prophylactic treatment of individuals in the population at risk was not
possible.
, - Data on the environmental occurrence of the pathogens and attack
rates have also been evaluated for waterborne outbreaks of giardiasis
and cryptosporidiosis (Table 5,1.7) (32). Of five outbreaks investigated,
Giardia cyst levels ranged from 0.87 to 21/100 L, and attack rates
ranged from 0.5 to 16 percent. Generally the higher cyst levels
corresponded with the higher attack rates. Cryptosporidium oocyst
levels were much higher (63/100 L) during the outbreak in.Carrollton,
Georgia (33), which resulted in a 40 percent attack rate and affected
13,000 people. Surveys during periods when outbreaks were not
occurring have reported that an average of 17 percent of drinking
water samples contained Giardia cysts or Cryptosporidium oocysts at
levels of 0.19 and 0.11/100 L, respectively (Table 5.1.7). This low
contamination level may be contributing to sporadic cases of disease in
the community, but serological surveys are necessary to confirm this
hypothesis, as these cases are not being detected through the current
waterborne disease surveillance system.
Table 5.1.7. Cryptosporidium Oocyst and Giardia Cyst Contamination in Drinking
Water during Waterborne Outbreaks
Giardia
Cysts/100 L (Mean Levels in Water)
Cryptosporidium
Oocysts/100 L (Mean Levels in Water)
From Five
Outbreaks3
Survey of Other
Potable Waters
Carrollton Outbreak15
Survey of Other
Potable Waters
11
0.19
63
0.11 •
_aMean. , , •
b Mean of nine samples.
Recommendations for Environmental Sampling
The collection of .microbial samples is one of the most important
steps in obtaining accurate results, particularly during an outbreak.
Considerations for sampling include proper collection procedures,
timing of sample collection, sampling sites, replicate samples, sample
volumes, proper storage, and handling and transportation of the
samples. During an outbreak situation, it is imperative that
environmental samples are collected as soon as an outbreak is
suspected (Figure 5.1.1).
The time of recognition of an outbreak depends on the incubation
period of the disease from a specific organism. Outbreaks having short
incubation periods are more likely to be reported and investigated
quickly because they are easier to recognize. Also, in the event of a
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Exposure
Disease
Incubation period variable
depends on type of
organism
Outbreak Recognition
Lag Time variable depends
on recognition and
response time
Organism's Survival and Persistence
Entry of
pathogen
Time period where water
sampling may be
productive
Time point where water is
generally sampled
Figure 5.1.1. Events In an outbreak.
short incubation period, investigators will probably be able to respond
to the outbreak faster, as the organism is more likely to still be present
in the water system. Lag time, however, is dependent primarily on
human factors, such as interest in the investigation, rather than the
length of the incubation period.
Many bacteria have a relatively short incubation period (2 to 5
days). The incubation period of many protozoans and viruses can be
much longer than for bacteria, i.e., 1 or 6 weeks for Cryptosporidium
and HAV, respectively. The amount of contamination and the lag time
for recognition will influence the ability to recover and detect the
bacteria, depending on their survival characteristics and
environmental transport patterns. Bacteria can die off within a few
days after the contamination event; and although viruses, and
particularly parasites, have a greater ability to survive and remain in
a system, even an immediate response may miss the exposure event.
Because it may be impossible to sample the water during the
contamination event, it is very important to take enough samples at
locations where pathogen residuals may be identified. If a
contaminated well is suspected, then a minimum of three replicate
samples per well should be collected. Seven to 10 samples should be
collected from a suspected distribution system, particularly at dead-
end mains where microorganisms may accumulate. In some cases,
investigators may be interested in sampling the sewage to further
document infection in the population and to demonstrate at a later
time that the outbreak is over.
230
-------
Samples for viruses, parasites (minimum volume of 400 L), and
bacteria (2-L volumes) all should be collected, if possible, unless there
is some immediate indication that one type of pathogen is responsible.
Sample analysis for viruses can wait if the filter concentrate is frozen.
Parasite samples may be stored at 4°C in 3.7 percent formaldehyde.
Bacterial samples should be assayed within 24 hours and stored at 4°C
until that time. All filters should be stored at 4°C until processed.
Finally, any laboratory can develop an action strategy and obtain
the proper equipment and training for environmental sampling of
microorganisms. This strategy could routinely be included in any
emergency plan or specific study.
Once samples are collected, they can be shipped to other
laboratories with the necessary capabilities for analysis.
Summary
•As the role of contaminated water in the transmission of viruses,
parasites, and bacteria was being elucidated, methods were under
development for the detection of these microorganisms in water.
Filtration methods have been developed for both the viruses and
parasites (volumes of 400 L). The samples are then assayed on cell
culture and using microscopic immunofluorescence techniques for
viruses and parasites, respectively. For bacteria such as
Campylobacter, filtration methods are also utilized (1-L samples),
followed by enrichment and selective culture techniques.
At issue with any of these methodologies is the effect of water
quality on the recovery efficiencies. Other limitations include 1) lack of
cell culture techniques for viruses such as the Norwalk virus, 2)
inability to assess viability of the protozoa and 3) the detection, of
noncultivatable and stressed bacteria.
Recommendations for environmental sampling include:
• Immediate collection of the water samples upon recognition of
an outbreak
• Collection of a minimum of 400 L for viruses and parasites and
1 L for bacteria
• Collection of 3 replicate samples of an individual well or 7 to 10
samples of treated water (distribution system and dead-end
mains)
• Collection and assay for a broad range of microorganisms
• Obtaining proper equipment and training
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References
1. Tyzzer, E.E. A sporozoan found in the peptic glands of the common
mouse. Proc. Soc. Exp. Biol. Med, 5:12-13,1907.
2. Feachem, R.G., Bradley, D.H., Garellick, H., and Mara, D.D.
Sanitation and Disease Health Aspects Excreta and Wastewater
Management. John Wiley and Sons, New York, NY, 1983.
3. Craun, G.F. Waterborne diseases in the United State.s. CRC.Press,
Boca Raton, FL, 1986.
4. Craun, G.F. Surface water supplies and health. J. Amer. Water
Works Assoc., 80:40-52, February, 1988. '
5. Rose, J.B. and C.P. Gerba. A review of viruses in drinking water.
In: Current Practices in Environmental Engineering. Vol. 2. ppl
119-143,1986.
6. Sawyer, W.A. An epidemiological study of poliomyelitis. Amer. J.
Trap. Dis. Preu. Med. 3:164-175,1915.
7. Gerba, C.P., Rose, J.B. and Singh, S.N. Waterborne gastroenteritis
and viral hepatitis. CRC Critical Rev. in Environ. Control. 15:213-
236,1985.
8. Ramia, S. and Sattar, S.A.. Concentration of seeded Simian
rotavirus SA-11 from potable waters by using talc-celite layers and
hydroextraction. Appl. Environ. Microbiol.,39:493,1980.
9., Lycke, E., Bloomberg, J., Berg, G., Eriksson, A., and Madsen, L.
Epidemic acute diarrhoea in adults associated with infantile
gastroenteritis. Lancet. 2:1056,1978.
10. Flewett, T.H., Beards, G.M. Brown, D.W.G- and Sanders, R,C. The
Diagnostic Gap in Diarrhoea Viruses. G. Bock and J. Whelan
(eds.), John Wiley and Sons Ltd., Chichester, U.K., 1987. PP 238-
249,1987.
11. Anderson, Y., and Stenstrom, T.A. Waterborne outbreaks in
Sweden-causes and etiology. Water Sci. Tech. 18:185-190,1986.
12. Benton, C., Forbes, G.I. Paterson, G.M., Sharp, J.C.M. The
incidence of waterborne and water associated disease in Scotland
from 1945-1987. In: Developments at the 14th International
Conference on Water Pollution Research, Brighton, England, U.S.
EPA, Cinncinati, OH. p. 157,1989.
13. Galbraith, N.S., Barrett, N.J., and Stanwell-Smith, R- Water and
disease after Croydon: a review of waterborne and water-
associated disease in the UK, 1937-86. J. Instlt:, of Water and
Environmental Management. 1:7-21,1987.
232
-------
14. Bennett, J.V., Homberg, S.D., Rogers, M.F., and Solomon, S.L.
Infectious and Parasitic Diseases. Amer. J. of Preventative
Medicine. 3:102-114,1987.
15. Water-related disease outbreaks. Morbidity Mortality Weekly
Report. 37:15-24,1988.
16. Gerba, C.P. and Goyal, S.M. Method in Environmental Virology.
Gerba, C.P. (ed). Marcel Dekker, Inc., New York, 1982.
17. Hejka'l, T.W., Keswick, B., LaBelle, R.L., Gerba, C.P., Sanchez, Y.,
Dressman, G., Hafkin, and Melnick, J.L. Viruses in a community
. watersupply associated with an outbreak of gatroenteritis and
infectious hepatitis. J. Amer. Water Works Assoc. 74:318-321,
1982.
18. Keswick, B.H., Gerba, C.P., DuPont, H.L., and J.B. Rose.
Occurrence of viruses in treated drinking water. Appl. Environ.
Microbiol. 47(6): 1290-1294,1984.
19. Sobsey, M.D., Oglesbee, S.E., and Waite, D.A. Detection of
hepatitis A virus (HAV) in drinking water. In: G.N. Vyas,
Dienstag, J.L., and Hoofnagle, J.H. (ed.), Viral Hepatitis and Liver
Disease. Grune and Stratton, Inc., 1984.
20. Moore, G.T., Cross, W.M., McGuire, D., Mollohan, C.S., Gleason,
N.W., Healy, G.R., and Newton, L.H. Epidemic giardiasis at a ski
resort. N. Engl. J. Med. 281:402-407,1969.
21. Jakubowski, W. and J.C. Hoff. Waterborne transmission of
giardiasis. Report No. EPA-600/9-001. U.S. Environmental
Protection Agency. Cincinnati, OH, pp. 104-126,1979.
22. Sauch, J.F. Use of immunofluorescence and phase-contrast
microscopy for detection and identification of Giardia cysts in
water samples. Appl. Environ. Microbiol. 50(6): 1434-1438.1985.
23. Musical, C.E., Arrowood, M.J., Sterling, C.R., and Gerba, C.P.
Detection of Cryptosporidium in water using polypropylene
cartridge filters. Appl. Environ. Microbiol. 53:687-692,1987.
24. Rose, J.B., Kayed, D., Madore, M.S., Gerba, C.P., Arrowood, M.J.,
and Sterling, C.R. Methods for the recovery of Giardia and
Cryptosporidium from environmental waters and their
comparative occurrence. In: P. Wallis and B. Hammond (ed),
Advances in Giardia Research. Univ. Calgary Press, pp. 205-209,
1988.
25. Vogt, R.L., Siurs, H.E., Barrett, R., Feldman, R.A., Dickinson,
R.J., and Witherell, L. Campylobacter enteritis associated with
contaminated water. Ann. Intern. Med. 96:292,1982.
233
-------
26. Schiemann, D.A. Isolation of Yersinia enterocolitica from surface
and well waters in Ontario. Canadian Jour. ofMicrobio. 24:1048,
1978.
27. Roszak, D.B., and Colwell, R.R. Metabolic activity of bacterial cells
enumerated by direct viable count. Appl. Environ. Microb.
53:2889-2983,1987.
28. LeChavallier, M.W., Shiemann, D.A., and McFeters, G.A. Factors
contributing to the reduced invasiveness of chlorine-injured
Yersinia enterocolitica. Appl. and Environ. Microbiol. 53:1358,
1987.
29. Richardson, K.J., Margolin, A.B., and Gerba, C.P. A novel method
for liberating viral nucleic acid for assay of water samples with
cDNA probes. J. Virol. Methods. 2:13-21,1988.
30. Somerville, C.C., Knight, I.T., Straube, W.L., and Colwell, R.R.
Simple, rapid method for direct isolation of nucleic acids from
aquatic environments. Appl. Environ. Microb. 55:548-554,1989.
31. Steffan, R.J. and Atlas, R.M. DNA amplification to enhance
detection of genetically engineered bacteria in environmental
samples. Appl. Environ. Microb. 54:2185-2191,1988.
32. Rose, J.B. Cryptosporidium in water; risk of protozoan waterborne
transmission. Report for American Association of Advancement of
Science/Environmental Protection Agency Science and
Engineering Fellow. 1988.
33. Hayes, E.B., Matte, T.D., O'Brien, T.R., McKinley, T.W., Logsdon,
G.S., Rose, J.B., Ungar, B.L.P., Word, D.M., Pinsky, P.P.,
Cummings, M.L., Wilson, M.A., Long, E.G., Hurwitz, E.S., and
Juranek, D.D. Contamination of a conventionally treated filtered
public water supply by Cryptosporidium associated with a large
community outbreak of cryptosporidiosis. New England Journ.
Med. 320:1372-1376,1989.
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Bacteriologic Analysis of Clinical Specimens in
Waterborne Disease Outbreaks
by: Julie Parsonnet, M.D.
Division of Geographic Medicine
HRP Building, Room 109
Stanford University
Stanford, California 94062
(415)725-4561
This chapter will describe bacteriologic laboratory support for
clinical samples, focusing on bacterial enteric outbreak investigations.
It will also briefly discuss norienteric waterborne diseases.
In the years 1971 to 1985, 61 waterborne disease outbreaks caused
by bacterial agents were reported to the Centers for Disease Control
(CDC). Bacteria implicated included Shigella, Campylobacter, non-
typhoidal Salmonella, Salmonella typhi, Vibrio cholerae, and Yersinia
enterocolitica. To determine the agents in these outbreaks, a
combination of basic epidemiologic and microbiologic principles were
applied.
Identifying Etiologic Agents
Human Specimens
The primary principle in the microbiologic investigation of an
outbreak is that human pathogens are found in people. One will
sometimes find suspicious organisms in environmental samples, which
suggest causation, but the key to identifying an etiologic agent is to
look in the patient rather than in the environment. For instance,
Yersinia species are not infrequently found in environmental samples
but one cannot assume that they have caused diarrheal disease unless
they are also found in the stool specimens of the ill persons.
In the bacteriologic analysis of human specimens three outcomes
are possible. First, quite commonly one may find nothing. Second, one
may find a known pathogen, such as Salmonella or Shigella. In that
case, if the clinical illness seen in-the outbreak is typical of that caused
by the organism, the etiologic agent has been identified. A third
outcome, which occurs fairly frequently, however, is that a suspicious
organism that is not necessarily a pathogen (e.g., Yersinia
enterocolitica), is identified. In such instances, the organism must be
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statistically more common in specimens from ill persons (cases) than
from well persons (controls) to be considered the outbreak etiologic
agent. An organism equally common in both cases and asymptomatic
controls must be presumed to be unassociated with the outbreak
illness.
Environmental Samples
Of secondary importance in waterborne outbreak investigations is
the culture of environmental samples. In general, finding a known
pathogen or a possible pathogen in water is not sufficient to
incriminate the organism as the cause of the outbreak. The agent has
to be isolated from human specimens, as well. Thus, if Yersinia is
found in a water source but not in human stool, it cannot be assumed
that the organism caused the illness. There are some exceptions to this
rule; diseases that have very specific clinical presentations may be
diagnosed from environmental samples without confirmatory human
specimens. Examples of agents that can cause such pathognomonic
symptoms are Staphylococcus aureus enterotoxin and Clostridium
botulinum toxin. If either of these agents are found in
epidemiologically suspect food vehicles, investigators would assume
that they caused the associated specific and dramatic human illnesses,
even in the absence of confirmatory human specimens.
In summary, the three routes to identifying the etiologic agent of
an outbreak are:
1. A known pathogen (e.g., Shigella) that causes symptoms
compatible with the outbreak illness is found in human specimens.
2. A possible pathogen (e.g., Yersinia) is found statistically more
commonly in specimens from ill persons than in those from well
persons.
3. A known pathogen that causes pathognomonic symptoms is found
in the implicated environmental source.
Specimen Collection
Because clinical specimens are the key to identifying the etiologic
agent of an outbreak, the most important part of the laboratory
investigation is proper specimen collection. In an outbreak
investigation, regardless of outbreak size, 10 specimens from ill
persons and 10 specimens from well persons usually suffice. Upon
receiving a fresh stool sample, an investigator should immediately
place two sterile swabs of stool in Gary-Blair or similar transport
medium and refrigerate them along with the remainder of the whole
stool sample. The swabs can remain refrigerated for up to 48 hours and
the whole stool for 4 hours before delivery to the laboratory; if longer
delays are anticipated, samples should be frozen, preferably at -70°F,
although -20°F will suffice. This protocol eliminates frequent freezing
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and thawing of the whole stool sample, minimizing compromise of the
specimens.
If the clinical illness suggests a viral or parasitic etiology, whole
stool specimens will need to be divided into aliquots so they can be
handled appropriately. Our current practice at CDC is to ask that the
aliquots held for viral examination be refrigerated indefinitely
without freezing; the aliquot for parasitic analysis should be
transferred to an appropriate parasitic transport medium.
The swabs should be used for routine bacterial analysis; the whole
stool can be reserved for necessary special studies. Stools from ill
persons should be analyzed first. If those cultures yield no likely agent,
the specimens from well persons need riot be tested.
Bacteriologic Study Techniques
The microbiologic studies performed in an outbreak investigation
should be tailored to the clinical illness and the suspected organism
involved. The analyses themselves can be divided into three
categories:
1. Bacterial culture and the use of epidemiologic markers
2. Pathogenicity assays
3. Serologic tests for antibacterial antibodies.
Tests from one or all of these methods may be applied to speciir jns
from any given outbreak (see Table 5.2.1).
Table 5.2.1. Bacteriologic Techniques in the Investigation
of a Waterborne Outbreak
1. Bacterial cultural and epidemiologic markers
a. Routine culture and biochemical identification
b. Serogroup/serotype
c. Antimicrobial resistance pattern
d. Plasmid, profile
••'"•• •-'. - e. , Isoenzyme type and/or biotype
2. Pathogenicity assays
a. Toxin assays
b. Adhesiveness, invasiveness assays ._. •
3. Serologic tests '
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Bacterial Culture and Epidemiologic Markers
Routine Culture
Routine bacterial culture of stool is the most common technique
used to detect an etiologic agent in enteric diseases and is essential to
any investigation. Culture techniques must be designed to include all
suspect organisms. In many outbreaks, however, culture alone is not
sufficient to trace the cases and the source. Tests which more
specifically identify the strain, such as serogrouping or serotyping, are
required.
Serogrouping and Serotyping
Serogrouping classifies the enterobacteria using one antigen,
usually the heat-stable somatic O antigen. Serotyping identifies ah
organism more specifically by defining many antigens on the bacterial
surface. As such, it requires a more complex collection of antisera and
is usually restricted to laboratories in major medical centers, state
public health departments, and reference centers such as the Centers
for Disease Control (CDC). Serotyping is helpful for classifying many
organisms, Salmonella chief among them.
Researchers have thus far identified approximately 2,000 different
serotypes of Salmonella with disparate environmental and clinical
prevalences. A steady increase in the enteriditis serotype was
recognized in 1984, when investigations by state health departments
in conjunction with CDC, U.S. Department of Agriculture, and U.S.
Food and Drug Administration identified intact grade A shell eggs as
the primary source of Salmonella enteriditis. This discovery is
resulting in major changes in the egg industry (1,2). Without
serotyping, the health risk would never have been uncovered.
Shigella strains demonstrate another example of the usefulness of
serotyping. Shigella are classified into four serogroups, A through D;
each of the first three groups is then subdivided into many serotypes.
Serotype Al, the Shiga bacillus, is quite rare in the United States but
causes severe, often life-threatening, illness. Serotyping by the state
public health laboratories recently enabled researchers to identify an
outbreak of serotype Al in tourists and to identify the source as
Cancun, Mexico. This strain is still being monitored using state
reports of Shigella serotypes (3).
Escherichia coli is another organism for which serotyping has been
incalculably helpful. Before the advent of serotyping, E. coli were
usually considered to be nonpathogenic to the bowel. Now, it is
apparent that certain serotypes are responsible for a wide diversity of
gastrointestinal illness, from chronic diarrhea in children to severe
hemorrhagic colitis and hemolytic uremic syndrome caused by
verotoxigenic strains such as E. coli 0157:H7.
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Antimicrobial Sensitivity
Antimicrobial sensitivity profiles can also be used to
epidemiologically trace strains associated with an outbreak. Although
organisms can rapidly change their antimicrobial sensitivity through
plasmid exchange, many times the antibiograms remain remarkably
stable. In 1986, a huge outbreak of Shigella sonnei among a gathering
of the Rainbow family in North Carolina was traceable because the
organism had a unique antibiotic sensitivity (4). Similarly, in 1983,
Salmonella newport was traced from patients to hamburger meat from
antimicrobially treated cattle because of the organism's unusual,
multiresistant antibiotic pattern (5).
Plasmid Profile
A more sophisticated epidemiologie tool is plasmid DNA
electrophoresis. This technique dissects the genetic composition of
bacterial strains and enables researchers to identify likely clones.
Using these molecular techniques, identical E. coli 0157:H7 strains
were identified in patients and in implicated hamburger meat from the
first recognized outbreak of hemorrhagic colitis in 1982 (6). Similarly,
illness caused by Salmonella newport was traced by plasmid profile to
ground beef (5).
Isoenzyme Type and/or Biotype
Other epidemiologie markers are helpful but less frequently used.
Biotyping is used for Vibrio cholera and Yersinia species. Isoenzyme
typing is the preferred strain identification technique for Legionella
pneumophila. Phage typing, frequently used for Staphylococcus
aureus, is also used for Salmonella typhi and more recently for
Salmonella enteritidis.
Pathogenicity Assays
With time, researchers of outbreaks have become increasingly
sophisticated in their understanding of disease mechanisms. This has
aided immeasurably in identifying new pathogens. However, even if
an organism is epidemiologically linked with illness, investigators will
be skeptical about causality unless a mechanism for disease has, or can
be, identified with a pathogenicity assay.
Toxin Assays
One way to use pathogenicity assays is in looking for an illness
mediator, such as a toxin. Toxins are identified in several ways.
Although bioassays are slowly becoming obsolete, they are still used
for identifying some bacterial pathogens. For example, enterotoxins
are often identified using a rabbit ileal loop model, in which bacterial
filtrate is injected into a blind ileal loop. Large volumes of fluid
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secretion indicate the presence of an enterotoxin. If the toxin's effects
can be neutralized by a specific antitoxin, the presence of the toxin is
confirmed.
More frequently, toxicity is assessed in cell culture. For example,
shigatoxin is cytotoxic to Vero cells in culture. This effect is
neutralized when shigatoxin antibody is added to the culture plates.
The shiga-like toxins, or verotoxins, produced by some E. coli strains,
have a similar effect on Vero cells.
DNA hybridization can be performed for any toxin for which the
gene has been cloned, and is now done for cholera toxin, shigatoxin,
shiga-like toxins, heat-labile and heat-stable enterotoxins (7)..
Enzyme-linked immunosorbent assays of culture filtrates have also
been successfully used to detect these toxins (8,9).
