United States Prepared by the EPA-570/9-78-006
Environmental Protection Environmental Research Center (ORD)
Agency Cincinnati, Ohio 45268
for the Office of Drinking Water (WH-550)
Water _^_^_
v>EPA Human Viruses in the
Aquatic Environment:
A Status Report With
Emphasis on the EPA
Research Program
Report To Congress
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EPA-570/9-78/006
HUMAN VIRUSES IN THE AQUATIC ENVIRONMENT:
A STATUS REPORT WITH EMPHASIS ON THE EPA
RESEARCH PROGRAM
REPORT TO CONGRESS
Prepared by the
Environmental Research Center (ORD)
Cincinnati, Ohio 45268
for the
Office of Drinking Water
U.S. Environmental Protection Agency
Washington, D.C. 20460
December 1978
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ABSTRACT
Enteric viruses enter water through fecal wastes from infected
individuals. These viruses abound in urban sewage due to the continual
occurrence of viral infections, usually in children and often without
symptoms. Viruses that infect humans are incapable of multiplying in the
envirorment, but are rather resistant and may survive for extended periods.
The health concern over their waterborne transmission stems primarily from
three factors: (1) much virus-laden waste ultimately contaminates surface
waters, the source for most drinking water, (2) only a few virus particles
may be required to produce an infectious dose, and (3) the enteric viruses
can produce a variety of serious diseases. Since water is consumed by all,
even a low level of viral contamination may significantly contribute to the
disease burden of a population.
In comparison to the traditional coliform bacteria indicators of water
quality, enteric virus levels in wastewater are extremely low. Waste and
water treatment processes further reduce the levels. Although viral detec-
tion methods have been dramatically improved over the past 15 years, this
technology is not adequate to assess the occurrence of the extremely low
levels possibly present in drinking water. Likewise, the role of drinking
water in maintaining the background level of viral disease is difficult to
evaluate epidemiologically because the more common person-to-person spread
of disease is continually occurring.
Viruses have been isolated from many surface water streams used as
drinking water sources and it now appears that full conventional treatment
may be required (and will be adequate) to produce reasonable assurance of a
virologically safe drinking water. Even so, it is of particular importance
that we remain diligent to the possible risk of the contamination of drink-
ing water supplies by viruses and other pathogenic microorganisms. Since
its inception, the U.S. Environmental Protection Agency has supported and
is continuing to support studies designed to fully evaluate the role of
water in viral disease transmission.
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PREFACE
The Safe Drinking Water Act, as amended (42 U.S.C. §300f et seq.),
states very succinctly [Section 1442 (a)(7)] that the Administrator of the
UjS. Environmental Protection Agency "shall carry out a study of virus
contamination." In a broad interpretation of this wording, the study of
the sources of viral contamination would require the tracking of viruses
from the infected individual, through the sewage system, the natural
aquatic environment and ultimately to the contamination of a glass of
drinking water. The development of the capability to conduct such a study
began about 15 years ago as the awareness of a potential virus-in-water
health question began to surface. This report will not review the
development of this capability, but rather will use the data thereby
obtained to put into focus the preventive-health questions and answers that
relate to the subject as they are currently perceived.
The report is divided into twelve sections that represent key areas of
interest and activity in the virus-in-water field. It concludes with a
discussion of the limitations of the current state of knowledge and
recommends nine specific areas for further research effort. Reference to
the older scientific literature is made frequently. However, the report
focuses on recent findings obtained from the ongoing research activity and
the drinking water survey of the EPA. The research program of EPA is
carried out through the support (both in-house and grants/contracts) of
projects by three laboratories within the Environmental Research Center-
Cincinnati: Health Effects Research Laboratory, Municipal Environmental
Research Laboratory and Environmental Monitoring & Support Laboratory. It
is hoped that this report accurately conveys the state of knowledge
regarding this potential health hazard and has clearly revealed the studies
engaged in by this Agency in response to the mandate of the Act and the
health needs of the American public.
ii
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ACKNOWLEDGEMENTS
The Office of Drinking Water gratefully acknowledges the preparation of
this report by the Health Effects Research Laboratory, the Municipal
Environmental Research Laboratory and the Environmental Monitoring and
Support Laboratory at the Environmental Research Center, U.S. Environmental
Protection Agency, Cincinnati, Ohio. The review efforts by the Office of
Research and Development in Washington, D.C. are also appreciated.