Adhesiveness/lnvasiveness Assays
Other useful pathogenicity assays involve detection of inherently
pathogenic traits of the bacteria themselves, such as their ability to
adhere to cells or invade tissue. The methods used for detecting these
traits are similar to those used to find toxin: bioassay, cell culture, and
DNA hybridization techniques.
An example of a bioassay is the now infrequently used Sereny test,
which detects bacterial invasiveness in guinea pig conjunctiva. Cell
cultures and molecular techniques have largely displaced bioassays.
For example, adherence can be detected by observing the effects of
bacteria on HeLa cell cultures. In addition, the genes for adherence
and invasiveness have been sequenced, allowing them to be detected
with DNA hybridization (10).
Serologic Tests
Serum antibody tests are the final method for diagnosing
outbreak-related disease. Serum antibacterial antibodies are most
useful for organisms that are difficult to culture. One of the best
examples of serologic testing for waterborne illness is
immunofluorescent antibody testing for L. pneumophila. Legionella is
often very difficult to isolate by routine sputum cultures. In a serologic
test, a fourfold rise between acute- and convalescent-phase titers
connotes an acute infection.
Investigators also rely on serodiagnosis when an acute outbreak
has passed. In a study of V. cholerae infection along the American Gulf
coast, vibriocidal antibody studies effectively demonstrated the extent
of the problem (11). Investigators are currently evaluating serologic
tests for E. coli- and Sh.igella-associa.ted shigatoxin and shiga-like
toxins.
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Recent Technology
One experimental technique that holds great promise in many
branches of microbiology is the polymerase chain reaction (PCR). PCR
is able to detect organisms of very low frequency in laboratory
specimens. In this technique, a gene or genetic sequence unique to a
specific organism is cloned and a labeled probe is made using this
sequence. DNA primers, sequences complementary to regions flanking
the cloned gene, are then added to a clinical specimen. These primers
bind to the regions adjacent to the cloned sequence and serve to attach
DNA polymerase. By alternately warming and cooling a surrounding
bath, the unique sequence can then be amplified thousands of times.
This enhancement allows probe detection of what otherwise may have
been an undetectably rare gene. Epidemiologic and diagnostic uses for
this emerging technique may be numerous.
Over the last 10 years, many new bacterial pathogens have been
identified using the microbiologic and epidemiologic methods
described above. Among these are E. coli 0157:H7, Campylobacter
jejuni, V. vulnificus, and L. pneumophila.
Although many of these can be identified using the routine culture
and bacterial identification techniques available in any standard
laboratory (Table 5.2.2), the more sophisticated tests now available in
reference laboratories are enabling researchers to define the
epidemiology of disease transmission on an increasingly specific
molecular level.
Table 5.2.2. Microbiologic Techniques Used for Specific Bacterial Pathogen-
Specific Bacterial Pathogens in Epidemiologic Investigations of
Waterborne Disease Control3
Agent
Campylo-
bacter
Salmonella
Shigella
Yersinia
E. coli
V. cholerae
Culture
X
X
X
X
X
X
Sensitivity Serotype
X
X X
X X
X
? X
X
Biotype Profile /Assay
X
X
X
X
X XX
X X
/Assay
X
a Culture and antimicrobial sensitivity testing are widely available. Serogrouping and
serotyping are available in many hospital laboratories and most state public health
laboratories. Toxin assays and pathogenicity assays are available in specific research
laboratories.
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References
1. St. Louis, M.E., Morse, D.L., Potter, M.E. et al. The emergency of
grade A eggs as a major source of Salmonella enteritidis infections:
New implications for the control of salmonellosis. JAMA.
259:2103-2107,1988.
2. Centers for Disease Control. Salmonella enteritidis infections and
grade A shell eggs, United States, MMWR. 37:490, 495-6,1988.
3. Centers for Disease Control. Shigella dysenteriae type 1 in tourists
to Cancun, Mexico. MMWR. 37:465,1988.
4. Centers for Disease Control. Nationwide dissemination of multiply
resistant Shigella sonnei following a common-source outbreak.
MMWR. 36:633-4,1987.
5. Holmberg, S.D., Osterholm, M.T., Senger, K.A. et al. Drug-
resistant salmonella from animals fed antimicrobials. N. Engl. J.
Med. 311:617-22,1984.
6. Riley, L.W., Remis, R.S., Helgerson, S.D. et al. Hemorrhagic colitis
associated with a rare Escherichia coli serotype. N. Engl. J. Med.
308:681-5,1983.
7. Venkatesan, M., Buysse, J.M., Vandendries, E. et al. Development
and testing of invasion-associated DNA probes for detection of
Shigella spp. and enteroinvasive Escherichia coli. J. Clin.
Microbiol. 26:261-6,1988.
8. Svennerholm, A.M. and Holmgren, J. Identification of Escherichia
coli heat-labile enterotoxin by means of a ganglioside
immunosorbent assay (GMi-ELISA) procedure. Curr. Microbiol.
1:19-23,1978.
9. Lockwood, E.D. and Robertson, D.C. Development of a competitive
enzyme-linked immunosorbent assay (ELISA) for Escherichia coli
heat-stable enterotoxin (STa). J. Immunol. Meth. 75:295-307,
1984.
10. Nataro, J.P., Baldini, M.M., Kaper, J.B. et al. Detection of an
adherence factor of enteropathogenic Escherichia coli with a DNA
probe. J. Infect. Dis. 152:560-65,1985.
11. Centers for Disease Control. Cholera in Louisiana - Update.
MMWR. 35:687-8,1986.
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Clinical Laboratory Diagnosis of
Enteric Viral Diseases
by: Christen J. Hurst, Ph.D.
Risk Reduction Engineering Laboratory
U. S. Environmental Protection Agency
Cincinnati, Ohio 45268
(513) 569-7331
Enteric viruses are those which replicate within, and are
subsequently shed from, the human enteric tract, There are presently
known to be more than 140 serologically distinct types, or serotypes, of
human enteric viruses. Enteric viral infections are very often
associated with stomach cramps, vomiting and diarrhea. Infections
caused by some of the enteric viruses can spread from the enteric tract
to other organs or tissues of the body. This can result in symptoms
associated with abnormality or disfunction of these secondary sites,
including fever, hepatitis, meningitis, and paralysis. Human enteric
viruses occasionally cause outbreaks of waterborne human- illness
because they are transmitted by the fecal-oral route, meaning that
they are passed from person to person via ingestion of fecally
contaminated materials. Of the different human enteric viruses which
have been recognized in association with waterborne illness, the most
prominent are two causative agents of infectious hepatitis (human
enterovirus 72, formerly hepatitis A virus; and hepatitis E virus, also
called the enterically transmitted non-A non-B hepatitis virus) plus
several causative agents of vomiting and diarrhea including the
Norwalk virus.
The process of laboratory diagnosis or confirmation of a viral
illness can be accomplished by demonstrating that collected specimens
or samples of body materials contain either identifiable virus particles,
viral structural components which can include proteins or nucleic
acids, or specific antiviral antibodies that have been produced by the
person's immunological defenses in response to infection. The types of
specimens that are most commonly collected in association with
suspected waterborne viral illnesses are stool solids and rectal swabs,
which are primarily examined for the presence of whole viruses or
viral specific antigens, and blood serum that is primarily examined for
the presence of antiviral antibodies.
Many of the techniques used for detecting viruses and viral
antigens are represented in Figure 5.3.1, where they are compared
243
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Assay Time
Days
Hours
Plaque Assay
Cytopathogenicity
Cell Immunofluorescence
Cell Enzyme Immunoassay
In-Situ Nucleic Acid Hybridization
Immune Electron Microscopy* Blot Nucleic Acid Hybridization
Direct Electron Microscopy
-t-
Blot Enzyme Immunoassay
—I H 1 1-
108
107
106
105 10" 103 102
Relative Sensitivity
" Physical Particles
Figure 5.3.1. Comparison of viral assay techniques.
Infectious Units
with respect to relative sensitivity and also the length of time that is
required for assay completion. This figure has been adapted from the
referenced publication by Hurst and Stetler (1). The relative
sensitivity ranges for these different techniques are indicated by
horizontal bars that appear under the name of each technique. These
sensitivity ranges are given in terms of quantitating physical virus
particles for the electron microscopy techniques, and in terms of viral
infectious units for the other assay techniques. Additional
methodologies such as radioimmunoassay, ELISA (enzyme-linked
immunosorbent assay), complement fixation, and hemagglutination
can also be used for identifying either whole viruses or viral materials
contained in collected specimens.
Serological assays are used to detect the presence of antiviral
antibodies rather than the presence of virus particles or viral
structural components. These serological tests utilize prepared viral
particles, virally infected cells, or viral antigens as targets that are
recognized by appropriate antibodies present in the patients serum.
The serological assays can employ the same general methodology as
used for viral detection, the difference being whether viral material or
antibody represents the unknown component in a test procedure. The
materials used for performing many of these techniques can be
purchased from biomedical companies in the form .of kits. Further
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details of these methods are provided in the referenced books edited by
by Rose et al. (2) and Lennette et al. (3).
References:
1.
3.
Hurst, C. J. and R. E. Stetler, 1988. Recent advances in the
detection of human viruses in drinking water. Pages 943-956 in
Proceedings of the 15th. Water Quality Technology Conference,
Baltimore, Maryland, November 15-20, 1987. American Water
Works Association, Denver.
Manual of Clinical Laboratory Immunology (3rd. edition), 1986
Editors: N. R. Rose, H. Friedman, and J. L. Fahey. American
Society for Microbiology, Washington.
Manual of Clinical Microbiology (4th. edition), 1985. Editors: E.
H. Lennette, A. Balows, W. J. Hausler, Jr., and H. Jean Shadomy.
American Society for Microbiology, Washington.
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Clinical Diagnosis of Enteric Protozoans
by: Prank W. Schaefer, III
Environmental Systems Monitoring Laboratory
U.S. Environmental Protection Agency
26 West Martin Luther King Drive
Cincinnati, Ohio 45268
(513)569-7222
. Enteric protozoan pathogens that have direct modes of trans-
mission in humans include Giardia lamblia, Entamoeba histolytica,
Cryptosporidium parvum, Isospora fyelli, and Balantidium coli. Of
these, B. coli is rarely seen, even through it is ubiquitous and
cosmopolitan in nature like the others. With the advent of the AIDS
epidemic, the importance of the first four organisms has been
reinforced.
Effective diagnostic procedures for these parasites require
implementation of a number of steps. Appropriate sample collection
must^ be followed by expeditious concentration, preservation, and
staining techniques. Ultimately, microscopic reading must be carried
out by personnel of considerable skill and experience. Furthermore,
each parasite has unique qualities that prevent the utilization of
universal procedures for all. Some parasites, like Giardia, are
intermittent in cyst production or produce no cysts in some infected
people. In addition to the necessity of doing multiple stool exams to
overcome this characteristic, sometimes invasive procedures, like
duodenal aspiration or biopsy, are required to make an accurate
diagnosis. An overall approach for carrying out these steps has been
published by the American Society of Parasitologists (1).
There is no consensus as to which of the published procedures for
diagnosing these parasites is optimal. Each laboratory has a tendency
to use those procedures with which they feel most comfortable,
whether or not they are the most effective. Presently the Centers for
Disease Control recommend the methods outlined by Melvin and
Brook (2), while the American Society for Clinical Pathologists
suggests procedures published by Ash and Orihel (3). The techniques
listed in these volumes and numerous other credible sources, which
space does not permit mentioning, are very labor intensive and require
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specialized skills which are rare to nonexistent in many clinical
laboratories.
Attempts have been made to employ molecular and immunological
procedures to simplify, expedite, and remove the high level of
microscope skill required of the present methods. Papers have been
published describing the use of monoclonal antibodies (4) to
differentially stain the organisms of interest. Genetic probes (5) and
enzyme-linked immunosorbent assays (6), which require only the
genetic material or the parasitic fecal antigenic epitope, have been
described as agents of potential clinical significance. Unfortunately,
these methods are not a panacea, as problems such as cross reaction
and sensitivity remain to be solved.
References
1. American Society of Parasitologists. 1977. Procedures for use in
examination of clinical specimens for parasitic infections. Journal
ofParasitology 63: 959-960.
2. Melvin, D.M. and Brooke, M.M. 1982. Laboratory Procedures for
the Diagnosis of Intestinal Parasites, 3rd ed. HHS Publication No.
(CDC) 82-8282!
3. Ash, L.R. and Orihel, T.C. Parasites: A Guide to Laboratory
Procedures and Identification. American Society of Clinical
Pathologists Press, Chicago. 328 p.
4. Sterling, C.R. and Arrowood, J.J. 1986. Detection of
Cryptosporidium sp. infections using a direct immunofluorescent
assay. Pediatric Infectious Diseases 5: S139-S142.
5. Samuelson, J., Acuna-Soto, R., Reed, S., Biagi, F., and Wirth, D.
1989. DNA hybridization probe for clinical diagnosis ofEntamoeba
histolytica. Journal of Clinical Microbiology 27: 671-676.
6. Janoff, E.N., Craft, J.C., Pickering, L.K., Novotny, T., Blaser, M.J.,
Knisley, C.V. and Reller, L.B. 1989. Diagnosis of Gardia lamblia
infections by detection of parasite-specific antigens. Journal of
Clinical Microbiology 27:431-435.
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Analysis of Water Samples for Bacterial Pathogens
by: Dr. Gerard N. Stelma, Jr.
Environmental Monitoring Systems Laboratory
Division of Microbiology Bacteriology Branch
U.S. Environmental Protection Agency
Cincinnati, OH 45268
(513) 569-7384
Introduction
Bacteria associated with waterborne illness outbreaks include two
types of pathogens, those present in water as a result of pollution and
some aquatic organisms that appear to be indigenous to drinking
water distribution systems. The pathogens acquired from polluted
waters are of fecal origin and are causes of gastrointestinal illness.
Pathogens acquired from polluted waters include recently recognized
species, such as Campylobacter jejuni and Yersinia enterocolitica, as
well as classical enteric pathogens, such as Salmonella and Shigella.
Most outbreaks of drinking water-associated gastroenteritis have
resulted from human fecal pollution; however, animal fecal pollution
is being increasingly implicated in outbreaks (1), The indigenous
aquatic organisms are opportunistic pathogens that rarely infect
healthy individuals but may cause serious (and sometimes fatal)
infections in those who are compromised. Legionalla pneumophila is a
good example of this type of pathogen. Not included in this review are
pathogens associated with rare sporadic illnesses in the United States
(e.g., Leptospira and Vibrio choleras) or those normally associated with
recreational water (e.g., Pseudomonas aeruginosa).
Investigations of waterborne outbreaks are complicated by 1) the
low numbers and transient nature of the fecal pathogens in drinking
water, 2) difficulties in detecting pathogens in water due to
interferences by natural competing flora, and 3) the fastidious nutrient
requirements of pathogenic bacteria (2). The methods used to isolate
pathogens from potable water are modifications of methods used in
clinical laboratories, with concentration and/or enrichment procedures
incorporated before the organisms are transferred to harsh selective
media.
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Although the role and relevance of follow-up laboratory
investigations of waterborne outbreaks have been questioned, the
cumulative knowledge obtained from these investigations may lead to
new preventative technical measures and may, therefore, be of great
value ha preventing future outbreaks (3). Furthermore, investigations
of these outbreaks have led to the identification of waterborne
pathogens not previously considered to be important, such as Y.
enterocolitica and C. jejuni, as well as pathogens not previously
recognized. L. pneumophila was not recognized until after the follow-
up investigation of a 1976 pneumonia outbreak (4).
Isolation and Enumeration of Enteric Pathogens
Salmonella
Good hygiene and modern sewage and potable water treatment
practices have dramatically reduced the incidence of illness caused by
Salmonella. Even though the number of outbreaks has declined since
the mid-20th century, Salmonella must still be considered a threat to
public health. The waterborne outbreak of salmonellosis that occurred
in Riverside, California, in 1965 verified the continued significance of
this organism. Over 16,000 cases of illness resulted from that single
outbreak (5).
Because of the low densities at which Salmonella occurs in water,
concentration and enrichment procedures are normally used for
isolating this organism. Usually water samples suspected to contain
Salmonella are concentrated by filtration either through membranes,
if the samples are relatively clear, or through diatomaceous earth if
the samples are highly turbid (6,7). Alternatively, absorbent pads
suspended in the water for long periods of time (8) or filtration through
fiberglass epoxy depth filters (9) may be used.
After concentration, the membranes or filter materials are
incubated in an enrichment medium to increase the number of
salmonellae relative to coliforms and enhance the probability of
isolating Salmonella. Quantitative estimates of the number of
salmonellae in drinking water or other waters containing low densities
of pathogens can be obtained by using filtration and subsequent
enrichment in conjunction with most probable number (MPN)
estimates. With these samples, three sets of decimal volumes are
substituted for decimal dilutions (9,10). The enrichment media
recommended for water samples are dulcitol-selenite broth or
tetrathionate broth. The advantages of using dulcitol-selenite broth
are that it allows the rapid growth of Salmonella while inhibiting
many nonpathogenic enterobacteria and that it causes cultures
containing salmonellae to develop a distinct orange or red color. The
advantage of tetrathionate broth is that it may yield greater numbers
of salmonellae (7).
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Primary enrichment is followed by secondary differentiation on
selective solid media, such as brilliant green agar (BG), bismuth
sulfite agar (BS), or xylose lysine deoxycholate agar (XLD).
Salmonella colonies appear pinkish-white with a red background on
BG, black (with or without a metallic sheen) on BS, and red with black
centers on XLD. Use of an incubation temperature of 41.5°C enhances
the isolation of many salmonellae (8). However, some serotypes,
including S. typhi, do not grow at this elevated temperature. When S.
typhi is suspected as the cause of an outbreak, BS agar and a growth
temperature of either 35 or 37°C should be used (6).
Because other enteric organisms of little or no pathogenicity share
some major biochemical characteristics with Salmonella, it is
necessary to confirm the identify of suspect colonies by biochemical
tests or serotyping. Serotyping is a particularly valuable technique
because it is highly specific. The isolation of a Salmonella from water
with the same serotype as that of patients' isolate confirms an
outbreak as waterborne.
Shigella
The shigellae are among the most common causes of diarrhea in
humans. All four species of the genus Shigella (S. dysenteriae, S.
flexneri, S. boydii, and S. sonnei) are pathogenic (11), and these
pathogens have been implicated in a number of waterborne outbreaks
(12). Because the shigellae are highly specific for humans, human
cases are always the source of infection (11).
The procedures used to isolate Shigella sp. and other enteric
pathogens from water are fundamentally the same as those used to
isolate the salmonellae: concentration by filtration, selective
enrichment, isolation on selective differential plating media, and
confirmation by biochemical and serological techniques.
Water samples suspected to contain shigellae are filtered as
described in the section on Salmonella. The filters or filter materials
are either placed in Gram-negative (GN) broth for nonselective
enrichment (13-15) or in the more effective autocytotoxic medium
developed by Park and colleagues (16,17). The autocytotoxic medium is
a lactose broth containing 4-chloro-2-cyclopentyl-phenyl-B-D-
galactopyranoside, an analogue of lactose. Coliforms and other
organisms that utilize lactose hydrolyze this compound to galactose
and a toxic moiety that subsequently kills them. Shigellae, which do
not utilize lactose, are not affected. The improved formula for this
medium allowed selective enrichment of 42 of 48 Shigella strains from
all four species in competition with a 1,000-fold higher population of
Escherichia coli (17). This medium can be used in a modified MPN
procedure to obtain a quantitative estimate of the number of shigellae
in water samples.
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The results of several studies indicate that XLD agar is the best
selective differential plating medium for isolating Shigella from
enrichment broths (13,15,18). Colonies of Shigella are red and can
easily be distinguished from Salmonella colonies, which are red with
black centers. Although the shigellae are very closely related to E. coli,
they can be differentiated from E. coli either by a battery of 13
biochemical tests or by serology (19).
Yers/n/a Enterocolitica
Y. enterocolitica is a relatively new addition to the list of bacterial
pathogens known to cause waterborne gastroenteritis. The first report
of waterborne illness attributed to Y. enterocolitica was in 1972 (20).
The requirements of Y. enterocolitica for special growth conditions
compared to other Enterobacteriaceae was no doubt partly responsible
for the failure to recognize this pathogen earlier (21). The most
common manifestation of infection is a diarrheal disease with
abdominal pain and fever. The occurrence of abdominal pain in the
lower right quadrant has sometimes confused the diagnosis as
appendicitis (21).
One of the characteristics of Y. enterocolitica that is unique among
the Enterobacteriaceae is the ability to grow well in suitable media at
refrigeration temperatures (21). The traditional method for
enrichment of Y. enterocolitica from fecal specimens, incubation at 4°C
for 3 weeks, took advantage of this characteristic and allowed Y.
enterocolitica to grow to higher densities than the other enteric flora
(22). Y. enterocolitica has been isolated from contaminated water after
only 9 days incubation at 4°C (23). However, this is still an
inconveniently long incubation time. Weagant and Kaysner (24)
developed a 22°C enrichment procedure that required incubation for
only 48 hours. Their enrichment medium may be inoculated directly,
or samples may be filtered and filters placed into the medium. Sorbitol
is the primary differential substrate in this medium.
A comparative study by Head et al. (25) identified the Cefsulodin-
irgasan- novobiocin (GIN) medium developed by Schiemann (26) as the
most efficient selective differential agar medium for isolating Y.
enterocolitica. This medium uses antibiotics and bile salts for
suppression of competing bacterial flora and a differential reaction
resulting from mannitol fermentation for discrimination of Y.
enterocolitica from most other Gram-negative bacteria able to grow oh
the medium. Y. enterocolitica colonies on GIN agar have a deep red
center surrounded by an outer zone that is usually translucent.
Although incubation at 32°C for 24 hours may be used, mannitol
fermentation is stronger and more complete with incubation at 22°C
for 48 hours. Further confirmation may be obtained by inoculation on
lysine-arginine-iron agar (LAIA) slants (27). Typical reactions of
Yersinia sp. on LAIA are alkaline slant (purple), acid butt (yellow), no
H2S (darkening of butt), or gas formation. Biotyping and serotyping
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procedures are also available (28,29) as is a recently developed DNA
probe that identifies virulent Y. enterocolitica colonies (30,31).
A membrane filter (MF) method is also available for enumerating
and isolating Y. enterocolitica from large volumes of low-turbidity
water (32). This method is particularly advantageous because it allows
tor presumptively identifying the organism without transferring
colonies to multiple confirmatory media. A sample is filtered through a
membrane filter, and the filter is placed on a cellulose pad saturated
with a recovery broth (mYE medium). After incubation for 48 hours at
^5 0, the membrane is asceptically transferred to a lysine-arginine
agar.substrate and incubated anaerobically at 35°C for 1 hour A
needle is then used to puncture a hole in the membrane next to each
yellow to yellow-orange colony, and the membrane is transferred to a
urease-saturated absorbent pad and incubated at 25°C for 5 to 10 min
All marked distinctly green or bluish colonies are counted
immediately. These colonies are sorbitol-positive, lysine- and arginine-
negative and urease-positive; and they may be presumptively
identified as Y. enterocolitica.
Pathogenic Escherichia Coli
There are five categories of Escherichia coli responsible for a wide
diversity of gastrointestinal illnesses: enterotoxigenic (ETEC)
f£™nvasive (EIEC)> enteropathogenic (SPEC), enterohemorrhagic
(fcHEC), and enteroadherent (EAEC). The ETEC are a major cause of
travelers diarrhea and infant diarrhea in less-developed countries
These strains produce heat-labile enterotoxin (LT), heat-stable
enterotoxin (ST), or both. The EIEC are a cause of dysentery and are
distinguished from other pathogenic E. coli by their capacity to invade
and proliferate within epithelial cells. The EPEC are an important
cause of infant diarrhea. EPEC strains are pathogens but cause
diarrhea by some mechanism distinct from LT,. ST, or Shieella-like
mvasiveness. The EHEC, a cause of hemorrhagic colitis and hemolytic
uremic syndrome, elaborate phage-encoded cytotoxins active on Vero
WAPO c
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either matching the serotype of the water isolate with that of the
clinical isolate or on verifying the pathogenicity of the isolate.
Traditional tests used to differentiate pathogenic E. coli include
suckling mouse and rabbit ileal loop bioassays and Y-l adrenal cell
and Chinese hamster ovary cell assays for ETEC and the Sereney test
for EIEC (36). One procedure for the identification of ETEC that
produce LT was developed specifically for isolating and enumerating
these organisms from water. ETEC colonies on a modified MF medium
were identified by transferring the membrane to Y-l adrenal cell
monolayers; colonies producing LT in approximately 2 days caused the
Y-l cells to round up (37).
Advances in biotechnology have led to more rapid and facile
techniques that may soon make all of the above assays obsolete.
Enzyme linked immunosorbent assay (ELISA) techniques are
available for identifying LT-, ST-, and VT-producing strains (38-41);
and DNA probes are available for identifying ETEC that produce
either LT or ST, EIEC, EPEC, and EHEC (42-47). DNA probe
methodology includes both dot-blot (43) and colony hybridization
(42,44,47) methods.
Campylobacter jejuni
Campylobacter jejuni is another recent addition to the growing list
of known enteric pathogens. This organism was not generally
recognized as a common cause of diarrhea until 1977 (48). The fairly
uncommon conditions for optimal growth of C. jejuni, i.e.,
microaerophilic atmosphere and 42°C, incubation apparently
contributed to the late recognition of this pathogen (49).
C. jejuni and closely related C. coli cause a gastroenteritis with
bloody diarrhea and abdominal cramps (50,51). Numerous animal
species have been cited as reservoirs of campylobacters (52). The most
common sources of human infection are unpasteurized milk, chicken,
and water (49). Although campylobacters are sensitive to chlorine,
municipal water systems have been the sources of outbreaks,
presumably due to breakdowns in treatment or contamination after
chlorination (1,53-55).
Methods originally developed for isolation of campylobacters from
fecal specimens have been successfully used to isolate these organisms
from water (55-57). The methods involve filtration of several liters of
the water sample, followed either by enrichment in a broth medium or
placing the filter on a selective agar medium, with subsequent
incubation under microaerophilic conditions at 42°C. Pressure
filtration in a stainless steel filtration device with a 1.5 L reservoir
may be necessary for samples that are turbid (7).
Methods for isolating campylobacters from environmental samples
are not standardized, and there is -still no specific method
recommended above all others. Several MPN methods have been used.
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The traditional medium, "Campythio broth," which was developed in
1979 by Blaser et al. (58), is still sometimes used in environmental
isolations (59). Another medium was developed by Oosterom et al. (60)
for detecting Campylobacter in sewage and river samples. However,
there is very little supporting information concerning the use of that
medium. Bolton and Robertson (61) developed a selective medium
which they called Preston medium. The Preston medium without agar
was used as an enrichment broth. Preston agar was compared with
Skirrow's (48) agar for recovery of Campylobacter from the feces of
humans and several other species. The results showed that the Preston
agar recovered more Campylobacter than the Skirrow agar, and if the
Preston enrichment broth was also used, considerably more
Campylobacter isolates were found. These investigators reported
successfully using the combination of Preston enrichment broth and
Preston agar to isolate campylobacters from coastal seawater samples,
drain swab effluent, and environmental specimens from local
abattoirs. Bolton et al. (62) reported that the use of Preston enrichment
broth followed by subculture on Preston agar allowed the estimation of
as few as 10 campylobacters per 100 mL of water. Mathewson et al. (63)
evaluated several types of filters and determined that a positive-
charged depth filter gave the highest percent recovery of
campylobacters from seeded tap and surface waters. This type of filter
should enhance the sensitivity of the filter enrichment MPN,
technique.
Several selective plating media, in addition to Preston medium,
are available for isolatihg Campylobacter from enrichment broth.
These include Skirrow's medium (48), Campy blood agar (58), Butzler's
medium (64), and a blood-free medium developed by Bolton et al. (65).
On these media, Campylobacter forms spreading colonies that range in
color from clear to white or tan. Suspect colonies should be verified
biochemically (49,66). Hutchinson et al. (67) and Dilworth et al. (68)
recommended further confirmation using a combination of serotyping
and biotyping.
Several different serotyping systems have been developed and
published, creating a very complicated situation. It was recommended
at the Third International Workshop on Campylobacter infections in
Ottawa, Ontario, in 1985 that only two of these systems should be
used. One is based on the heat-labile antigens using slide
agglutination as described by Lior et al. (69) and the other is based on
heat-stable antigen using passive hemagglutination as described by
Penner etal. (70). Biotyping schemes have also been developed by Lior
(71) and Bolton et al. (72).
Campylobacter sp. may also be directly isolated by the MF
procedure using any of the selective media described above. Rosef et al.
(73) reported that enrichment did not significantly enhance the
recovery of thermotolerant campylobacters from naturally
contaminated water samples over that produced by direct plating of
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the membrane filters onto Preston agar or colistin-amphotericin-keflin
(CAK) agar. However, several other researchers have reported that
the MT procedure was less effective than selective enrichment
procedures. Bolton et al. (62) reported that the MF procedure was
unsuitable for the enumeration of campyldbacters because of the
tendency of colonies developing on the membrane to spread and
coalesce. Ribeiro et al. (74) reported that 12 out of 64 water samples
that were Campylobacter-negative by direct plating were positive if
the filters were placed in Preston enrichment broth and subsequently
subcultured onto a selective medium. Bolton et al. (75) compared
Skirrow's, Butyle's, Blaser's, Campy-BAP, and Preston medium for
efficiency in isolating Campylobacter from fecal and environmental
specimens. They found that Preston agar, which was the most
selective, gave the highest isolation rate and that preenrichment in
Preston broth gave a higher isolation rate than direct plating on
Preston agar. Goosens et al. (76) found that the MF method was easy
and cheap, but low in sensitivity. The low sensitivity of the MF method
reported by Goosens et al. (76) may be partially due to their use of 0.45
pm filters. Researchers from the U.S. Food and Drug Administration
report that recovery is significantly enhanced if 0.22 urn filters are
used (J.M. Hunt, personal communication).
Isolation of campylobacters from source waters in the absence of an
outbreak should be interpreted with caution. Campylobacter
organisms that closely resembled C. jejuni but were apparently
nonpathogenic were isolated from a variety of water samples by
Mawer (77,78). These atypical strains can "be distinguished front C.
jejuni by their inability to hydrolyze hippurate.
Indicator Organisms
Total Conforms
Sometimes the pathogenic bacterium that caused a waterborne
outbreak will have died off or reached too low a density to be recovered
by the time the water is sampled and analyzed. When this situation
occurs, isolation of an indicator of fecal contamination from
epidemiologically implicated water can provide further evidence that
the outbreak was waterborne. Indicators are normally present in
fecally contaminated waters in higher numbers than pathogens and
consequently persist after the pathogen can no longer be isolated.
The coliforms are defined as the group of bacteria that are Gram-
negative, aerobic or facultatively anaerobic, nonsporeforming rods
that ferment lactose with the production of gas within 48 hours at 35°C
(7). This group of organisms was originally believed to indicate the
presence of fecal pollution, and they are still used for that purpose (2).
The finding of coliforms in source waters or drinking water is
interpreted as either a degradation of the quality of water,or
ineffective treatment, even though some coliforms are widely
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distributed in nature and are not associated with the intestinal tract of
warm-blooded animals (79).
The methods approved by the U.S. Environmental Protection
Agency (U.S. EPA) and recommended in Standard Methods for the
Examination of Water and Wastewater (7) are the lauryl sulfate
tryptose broth (LTB) MPN method and the MF procedure employing
m- Endo or m-Endo LES agar.
The MPN procedure uses five fermentation tubes for each of three
decimal multiples of 1 mL. Formation of gas in the inner fermentation
tubes constitutes a presumptive positive test. Presumptive positive
cultures must be verified by growth and gas production in brilliant
green lactose broth. A completed test requires transfer onto m-Endo
LES or eosin methylene blue (EMB) agar slants and Gram-stainine
(7).
A new method called the "Autoanalysis Colilert" (80) apparently
does not require confirmatory or completed tests. To perform this test,
one only has to add the water sample to the powdered ingredients in a
tube or flask and incubate for 24 hours. If total coliforms are present in
the water sample, the solution will change from its colorless state to
yellow. This test can be used as a MPN test and will detect 1 total
coliform per 100 mL in a maximum of 24 hours.
The standard MF procedure utilizes m-Endo broth or agar.
Organisms that produce a colony with a golden-green metallic sheen
on this medium within 24 hours of incubation are considered
presumptive members of the coliform group. Coliform organisms may
occasionally produce atypical colonies. The identity of these colonies as
coliforms can be verified by inoculating them into LTB and brilliant
green lactose bile broth and observing the tubes for gas formation
within 48 hours of incubation at 35 + 0.5°C (7). To save time, both
media may be inoculated simultaneously.
Observation of many false-positive and false-negative results and
poor recovery of stressed cells on m-Endo agar (81,82) led to the
development of m-T7 medium by LeChevallier et al. (83). They
reported recovering an average of 43% more verified coliforms from
surface and drinking water samples than were recovered on m-Endo
medium, due to the recovery of higher numbers of injured coliforms.
Coliform organisms produce smooth, yellow, convex colonies on M-T7.
LeChevallier et al. (84) also proposed a new verification test for
coliforms from MF methods to substitute for the subculturing of
colonies to lactose broth and brilliant green bile broth. This method,
which utilizes testing for the presence of {3-galactosidase and absence
of cytochrome oxidase, increases the confirmation rate by 87 percent
over the currently accepted methods.
Another new MF medium (m-HARC) developed at the U.S. EPA
Cincinnati, Ohio, laboratories has excellent potential. Although this
257
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medium does not recover significantly more coliforms than are
recovered on m-Endo, it has the advantages of allowing coliforms to be
counted more easily and of not requiring proprietary ingredients.
Coliform colonies on m-HARC are yellow against a cobalt blue
background, whereas noncoliform colonies are blue or green (J.R.
Haines, personal communication).
Fecal Coliforms
Fecal coliforms are a subgroup of the total coliforms that can be
differentiated by their ability to grow at 44.5 + O.2°C (7). This
thermotolerant characteristic usually separates coliforms that may be
found in the GI tract of warm-blooded animals from the other
coliforms. Therefore, a positive test for fecal coliforms is a more specific
indication of fecal contamination than a positive test for total
coliforms. The fecal coliform test is not recommended for routine
examination of potable waters, because no coliform of any kind should
be tolerated in treated water (7). However, this test may be quite
useful in an outbreak investigation to aid in establishing the vehicle of
the outbreak.
This group of organisms is identified either by the MPN procedure
using EC broth and fermentation tubes or the MF method using m-FC
medium. Gas production in a fermentation tube within 24 hours or less
is considered a positive MPN reaction, indicating coliforms of fecal
origin (7). A positive test on m-FC medium is indicated by the
appearance of dark blue colonies; all other colonies are light blue to
gray to cream colored (85). Few nonfecal coliform colonies will be
observed on m-FC medium because of the selective action of the
elevated temperature and the presence of a rosolic acid salt reagent (7).
Stuart et al. (86) compared a two-layer MF medium (IM-MF) for
injured fecal coliforms with the EC broth MPN procedure and the
standard m-FC method using various types of water samples. They
observed that recoveries by the IM-MF method were equal to or
greater than those by the MPN method in 50% of the samples tested
and greater than the m-FC method in all of the samples.
Escherichia coli
A positive test for E. coli in a water sample is a more definite
indication of fecal contamination than is a positive fecal coliform test.
E. coli, one of the members of the fecal coliform group, is an
unquestionable inhabitant of the GI tract of humans and other warm-
blooded animals, whereas some of the other so-called fecal coliforms
are of environmental origin. E. coli is easily distinguished from the
other members of the fecal coliform group by the urease test. E. coli
does not possess this enzyme, whereas the other members of the
coliform group do. Dufour et al. (87) used this characteristic to develop
a MF method that was 90 percent specific for E. coli from marine,
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estuarine, and fresh water supplies. The method incorporates a
lactose-base selective differential medium (mTEC), a 2-hour
resuscitation of weakened organisms at 35°C before incubation at
44.5°C for 18 to 22 hours and an in situ test for urease. Yellow colonies
obtained after the urease test are presumed to be E. coli.
The developers of the new "Autoanalysis Colilert" test for total
coliforms contend that the method can also be used as a MPN method
for E. coli. The medium contains 4-methylumbelliferyl-p>-D-glucuro-
nide (MUG), which is hydrolyzed by E. coli but not by other members of
the coliform group. One of the metabolites of MUG fluoresces when
exposed to ultraviolet light. Species in other genera, such as
Aeromonas, Pseudomonas, and Flavobacterium, that could be positive
in a MUG assay do not grow in the restrictive medium and will not be
positive in this growth-based test unless a high density (> 20,000
bacteria per mL) is present (80).
Opportunistic Pathogens
Legionella Species
Legionella pneumophila is a Gram-negative pleomorphic
bacterium that was identified in 1977 as the causative agent of the
pneumonic illness, Legionellosis (Legionnaires' disease) (88). L.
pneumophila and related organisms have been assigned to the family
Legionellaceae, which consists of a single genus (Legionella) and a
number of species (89). The recently described L. cincinnatiensis is the
25th species and the 41st serogroup described in the genus and the
14th species shown to be pathogenic for humans (90). Epidemiological
data suggest that about two thirds of reported cases occur among
individuals who smoke and/or have significant underlying conditions
The Legionellaceae are ubiquitous in aquatic environments,
including potable water (92-94). Even water taps, water storage tanks,
and showerheads may provide suitable environments for growth of
legionellae (95). The usual mechanism of transmission appears to be
aerosolization of water containing legionellae (96).
Legionella is also the etiologic agent for a second type of illness
called Pontiac fever. This illness is characterized by a short (1- to 2-
day) incubation period, a self-limited grippelike illness, without
pneumonia, and a high attack rate (100% in one outbreak) (97-99).
.Three media are available for isolating legionellae. All three use
the charcoal, yeast extract, cysteine, ferric pyrophosphate agar base
developed by Feeley et al. (100). Bopp et al. (101), Wadowsky and Yee
(102), and Edelstein (103) modified this basal medium by adding
various combinations of antibiotic inhibitors. Calderon and Dufour
(104) compared the efficiencies of these media for eliminating
background flora and recovering Legionella. They found that the
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modification developed by Edelstein was the most efficient of the three
media for recovering legionellae seeded into environmental samples.
Recommended incubation conditions are 35°C and 90 percent relative
humidity if the buffered medium developed by Edelstein is used, and
the above conditions plus 2.5 percent CO2 if unbuffered media are used
(103). Legionella colonies should be visible on all of the charcoal, yeast
extract-based media in 3 to 5 days, appearing light blue and having a
cut-glass texture. On continued incubation, colonies will become
whiter, smoother and larger in diameter (105).
Several cultural and biochemical characteristics are helpful In
distinguishing legionellae from other bacteria (105). Gram-negative
organisms that resemble L. pneumophila in colonial morphology and
grow only on L-cysteine-HCl-containing media are probably legion-
ellae, whereas organisms that grow on ordinary blood agar are defin-
itely not. Although the legionellae are positive for enzymes such as
catalase and oxidase, they give negative reactions for most
biochemical tests; and an isolate can only be definitively identified by
results from highly specific methods such as the direct fluorescent-
antibody test (DFA) (106), DNA probes (107,108), or monoclonal
antibody techniques (109,110).
If rapid presumptive identification is required, sedimented
material from environmental samples can be examined directly by
DFA. However, the DFA procedure gives some cross-reactivity with
other species (111) and does not distinguish between viable and
nonviable organisms. Therefore, the presence of Legionella in an
environmental sample should be confirmed using direct isolation
procedures (7).
Barbaree et al. (112) developed a detailed protocol for sampling
environmental sites for legionellae. Their protocol included
recommended numbers of samples and sample volumes for a variety of
sites, including water treatment plants, different sites within hospital
water systems, air-conditioning systems and whirlpools. They
recommended inoculating duplicate plates of each of two kinds of
media with 0.1 mL of each concentrated or nonconcentrated sample.
The recommended media were buffered charcoal-yeast extract agar
containing alpha-keloglutarate (aBCYE) and BCYE supplemented
with glycine, polymyxin B, anisomycin and vancomycin (GPAV). They
also suggested inoculating one plate of aBCYE and one plate of GPAV
without L-cysteine as controls.
It may be advantageous to add a heat-shock step to the isolation
procedure when cold waters are examined. A recent study by Hussong
et aL (113) provided evidence that L. pneumophila sometimes exists in
a viable but not culturable state. Colbourne et al. (114) discovered that
culturability was related to temperature above 20°C and that heat-
treatment of culture-negative distribution water samples at 45°C for
either 10 or 20 min allowed them to recover viable L. pneumophila:
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There are several methods that have been used to correlate
environmental isolates of Legionella with those from patients.
Serpgrouping has typically been used as an epidemiological marker.
However, additional markers such as plasmids (115) and panels of
monoclonal antibodies (109,110) are useful when the same serotype is
isolated from two or more discrete environmental points.
Mycobacterium Sp.
Several of the nontuberculosis mycobacteria have been associated
with (opportunistic infections. Species implicated in this type of
infection include M. kansasii, M. xenopi, M avium, the intracellular
variant of M. avium. and M. scrofulaceum (116), Two of these species or
complexes M kansasii, and M. avium complex (MAC), are the
prominent cause of disseminating mycobacteriosis in the United
States (117). The rate of occurrence of MAC has increased markedly in
recent years, primarily because the epidemic of acquired
immunodeficiency syndrome (AIDS) has caused an increase in the
number of susceptible individuals (118). Data collected in
Massachusetts showed a five-fold increase in the incidence of isolation
of MAC from clinical specimens between 1972 and 1983 (119).
The etiology and pathophysiology of mycobacteriosis has not been
fully elucidated. However, water has been suspected for many years as
a .source of potentially pathogenic mycobacteria (116,120).
Aerosolization may be a mechanism for transmission. These organisms
are relatively resistant to chlorine (121); and have been isolated from
chlorinated sources, including swimming pools, taps, drinking
fountains, and aquariums (120). Du Moulin et al (122) found that
mycobacteria survive and proliferate in water with temperature
ranges of 52 to 57°C and that potable hot water systems, particularly
those that generate aerosols, may contain concentrations of M. avium
greater than those found in cold water. They speculated that these
systems could serve as environmental sources for colonization and'
infection of immunocompromised persons.
The first step in isolating mycobacteria from water is
concentrating the sample either by centrifugation or filtration. The
next step is decontamination to eliminate unwanted competing
bacteria. For this, several decontamination procedures have been used.
Initially either sodium hydroxide (123-125) or a mixture of sodium
hydroxide and sodium hypochlorite (120) was used. Later, du Moulin
and Stottmeier (126) developed a method more suitable for large
volume water samples. Their method substitutes overnight exposure
to 0.4% cetylpyridinium chloride for alkali treatment. This method
eliminates the adverse effects of the alkali on membrane filters and
the suppress! ve effect of alkali on the growth of mycobacteria.
The final step of the isolation procedure involves placing the,
concentrated sample or the membrane filter on a selective medium.
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Middlebrook 7H10 is the basal medium most frequently used. Studies
have employed several modifications of this medium, with various
combinations of antibiotics added to the basal medium. Petran and
Vera (127) added cyclohexamide, lincomycin, and nalidixic acid to
inhibit Gram-negative competitors. Lorain and Haddock (128)
incorporated colimycin, spiramycin, and amphotercin B. Mitchison et
al (129) developed a 7H11 medium that contained polymyxin B,
carbenicillin, amphotercin B, and trimethoprim. Du Moulin et al. (122)
reported that adjusting the pH of Middlebrook Cohn 7H10 to 5.5 gave
optimal recovery of waterborne mycobacteria.
Growth of mycobacteria is slow on all laboratory media. M.
fortuitum and M. chelonii may form colonies in < 1 week. However,
most strains of Mycobacerium require 2 or more weeks to form visible
colonies (130).
Species identification and confirmation may be accomplished by a
combination of traditional methods such as colony morphology (130),
biochemical tests (120,130), antimicrobial susceptibility tests (118,
131), and seroagglutination (118); or by more sophisticated techniques
such as monoclonal antibodies (132), DNA probes (133,134); or
analysis of mycolic acids by high-performance liquid chromatography
(HPLC) (135). '
Summary
Two types of pathogens have been implicated in illness outbreaks
attributed to potable water. The first type consists of pathogens of fecal
origin, including Salmonella, Shigella, and Campylobacter. These
pathogens are normally inactivated by water treatment processes but
may get into distribution systems through temporary breakdowns in
treatment or through post-treatment contamination with sewage.
Isolation of these pathogens from potable water is difficult because of
their low numbers and transient existence in drinking water and
because of competition by numerous organisms with less fastidious
nutrient requirements. These difficulties are usually minimized by
using selective enrichment procedures prior to isolation on selective
differential agar media.
The isolation procedures for all of the fecal pathogens are
fundamentally the same: concentration by filtration, selective
enrichment in broth media, isolation on selective differential agar
media, and verification of the species by biochemical and serological
tests. In some cases, DNA probes can now be used for verification.
In some outbreaks, the suspected pathogen may no longer be
detectable when the water is analyzed. When this occurs, isolation of
organisms that are indicators of fecal contamination can provide
evidence that epidemiologically implicated drinking water was the
vehicle for infection. The presence of total coliforms, the traditional
indicator used for measuring degradation of potable water quality, can
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be measured either by MPN or MF techniques. Similar techniques are
available for estimating the numbers of fecal coliforms, a subgroup of
total coliforms more likely to be of fecal origin, and for E. coli, an
organism of undisputed fecal origin.