iii
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CONTENTS
Abstract i
Preface ii
Acknowledgements ill
Contents iv
General Overview v
Historical Perspective 1
Introduction 3
Sources of Enteric Viruses 4
Viruses in Sewage and Sewage Effluents 5
Viruses in Surface Waters and Treatment Plant Intakes 9
Theoretical Concentrations of Viruses in Treated Drinking
Waters 10
Disinfection of Drinking Water 11
Testing of Drinking Water for Viruses 16
Results of Viral Testing of Drinking Water Systems 22
Viruses in Ground Water 23
Risk of Viral Exposure from Drinking Water 24
Conclusions and Recommendations for Future Work 26
References 28
Appendix I - List of Virus-in-Water Related Projects Funded
by Environmental Research Center-Cincinnati During FY 77
and 78 35
Appendix II - Glossary 37
iv
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GENERAL OVERVIEW
Human viruses enter the aquatic environment primarily through sewage
discharges. Some viruses are excreted in large numbers in the feces of
infected individuals. These viruses are grouped under the heading of
"enteric viruses" and about 100 such virus types have been identified.
These are the ones of primary concern in the waterborne transmission of
viral disease. Although enteric viruses frequently produce asymptomatic
infections, they are capable of producing paralysis, heart anomalies,
infection of the brain and eyes, upper respiratory infections, gastro-
enteritis and many less serious symptoms. Children under the age of 10
years appear to be the most susceptible to infection. In the aquatic
environment, the viruses are unable to multiply but may remain viable for
an extended period of time, especially at low temperatures, thereby posing
a health risk to users of these waters.
The occurrence of viruses in the environment and the related health
risk have been a subject of EPA research programs for several years.
Outbreaks of viral disease caused by the ingestion of sewage-contaminated
waters have been well-documented by epidemiological studies. However, the
role of "acceptable" drinking water in spreading and maintaining the
endemic level of viral disease has not been elucidated. One major
objective of EPA-funded work as well as work supported by others has been
the development of methods for the recovery of low numbers of viruses from
large water samples. Fairly sensitive methods have been developed in the
past few years and these have been used to quantitate the number of viruses
in environmental samples from various locations. In addition, these
methods have been used to evaluate the effectiveness of sewage and water
treatment processes, including disinfection, for viral inactivation and
removal.
Even though the amount of data available from these studies is very
limited, an estimate of the level of contamination that could occur in
drinking water can be made. However, viral exposure levels predicted from
such models are highly speculative in that the accuracy of the data and
assumptions concerning the effectiveness of water treatment are open to
question. Many virologists feel that the actual environmental concentra-
tions of viruses are much higher than those currently reported because of
known and suspected limitations of the virus-recovery methods. There is
also a fairly widespread view that water treatment procedures are not
continually applied in all plants. It is also felt that the procedures are
not as effective in removing or inactivating naturally-occurring viruses as
they are in removing the laboratory strains used in treatment-efficiency
studies. Therefore, it is possible that viruses may be present in some
treated drinking waters at concentrations near or below the level
detectable with current methodology.
The EPA has applied state-of-the-art virus recovery methods to drinking
water samples collected from community systems in many locations. A total
of 225 samples with a maximum volume of 1,900 liters/sample has been
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collected from 56 systems in the past six years. No confinned viral
isolations have been made from these samples. Other laboratories have
conducted viral testing of water supplies, and viral isolations from two
treated supplies in the United States have been reported in the technical
literature. More testing and evaluation is needed to confirm the occur-
rence of viruses in such relatively small volumes of a fesw hundred liters
of treated drinking water. In addition to the techniques that must be
developed or improved to obtain an accurate indication of the occurrence of
viruses in water, the actual number of viruses needed to produce infection
is currently under investigation.
Conventional water treatment (coagulation-sedimentation, filtration and
disinfection) is believed by most authorities to be capable of producing
virologically-safe drinking water. However, the adequacy of disinfection
(the final water treatment to defend against microbial-disease transmis-
sion) to assure complete inactivation of naturally-occurring viruses has
been questioned in recent years. Based upon the information presently
available, it is recommended that research should be initiated or continued
in nine areas in order to more fully evaluate the possible health risk from
viruses which might be in drinking water. These recommendations are:
1. The development of improved recovery methods with emphasis on
increased sensitivity for the total number and types of viruses that
may be present in water and wastewaters.
2. The further evaluation of the disinfection capability of chlorine
and other disinfectants on natural viruses under field conditions
and new viruses implicated in waterborne disease outbreaks (e.g.
gastroenteritis virus).