Other pathogens implicated as causes of waterborne illness are
opportunistic pathogens that are capable of long-term survival and
growth within potable water distribution systems. Known pathogens
of this type include many of the legionellae and nontuberculosis
mycobacteria. These organisms are resistant to chlorine and are
capable of growth at high water temperatures. They are probably
disseminated by aerosols. Isolation and identification of these
pathogens are difficult due to their slow growth rates, their
requirements for highly specialized growth media, and the negative
reactions they give in most biochemical tests. Sophisticated rapid
methods, such as DNA probes, panels of monoclonal antibodies, and
HPLC, are now available as alternatives to traditional biochemical
tests.
References
1. Craun, G.F. Recent statistics of waterborne disease outbreaks
(1981-1983). In: G.F. Craun (ed.), Waterborne Diseases in the
United States, CRC Press Inc., Boca Raton, Florida, 1986.
2. Ericksen, T.N. and Dufour, A.P. Methods to identify
waterborne pathogens and indicator organisms. In: G. F.
Craun (ed.), Waterborne Diseases in the United States, CRC
Press, Inc., Boca Raton, Florida, 1986.
3. Anderson, Y. and Senstrom, T.A. Waterborne outbreaks in
Sweden-causes and etiology. Wat. Sci. Tech. 19:575,1987.
4. McDade, J.E., Shepard, C.C., Fraser, D.W., Tsai, T.R., Redus,
M.A., and Dowdle, W.R. Legionnaires disease: isolation of a
bacterium and demonstration of its role in other respiratory
disease. New England. J. Med. 297:1197,1977.
5. A waterborne outbreak of salmonellosis in Riverside,
California, 1965: epidemiological aspects. Am. J. Epldemiol.
93:33,1971.
6. Cheng, C.M., Boyle, W.C. and Goepfert, J.M. Rapid
quantitative method for Salmonella detection in polluted
waters. Appl. Microbiol. 21:662,1971.
7. Standard Methods for the Examination of Water and
Wastewater, 16th ed. American Public Health Association,
Washington, D.C., 1985.
8. Spino, D.F. Elevated temperature technique for the isolation of
Salmonella from streams. Appl. Microbiol. 14:591,1966.
263
-------
9. Levin, M.A., Fischer, J.R., and Cabelli, V.J. Quantitative
large-volume sampling technique. Appl. Microbiol. 28:515,
1974.
10. Grunnet, K. Quantitative Salmonella demonstration by pad
technique. Rev. Inter. Oceanogr. Med. 34:155,1974.
11. Orskov, F. and Orskov, I. Enterobacteriaceae In: A.I. Braude,
C.E. Davis, and J. Fierer (ed.), Medical Microbiology and
Infectious Diseases. W.B. Saunders Co., Philadelphia,
Pennsylvania, 1981.
12. Craun, G.F. Statistics of waterborne outbreaks in the U.S.
(1920-1980). In: G.F. Craun (ed.), Waterborne Diseases in the
United States. CRC Press, Inc., Boca Raton, Florida, 1986.
13. Dunn, C, and Martin, W.T. Comparison of media for isolation
of salmonellae and shigellae from fecal specimens. Appl.
Microbiol. 22:17,1971.
14. Taylor, W.I. and Schelhart, D. Isolation of shigellae. VIII.
Comparison of xylose lysine deoxycholate agar, hektoen
enteric agar, Salmonella-Shigella agar and eosin methylene
blue agar with stool specimens. Appl. Microbiol. 21:32,1971.
15. Taylor, W.I. and Schelhart, D. Effect of temperature on
transport and plating media for enteric pathogens. J. Clin.
Microbiol. 2:281,1975.
16. Park, C.E., Rayman, M.K., Szabo, R. and Stankiewicz, Z.K.
Selective enrichment ofShigellain the presence ofEscherichia
coli by the use of 4-Chloro-2-Cyclopentylphenyl-p'-D-
galactopyranoside. Can. J. Microbiol. 22:654,1976.
17. Park, C.E., Rayman, M.K., and Stankiewicz, Z.K. Improved
procedure for selective enrichment of Shigella in the presence
of Escherichia coli by use of 4-Chloro-2-Cyclopentylphenyl-g-
D-galacctopyranoside. Can. J. Microbiol. 23:563,1977.
18. Taylor, W.I. and Harris, B. Isolation of shigellae. II.
Comparison of plating media and enrichment broths. Amer. J.
Clin. Pathol. 44:476,1965.
19. Martin, W.J. and Washington, J.A. II. Enterobacteriaceae. In:
E.H. Lennette, Balows, A., Hausler, W.J. and Truant, J.P.
(eds.), Manual of Clinical Microbiology,, 3rd ed. American
Society for Microbiology, Washington, D.C., 1980.
20. Lassen, J. Yersinia enterocolitica in drinking water. Scand. J.
Infect. Dis. 4:125,1972.
21. Schiemann, D.A. A synoptic review on Yersinia enterocolitica.
J. Environ. Health. 44:183,1982.
264
-------
25.
26.
27.
28.
.22, r. Highsmith, A.K., Feeley, J.C. and Morris, G.K. Yersinia
enterocolitica: a review of the bacterium and recommended
laboratory methodology. Health Lab. Sci. 14:253,1979.
23. Thompson, J.S. and Gravel, M.J. Family outbreak of
gastroenteritis due to Yersinia enterocolitica serotype 0:3 from
well water. Can. J. Microbiol. 32:700,1986.
24. Weagant, S.D. and Kaysner, C.A. Modified enrichment broth
for isolation of Yersinia enterocolitica from nonfood sources.
Appl. Environ. Microbiol. 45:468,1983.
Head, C.B., Whitty, D.A. and Ratnam, S. Comparative study of
selective media for recovery of Yersinia enterocolitica. J. Clin.
Microbiol. 16: 615,1982.
Schiemann, D.A. Synthesis of a selective agar medium for
Yersinia enterocolitica. Can. J. Microbiol. 25:1298,1979.
Weagant, S.D. Medium for presumptive identification of
Yersinia enterocolitica. Appl. Environ. Microbiol. 45:472,1983.
Sonnenwirth, A.C. Yersinia. In: E.H. Lennette, Spaulding,
E.H. and Truant, J.P., (eds.), Manual of Clinical Microbiology,
2nd ed. American Society for Microbiology, Washington, D.C.,
1974.
29. Swaminathari, B., Harmon, M.C. and Mehlman, I.J. A review:
Yersinia enterocolitica. J. Appl. Bacterial. 52:151,1982.
30. Jagow, J. and Hill, W.E. Enumeration by DNA colony
hybridization of virulent Yersinia enterocolitica colonies in
artificially contaminated food. Appl. Environ. Microbiol.
51:441,1986.
31. Jagow, J. and Hill, W.E. Enumeration of virulent Yersinia
enterocolitica colonies by DNA colony hybridization using
alkaline treatment and paper filters: Molec. Cell. Probes.
2:189,1988.
32. Bartley, T.D., Quan, T.J., Collins, M.T., and Morrison, S.M.
Membrane filter technique for the isolation of Yersinia
enterocolitica. Appl. Environ. Microbiol. 43:829,1982.
33. Levine, M.M. Escherichia coli that cause diarrhea:
enterotoxigenic, enteropathogenic, enteroinvasive, entero-
hemorrhagic, and enteroadherent. J. Infect. Dis. 155:377,1987.
34. Ewing, W.H. Sources of Escherichia coli cultures that belong to
0-antigen groups associated with infantile diarrheal disease. J.
Infect. Dis. 110:114,1962.
35. O'Mahony, M.C., Noah, N.D., Evans, B., Harper, D., Rowe, B.,
Lowes, J.A., Pearson, A., and Goode, B. An outbreak of
265
-------
gastroenteritis on a passenger cruise ship. J. Hyg. Camb.
97:229,1986.
36. Sack, R.B. Enterotoxigenic Escherichia coli: identification and
characterization. J. Infect. Dis. 142:279,1980.
37. Calderon, R.L. and Levin, M.A. Quantitative method for
enumeration of enterotoxigenic Escherichia coli. J. Clin.
Microbiol. 13:130,1981.
38. Sack, D.A., Huda, S., Neogi, P.K.B., Daniel, R.R., and Spira,
W.M. Microtiter ganglioside enzyme-linked immunosorbent
assay for Vibrio and Escherichia coli heat-labile enterotoxins
and antitoxin. J. Clin. Microbiol. 11:35,1980.
39. Thompson, M.R., Brandwein, H., Labine-Racke, M., and
Gianella, R.A. Simple and reliable enzyme-linked
immunosorbent assay with monoclonal antibodies for
detection of Escherichia coli heat-stable enterotoxins. J. Clin.
Microbiol. 20:59,1984.
40. Karch, H., Strockbine, N.A., and O'Brien, A.D. Growth of
Escherichia coli in the presence of trimethoprim-
sulfamethoxazole facilitates detection of shiga-like toxin-
producing strains by colony blot assay. FEMS Microbiol. Lett.
35:141,1986.
41. Kongmuang, U., Honda, T., and Miwatani, T. Enzyme-linked
immunosorbent assay to detect shiga toxin of Shigella
dysenteriae and related toxins. J. Clin. Microbiol. 25:115,1987.
42 Hill, W.E., Madden, J.M., McCardell, B.A., Shah, D.B., Jagow,
J.A., Payne, W. L., and Boutin, B . K. Foodborne
enterotoxigenic Escherichia coli: detection and enumeration
by DNA colony hybridization. Appl. Environ. Microbiol.
45:1324,1983.
43. Echeverria, P., Taylor, D.N., Seriwatana, J., and Moe, C.
Comparative study of synthetic oligonucleotide and cloned
polynucleotide enterotoxin gene probes to identify
enterotoxigenic Escherichia coli. J. Clin. Microbiol.
25:106,1987.
44. Hill, W.E., Payne, W.L., Zon, G., and Mosely, S.L. Synthetic
oligonucleotide probes for detecting heat-stable enterotoxin-
producing Escherichia coli by DNA colony hybridization. Appl.
Environ. Microbiol. 50:1187,1985.
45. Wood, P.K., Morris, Jr./J.G., Small, P.L.C., Sethabutr, 0.,
Toledo, M.R.E., Trabulsi, L., and Kaper, J.B. Comparison of
DNA probes with the Sereny test for identification of invasive
Shigella and Escherichia coli strains. J. Clin. Microbiol.
24:498,1986.
266
-------
46. Nataro, J.P., Baldini, M.M., Kaper, J.B., Black, R.E., Bravo,
N., and Levine, M.M. Detection of an adherence factor of
enteropathogenic Esckerichia coli with a DNA probe. J. Infect.
Dis. 152:560,1985.
47. Levine, M.M., Xu, J., Kaper, J.B., Lior, H., Prado, V., Tall, B.,
Nataro, J., Karen, H., and Wachsmuth, K. A DNA probe to
identify enterohemorrhagic Escherichia coli of 0157:H7 and
other serotypes that cause hemorrhagic colitis and hemolytic
uremic syndrome. J. Infect. Dis. 156:175,1987.
48. Skirrow, M.B. Campylobacter enteritis: a "new" disease. Brit.
Med. J. 2:9,1977.
49. Kaijser, B. Campylobacter jejuni/coli. APMIS. 96:283,1988.
50. Butzler, J.P. and Skirrow, M.B. Campylobacter enteritis. Clin.
Gastroenterol. 8:737,1979.
51. Karmali, M.A. and Fleming, P.C. Campylobacter enteritis in
children. J. Pediatr. 94:527,1979.
52. Blaser, M.J. and Keller, L.B. Campylobacter enteritis. N. Engl.
J. of Med. 305:1444,1981.
53. Centers for Disease Control. Waterborne Campylobacter
gastroenteritis -Vermont. Morbid. Mortal. Weekly Rep. 27:207,
1978.
54. Mentzing, L.O. Waterborne outbreaks of Campylobacter
enteritis in central Sweden. Lancet, ii: 352,1981.
55. Palmer, S.R., Gully, P.R., White, J.M.,Pearson, A.D., Suckling,
W.G., Jones, D.M., Rawes, J.C.L., and Penner, J.L. Waterborne
outbreak of Campylobacter gastroenteritis. Lancet, ii: 1002,
1978.
56. Knill, M., Suckling, W.G., and Pearson, A.D. Environmental
isolation of heat-tolerant Campylobacter in the Southampton
area. Lancet, ii: 1002,1978.
57. Taylor, D.N., Brown, M., and McDermott, K.T. Waterborne
transmission of Campylobacter enteritis. Microb. Ecol. 8:347,
1982.
58. Blaser, M.J., Berkowitz, I.D., La Force, P.M., Cravens, J.,
Cravens, L.B., Reller, L.B., and Wang, W.L.L. Campylobacter
enteritis: clinical and epidemiological features. Ann. Intern.
Med. 91:179,1979.
59. Carter, A.M., Pacha, R.E., Clark, G.W., and Williams, E.A.
Seasonal occurrence of Campylobacter spp. in surface waters
and their correlation with standard indicator bacteria. Appl.
Environ. Microbiol. 53:523,1987.
267
-------
60. Oosterom, J., Vereijken, M.J.G.M., and Engels, G.B.
Campylobacter isolation. Vet. Q. 3:104,1981.
61. Bolton, F.J. and Robetson, L. A selective medium for isolating
Campylobacter jejunilcoli. J. Clin. Pathol. 35:462,1982.
62. Bolton, F.J., Hinchliffe, P.M., Coates, D., and Robertson, L. A
most probable number method for estimating small numbers of
campylobacters in water: J. Hyg. Camb. 89:185,1982.
63. Mathewson, J.J., Keswick, B.H., and Du Pont, H.L. Evaluation
of filters for recovery ofCampylobacter jejuni from water. Appl.
Environ. Microbiol. 46:985,1983.
64. Lauwers, S., De Boeck, M. and Butzler, J.P. Campylobacter
enteritis in Brussels. Lancet. i:604,1978.
65. Bolton, F.J., Hutchinson, D.N., and Coates, D. Blood-free
selective medium for isolation of Campylobacter jejuni from
feces. J. Clin. Microbiol. 19:169,1984.
66. Kaplan, R.L. Campylobacter. In: Lennette, E. (ed.), Manual of
Clinical Microbiology, 3rd ed. American Society for
Microbiology, Washington, D.C., 1980.
67. Hutchinson, D.N., Bolton, F.J., Jones, D.M., Sutcliffe, E.M.,
and Abbott, J.D. Application of three typing schemes (Penner,
Lior, Preston) to strains of Campylobacter spp. isolated from
three outbreaks. Epidem. Inf. 98:139,1987.
68. Dilworth, C.R., Lior, H., and Belliveau, M.A. Campylobacter
enteritis acquired from cattle. Can. J. Publ. Hlth. 79:60,1988.
69. Lior, H., Woodward, D.J., Edgar, J.A., Laroche, I.J., and Gill,
P. Serotyping of Campylobacter jejuni by slide agglutination
based on heat-labile antigenic factors. J. Clin. Microbiol.
15:761,1982.
70. Penner, J.L., Hennessy, J.N., and Congi, R.V. Serotyping of
Campylobacter jejuni and Campylobacter coli on the basis of
thermostable antigens. Eur. J. Clin. Microbiol. 2:378,1983.
71. Lior, H. New, extended biotyping scheme for Campylobacter
jejuni, Campylobacter coli, and Campylobacter laridis. J. Clin.
Microbiol. 20: 636,1984.
72. Bolton, F.J., Holt, A.V., and Hutchinson, D.V. Campylobacter
biotyping scheme of epidemiological value. J. Clin. Pathol.
37:677,1984.
73. Rosef, 0., K.apperud, G., and Skjerve, E. Comparison of media
and filtration procedures for qualitative recovery of thermo-
tolerant Campylobacter spp. from naturally contaminated
surface water. Int. J. Food Microbiol. 5:29,1987.
268
-------
74. Ribeiro, C.D., Gray, S.J., and Price, T.H. A new medium for
isolating Campylobacter jejunilcoli. J. Clin. Pathol. 35:1036,
1982.
75. Bolton, F.J., Coates, D., Hinchliffe, P.M., and Robertson, L.
Comparison of selective media for isolation of Campylobacter
jejunilcoli. J. Clin. Pathol. 36:78,1983.
76. Goosens; H., De Boeck, M., Coignau, H., Vlaes, L., Van Den
Borre, C., and Butzler, J.P. Modified selective medium for
-isolation of Campylobacter spp. from feces: a comparison with
Preston medium, a blood-free medium and filtration system. J.
Clin. Microbiol. 24:840,1986.
77. Mawer, S.L. Campylobacters in man and the environment in
Hull and East Yorkshire. Epidem. Inf. 101:287, 1988.
78. Mawer, S.L. The pathogenicity of environmental
Campylobacters -a human volunteer experiment. Epidem. Inf.
101:295, 1988.
79. Dutka, B.J. Coliforms are an inadequate index of water
quality. J. Environ. Health. 36:39,1973.
80. Edberg, S.C., Allen, M.J., Smith, D.B., and the National
Collaborative Study. National field evaluation of a defined
substrate method for the simultaneous enumeration of total
coliforms and Escherichia coli from drinking water:
comparison with the standard multiple tube fermentation
method. Appl. Environ. Microbiol. 54:1595,1988.
81. Evans, T.M., Seidler, R.J., and Le Chevallier, M.W. Impact of
verification media and resuscitation on accuracy of the
membrane filter total coliform enumeration technique. Appl.
Environ. Microbiol. 41:1144,1981.
82. Camper, A.K. and McFeters, G.A. Chlorine injury and the
enumeration of waterborne coliform bacteria. Appl. Environ
Microbiol. 37:633, 1979.
83. Le Chevallier, M.W., Cameron, S.C., and McFeters, G.A. New
medium for improved recovery of coliform bacteria from
drinking water. Appl. Environ. Microbiol, 45:484,1983.
84. Le Chevallier, M.W., Cameron, S.C., and McFeters, G.A'.
, Comparison of verification procedures for the membrane filter
total eoliform technique. Appl. Environ. Microbiol. 45:1126
1983.
85. Gejdreich, E.E., Clark, H.F., Huff, C.B., and Best, L.C. Fecal
coliform organism medium for the membrane filter technique.
. .' J. Am. Water Works Assoc. 57:208,1965.
269
-------
86. Stuart, D.G., McFeters, G.A., and Schillinger, J.E. Membrane
filter technique for the quantification of stressed fecal
coliforms in the aquatic environment. Appl. Environ.
Microbiol. 34:42,1977.
87. Dufour, A.P., Strickland, E.R.,and Cabelli, V.J. Membrane
filter method for enumerating Escherichia coll. Appl. Environ.
Microbiol. 41:1152,1981.
88. McDade, J.E., Shepard, C.C., Fraser, D.W., Tsai, T.R., Redus,
M.A., and Dowdle, W.R. Legionnaires disease: isolation of a
bacterium and demonstration of its role in other respiratory
diseases. N. Engl. J. Med. 297:1197,1977.
89. Brenner, D.J., Steigerwalt, A.G., Gorman, G.W., Wilkinson,
H.W., Bibb, W.F., Hacket, M., Tyndall, R.L., Campbell, J.,
Feeley, J.C., Thacker, W.L., Skaliy, P., Martin, W.T., Brake,
B.J., Fields, B.S., NcEachern, H.V., and Corcoran, L.K. Ten
new species of Legionella. Int. J. Syst. Bacterial. 35:50,1985.
90. Thacker, W.L., Benson, R.F., Staneck, J.L., Vincent, S.R.,
Mayberry, W.R., Brenner, D.J., and Wilkinson, H.W.
Legionella cincinnatiensis sp. nov. isolated from a patient with
pneumonia. J. Clin. Microbiol. 26:418,1988.
91. Kirby, B.D., Snyder, K.M., Meyer, R.D., and Finegold, S.M.
Legionnaires' disease: clinical features of 24 cases. Ann.
Intern. Med. 89:297,1978.
92. Broome, C.V. Epidemiological assessment of methods of
transmission of Legionellosis. Zbl. Bakt. Hyg. I. Abt. Orig A
225: 52,1983.
93. Fliermans, C.B., Cherry, W.B., Orrison, L.H., Smith, S.J.,
Tison, D.L., and Pope, D.H. Ecological distribution of
Legionellapneumonhila. Appl. Environ. Microbiol. 41:9,1981.
94. Tobin, J.O., Bartlett, C.L.R., Waitkins, S.A., Barrow, G.I.,
Macrae, A.D., Taylor, A.G., Fallon, R.J., and Lynch, F.R.N.
Legionnaires' disease: further evidence to implicate water
storage and distribution systems as sources. Br. J. Med
282:573,1981.
95. Dufour, A.P. and Jakubowski, W. Drinking water and
Legionnaires' disease. Journal AWWA. 74:631,1982.
96. Meyer, R.D. Legionella infections: a review of five years of
research. Rev. Infect. Dis. 5:258,1983.
97. Friedman, S., Spitalny, K., Barbaree, J., Faur, Y., and
McKinney, R. Pontiac fever outbreak associated with a cooling
tower. Am. J. Publ. Hlth. 77:568,1987.
270
-------
98. Kaufmann, A.F., McDade, J.E., Fatten, C.M., Bennett, J.V.,
Skaliy, P., Feeley, J.C., Anderson, B.C., Potter, M.E.,
Newhouse, V.F., Gregg, M.B., and Brachman, P.S. Pontiac
fever: isolation of the etiologic agent (Legionella pneumophila)
and demonstration of its mode of transmission. Am. J.
Epidemiol. 114:337,1981.
Girod, J.C., Reichman, R.C., Winn, W.C., Jr., Klaucke, D.N.,
Vogt, R.L., and Dolin, R. Pneumonic and nonpneumonic forms
of legionellosis. Arch. Intern. Med. 142:545,1982.
Feeley, J.C., Gibson, R.J., Gorman, G.W., Langford, M.C.
Rasheed, J.K. Mackel, D.C., and Bain, W.B. Charcoal yeast
extract agar: a primary isolation medium for the Legionnaires'
disease bacterium. J. Clin. Microbiol. 16:437,1979.
Bopp, C.A., Sumner, J.W., Morris, G.K., and Wells, J.G.
Isolation of Legionella spp. from environmental water samples
by low-pH treatment and use of a selective medium. J. Clin.
Microbiol. 13:714,1981.
Wadowsky, R. and Yee, R.B. Glycine-containing selective
medium for isolation of Legionellaceae from environmental
specimens. Appl. Environ, Microbiol. 42:768,1981.
Edelstein, P.H. Improved semiselective medium for isolation of
Legionella pneumophila from contaminated clinical and
environmental specimens. J. Clin. Microbiol. 14:298,1981.
Calderon, R.L. and Dufour, A.P. Media for detection of
Legionella spp. in environmental water samples. In: C.
Thronsberry, Balows, A., Feeley, J., and Jakubowski, W. (eds.),
Legionella, Proceedings of the 2nd International Symposium.
American Society for Microbiology, Washington, D.C., 1984.
105. Feeley, J.C. and Gorman, G.W. Legionella. In: E. Lennette
(ed.), Manual of Clinical Microbiology, 3rd ed. American
Society for Microbiology, Washington, D.C., 1980.
106. Cherry, W.B. and McKinney, R.M. Direct immunofluorescence
tests for diagnosis of legionellosis. Am. J. Pathol. 103:448,
1981.
107. Edelstein, P.H. Evaluation of the Gen-Probe DNA probe for
the detection of legionellae in culture. J. Clin. Microbiol.
23:481,1986.
108. Pfaller, M.A. Laboratory diagnosis of infections due to
Legionella species: practical application of DNA probes in the
clinical microbiology laboratory. Lab. Med. 19:301,1988.
99.
100.
101.
102.
103.
104.
271
-------
109. Para, M.F. and Plouffe, J.F. Production of monoclonal
antibodies to Legionella pneumophila serogroups 1 and 6. J.
Clin.Microbiol. 18:895,1983.
110. Joly, J.R. and Winn, W.C. Correlation of subtypes of Legionella
pneumophila defined by monoclonal antibodies with
epidemiological classification of cases and environmental
sources. J. Infect. Dis. 150:667,1984.
111. Edelstein, P.H., McKinney, R.M., Meyer, R.D., Edelstein,
M.A.C., Krause, C.J., and Finegold, S.M. Immunologic
diagnosis of Legionnaires' disease: cross reactions with
anaerobic and microaerophilic organisms and infections
caused by them. J. Infect. Dis. 141:652,1980.
112. Barbaree, J.M., Gorman, G.W., Martin, W.T., Fields, B.S., and
Morrill, W.E. Protocol for sampling environmental sites for
legionellae. Appl. Environ. Microbiol. 53:1454,1987.
113. Hussong, D., Colwell, R.R., O'Brien, M., Weiss, E., Pearson,
A.D., Weiner, R.M., and Burge, W.D. Viable Legionella
pneumophila not detectable on agar media. Biotechnol. 5:947,
1987.
114. Colbourne, J.S., Dennis, P.J., Trew, R.M., Berry, C. and Vesey,
G. Legionella and public water supplies In: Proc. Int. Conf. on
Water and Wastewater Microbiol. 1:3-1,1988. ,
115. Nolte, F.S., Conlin, C.A., Roisin, A.J.M., and Redmond, S.R.
Plasmids as epidemiological markers in nosocomial
Legionnaires' disease. J. Infect. Dis. 149:251,1984.
116. Collins, C.H., Grange, J.M., and Yates, M.D. A review:
mycobacteria in water. J. Appl. Bacteriol. 57:193,1984.
117. Wolinsky, E. Nontuberculosis mycobacteria and associated
diseases. Am. Rev. Respir. Dis. 119:107,1979.
118 Kiehn, T.E., Edwards, F.F., Brannon, P., Tsang, A.Y., Maio,
M., Gold, J.W.M., Whimby, E., Wong, B., McClatchy, J.K. and
Armstrong, D. Infections caused by Mycobacterium avium
complex in the immunocompromised patients: diagnosis by
blood culture and fecal examination, antimicrobial
susceptibility tests, and morphological and seroagglutination
characteristics. J. Clin. Microbiol. 21:168,1985.
119. Du Moulin, G.C., Sherman, I.H., Hoaglin, B.C., and
Stottmeier, K.D. Mycobacterium avium complex, an emerging
pathogen in Massachusetts. J. Clin. Microbiol. 22:9,1985.
120. Goslee, S. and Wolinski, E. Water as a source of potentially
pathogenic mycobacteria. Am. Rev. Respir. Dis. 113:287,1976.
272
-------
121.
122.
123.
124
125.
126.
127.
128.
129.
130.
131.
132.
133.
Pelletier, P.A., du Moulin, G.C., and Stottmeier, K.D.
Mycobacteria in public water supplies: comparative resistance
to chlorine. Microbiol. Sci. 5:147,1988.
Du Moulin, G.C., Stottmeier, K.D., Pelletier, P.A., Tsang, A. Y.
and Hedley-White, J. Concentration of Mycobacterium avium
by hospital hot water systems. JAMA. 260:1599,1988.
Beerworth, W. Culture of mycobacteria from droppings of
domestic animals and the role of these bacteria in
epizootiology. Prax. Pneumol. 25:189,1967.
Bailey, R.K., Wyles, S., Dingley, M., Hesse, F., and Kent, G.W.
The isolation of high catalase Mycobacterium kansasii from tap
water. Am. Rev. Respir. Dis. 101:430,1970.
McSwiggan, D.A. and Collins, C.H. The isolation of M.
kansasii and M. xenopi from water systems. Tubercle 55-291
1974.
Du Moulin, G.C. and Stottmeier, K.D. Use of cetylpyridinium
chloride in the decontamination of water for culture of
mycobacteria. Appl. Environ. Microbiol. 36:771,1978.
Petran, E.I. and Vera, H.D. Media for selective isolation of
mycobacteria. Health Lab. Sci. 8:225,1971.
Lorain, V. and Maddock, S. The effect of. anticontamination
agents in media for the isolation of mycobacteria. Dis. Chest
50:630,1966.
Mitchison, D.A., Allen, B.W., Carrol, L., Dickinson, J.M. and
Aber, V.R. A selective oleic acid albumin agar medium for
tubercle bacilli. J. Med. Microbiol. 5:165,1972.
Sommers, H.M. and McClatchy, J.K. Mycobacterium. In: E.
Lennette (ed.), Manual of Clinical Microbiology, 3rd ed.
American Society for Microbiology, Washington, D.C., 1980.
Tsukamura, M. In vitro antimycobacterial activity of a new
antibacterial substance DL-8280 - differentiation between
some species of mycobacteria and related organisms by the DL-
8280 susceptibility test. Microbiol. Immunol. 27:1129,1983.
Paramasivan, C.N., Jackett, P.S., Coates, A.R.M., Lowrie, D.B.
and Mitchison, D.A. Monoclonal antibodies against
Mycobacterium aviumlintracellulare. Indian. J. Med. Res
88:13,1988.
Hampson, S.J., Thompson, J., Moss, M.T., Portaels, F., Green,
E.P., Herman-Taylor, J., and McFadden, J.J. DNA probes
demonstrate a single highly conserved strain of
Mycobacterium avium infecting AIDS patients. Lancet. i:65,
1989.
273
-------
134. Roberts, M.C., McMillan, C. and Coyle, M.B. Whole
chromosomal DNA probes for rapid identification of
Mycobacterium tuberculosis and Mycobacerium avium
complex. J. Clin. Microbiol. 25:1239,1987.
135. Butler, W.R. and Kilburn, J.O. Identification of major slowly
growing pathogenic mycobacteria and Mycobacterium
gordonae by high performance liquid chromatography of their
mycolic acids. J. Clin. Microbiol. 26:50,1988.
274
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Virological Analysis of Environmental Water
Samples
by: Christen J. Hurst, Ph.D
U. S. Environmental Protection Agency
Cincinnati, Ohio 45268
513-569-7331
Introduction
Those viruses which are of greatest concern regarding waterborne
disease transmission are the human enteric viruses. These viruses
replicate within, and are subsequently shed from, the human enteric
tract. Their replication is not necessarily limited to the enteric tract
and in many instances an infection can spread to other organs of the
body thereby resulting in different recognizable disease syndromes.
Table 5.6.1 presents a list of the different groups of human enteric
viruses and the illnesses they cause. After being shed in feces, these
viruses are transmitted from person to person through the ingestion of
fecally contaminated food or water. Enteric viruses are naturally
found in municipal sewerage and domestic waste drainage; from there
they may enter environmental surface and ground waters. Human
recreational activities both within and around bodies of water are
another possible source of viral contamination. Once in the water these
viruses might survive long enough, for periods of up to several weeks
or months depending upon climatic conditions, to complete a cycle of
disease transmission back to humans.
Human health concerns about such a waterborne cycle of viral
disease transmission have resulted in the development of methods for
detecting viruses present in environmental waters and drinking
water. Of the human enteric virus groups listed in Table 5.6.1,
detection efforts have largely been limited to the adenoviruses,
enteroviruses, and rotaviruses. Table 5.6.2 presents a list of the steps
that are involved in detecting these and other viruses contained in
water samples.
Preparation of Virus Samples
The first two steps in virus monitoring, sample collection, and on-
site processing, are often combined into a single operation in which
275
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Table 5.6.1. Human Enteric Viruses
Virus Group
Illness Caused
Adenovirus
Astrovirus
Calicivirus
Coronavirus
Enterically transmitted non-A, non-B
hepatitis virus (hepatitis E)
Enterovirus (includes polio, echo,
coxsackie groups A & B and hepatitis A)
Norwalk virus (possibly a calicivirus)
Parvovirus
Reovirus
Rotavirus
"Small round viruses"
(possibly enteroviruses)
Pharyngitis, conjunctivitis, respiratory
illness, vomiting, diarrhea
Vomiting, diarrhea
Vomiting, diarrhea
Vomiting, diarrhea
Hepatitis
Congenital heart anomalies, herpangina,
myocarditis, meningitis, encephalitis,
paralysis, rash, fever, hemorrhagic
conjunctivitis, respiratory illness, hepatitis
Epidemic vomiting and diarrhea'
One of two types possibly associated with
enteric infection
Fecally shed, but symptoms of illness not
well defined
Vomiting, diarrhea
Vomiting, diarrhea
Table 5.6.2. Steps Involved in Virus Monitoring
1. Collection of the water sample.
2. On-site processing of the sample.
3. Preservation and transport of the filter or resulting eluate fluid.
4. Laboratory processing including elution of the filter, if not already done in the
field, and secondary concentration of viruses from the resulting eluate fluid.
5. Titration and classification of the viruses. ,
6. Analysis and assessment of the results.
viruses are concentrated from a water sample through surface
adsorption onto the matrix of a cartridge filter. This adsorption occurs
during passage of the water sample through an appropriate filter, of
which several types are available and these differ in their
configuration and chemical composition. While bacteria are large
enough that they can be concentrated using filtration methods that
rely upon particle size exclusion, common to the "membrane filter"
(MF) methods, such techniques are not practical for use in isolating the
far smaller enteric viruses from environmental water samples.
The fact that viruses generally have a net negative electrostatic
charge at neutral pH is one of the most important considerations when
276
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utilizing adsorption methods for concentrating viruses from water
samples. In contrast, filter materials have either a net negative or
positive charge, which is established primarily by the filters' chemical
composition. If the filter material being used for virus concentration
has a net positive charge, then efficient viral adsorption may be
achievable without chemically altering the water sample. The
adsorption of viruses to positively charged filters is accomplished using
a flow-through system of the general design shown in diagram 1 of
Table 5.6.3. If the filter material instead has a net negative charge, it
is generally necessary to chemically modify the water samples prior to
filtration. The most commonly used modification consists of adding a
multivalent cationic salt such as aluminum chloride, and a sufficient
amount of dilute hydrochloric acid to lower the water sample's pH to
3.5. Required chemical additions can easily be performed using a flow-
through system incorporating either a proportioning pump or an in-
line injector ahead of the virus adsorbing filter, as shown in diagram 2
of Table 5.6.3. An alternative approach for achieving the required
chemical modification consists of batch adjustment of water samples
prior to filtration. This latter approach is shown in diagram 3 of Table
5.6.3. If the water sample being examined contains chemical
disinfectants such as chlorine, it is necessary to neutralize the residual
disinfectant activity prior to filtration of the water sample. Chemicals,
such as sodium thiosulfate, can serve to neutralize residual
disinfectant activity and are presumed not to have deleterious effect
upon the viruses.
The next stage of the virus concentration process consists of
recovering adsorbed viruses from the filters. Viral recovery is
normally accomplished by a reversal of the adsorption process,
achieved during the passage of an eluant fluid, such as beef extract^
through the filters. Following its passage through a filter, the eluant
solution is termed an.eluate, and should contain those viruses'which
had been adsorbed. This elution process can be performed either in the
field, or following transport of the filter to a laboratory. In the latter
case, it is necessary that the filter be kept in a sealed sterile container
during transport to prevent possible drying and contamination. As a
further precaution, the filter should be kept on ice during transport to
help protect against thermal inactivation of the adsorbed .viruses.
Viruses contained in filter eluates are normally subjected to a
secondary concentration procedure prior to their being assayed. Table
5.6.4 presents an outline of the steps used to elute viruses from filters
with a beef extract solution, and theft to secondarily concentrate them
via organic flocculation, a process which involves a low-pH
precipitation of proteinaceous compounds that are naturally present in
beef extracts. The mechanism by which viruses are concentrated
during organic flocculation relies upon their becoming associated with
the precipitate that is produced at low pH. This precipitate is collected
by centrifugation and then dissolved. The resulting solution is assayed
for presence of viruses. Overall, the recovery process concentrates
277
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Table 5.6.3. Equipment Set-up for Achieving Adsorption of Viruses onto Filters
1. Positively charged filters, flow-through system:
Pump —*- Prefilter -
Water .
Source
Virus
adsorbing
filter
Flow
meter
Discharge
' to waste
2. Negatively charged filters, flow-through system:
Aluminum
Dilute
chloride hydrochloric
solution acid
Water .
Source
Pump -
Prefilter
Proportioning
pump or In-
line injector
Virus
adsorbing
filter
Discharge
to waste
Flow
meter
3. Negatively charged filters, batch adjustment and filtration:
Volume-calibrated
container used to
combine the water
sample with
aluminum chloride
and dilute
hydrochloric acid
Pump
'Prefilter
Virus
adsorbing
filter •
Discharge
to waste
viruses from volumes of 100 to 400 L of water into approximately 1.5 to
2.0 L of filter eluate, and then secondarily concentrates the filter
eluate to between 75 and 100 mL of dissolved Ipw-pH precipitate. This
represents net concentration factors of approximately 1,00.0- to 5,000-
fold. '...''":'.'',." -.,'.'
Viral Assay Techniques (
The next step in virus monitoring consists of assaying the
dissolved low-pH precipitates for presence of infectious enteric viruses.
Because these viruses are obligate intracellular parasites, their assay
requires the use of live mammalian cells in order to achieve a
determination of infectiousness. Mammalian cells can be maintained
as cultures in the laboratory, and numerous established cell "lines" are
available. A great number of these cell lines are deemed continuous in
nature and can be kept in cultivation for a period of years during which
time the cells will continue to replicate, allowing the culture to be
278
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Table 5.6.4. Steps in the Elution of Viruses from Filters Using a Beef Extract
Solution Plus Subsequent Secondary Concentration of Viruses
from the Eluate Fluid
Air pressure
Eluant
reservoir
Virus
adsorbing
filter
Eluate
collection
container
(induce precipitation by
lowering eluate's pH to 3.5,
then centrifuge at low speed)
collect
precipitate
discard
supernate
dissolve and
assay for
viruses ,
periodically subdivided. Separate subcultures of the cell lines can then
be prepared in commercially available, specially designed tubes,
flasks, chambered microscope slides, or petri dishes. Table 5.6.5
contains brief descriptions of four different types of viral assays that
have been used for detecting indigenous viruses present in
environmental samples. Each of these assay types detects a different
viral effect or material that results from the replication of inoculated
viruses in cultures of susceptible cells. These assay types differ in the
speed with which a successful endpoint can be reached^ corresponding
to the time required for the virus to produce the particular detected
effect or material.
Traditional plaque formation assays require the production of
expanding focal areas of cell death, termed "plaques," within cultures
of cells. The results of such assays are calculated in terms of plaque
forming units per volume of sample. Determination of cell death may
be assisted by the incorporation of a vital stain, such as neutral red,
into the cell culture medium. This type of assay requires that an
infecting virus complete its cycle of replication, killing the initial host
cell during the process, and generate progeny viruses which then infect
and kill neighboring host cells. Gelling agents, such as agar, are often
added to the cell culture media used in plaque formation assays. These
gelling agents serve to reduce the rate of viral spread throughout the
infected cell cultures, in turn facilitating the detection of more than a
single center of infection per culture. While plaque assay techniques
are often considered to be traditional for detecting infectious viruses
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Table 5.6.5. Comparative Description of Viral Assay Techniques
Type of Assay
Technique
Viral Effect or
Material which is
Detected
Comments
Plaque
formation
Cytopatho-
genicity
Cell Immuno-
fluorescence
or Enzyme
Immunoassay
In Situ Nucleic
Acid
Hybridization
Focal areas of cell
death induced in a
culture of cells •
Characteristic
alterations in cell
morphology resulting
from viral infection
Viral proteins
produced in cells
during the course of
infection
Viral nucleic acids
produced in cells
during the course of
infection
Traditional and sensitive but slow.
Assay titers may vaiy depending ori
factors involved with the assay,
including whether improvements like
the use of a suspended cell culture
technique are employed. Assay
results are determined with unaided •
eye and facilitated by incorporation of
a vital stain into the cell culture
medium.
This assay technique is older, more
rapid, and potentially more sensitive
than plaque formation. Results are
determined visually by using light
microscopy.
Faster than either plaque formation or
cytopathogenicity and considered
approximately equal in sensitivity.
Range of sensitivity is .determined by
the specificity of antibody
preparations used to locate the
position of viral proteins within
infected cells. Assay results can be
determined visually using light
microscopy. ,
Recently developed and either equal
• in speed or faster than any of the
above assay techniques. Range of
sensitivity is determined by the
specificity of nucleic acid strands
used to locate the position of viral .,
materials produced within infected '
; cells. Assay results can be
determined visually using light
< microscopy,
isolated from environmental samples, this type of assay is
operationally slow.
Cytopathogenicity assays are based upon the detection of
characteristic morphological changes, termed "CPE", as an
abbreviation for "cytopathogenic effects," resulting from the
replication of viruses in host cells. Such morphological changes often
appear prior to the actual death of the host cell; partially for this
reason, cytopathogenicity assays generally require less time for
completion than do plaque formation assays. The development of•
cytopathogenicity assays predates that of plaque formation, and
cytopathogenicity assays are in some ways more cumbersome. While it
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is often possible to distinguish several discrete plaques within a single
culture of cells, the same individual cell cultures could only be rated as
either positive or negative if used for performing viral
eytopathogenicity assays. This is due to the fact that viral infection
rapidly spreads throughout the entire culture of cells during
cytopathogenicity assays. Gelling agents cannot successfully be used
to limit viral spread in cytopathogenicity assays because their
presence may obscure the development of viral cytopathogenic effects.
For these reasons, quantitative cytopathogenicity assays require a
greater number of cell cultures than do plaque assays, although for
cytopathogenicity assays each individual culture can be smaller in
size. Results of cytopathogenicity assays are determined by calculating
the most probable number of viral infectious units per volume of
sample.
Cell immunofluorescence and cell enzyme immunoassay
procedures detect progeny viral proteins produced during the course of
viral replication in infected cells. Both of these approaches expose the
infected cells to aqueous preparations containing antibodies used as
"probes," which specifically recognize and bind to the produced cell-
associated viral proteins serving as their "targets." Subsequent
visualization of bound antibodies relies upon fluorescing compounds or
enzymes that are covalently linked to either the probe antibodies
themselves or to "third party" compounds sequentially applied to the
infected cells and which adhere to the viral-specific probe antibodies.
In situ nucleic acid hybridization assays detect progeny viral nucleic
acids that are produced in infected cells during the course of virus
replication. This approach is operationally similar to those of cell
immunofluorescence and enzyme immunoassay, the major difference
being that the hybridization techniques utilize nucleic acid strands
rather than antibodies as the probes. The nucleotide sequence of these
probe nucleic acid strands is complementary to the sequence of the
targeted progeny viral nucleic acid material. Following the application
of a solution containing the appropriate nucleic acid probe to virally
infected cells, the probe and target viral nucleic acids form "hybrid"
double stranded nucleic acid molecules. Subsequent visualization of
the bound probe nucleic acid strands can be accomplished utilizing
either fluorescence- or enzyme-based detection systems similar to
those employed for the antibody-based assays.
Both the antibody-based and nucleic acid-based assay techniques
require shorter time periods for completion than either the plaque
formation or cytopathogenicity assays. This shorter time period is a
great advantage in environmental monitoring situations, and results
because progeny viral proteins and nucleic acids can be detected early
in the viral replicative cycle, often prior to the development of visual
cytopathogenic effects or cell death. Results from the antibody or
nucleic acid assay techniques can be calculated directly in terms of
infectious units per volume of sample by microscopically counting the
number of viral positive cells present in the cultures used for the
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assays. Alternatively, the inoculated cell cultures may be judged
positive versus negative and the associated viral titer calculated as a
most probable number. The antibody and nucleic acid assay techniques
described above are newer to science than are either plaque formation
or cytopathogenicity assays. However, the history of using
immunofluorescence assays for viral detection spans more than 30
years, and viral applications for in situ nucleic acid hybridization span
more than a decade.
Assessing the Results of Virological Testing
Enteric viruses can be found both in environmental waters and
drinking waters. Viral pollution may be presumed to constitute a
potential health hazard for persons consuming or otherwise coming
into physical contact with the water. While it is possible to sample
waters for the presence of human enteric viruses, practical laboratory
techniques are not yet available for determining infectivity of all the
viral groups listed in Table 5.6.1. Practical infectivity techniques do
exist for members of the adenovirus, enterovirus (including hepatitis A
virus), reovirus, and rotavirus groups. The other viral groups listed in
Table 5.6.1 can readily be detected by antibody- or nucleic acid-based
in, vitro assays, i.e., assay techniques which do not utilize live host
cells. Immune electron microscopy techniques can also be used to
detect all of the different virus groups. However, in vitro assays and
electron microscopy techniques are relatively insensitive when
compared with the viral infectivity assays described in this paper and,
in their currently available form in vitro assays are impractical for use
when detecting viruses in environmental water samples. It should also
be recognized that, by themselves, the in vitro and electron microscopy
techniques cannot yield information regarding viral infectiousness. It
is hoped that suitable viral infectivity assays will in the future be
developed for the other human enteric virus groups listed in Table
5.6.1.
Temporal and Epidemiological Considerations in Virus Sampling
An important factor to consider when assessing the relative merits of
examining a water source for the presence of enteric viruses is the
likelihood that causative agents might still be in the suspected water
source when it is sampled. All viral diseases involve an incubation
period between the time a person is exposed to the virus and the
subsequent onset of symptoms. The length of incubation for the enteric
viruses varies from one virus type to another, ranging from a few days
for Norwalk virus to more than one month for human enterovirus 72
(hepatitis A virus). These incubation periods can result in substantial
time delays between the exposure of a population to the suspected viral
agent and subsequent identification of an ongoing disease epidemic.
Another factor to be considered is whether the outbreak pattern
suggests that exposure of the population was to acute versus chronic
water contamination. Suggestion of chronic contamination involving a
282
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virus whose illness has a short incubation period would yield the
greatest chance of finding the organism still present in the water
source at the time of the outbreak investigation.
Suggested References
1. Akin, E.W. Occurrence of viruses in treated drinking water in
2.
3.
4.
6.
7.
8.
9.
10.
11.
the United States. Water Science and Technology. 17:689-700,
Belshe, R.B. Textbook of Human Virology. PSG Publishing
Company, Littleton Massachusetts, 1984.