3. The development of practical methods to remove/inactivate all
detectable viruses from treated sewage and sludge.
4. The further evaluation of the viral contamination of surface and
ground water as one of the factors to be considered in the land
application of wastewater and sludge.
5. The development of a broader data base for estimating the minimum
infective dose for ingested viruses.
6. The development of methods for the laboratory cultivation of
hepatitis A virus and agents of acute viral gastroenteritis.
7. The evaluation of the role of lower animals as reservoirs of viruses
that may infect humans.
8. The development of epidemiological approaches to determine the
extent of endemic waterborne viral transmission.
9. The elucidation of specific factors and mechanisms responsible for
viral inactivation and destruction in natural waters and soils.
vi
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HISTORICAL PERSPECTIVE
The field of environmental virology came into being in the 1950's
largely as a result of the tremendous expansion of virological research
made economically and technically feasible by the development of animal
cell culture techniques. This led to initiation of research which showed
that:
1. Enteroviruses were more resistant to chlorine than the coliform
bacteria group used to indicate the sanitary quality of water.
2. Enteroviruses had high survival capabilities under various
environnental conditions including wastewater treatment processes
and were not efficiently removed by these processes.
3. The minimum infectious dose for enteroviruses was very low (on the
order of one plaque-forming unit under certain experimental
conditions). This was four to six orders of magnitude lower than
the number of enteric bacteria needed to initiate infection.
These findings, coupled with epidemiological studies incriminating water as
a vehicle for transmission of infectious hepatitis, led to increased
concern about possible spread of other viral diseases by drinking water.
As a result, efforts to isolate viruses from water and consequent efforts
to improve these methods were initiated.
By 1965, interest in the subject was widespread and considerable
information had been developed. At an international symposium convened
that year, reports on various aspects of the waterborne virus question were
presented. The symposium proceedings (Berg, 1967) were widely distributed
and undoubtedly played a key role in enhancing interest in environmental
virological research. The divergence of opinion on the seriousness of the
problem of the spread of viral disease by drinking water was brought out in
the first and last paragraphs of the conference summary statement as
follows:
We have been told that the number of proved outbreaks of waterborne
diseases does not pose a serious public health problem. But, we know
also that detection of infections resulting from waterborne
transmission would be difficult if any of the infections were
inapparent, even if subsequent contact transmission should produce a
high disease rate.
Finally, we must decide upon the direction of our future efforts. To
this end, I would make this final admonition: knowing that viruses are
present in our sewage and in our rivers, that viruses have been
demonstrated in the water supply of a major city,* that it is possible
for waterborne transmission to occur without being readily detected, I
would say that if we are to err in the direction we take, we must err
on the side of safety. We must do sufficient research to be certain
*Paris, France
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that there is no important waterborne transmission, and should we find
instead that there is a danger, then, at least we can do something
about it.
Despite much additional research and subsequent major national and
international conferences on the subject (Snoeyink, 1971; Malina and Sagik,
1974; APHA, 1976), the controversies conveyed by the above statements have
not been resolved and are still with us today. The importance of the so
called "focal infection" (caused by ingestion of water containing extremely
low levels of virus) in the spread of enteric viral diseaise in a community
has been neither proven nor disproven. This is mainly because
epidemiological research methods are not sufficiently sensitive to
determine whether or not waterborne "focal infections" occur. How far we
must go in research "to be certain that there is no important waterborne
transmission. . ." has not been determined. Despite technological
improvements which have significantly increased the sensitivity of virus
detection methods, calculations based on the number of viruses which might
be expected in conventionally treated drinking water, if treatment is truly
as effective as indicated by virus-seeded studies, indicate that these
methods must be more sensitive by many more orders of magnitude in order to
detect the viruses at the levels in which they may be present. In the few
instances when viruses have been found in treated drinking water, the
validity of the results has been challenged, in some cases on direct
evidence, e.g. virus contamination problems with controls, and in other
cases on what may be called circumstantial evidence, e.g. presence of high
free-chlorine residuals or only a few viruses found in the source water.
It is against this background that this report has been prepared. The
concerns and controversies described above are further delineated and
expanded upon in the body of this report. The objective has been to place
the problem in perspective, based on the information currently available,
and to point out areas in which more information is needed to resolve the
present dilemma.