Berg, G.( Safferman, R.S., Dahling, D.R., Berman, D., and
Hurst, C.J. U.S. EPA Manual of Methods for Virology Report
No. EPA- 600/4-84-013. U. S. Environmental Protection
Agency, Cincinnati, Ohio, 1984.
Dahling, D.R. and Wright, B.A. Optimization of the BCM cell
line culture and viral assay procedures for monitoring viruses in
the environment. Appl. Env. Microbiol. 51:709-812, 1986.
Hurst, C.J., and Benton, U.N., and R.E. Stetler. Detecting
viruses in water. A WWA. 81(9) 71-80, 1989.
Hurst, C.J. and Stetler, R.E. Recent advances in the detection of
human viruses in drinking water. In: Proceedings of the 15th
Water Quality Technology Conference. American Water Works
Association, Denver, Colorado, 1988. pp. 943-956.
Hurst, C.J., McClellan, K.A., and Benton, U.N. Comparison of
cytopathogenicity, immunofluorescence, and in situ DNA
hybridization as methods for the detection of adenoviruses
Water Research. 22:1547-1552, 1988.
Melnick, J. L., Safferman, R., Rao, V.C., Goyal, S., Berg, G
Dahling, D,R, Wright, B.A., Akin, E., Stetler, R., Sorber C '
Moore, B., Sobsey, M.D., Moore, R., Lewis, A.L., and Wellings'
F.M. Round robin investigation of methods for the recovery of
poliovirus from drinking water. Appl. Env. Microbiol. 47:144-
150, 1984.
Payment, P., Morin, E., and Trudel, M. Coliphages and enteric
viruses in the particulate phase of river water. Canadian
Journal of Microbiology, 34:907-910, 1988.
Payment, P. and Trudel, M. Immunoperoxidase method with
human immune serum globulin for broad-spectrum detection of
cultivable human enteric viruses: application to enumeration of
cultivable viruses in environmental samples. Appl Env
Microbiol. 50:1308-1310, 1985.
Payment, P. and Trudel, M. Wound fiberglass depth filters as a
less expensive approach for the concentration of viruses from
water. Canadian Journal of Microbiology. 34:271-272, 1988.
283
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Field Method for Concentrating Viruses from Water
Samples
by: Christen J. Hurst, Ph.D.
U. S. Environmental Protection Agency
26 Martin Luther King Drive
Cincinnati, Ohio 45268
(513) 569-7461
Purpose
The procedure described can be used to concentrate suspected
human enteric viruses from both environmental freshwaters and tap
waters in conjunction with waterborne outbreaks. The produced
concentrated sample materials can then be examined for the presence
of viruses using other laboratory procedures as desired. The
concentration technique described here utilizes a three-stage process
consisting of directed virus adsorption onto a solid surface, using in
this case a cartridge membrane filter, combined with subsequent use of
a proteinaceous eluant fluid to recover the adsorbed viruses and
secondary concentration of the eluant fluid. The basic theory behind
these methods has been described by Hurst, Benton and Stetler (J
Amer. Water Works Assoc., Vol. 81, No. 9, pp. 71-80, 1989) The
volume of water sample that is processed when looking for presence of
viruses is not fixed by convention, although a minimum useful
quantity would probably be 25 gallons, and an amount of 30 to 50
gallons preferred. For some very low turbidity waters, such as sand-
filtered tap waters, the apparatus described here could be used for
processing volumes as great as 100 gallons.
Equipment and Reagents
The following materials will be needed and can be prepared ahead
of time. Some will be required at the field site where the water sample
is to be processed using the filters, others will be used in the laboratory
for elution and secondary concentration of the filter eluant All parts of
the equipment and laboratory apparatus that will come into direct
contact with either the water sample, beef extract eluant fluid or
final virus concentrate" must first be thoroughly cleaned and
sterilized. All reagents that will be used during any of these operations
must also be sterilized. Methods which can be used for sterilization
include autoclaving, ethylene-oxide gas treatment, sterile filtration
(solutions only!) and chemical treatment using solutions such as
calcium hypochlorite or sodium hypochlorite. Sodium hypochlorite
285
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solution can be purchased as standard household liquid bleach. Either
of these hypochlorite solutions can be used for field sterilization of
water storage containers and the other equipment described in this
procedure with exception of the filters. The filters described in this
study should be sterilized by ethylene oxide gas treatment.
Items to be shipped to the FIELD in preparation for sampling, or
otherwise made available there:
(Except for the insulated shipping container and optional stands
for supporing the filter holders, which do not need to be sterile, all
of the following filtration apparatus components and reagents can
be sterilized before they are transported to the field site. The
eluant can be sterilized within the 1- gallon polypropylene storage
container by autoclaving, the optional sodium thiosulfate solution
can be sterilized either by autoclaving or sterile filtration, and all
of the equipment except for gasoline powered pumps can generally
be sterilized by ethylene oxide treatment. In addition to the
equipment listed below, other items that should be available in the
field are a compact field type DPD chlorine test kit if you are
sampling chlorinated tap waters and therefore need to confirm
that you have completely neutralized all free chlorine or other
chlorine compounds present in the water sample by adding the
prescribed quantity of sodium thiosulfate solution. A portable pH
meter would also be needed if you will be sampling waters that
need to have their pH adjusted prior to the filtration process. The
procedure described relies upon virus-adsorbing filters that are
considered electropositive, and as such the only time that pH
adjustment would likely be required is when using filters of the 1-
MDS type for recovering viruses from waters whose pH is above a
value of approximately 7.5. Filters of the Posidyne N-66 type
appear to work well at pH levels up to 8.5.
1. Container for storage of water sample prior to concentration
(If necessary, a new plastic garbage can with fitting cover can be
used for this purpose.)
2. Light weight portable electric or gasoline powered water pump
fitted for use with either quick-disconnect plumbing adaptors or
garden hose couplings. All plumbing adaptors or couplings should
either be brass or stainless steel to reduce problems associated
with their corrosion during sterilization using chlorine compounds
or autoclaving.
(The style of pumps that you will want to use are commonly
referred to as "portapumps" and weigh less than twenty pounds.
Larger pumps are not required for this procedure and their use
would not offer appreciable advantage since the filtration
apparatus has a limited maximum flow rate. If electricity will be
available at the site where the sample is to be processed, then take
286
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advantage of this fact and plan on using an electric pump of 1/6 to
1/2 hp. Electric pumps are often lighter in weight and easier to
handle as compared to gasoline powered pumps. Also, some electric
pumps can be sterilized in advance using ethylene oxide gas
treatment. The pump chosen for use should be self-priming and, if
electric, should be thermally protected. In this case, thermal
protection means that the pump will automatically shut off if it
overheats. The only parts of a pump which need to be sterile are
those which come into contact with the water sample, and these
parts of a gasoline powered pump should be sterilized by treatment
with hypochlorite solution.)
3. Two filter holders designed for use with standard 10-inch length
cartridge water filters, fitted for use with either quick- disconnect
plumbing adaptors or garden hose couplings.
(The filter holders should have clear base sections so that the
filtration process can be visually monitored, particularly for air
pockets which occasionally develop within the filter holder and
impede the water flow. Suitable holders should be available from
any of the manufacturers and distributors for the cartridge filters
that are listed below. The top of each filter holder should be fitted
with a stainless steel finger- operated air pressure release valve
connecting to that portion of the filter holder which will be outside
the resting position of the cartridge filter. The installation of air
pressure release valves in this manner allows for easy elimination
of air pockets from the filter holder. Figure 5.7.1 is a diagram of a
filter holder equiped with a pressure release valve. These air
pressure release valves can be purchased from filtration
equipment supply companies and, if necessary, installed by a
maintenance shop.)
4. Portable water meter fitted for use with either quick-disconnect
plumbing adaptors or garden hose couplings.
(Light weight water meters with sturdy plastic housings are
available from plumbing suppliers.)
5.
6.
Four lengths of fiber-reinforced garden hose fitted for use with
either quick-disconnect plumbing adaptors or garden hose
couplings.
(Three of the hoses will be needed for connecting the pump, filter
holders containing their cartridge filters, and water meter to form
the virus concentration apparatus shown in Figure 5.7.2. The
fourth piece of hose will be used to direct outflow from the water
meter.)
One length of strong-walled water supply hose fitted for use with
either quick-disconnect plumbing adaptors or garden hose
couplings.
287
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Air Pressure Release
Valve
(Finger Operated)
Inflow
\
Outflow
Figure 5.7.1. Filter holder with cartridge filter in place.
(For this hose a coupling will be required on only that end which
attaches to the pump. Garden hose would tend to collapse inward
during operation of the pump and thus may not be suitable for this
particular usage. It may be helpful to have a stainless steel or
brass wire strainer fitted to the intake end of the water supply hose
when concentrating environmental waters by pumping directly
from their source through the filters. Use of a strainer in this
instance will help to prevent the water supply hose from clogging.)
7. Two 10-inch cartridge filters.
(For each water sample that is to be processed you will need both a
prefilter and a virus-adsorbing filter. These filters should not be
reused. The prefilter should be a 3-micron nominal porosity wound
polypropylene yarn filter with a hollow perforated stainless steel
core, available as stock number M27R10S from Commercial
Filters Division, Carborundum Company, Lebanon, Indiana. The
virus-adsorbing filter should be either a pleated 0.20 micron
porosity charged nylon membrane type, or a pleated 0.45 micron
"288
-------
I
O)
ill
o>
Is
-------
porosity charged glass fiber membrane type. The first of these two
is available as a Posidyne N-66 filter cartridge from Pall Trinity
Micro Corporation, Cortland, New York, the second is available as
a 1-MDS filter cartridge from AMF Cuno Division, Meriden,
Connecticut. Both of these virus-adsorbing filters are considered to
be positively charged relative to untreated cellulose ester or glass
fiber filters, and are therefore suitable for concentrating viruses
from water at most ambient pH levels. The charged nylon filter
would be preferred for use with water samples of moderately
alkaline pH level. If you are instead using the charged glass fiber
filter, it would be preferable that dilute hydrochloric acid be used
to adjust the pH of the any alkaline water samples to less than 7.5
before filtration. The wound polypropylene cartridge filters and
pleated membrane cartridge filters can be presterilized within the
filter holders using ethylene oxide gas before they are transported
to the field site.)
8. Eluant in plastic storage container.
(For each water sample to be processed, you will need 1600 mL of
sterile, pH 7, 3-percent beef extract solution contained in a
separate water-tight, 1-gallon capacity, wide-mouth, screw-
capped autoclavable polypropylene container. Polypropylene is
preferred for this purpose because it is autoclavable and viruses do
not readily adsorb to polypropylene. The eluant solution can be
sterilized by autoclaving it inside the polypropylene containers.
Containers of this type are available as stock number 2121-0010
from Nalge Company, Division of Sybron Corporation, Rochester,
NY. The beef extract solution should consist of 48 grams of
microbiological grade beef extract powder dissolved in distilled
water. It would be best if at least one half of this powdered beef
extract were of the type sold as Beef Extract V by BBL
Microbiology Systems, Cockeysville, Maryland. This will help to
assure that an easily visible precipitate is produced during
subsequent secondary concentration of the filter eluant.)
9. Light-weight insulated container, with ice packs, suitable for
mailing.
(This will be used to ship the used virus-adsorbing filter, inside the
polypropylene container of eluant, by overnight delivery service to
the laboratory where the elution and secondary concentration
steps are to be performed.)
10. Stands to support the filter holders in upright position during the
water filtration process. These are optional.
(Stands of this type can be made of clear plastic or of wood provided
that, if made of wood, it is still possible to see the clear base portion
of the filter holder while in use. The stands do not need to be
sterile.)
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11. Solutions of calcium hypochlorite (0.5% w/v) and sodium
thiosulfate (50% w/v). These are optional unless you will need to
field sterilize either a storage container or other equipment. The
thiosulfate solution would also be needed if you are intending to
process a sample of previously chlorinated water such as tap water.
(The calcium hypochlorite solution will be used for field
sterilization of equipment as necessary and does not, itself, need to
be sterilized. Standard household liquid bleach, sold as 5.25%
sodium hypochlorite by weight, can be used as a substitute for the
calcium hypochlorite solution with the realization that this bleach
is appproximately 10-fold stronger than the recommended calcium
hypochlorite solution. The sodium thiosulfate solution can be
sterilized either by filtration or autoclaving and will be used to
neutralize residual chlorine present on field equipment and also to
neutralize residual disinfectant activity in samples such as tap
water which may already contain free chlorine or chlorine
compounds. Pipettes or other means of measuring the hypochlorite
and thiosulfate solutions will also be needed.)
Items to be used in the LABORATORY upon return of the virus
adsorbing filter and eluate:
1. pH meter, standardization buffers used for calibration of the
meter, dilute (1 M) solutions of hydrochloric acid and sodium
hydroxide used for pH adjustment, and mechanical stirring
apparatus.
(These items will be used for adjusting the pH of the beef extract
eluant and also the dissolved low-pH precipitate, the latter being
the end product of the secondary concentration process.)
2. Centrifuge with centrifuge bottles capable of being used at
1350 xg.
(These items will be used to collect precipitate produced during the
secondary concentration process. It is preferable to use a swinging
bucket rotor head in the centrifuge. This will deposit the collected
precipitate in a layer at the bottom of the centrifuge bottle and
thus can simplify dissolution of the precipitate.)
3. Sterile, pH 9.0, 0.15 M solution of dibasic sodium phosphate.
(This is used to dissolve the low-pH precipitate produced during
the secondary concentration procedure.)
4. Sterile filter holder identical to those used in the field for the virus
adsorbing filters.
5. Low-capacity electric pump for recirculating pH adjusted eluant
through the virus-adsorbing filter contained in a filter holder.
(A peristaltic pump may be adapted for this purpose. The
alternative would be to use a 1-gallon capacity reservoir container
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or "pressure can", designed for positive pressure filtration of fluids,
plus a suitable hose connecting the pressure can to a source of
compressed air or nitrogen gas used for pushing the eluant fluid
through the filter. Carbon dioxide gas should not be used for this
purpose since it may change the pH of the eluant fluid. In common
practice only a very limited amount of pressure is required. The
amount of pressure applied must never exceed that recommended
by the manufacturers of either the filters, filter holders, hoses,
couplings, or fluid reservoir. Be mindful that using pressurized gas
will necessitate the use of a suitable gas pressure regulator.)
6. Two hoses with fittings that match those on the filter holder to the
pump or pressure can.
(Items 4, 5 and 6 will be used to circulate the eluant fluid through
the filter after the eluant has been adjusted to pH 9.5. The use of an
antifoaming agent, such as Antifoam C from Dow-Corning Corp.,
Midland, Michigan, added directly to the eluant may be helpful if
foaming is a problem when eluting viruses from the filter. This
particularantifbaming compound should be used at a ratio of 0.1
mL antifoam per 100 mL of beef extract eluant solution, for a total
amount of 1.6 mL antifoam C in 1600 mL of eluant).
7. Other routine laboratory supplies such as pipettes and beakers
will also be needed in addition to a suitable container for storing
the final virus concentrate.
Procedure for Concentrating Viruses from Water Samples
The procedure listed below can be used for concentrating viruses
directly from unchlorinated surface water or groundwater sources, or
else from a stored water sample representing either of these. If the
sample being examined instead consists of tap water, then the sample
will first need to be collected into a storage container and immediately
dechlorinated using sodium thiosulfate stock solution. The latter point
is very important since it would be unlikely to find viable viruses in
low turbidity water that had been exposed for a prolonged time to even
•the relatively modest concentrations of chlorine as are normally added
to drinking water. If the water sample is to be placed into a storage
container for any reason, then that container must first be sterilized. A
method for sterilizing water storage containers is described below.
Preparation of water storage containers prior to their use:
The container used for temporary storage of a water sample should
be visably clean and sterile, and should not previously have been used
for any purpose other than the short term storage of water samples.
Large, up to 50-plus gallon capacity, polyethylene containers are
available from scientific supply companies and can be used for storage
of water samples. In an emergency situation, as mentioned above, a
new plastic garbage can may be used for this purpose. Field
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sterilization of the container should be, done by first completely filling
it with clear water, and then adding calcium hypochlorite solution (0.5
% w/v) to the water at a rate of 3.8 mL per gallon. A 10% solution of
standard household liquid bleach (sold as 5.25 % sodium hypochloride
by weight) can be used as a substitute for the calcium hypochlorite
solution. The hypochlorite solution should be throughly mixed into the
water, and this water then allowed to remain in the storage container
for at least 30 minutes. The chlorinated water should then be emptied
from the storage container, the container rinsed very thoroughly with
clear water, arid again completely filled with clear water. Sodium
thiosulfate solution should be added to this second filling of water at a
rate of 10 mL per gallon. The sodium thiosulfate solution should
thoroughly be mixed into the water, and the water then allowed to
remain in the storage container for at least 30 minutes. The container
can then be emptied and filled with the intended water sample, If the
intended water sample consists of tap water or water that for other
reasons may have received either chlorine or a chlorinated compound
serving as a disinfectant, then the collected water sample should also
be dosed with sodium thiosulfate solution at a rate of 10 mL per gallon
before it is processed for concentrating any viruses which it may
contain. If possible, the water sample should be processed immediately
upon its collection using the virus concentration apparatus. If it is
necessary to store the water sample, the storage container should be
kept covered to prevent contamination and in a cool location to reduce
viral inactivation until such time as the water sample can been
processed. Prolonged storage of water samples should be done in a
refrigerated room.
Field Sterilization of Equipment:
All of the equipment used in the virus concentration apparatus, except
for the filters, can be field sterilized if such proves to be necessary. The
individual parts should be sterilized separately in order to make
certain that all surfaces receive thorough exposure to the hypochlorite
disinfectant and that all residual hypochlorite is then neutralized
before the water sample is processed through the system. Hoses and
assembled filter holders (without filters), with their air pressure
release valves open, should be carefully, submerged in a solution of
clear water containing hypochlorite prepared as described above for
treatment of storage containers. Be certain that there are no pockets of
trapped air in either the hoses or filter holders, as this may preclude
complete sterilization. Keep the hoses and filter holders completely
submerged in this chlorinated water for 30 minutes, then remove and
drain the hoses and filter holders. Next, similarly treat the hoses and
filter holders by submersion for 30 minutes in clear water containing
s6diu,m thiosulfate solution prepared as described above for treatment
of sample storage containers. The hoses and filter holders should be
drained and can then'be used immediately or, if to be used at some
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latter time, their openings should be wrapped with sterile aluminum
foil or some other type of sterile covering to prevent contamination.
Field sterilization of pumps can be done by first continuously
recirculating clear water containing hypochlorite solution, prepared as
described above, through them for 30 minutes. Residual chlorinated
water should then be drained from the pump and its necessarily
attached hoses. Clear water containing sodium thiosulfate solution,
likewise prepared as described above, should then be recirculated
through the pump and attached hoses for 30 minutes. Residual water
containing thiosulfate solution should then be drained from the pump
and attached hoses. The pump and hoses can then immediately be used
to process a water sample. If the pump and its attached hoses are not to
be used immediately, their openings should receive a sterile covering
as described above to prevent contamination.
Concentration of the water sample:
Assemble the virus concentration apparatus as shown in Figure 2,
making certain to properly align the pump, filter holders containing
their respective filters, and water meter, with respect to their
indicated directions of water flow. Be certain that the tops of the filter
holders are fully tightened to their bottom sections, that the filter
holders contain the correct filters, and that the air pressure release
valves on the filter holders are closed. Connect one end of the water
supply hose to the pump, and place the other end of this hose into the
water being tested, whether it is being taken directly from an
environmental water source or from a storage container prepared as
described above. Note the reading on the water meter, as water meters
usually cannot be reset, and in order to know how much water sample
you have filtered it will be necessary to read the meter both before and
after the filtration. Turn on the pump and let the sample water pass
through the filters and meter. It may be necessary to occasionally
release trapped air pockets from the filter holders. The presence of air
pockets that form within the filter holders can easily be seen by
looking at the clear bottom parts of the holders. Such air pockets
should be
released as they will otherwise reduce the rate at which water is able
to flow through the filters. To release air, first make sure that the
holder in question is being held upright so that the trapped air can
pass through the release valve. While keeping the holder upright,
slightly open the valve until the air has passed through, and then close
the valve to keep the water sample from leaking out. After you have
filtered the water sample, disassemble the apparatus and allow any
remaining water to drain from the filter holders. The filter holders
should then be opened carefully, the wound polypropylene pre-filter
discarded, and the virus-adsorbing membrane filter aseptically placed
into the container of beef extract solution (eluant). The virus adsorbing
filter should be kept in the container of eluant and both immediately
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placed into the insulated container, surrounded with ice packs, and
shipped to the laboratory by overnight delivery service.
Laboratory procedure for elution of adsorbed viruses from the filter and
secondary concentration of the filter eluant:
Aseptically remove the virus-adsorbing filter from its container of
eluant and place the filter into a filter holder that has been
presterilized (remember that you must presterilize all reagents and
parts of equipment which will come into direct contact with either the
water sample, beef extract eluant fluid, or final virus concentrate). The
top of the filter holder should be fully tightened to its bottom section
and the air pressure release valve closed. The filter holder should then
be connected by hoses to either the pump or the pressure can which
will hold the eluant. Next, while continuously stirring the filter
eluant, adjust it to a pH of 9.5 by addition of sodium hydroxide and
hydrochloric acid as necessary. The eluant should then be passed
through the filter three times sequentially with the flow of eluant
being opposite of that used during the virus adsorption process. It may
be necessary to eliminate air pockets from the filter holder using the
air pressure release valve. This represents a reversal of the virus
adsorption technique described above. The filter can now be discarded.
The eluant, including any remaining in the filter holder, pump
chamber, pressure can or hoses should then be lowered to pH 3.5 by
addition of hydrochloric acid using continuous stirring. During this
latter pH adjustment the eluant should become cloudy as an organic
precipitate forms. The pH 3.5 eluant should be stirred for 30 minutes,
and then centrifuged for 20 minutes at 1350 x g to collect the
precipitate. The supernate resulting from this centrifugation should be
discarded and the precipitate from all 1600 mL of eluant dissolved in
place within the centrifuge bottles using a total volume of 80 mL of
sterile, pH 9.0, 0.15 M dibasic sodium phosphate solution prepared in
distilled water. The pH of the dissolved precipitate, now called "final
virus concentrate", should then be checked and if not already between
7.0 and 7.2, adjusted to within that range by adding sodium hydroxide
or hydrochloric acid. Antibiotics can be added to the final virus
concentrate if such is desired, and the concentrate should then be
stored either under refrigeration (for a very few days) or frozen at -70
to -80°C. The final virus concentrate can then be assayed as desired for
the presence of viruses. The most commonly used assays for this type of
sample are plaque formation, cytopathogenicity (CPE), infected cell
immunofluorescence, and in-situ nucleic acid hybridization.
(Note: the filter and its eluant should be processed immediately
upon their receipt by the laboratory. If this is not possible, then the
filter should be stored refrigerated inside its container of eluant
until such time as the elution and secondary concentration
processes can be performed. This period of storage should not be
longer than 2 or 3 days.)
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Analysis of Water Samples for Protozoans
by: Jan L. Sykora
Graduate School of Public Health
University of Pittsburgh
Pittsburgh, PA 15261
(412) 624-3516
Introduction
This study includes two sections. Section I is intended as an
introduction to analytical procedures for recovery, isolation, and
identification of protozoans from water. Section II focuses on
laboratory experiments related to the analytical recovery of Giardia
cysts from water and sewage.
Most early studies of microbial contaminants in drinking and
ambient waters focused on pathogenic bacteria, the exception being
the protozoan Entamoeba histolytica which causes amoebiasis. This
disease was recognized long ago to be waterborne; however,
waterborne outbreaks of amoebic dysentery are now rare in the United
States and none have been reported in the last few years (1). Recently,
the significance of previously unrecognized waterborne illnesses such
as primary amoebic meningoencephalitis, giardiasis, and crypto-
sporidiosis, as well as improvements in analytical techniques, have
drawn attention to protozoans.
This chapter focuses on protozoan parasites of major medical
significance that are spread by drinking or ambient water. Some of
these protozoans are of concern because they are known parasites
and/or cause allergic responses, while others may support the growth
of disease-causing pathogens (2).
Entamoeba histolytica, free-living pathogenic amoebae, Giardia
lamblia, Cryptosporidium sp., and Balantidium coli are the primary
parasitic protozoans known to cause a variety of symptoms (3,4,5,6).
Some of the ubiquitous protozoans, including nonpathogenic amoebae
(Acanthamoebae), may cause allergic reactions, such as humidifier
fever or hypersensitivity pneumonitis when inhaled (7,8). The role of
amoebae and other free-living protozoa in supporting Legionella
bacteria has been well documented and discussed (9,10,11).
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Section I - Analytical Methods
Recovery and isolation of protozoans from water samples utilizes
two basic techniques: direct count and cultivation.
The direct count technique is based on microscopic counts of
protozoan parasites. It is a complex process that includes filtration,
concentration, sample processing by flotation, and microscopic
examination. However, highly contaminated samples (such as raw
sewage) do not require filtration and some organisms can be counted
directly in aliquots concentrated by centrifugation. This technique is
used for isolating Giardia cysts and Cryptosporidium oocysts.
The second isolation technique requires cultivation of protozoans
concentrated by membrane filtration on special media (e.g.,
nonnutrient agar seeded with Gram-negative bacteria). Isolation of
free-living amoebae such as Naegleria sp. and Acanthamoeba sp. is
usually accomplished by this method.
Free-Living Amoebae
Several free-living amoebae are pathogenic, including Naegleria
fowleri, the causative agent of the acute human disease, primary
amoebic meningoencephalitis (PAM), a fulminant, usually fatal
illness affecting the central nervous system.
N. fowleri is a ubiquitous, free-living amoeba found in diverse
freshwater environments such as lakes, ponds, and rivers. Most cases
of PAM occur during the summer within 1 week after the victims swim
in warm, fresh or brackish waters. The disease is generally associated
with diving. From 1965, when this disease was first discovered in
Australia, through December 1980, a total of 49 cases have been
reported in the United States (12,13).
Pathogenic N. fowleri is a thermophilic amoeba, relatively
common in warm lakes. This organism has been isolated from lakes
and rivers in Florida and manmade reservoirs in Texas (14). Wellings
et at. have reported that over 50 percent of freshwater lakes in Florida
yielded pathogenic N. fowleri when their temperatures were 30°C or
greater (15).
A new species (Naegleria australiensis) has been described and
characterized as pathogenic to mice by De Jonckheere (16). However,
this amoeba does not grow at temperatures above 45°C and its
pathogenicity to mice is lower than that of N. fowleri.
In temperate climates the heated effluents from power plants and
other industrial facilities discharging cooling waters have been found
to provide a perfect environment for maintenance and reproduction of
Naegleria. In 1974, De Jonckheere and coworkers isolated pathogenic
N. fowleri from a canal receiving warm effluent from a lead and zinc
smelter in Belgium, where a boy swimming in this water contracted
PAM in 1973 (16). Additional information on the occurrence of N.
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fowleri in Florida, Texas, and other parts of the United States was
provided by Wellings et al. (17), Stevens et al. (14), Tyndall et al. (18),
and Duma (19). Sykora et al. detected N. fowleri in discharges and/or
recipient waters from five out of eight power plants surveyed in
western Pennsylvania (5).
In the early 1970s, Acanthamoeba was identified as the causative
agent of a disease known as granulomatous amoebic encephalitis
(GAE). Also at this time, skin and ocular infections (keratitis) caused
by Acanthamoebae were reported. Due to the current popularity of
contact lenses, the incidence of Acanthamoebic keratitis has risen.
Some free-living amoebae isolated from humidifiers and air
conditioning systems have also been reported as likely causative
agents of hyper sensitivity pneumonitis (8,20).
The role of amoebae in supporting other waterborne pathogens
was first documented by Rowbotham, who showed that free-living
pathogenic amoebae (Naegleria and Acanthamoebae) can be infected
with Legionella organisms (9,21). Tyndall and Dominique established
not only that L. pneumophila may be used as a food source for free-
living amoebae but also that some amoebae cultures become
chronically infected and support growth of Legionellae (10). A recent
report demonstrated that Acanthamoeba sp. supported multiplication
of L. pneumophila in drinking water and that free-living amoebae are
frequently present in drinking water in association with Legionellae
(22). Wadowsky et al. demonstrated that hartmanellid amoebae are
important determinants of L. pneumophila multiplication in some tap
water cultures (23),
Detection and Identification of Naegleria fowleri
Cultivation - For each water sample, 250 mL is filtered through a
1.2-um membrane filter. The exposed filter is cut into halves and
placed in an inverted position on nonnutrient agar seeded with
Enterobocter oerogenes (NNAEa). Bottom sediments including algae,
mud, and debris are placed directly into the middle of the same
medium. The inoculated plates are sealed, inverted, and incubated at
45°C for up to 8 days. When growth is observed, the amoebae are
transferred immediately onto a NNAEa and again incubated for up to
8 days at 45°C to purify the isolate and partially prevent growth of
other species of amoebae (Figure 1).
Enterobacter aerogenes Seed - Test tubes of nutrient broth are
inoculated with Enterobacter aerogenes and incubated at 35°C for 24
hours. This broth (0.5 mL) is then pipetted onto trypticase soy agar
plates. Plates are allowed to dry and incubate at 35°C for 24 hours. Two
mL of sterile water is added to each trypticase soy agar plate, and
spread over the agar surface by gently tilting the plate. The bacterial
suspension is scraped by a sterile spreader, and pipetted off into a
sterile centrifuge tube. The suspension is washed twice by
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250 mL
Water
Sample
NNAEa Plate
3-8 Days
Incubation 45°C
Moribund or Dead
Mice Dissectected
3-8 Days
Incubation
45°C
Positive
Flagellation
3 Days, 37°C
Instillation into
Mice
8 Days. 40°C Incubation
Brain for
Histopathological Examination
Figure 5.7.1. Isolation and identification of pathogenic Naegleria fowleri.
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centrifugation for 25 minutes. The resulting bacterial suspension is
boiled for 10 to 15 minutes and then cooled. Four drops of the solution
are spread onto each nonnutrient agar plate.
Flagellation Test - Usually 3 to 4 days after the start of the
second incubation, a small piece of agar with pure amoebic culture is
transferred onto a new NNAEa plate and again incubated for 2 to 3
days at 37°C. A piece of agar with the amoebae from this plate is placed
in a tube containing 0.75 mL of sterile distilled water and incubated at
37°C. Each tube is examined hourly for 4 consecutive hours using an
inverted microscope. Positive amoeba flagellate forming strains are
transferred onto a new NNAEa plate and incubated for 3 days at 37°C.
These plates are for use in mice pathogenicity tests (Figure 1).
Mouse Pathogenicity Test - A suspension of amoebae, scraped
from the plate by a sterile spreader, is prepared in sterile distilled
water. The trophozoites are counted using a hemacytometer. Five
weanling, white (female) Swiss Webster mice are anesthetized and 30
uL of the amoebic suspension (103 to 104 of organisms per inoculum) is
instilled into their nostrils. Five mice instilled with sterile distilled
water are used as controls. The mice are fed and provided with water
ad libitum, and kept up to 16 days. Mice with severe neurological
symptoms, or those which die, are aseptically dissected. Small sections
of the brain are placed in NNAEa and incubated at 40°C to reisolate
the pathogenic Naegleria. The remainder of the brain can be preserved
in buffered formaldehyde, stained, and histopathologically examined.
Reisolation of amoeba from the CNS tissue of mice and optional
histopathological confirmation are considered to be definitive evidence
of the presence of pathogenic Naegleria in the tested water or sediment
samples.
Detection and Identification of Acanthamoeba
Cultivation - Water samples filtered through 1.2 um membrane
filters or sediment samples are cultivated on 1.5 percent nonnutrient
agar containing 2 percent NaCl seeded with E. aerogenes (see
Detection and Identification of Naegleria fowleri for details). All plates
are sealed'and placed in plastic bags to prevent drying and
contamination, and the plates are incubated at 406 for 3 to 8 days. All
positive plates are transferred by removing a small piece of agar
containing trophozoites and placing it in the center of a new plate (1.5
percent agar without NaCl and incubated at 37°C). Each sample is
examined for the presence of Acanthamoeba based on its morphological
criteria (24, 25).
Pathogenicity Test - Ten young female white Swiss Webster
mice are anesthetized with ether and instilled intranasally with
known concentrations of amoebae using a Finn-pipette with sterilized
plastic tips. Sixty to 90 p.L of the suspension are instilled with a known
concentration of trophozoites (IQSto 104 trophozoites/30 uL). A known
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pathogenic strain of Acanthamoeba (e.g., A. polyphaga) is instilled into
a positive control group consisting of 16 mice (each mouse is instilled
with 60 uL with approximately 103 to 104 trophozoites/30 uL). Sixty uL
per mouse of sterile distilled water should be instilled into the control
group of four mice. The infected mice are observed for 30 days. The
presence of Acanthamoebae in dead mice is confirmed by cultivating
sections of lungs and brains on NNAEa plates.
Entamoeba histolytica
Entamoeba histolytica has been implicated as a causative agent of
human disease since 1875 (26). However, not until 1903 was this
amoeba differentiated from previously described E. coli, a confusion
which created a controversy concerning the pathogenicity of E.
histolytica. Most investigators now believe that E. histolytica, also a
potential pathogen, usually is a commensal inhabitant of the
intestinal tract (27).
The life cycle of E. histolytica is characterized by three stages, i.e.,
trophozoite, precyst, and cyst, with cyst being the infective stage. The
cystic stage is resistant, but it cannot withstand temperatures above
50°C, sunlight, or extended exposure to disinfectants. All three stages
of the organism may be found in stool of infected persons.
The pathology of E. histolytica is complex and the infection caused
by trophozoites inhabiting the lower part of the small intestine may
result in 1) a carrier state without symptoms (majority of cases), 2)
invasion of the bowel wall without systemic spread, or 3) invasion of
the bowel wall followed by systemic spread which could result in
lesions in a variety of other organs, most often the liver. Clinical
response is variable but most commonly involves acute amoebic
dysentery or amoebic diarrhea.
Humans and subhuman primates are the only reservoirs of E.
histolytica, and the organism is most often transmitted by the fecal-
oral route. Sewage contaminated water has been responsible for
waterborne outbreaks. The last waterborne outbreak of amoebiasis
reported in the United States, which affected 31 individuals, occurred
in 1953 (1).
E. histolytica can be identified in environmental samples by direct
microscopic count in the sample concentrate or by cultivation. In the
past, several in vitro cultivation diphasic media have been formulated,
including horse serum, ringers solution, and starch (28). A number of
investigators developed monobasic media useful for isolation of
amoebae from fecal material.
These media usually contain serum, egg products, bacteria, and
starch (Balamuth medium) (29). Balamuth medium is now
commercially available. Standard Methods recommends the liver
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infusion medium as modified by Chang and Kabler (30) for E.
histolytica cultivation (31).
Detect/on and Identification of E. histolytica
Concentration - Four liters or more of water are filtered through
membrane filters (7 to 10 um pore size), if the concentration of
suspended matter is low. Wet filters are flushed with several
milliliters of sterile distilled water. The washing could be concentrated
by centrifugation. High turbidity water or water suspected of
containing a low concentrations of cysts can be processed using high
volume filtration described in Detection and Identification of Giardia
cysts.
Direct Microscopical Examination - Aliquots of concentrated
samples are placed in a Palmer-Maloney nanoplankton counting
chamber or a Sedwick-Rafter chamber and are examined under low
power magnification for cysts and/or trophozoites.
Cultivation - A concentrated sample is inoculated into a modified
liver infusion medium, as proposed by Chang and Kabler (30),
incubated for 3 to 6. days at 37°C, and examined microscopically for
trophozoites. Concentration of E. histolytica in the original water
sample could be accomplished using the most probable number
technique described in Standard Methods (31).
Giardia
Giardia intestinalis is a common human intestinal parasite and
has been the most frequently identified etiological agent of waterborne
disease in recent years. The causative agent is a flagellate protozoan
characterized by a simple life cycle involving two stages - a cyst and a
trophozoite. Giardia is shed in the feces of humans and animals, most
often in a resistant cyst stage. Each viable, ingested cyst produces two
flagellate trophozoites that attach themselves to the epithelial cells of
the duodenum and jejunum. The cysts can survive in water for 2
months at 8°C and are more resistant to chlorine than bacteria and E.
histolytica (32). However, they are highly vulnerable to dessication
and temperatures higher than 50°C. The cyst is football shaped,
averaging 8 to 12 um in length by 7 to 10 um in width. It contains
inner structures specific to Giardia, called median bodies and an
axostyle, which are used for the positive identification of cysts.
At present, since no reliable method exists for culturing Giardia
cysts from water samples, the only practical technique for Giardia
detection in water relies on microscopical examination of the sample
concentrates. Jakubowski (33), Jakubowski and Ericksen (34), and
Vasconcelos (35) reviewed the methods for detecting the Giardia cysts
in water in great detail and described state-of-the-art sampling and
detection of Giardia in drinking water. These authors concluded that
an ideal technique for the detection of Giardia cysts in water would be
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one that recovers all cysts in a sample simply and inexpensively,
enables rapid identification and quantification, and provides
information on the infectivity potential of the cyst.
The techniques currently used by most laboratories engaged in
detection of Giardia cysts in water employ microscopical examination
of concentrates derived from several hundred gallons of water. Thus,
cyst recovery and positive identification depend on such factors as the
amount of water pumped through the filter and the environmental
factors affecting the distribution of the cyst in the aquatic
environment. More importantly, cyst identification relies on the
experience, skill, and persistence of the person analyzing the sample
(35). The current sampling and detection techniques for Giardia cysts,
as described in Standard Methods (31) or as utilized by EPA (36,37)
and modified by Schaefer (38), are employed mostly to detect the cyst
in drinking water supplies. All methods presently in use involve high
volume filtration, cyst concentration, and a microscopical cyst
enumeration step. A Lugol's iodine solution is conventionally used to
stain the cysts and distinguish them from similar objects such as algal
cells. Recently, immunofluorescent techniques have been developed to
facilitate cyst identification. These techniques are currently being
used by a majority of researchers (39,40,41,37,42) while others still
prefer the Lugol's staining procedure (43,44).
The Giardia sampling and testing procedure as described in the
following section provides general information on techniques which
could be modified based on the availability of equipment and the skill
of laboratory personnel.
Detection and Identification of Giardia Cysts
Sampling Procedure - In 1976, EPA developed a large volume
sampling technique based on virus concentration apparatus then in
use (34). Because of the success of this procedure, most investigators
now collect samples using Giardia sampling equipment consisting of
an inlet hose, a plastic filter holder with a 25-cm long, orlon yarn-
wound filter (1 um porosity), an outlet hose, and a water meter. To
collect a sample, the inlet hose may be connected to a tap or, if needed,
a portable pump can be utilized to pump the water through the
cartridge. The volume of water pumped through the filter depends on
the purpose of the investigation. A minimum of 380 L (100 gal) is
recommended. After sampling is completed, the filter cartridge is
placed in a sealed Ziplock® bag containing the water drained from the
filter holder, double-bagged, labeled, placed on ice, and processed
within 48 hours.
In the laboratory, the filters could, be cut in half or divided into
fourths by unwinding the yarn into four skeins. The procedure selected
depends on the type of filter used. The individual filter sections are
rinsed in successive 1-L aliquots of 0.01 percent Tween 20® until the
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filters appear clean. One percent Tween solution is added as needed to
maintain suds. The alternative procedure preferred by some
investigators involves backwashing the filter cartridge and using
Tween 80® instead of Tween 20®
The sample washed from the filter is concentrated by
sedimentation under refrigeration for 24 hours followed by
centrifugation or by centrifugation at 600 xg for 3 minutes. One to 2
mL of combined sediment pellets suspended in 70 to 90 mL of 0.01
percent Tween 10® are layered on top of 70 mL of 1.5 M sucrose
solution (specific gravity 1.18) in a 250-mL centrifuge bottle. After
centrifugation at 800 xg for five minutes the supernatant above the
interface and the pellet are discarded. The interface and all underlying
sucrose solution are diluted five times with 0.01 percent Tween 20®
and further processed by centrifugation at 600 xg for 2 minutes. The
resultant sediment is washed twice with Tween 20® in 50-mL
centrifuge bottles, transferred into 15-mL centrifuge tubes,
concentrated by centrifugation, and stained by Lugol's solution using
the Jakubowski and Ericksen procedure (32).
Giardia Cyst Identification - The sediment obtained by this
flotation process is examined microscopically using a Palmer-Maloney
nanoplankton counting chamber (0.1 mL) and a 45x objective equipped
with phase contrast. Positive identification of the Giardia cyst is based
on dimension, shape, and internal morphological features. For positive
identification of a cyst, at least two internal structures must be
observed.
Several modifications of this technique involve a membrane
filtration procedure proposed by Ongerth (42) and different flotation
fluids with a specific gravity of approximately 1.2. In addition to
sucrose (Sheather's solution), zinc sulfate and potassium citrate are
frequently used by various researchers (31,36,37,40,42,45).
In the early 1980s, a fluorescent antibody technique for
identification of Giardia cysts in stool samples was developed by Riggs
et al. (46), who also demonstrated the application of this technique to
analyses of large volume water samples (45). Sauch developed an
indirect immunofluorescence/phase contrast technique that was
successfully used during several giardiasis outbreaks (41).
Initially, immunofluorescent techniques were not widely available
because they involved complex procedures for cyst purification and
antisera production. However, due to recent commercial availability of
the Giardia antibodies, this technique is becoming routine.
The indirect immunofluorescent technique described by Sauch
utilizes application of anti-Giardia cyst antiserum and fluorescein
conjugate to membrane filters through which the concentrated
samples are filtered (41). The cysts fluoresce bright green when viewed
with ultraviolet light. Rose used direct immunofluorescent techniques
305
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consisting of the application of monoclonal antibodies directly
conjugated to fluorescein isothiocyanate (37). In both techniques, the
filters are contrastained by Evans blue and cleared with glycerol, and
the cysts are counted using epifluorescent microscopy.
Cryptosporidium
Giardia and Cryptosporidium are enteric parasites that frequently
occur in the same environment, and both have high potential for
causing waterborne illness. The principal mode of transmission
appears to be the fecal-oral route. Both parasites also produce cyst
stages that are resistant to standard disinfection.
Cryptosporidium has been classified as a coccidian parasite, but
very little is known about its taxonomy and host specificity. In
comparison with Giardia, it has a relatively complicated life cycle
involving sexual and asexual stages. According to Rose (4), two aspects
of the taxonomy and life cycle of Cryptosporidium increase the
possibility of its transmission by water. First, a single species may be
responsible for the diarrheal illness in mammals, including humans.
Secondly, an environmentally stable oocyst is excreted by infected
animals, which may contaminate water.
While the first documented waterborne giardiasis outbreak in the
United States occurred in 1965, Cryptosporidium received little
attention until 1976 when the first human cases were reported and
associated with exposure to farm animals (47). Cryptosporidiosis is
considered to be a zoonotic disease, transmitted from a variety of
domestic and wild animals including calves, deer, and beaver. Prior to
1982, only seven cases of cryptosporidiosis were reported. During that
period, most reported infections were in patients with
immunodeficiencies other than the acquired immunodeficiency
syndrome (AIDS). After that period, however, a significant percentage
of AIDS patients developed cryptosporidiosis at some time during their
illness. Recent reports show that Cryptosporidium is now frequently
identified in cases of acute, self-limited diarrheal illness even in
immunocompetent hosts in both developed and developing nations.
Fecal-oral spread among humans and animals and ingestion of
contaminated water seem to be the principal modes of infection.
Recently three Cryptosporidium-caused waterborne outbreaks
were reported in the United States. The first, caused by a sewage-
contaminated well, resulted in 117 cases (4). The second occurred in
winter 1986-1987 in Carrolton, Georgia, when over 10,000 people were
affected and 5,000 became ill. due to water contaminated with this
pathogen (4). In early 1987, another waterborne outbreak was
investigated in Arizona. The illness was widespread throughout a
community of 27,0.00 to 36,000 people with the attack rate
approximately 71 percent (37,48). The most recent waterborne
outbreak of cryptosporidiosis occurred in England in Oxford and
306
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Swindon during February and March 1989. Water treatment plants in
these two cities serve approximately 600,000 people.
An investigation of Cryptosporidium distribution in
environmental samples showed that this parasite is widely distributed
in sewage and in surface waters in Arizona, and some cysts were even
recovered from water supplies treated by filtration (37,48).
Detection and Identification of Cryptosporidium Oocyst
Sample Collection and Processing - The following methodology
is based on a technique described by Rose (37). Water samples for
Cryptosporidium analysis are collected using standard Giardia
sampling equipment and filtered through 10-inch spun polypropylene
cartridge filters (Micro Wynd II, AMF/CUNO Division, Meriden CT)
with a nominal porosity of 1 um. One hundred to 400 gallons of water
are filtered at flow rates of 4 to 5 gallons per minute (37). Facilities
with pressurized lines can be sampled without the pump by attaching
the filter inlet hose directly to a faucet. The filter cartridges containing
the sample are double bagged in Ziplock® bags with water from the
filter holder, chilled immediately, iced, and processed within 48 hours.
Filter Elution - Filters are processed by backflushing the filter
with 2,700 mL of deionized (DI) water eluent containing 0.1 percent
Tween 80®. The filters are cut longitudinally, separated from the core,
teased apart, and washed three times; each time in one third (900 mL)
of the eluent. The washing is performed on an automatic shaker for 10
minutes in a 1-gallon container. The sample is concentrated and
combined into a single pellet by centrifugation (1,200 xg for 10
minutes). The final pellet is divided in half and resuspended in io
percent formalin or 2.5 percent buffered potassium dichromate.
Pellet Processing - Pellets suspended in buffered 2.5 percent
potassium dichromate are washed and resuspended with a detergent
solution (water with 1 percent Tween 80® and 1 percent sodium
dodecyl sulfate) and then homogenized. One drop of antifoam is added
to facilitate total sample recovery. Next, the sample is washed and
resuspended with the detergent solution, or DI water, and finally
sonicated immediately prior to the layering onto a flotation solution.
Centrifugation on Sheather's 500 g sucrose, 320 mL of DI water,
and 9.7 mL liquid phenol are used to separate oocysts from sediments.
Five mL aliquots of sample are layered onto 10 mL of Sheather's
solution. The tubes are centrifuged at 1,200 xg for 10 minutes, and the
supernatants are recovered. Additionally, potassium citrate (40
percent solution) could be used to separate oocysts from sediments. The
samples suspended in DI water are used for this centrifugation. A 1:3
ratio of sample to media (10 mL to 30 mL) is used. The tubes are
centrifuged at 800 xg for 2 minutes and supernatants are recovered..
307
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These recovered supernatants are washed and analyzed using an
indirect immunofluorescent antibody technique.
Cryptosporidium Oocyst Identification - The indirect immuno-
fluorescent antibody technique has been modified from previously
published immunofluorescent procedures for detection of Crypto-
sporidium oocysts (42,49). Alcohol-cleaned microscope slides con-
taining 10-mm inscribed wells are coated with a 0.01 percent (w/v)
solution of poly-L-lysine to improve adherence of Cryptosporidium
oocysts to the microscope slides (50). Smears from the final resus-
pended sample pellet are prepared by spreading 0.05-ml portions
within the inscribed wells, then air drying and heat fixing them. The
entire resuspended sample pellet is processed to maximize sensitivity
of the procedure. Smears are covered with 0.05 mL of a murine anti-
monoclonal antibody solution, and the slide is incubated in a humidity
chamber for 30 minutes at room temperature. Following incubation,
slides are washed for 5 minutes in phosphate- buffered saline (pH 7.6).
A 0.05-ml portion of fluorescein-conjugated goat anti-murine antibody
is added to the wells to detect the presence of bound monoclonal
antibodies. The slides are then incubated in a humidity chamber,
shielded from light, for 30 minutes at room temperature. Following
incubation, they are washed with the phosphate-buffered saline and
mounted with buffered glycerol mounting medium. Smears are
immediately examined for fluorescent Cryptosporidium oocysts, 4 to 6
um in diameter, using an epifluorescent microscope. The monoclonal
and fluorescent conjugate antibody preparations can be obtained
commercially from Meridian Diagnostics, Inc. (Cincinnati, Ohio). The
working dilutions of the reagents should be those determined by the
manufacturer.
The monoclonal antibody supplied by the manufacturer has two
advantages over a monoclonal antibody used in a previous study (49).
First, the monoclonal antibody preparation supplied by Meridian
Diagnostics, Inc., is an IgM antibody and has been found to be more
specific than the previously used IgG monoclonal antibody. It is
necessary, however, to use the IgM monoclonal antibody in an indirect
rather than a direct fluorescent antibody test, since specificity has
decreased when attempts have been made to directly conjugate the
IgM monoclonal antibody with fluorescein. Secondly, the IgM antibody
reacts only with mammalian strains of Cryptosporidium and not with
avian strains. Since avian strains are not pathogenic to mammals, this
additional feature further improves specificity (50).
Section II - Laboratory Studies
It is important to determine the efficiency of recovery from water
samples for various organisms. Studies were conducted in our
laboratory to determine the efficiency of Giardia cyst recovery in
distilled water and from sewage samples (51,52).
308
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For Giardia cyst recovery experiments in distilled water, raw
sewage and activated sludge, trickling filter, and biodisc effluents, 900
mL,samples were each placed in separate glass jars and seeded with
equal and known concentrations of Giardia lamblia cyst stock. Each
jar was mixed thoroughly by inversion and shaking* after which a 250-
ml sample was taken from each jar. For Giardia-settling experiments,
samples were collected after 2 and 24 hours. These tests were
performed at two different temperatures (4° and 21°) in distilled water.
Grab samples of raw sewage and activated sludge, trickling filter,
and biodisc effluents were collected from three different sewage
treatment plants located in the Pittsburgh region. All samples were
taken from a point prior to their entrance into the clarifier. Samples
were stored under refrigeration until used and experiments were run
within 48 hours of a sample's collection.
In; addition, flow composite raw sewage and final effluent samples
were collected from a sewage treatment plant also in Pittsburgh.
Giardia cysts were counted in three samples each consisting of 250 mL
of raw sewage, layered on sucrose. Another 250-mL sample of raw
sewage was counted without layering on sucrose.
For sample concentration and microscopic examination, samples
were centrifuged at 600 xg for 3 minutes. After aspiration of the
supernatant, the sediment was stained with two to three drops of
Lugol's solution. The sample was then resuspended in 15 mL 0.01
percent Tween 20®, centrifuged again at 600 xg for 3 minutes and
aspirated to remove residual Lugol's solution. The remaining sediment
was then diluted with 0.01 percent Tween 20® to a concentration
appropriate for microscopic examination. Concentrated samples were
examined using a hemacytometer (.02 mL) and a light microscope
equipped with a 40x objective.
Field samples were also collected from raw water and effluents
from the settling tank prior to filtration and postchlorination at the
McKeesport, Pennsylvania, water treatment plant. A total of 12
samples were collected in March, April, May, and December of 1987.
Samples collected at the water treatment plant were obtained using
standard Giardia filtration equipment (31). One hundred to 300
gallons of settled water were filtered at a rate of 1 to 2 GPM, while less
than 200 gallons of raw water were filtered due to clogging. Following
sample collection, filters were refrigerated and maintained at 5°C until
analyzed, using the technique by Schaefer and Rice described in the
Giardia section (36).
Results
Laboratory experiment results show substantial Giardia cyst
reduction due to layering and flotation procedures. Layering and
flotation techniques resulted in 10 to 18 percent of the recovery
achieved by direct counts. Additional experiments with two aliquots of
309
-------
the final effluent seeded with a known concentration of cysts (400
cysts/L) showed that 55 percent of the cysts were lost by centrifugation
and 92 percent were lost by centrifugation and layering on sucrose
(Table 5.7.1).
Table 5.7.1. Effect of Layering for Recovery of Giardia Cysts, "-,•".
Raw Sewage
Procedure
Layered8
Giardia
"Seed"/L
None
Date
10/8/87
Cysts/L
120
Percent
Recovery
10
Final Effluent'
Cysts/L
4
Percent
Recovery
-
Layered" None 10/8/87 140 12
"Layered8" ~ None 10/8787 220 18 -
Not layered8 None 10/8/87 1,200
"NoUayered8 400^ 11/5/87' ' j.- _.- 180._
layered'8400* 1 i/5/87 ' - :j- ——— ' " 32
45
""a"
8 Flow composite samples.
*» 250 mL samples were seeded with 100 G. lamblla cysts.
In experiments to investigate cyst recovery from different
biological treatment processes, raw sewage and distilled water
samples were seeded with Giardia lamblia cysts and concentrated
using centrifugation without the sucrose layering step. Direct
microscopical examination of subsamples revealed relatively high
recovery rates ranging from 38 to 72 percent (Table 5.7.2) when
compared with a layered sample (8 percent) from Table 5.7.1.
Table 5.7.2. Recovery Of G/ard/a Cysts without Sucrose
Flotation
Sample Replicate Percent Recovery
Distilled water
Activated sludge
Raw sewage
,
Trickling filter effluent
Biodisc effluent
Activated sludge effluent
1
2
3
1
2
3
4
1
2
1
2
1
2
1
40
63
50
38
47
60
44
72
47
67
55
53
56
44
310
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. Giardia removal by settling in distilled water at two different
temperatures was also investigated. After 2 hours of quiescent settling
at rooni temperature (21°C), only 40 to 50 percent of the cysts were
removed from the surface, indicating that settling is not a very
efficient means of cyst removal. The removal rate via settling was
much lower at the colder temperature, only 0 to 8 percent of cysts were
removed from the surface layers after 2 hours of settling at 4°C. After
24 hours of settling, approximately 60 percent of the cysts were
removed (Table 5.7.3).
Table 5.7.3. Removal of Giardia lamblia Cysts Via Sedimentation in
Distilled Water
Temperature (°C)
21 ,
21
4
4
4
' 4
Settling Time (hr)
.. ,2 . . ,
2
2
2
24
24
Sample Level3'
... .... ..1. . .
2
2
1
, - 2
Percent Removal
,b Giardia
,40
50
8
0
58
64
a Level 1 is 0.5 cm below the surface.
•? Level 2 is 7.5 cm bebw the surface.
To study the removal efficiency by sedimentation and flocculation,
field samples were obtained from raw and settled water from the
McKeesport water treatment plant. Higher concentrations of cysts in
settled water were observed in winter months (December and March),
indicating that colder water may affect the sedimentation of the cysts,
as noted in the laboratory study (Table 5.7.4).
Table 5.7.4. Giardia Cysts in Settled and Raw Water (McKeesport)
Settled Water Raw Water Percent Cyst Raw Water Settled Water
Date Cyst/100 gal Cyst/100 gal Removal Turb, NTU Turb, NTU
3/4/87
3/18/87
4/2/87
4/15/87
5/18/87
12/10/87
18
9
0
7
0
10
60
126
193
45
4
62
70
93
100
84
100
84
18
48
38
15
37
-
3.1
3.7
2.0
2.5
2.4
• -
311
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Discussion
The reported laboratory experiments showed that cyst recovery
from raw and treated sewage samples is variable and may range from
8 to 45 percent. The results also indicate that recovery is usually quite
reproducible when aliquots of one sample are compared (Table 5.7.1).
Furthermore, Tables 5.7.1 and 5.7.2 show that more than 50 percent of
the cysts could be lost due to concentration by centrifugation and that
the layering' procedure is responsible for substantial cyst loss.
However, results obtained by Sykora et al. (53) demonstrated that in
spite of the low recovery rate, monitoring of public water supplies with
a past history or potential for Giardia contamination provides
additional information for public water supply protection if Giardia
cysts are found. A positive result, even without establishing infectivity
or viability should stimulate action to modify or provide additional
water treatment or water source protection. Such monitoring, though
informative, is, however, no substitute for complete and effective
treatment of public water supplies. Also, negative results of ,such
monitoring may be misleading due to low recovery of cysts.
Laboratory and field data show that temperature may have a
significant effect on the rate of cyst settling. Thus, the higher densities
of water in winter may affect the cyst removal efficiency in treatment
plants. In addition, the results showing the effect of temperature on
cyst settling rate have practical significance. The method for Giardia
cyst detection published by the American Public Health Association
recommends that water samples first be concentrated by
centrifugation or by sedimentation under overnight refrigeration (31).
As presented in Table 5.7.3, however, after 24 hours of settling under
refrigeration (4°C), surface samples contained only approximately 40
percent of the initial concentration of cysts. This indicates that the
usefulness of sedimentation under overnight refrigeration as a method
of preconcentration should be reevaluated, especially for samples of
low turbidity (i.e., drinking water samples). Under these conditions,
only centrifugation is recommended for preconcentration of water
samples to ensure adequate cyst concentration and better cyst
recovery.
References
1. Craun, G.F. 1986. Waterborne Diseases in the United States CRC
Press, Inc. Boca Raton, Florida, pp. 1-295.
2. States, S.J. et al. 1989.' Free-living amoebae and implications for
Legionella, Giardia and Crvptosporidia. In: Public Water Supplies.
Proceedings AWWA Water Quality Technology Conference, pp.
109-125.
3. Craun, G.F. 1986. Waterborne giardiasis in the United States.
Lancet. 2:513-514.
312
-------
4. Rose, J.B. 1988. Occurrence and significance otCryptosporidium in
water. J. AWWA. 80:(2):53-58.
5. Sykora, J.L. etal. 1983. Occurrence and pathogenicity of Naegleria
fowleri in artificially heated waters. Appl. Environ. Microbiol.
45:974-979.
6. Sykora, J.L. et al. 1988. Giardia cyst in raw and treated sewage.
In: G.S. Logsdon (ed.), Controlling Waterborne Giardiasis. ASCE,
New York.
7. Edwards, J.H. et al. 1976. Protozoa as sources of antigens in
"humidifier fever." Nature. 206:438-439.
8, Sykora, J.L. et al. 1982. Amoebae as sources of hypersensitivity
pneumonitis. Environment International. 8:343-347.
9. Rowbotham, T.J. 1980. Preliminary report on the pathogenicity of
Legionella pneumophila for freshwater and soil amoebae. J. Clin.
Pathol. 33:1179-1183.
10. Tyndal, R.L. and E.L.Dominique. 1982. Cocultivation of Legionella
pneumophila and free-living amoebae. Appl. Environ. Microbiol
' 44:954-959.
11. Fields, B.S. etal. 1984. Proliferation of Legionella pneumophila as
an intracellular parasite of the ciliated protozoan Tetrahymena
pyriformis. Appl. Environ. Microbiol. 47:467-471.
12. Martinez, J.A. 1981. Free-living amoebas in human infections.
Eastern Pennsylvania Branch of ASM, Philadelphia, PA. p. 57.
13. Martinez, J.A. 1985. Free-Living Amoebas: Natural History,
Prevention, Diagnosis, Pathology, and Treatment of Disease. CRC
Press, Boca Raton, FL.
14. Stevens, A.R. et al. 1977. Isolation of the etiological agent of
primary amoebic meningoencephalitis from artificially heated
waters. Appl. Environ. Microbiol. 34:701-705.
15. Wellings, F.M. et al. 1977. Isolation and identification of
pathogenic Naegleria from Florida lakes. Appl. Environ. Microbiol.
34:661-667.
16. De Jonckheere, J.F. 1981. Naegleria australiensis sp, nov., another
pathogenic Naegleria from water. Protistologica. 17(3):423-429.
17. Wellings, F.M. et al. 1979. Pathogenic Naegleria: Distribution in
nature. Report No. EPA-600/1-79-018, U.S. EPA, Cincinnati, Ohio.
18. Tyndal, R.L. et al. 1978. Isolation of pathogenic Naegleria from
artificially heated waters. In: R.M. Gerhold (ed.), Proceedings of
Symposium on Microbiology of Power Plant Thermal Effluents,
University of Iowa, Iowa City, pp. 117-123.
313
-------
19. Duma, R.J. 1981. Study of pathogenic free-living amoebas in
freshwater lakes in Virginia. Project Summary. Report No. EPA-
800151-80-037. U.S. EPA Cincinnati, Ohio. pp. 1-5.
20. Shapiro, M.A. et al. 1983. The role of free-living amoebae occurring
in heated effluents as causative agents of human disease. Wat. Sci.
Techn. 15:135-147.
21. Rowbotham, T.J. 1986. Current views on the relationships
between amoebae, Legionellae, and Man. 7sr. J. Med. Sci. 22:678-
689.
22. Henke, M., and K. Seidel. 1986. Association between Legionella
pneumophila and amoeba in water. Isr. J. of Med. Sci., 22:690-695.
23. Wadowsky, R.M. et al. 1988. Growth supporting activity for
Legionella pneumophila in tap water cultures and implication of
hartmanellid amoebae as growth factors. Appl. Environ. Microbiol.
54(11)2677-2682.
24. Page, F.C. 1967. Taxonomic criteria for Limax amoebae. J.
Protozool. 14:499-521.
25. Page, F.C. 1976. An illustrated key to freshwater and soil
amoebae. Freshwater Biological Association. No. 34.
26. Loesch, F. 1875. Massenhaft Entwickelung von Amoeben in
Dickdarm. Arch. Pathol. Anat. Physiol. Klin. Med. 65:196-211.
27. Albach, R. A. and Booden T. 1948. Amoebae. In: J.P. Kreier (ed.),
Parasitic Protozoa, Vol II. Academic Press, New York. pp. 455-
506.
28. Dobell, C., and P.P. Laidlow. 1926. On the cultivation of
Entamoeba histolytica and some other entozoic amoebae.
Parasitology. 18:283-318.
29. Balamuth, W. 1946. Improved egg yolk infusion for cultivation of
Entamoeba histolytica and other intestinal protozoa. Am. J. Clin.
Pathol. 16:380.
30. Chang, S.L. and P.W. Kabler. 1956. Detection of cysts of
Entamoeba histolytica in tap water by the use of membrane filter.
Amer.
-------
33. Jakubowski, W. 1984. Detection of Giardia cysts in drinking
water. In: S.L. Erlandsen and E.A. Meyer (eds.), Giardia and
giardiasis, Plenum Press, New York, pp. 263-286.
34. Jakubowski, W. and T.N. Ericksen. 1979. Methods for detection of
Giardia cysts in water supplies. In: Waterborne Transmission of
Giardiasis. Report No. EPA-600-9-79-001.
35. Vasconcelos, O. 1984. Methods for testing for Giardia in water.
Paper presented at Portland Giardia conference No. 14-16, 1983.
U.S. Environmental Protection Agency Report, pp. 14-73. April
36 Schaefer, F.W., III, and E.W. Rice. 1982. Giardia methodology for
water supply analysis. In: Proceedings AWWA Water Quality
Technology Conference, Seattle, Washington, pp. 143-147.
37. Rose, J.B.. etal. 1987. Detection ofCryptosporidium and Giardia in
environmental waters. In: Advances in Water Analysis and
Treatment. AWWA, Denver, CO. pp. 417-424.
38. Schaefer, F.W, III. 1984. Personal communication.
39. Riggs, J.L. et al. 1988. Recovery, detection, and enumeration of
Giardia cysts in water. In: 6.5. Logsdon (ed.), Controlling
Waterborne Giardiasis. ASCE, New York. pp. 13-21.
40. Sauch, J.F. 1984. Purification of Giardia muris cysts by velocity
sedimentation. Appl. Environ. Microbiol. 48(2)454-455.
41. Sauch, J.F. 1985. Use of immunofluorescence and phase-contrast
microscopy for detection and identification of Giardia cysts in
water samples. Appl. Environ. Microbiol. 50(6)1434-1438.
42. Ongerth, J.E. et al. 1988. Giardia cyst concentrations in river
water. In: Issues and Answers for Today's Water Quality
Professional. AWWA. Baltimore, MD., pp 243-261.
43. Sykora, J.L. et al. 1987. Monitoring of water and wastewater for
Giardia. In: Advances in Water Analysis and Treatment,
Proceedings AWWA Water Quality Technology Conference, pp.
1043-1054.
44. Mai-rocco, F. et al. 1987. Giardia monitoring and regulation in
Pennsylvania surface water supplies. In: Advances in Water
Analysis and Treatment, AWWA, Portland, OR. pp 1055-1066.
45. Riggs, J.L. et al. 1984. Identifying Giardia lamblia by
Immunofluorescence. In: M. Pirbazari and I.S. Devinney (eds.),
Proceedings of the 1984 ASCE Environmental Engineering
Specialty Conference, Los Angeles, California, pp. 234-238. June.
46. Riggs, J.L. et al. 1983. Detection of Giardia lamblia by
Immunofluorescence. Appl. and Environ. Microbiol. 45(2) 698-700.
315
-------
47. Janoff, E.N. and L.B. Reller. 1987. Cryptosporidium species, a
protean protozoan. Journal of Clinical Microbiology. 25(6) 967-
975.
48. Rose, J.B.. et al. 1988. Method for the recovery of Giardia and
Cryptosporidium from environmental waters and their
comparative occurrence. In: R.M. Wallis and B.R. Hammond (eds.),
Advances in Giardia Research. University of Calgary Press,
Calgary, Canada, pp. 205-209.
49. Musial, C.E. et al. 1987. Detection of Cryptosporidium in water
using polypropylene cartridge filters. Appl. Envir. Microbiol.
53:687.
50. Sterling, C.R. 1987. Personal communication.
51. Sykora, J.L. et al. 1989. Monitoring of environmental samples for
Giardia cysts. In: Proceedings of Fourth Conference on Progress in
Chemical Disinfection, April 11-13, 1988, SUNY, Binghamton,
New York, pp. 61-74.
52. Kim, R.P. and Sykora, J.L. 1989. Removal of Giardia lamblia cysts
by coagulation and sedimentation processes. In: Proceedings of
Fourth Conference on Progress in Chemical Disinfection, April 11-
13,1988, SUNY, Binghamton, New York, pp. 75-98.
53. Sykora, J.L. et al. 1987. Monitoring as a tool in waterborne
giardiasis prevention. In: P.M. Wallis and B.R. Hammond (eds.),
Advances in Giardia Research. University of Calgary Press,
Calgary, Canada, pp. 103-106.
316
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Epilogue
The emphasis of this volume is on the prevention of infectious
waterborne diseases and the improvement of surveillance, reporting,
epidemiologic investigations, and laboratory analyses. In these areas,
we cannot rest on past accomplishments. Continuing microbiological
challenges are a reminder that our task is yet unfinished; Although
the focus of this volume has been on drinking water systems, enteric
infections have also been associated with swimming and other
recreational activities where water is accidentally ingested.
Pseudomas folliculitis, pharyngitis, external otitis, and schistosomal
dermatitis have been associated with recreational activities involving
water contact in hot tubs, whirlpools, and swimming pools or lakes.
There .is evidence that inhalation exposure is important as well;
Pontiac fever caused by Legionella has been associated with use of
whirlpools. Organisms such as Legionella and nontuberculous
Mycobacteria can contaminate drinking water systems, find conditions
favorable for growth, and may be aerosolized into the indoor air
through showering and other household uses of water. Legionella has
also been disseminated in the air environment through cooling towers.
Not only is more work required on infectious waterborne diseases
but also on the noninfectious diseases associated with chronic
waterborne exposures. In addition to inorganic constituents and
radionuclides, more and more organic contaminants are being
identified in our drinking water. These organic compounds are
contaminants from our industrial society but, as previously noted, can
be formed during the disinfection of drinking water. The routes of
exposure can be oral, dermal, or inhalant; for some volatile organics,
inhalation can be an important route of exposure. The offgassing of
radon from some ground-water supplies into the home environment
can be as significant a source of exposure in addition to radon entering
household air from the decay of radium in underlying bedrock or soils.
Although toxicological data can provide risk estimates for
exposures to chemicals and radioactivity, uncertainties still exist -
uncertainties which can be minimized only when data are available
from human populations. Epidemiology, which is required for the
investigation of outbreaks, is also used for establishing associations
and determining the magnitude of risk from these chronic exposures.
Case-comparison and cohort studies must be carefully designed,
conducted, and interpreted if they are to contribute to our
understanding of the etiologies of noninfectious disease. Epidemiologic
methods have been developed specifically to study chronic exposures
and diseases of long latency periods. While certain statistical methods
317
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cannot be strictly applied to the study of infectious diseases, these
epidemiologic concepts must be considered and used appropriately to
help avoid bias and improve inferences from outbreak investigations.
G.F.C.
4U.S. GOVERNMENT PRINTING OFFICE: a 9 91 - 51*e- 18? l»05't3
318
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