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3
INTRODUCTION
The persistent fear of death and debilitation from the ravages of
infectious agents of past generations was apparently allayed in the United
States with the control of the remaining dreaded disease—paralytic
poliomyelitis—in the mid-1950's by the widespread administration of the
newly-developed vaccine. The lack of concern over the epidemic return of
this viral disease is reflected in the increasing number of children who
are not receiving the protective vaccine. In 1976, 23 years after the
introduction of a vaccine, 38 percent of the children in this country
between the ages of 1 to 4 years had not received a primary vaccination
(MMWR, 1977). Yet untreatable infections with numerous viral agents remain
a major cause of morbidity in this country. Few communities escape the
gastroenteritis and upper respiratory infections that sweep through the
country in autumn or early winter each year. The actual number of cases
that occurs is difficult to quantitate because reporting of cases by
medical personnel to the health agencies is not required. In addition,
many affected people do not seek medical treatment during the relatively
short-term illness. Nonetheless, these outbreaks, thought to be primarily
of viral etiology, are a significant drain on the health of the nation and
these outbreaks are among the most common diseases experienced in the U.S.
today (Dingle, et al., 1953). In addition, viral diseases not uncommon in
this country, e.g. aseptic meningitis, myocarditis, hepatitis and
influenza, may cause death or long-term illness.
The transmission of the common viral infections is thought to be
primarily from person to person through direct contact. This mechanism
would appear to explain the spread of the vast majority of the respiratory
infections in which the primary portals of exit of the virus are through
the nose and mouth. However, approximately 100 viruses have been
identified that infect the lining of the stomach and intestine as well as
the pharynx. The primary portal of exit for these enteric viruses is the
intestinal tract. Thus, the virus is excreted in the feces of infected
individuals with viral concentrations reported to be as high as 10? and
10° infectious units per gram (Sabin, 1956). These enteric viruses
abound in sewage from larger communities where the likelihood of some
degree of viral transmission is occurring continually. The isolation of
infectious virus from sewage was first reported in the early 1940's and
since that time numerous studies have found a wide variety of viruses
present in sewage. These viruses are rather resistant to inactivation in
wastewater and surface water, and depending upon temperature and other
factors, may survive for days to months in this environment (Akin, et al.,
1971). Since most domestic wastes are discharged directly or indirectly
into the surface water systems that supply much of the country's drinking
water, viral contamination of these waters is continuous. Thus, the
waterborne transmission of the enteric viruses could significantly augment
the direct person-to-person spread of these viral infections.
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SOURCES OF ENTERIC VIRUSES
Man is thought to be the only important reservoir for members of the
human enteric viruses. However, viruses that appear to be human viral
serotypes have been isolated from the feces of domestic animals (Grew, et
al., 1970; Graves and Oppenheimer, 1975). It is assumed that these animals
are only passive shedders of viruses ingested via grossly contaminated food
or water and that the viruses do not multiply in these hosts. An exception
to this view may be the situation that appears to exist with the newly
identified rotaviruses. Serologically related rotaviruses have been
isolated from the feces of children, calves, piglets, mice and foals having
acute gastroenteritis (Woode, et al., 1976). The role of the animals in
the natural transmission of rotavirus disease in man has not been explored.
In fact, the role of domestic and wild animals in the transmission of
enteric viral disease to man has not been extensively studied and further
work in this area is needed. Nonetheless, domestic waste from humans would
appear to be the primary source of viruses in surface water as indicated by
the close association of virus isolations and domestic pollution sources
(Berg, et al., 1971).
Enteric viral infections are common in children, especially those below
five years of age. The high frequency of occurrence is obscured by the
fact that only a small percent of those infected manifest serious disease.
Prior to the introduction of the poliovirus vaccine, it has been estimated
that only one of every 1,000 children infected with the wild virus
contracted paralytic disease (Mslnick and Ledinko, 1951). A similar
pathogenic ratio probably exists for many of the other enteric viruses.
Therefore, an indication of the prevalence of enteric viruses in a
community must be determined from viral excretion data. The findings of a
recent study conducted in Seattle, Washington, indicated a fecal isolation
rate of enteroviruses (other than poliovirus) of two to four percent among
family members selected for the study (Cooney, et al., 1972). Poliovirus
isolates were not considered in the evaluation because they were assumed to
be primarily of vaccine origin. Gelfand, et al. (1957) in earlier work in
Louisiana found that as many as 16 percent of the healthy children included
in their study were excreting viruses other than polioviruses during the
summer months. They also found excretion rates to be inversely related to
socio-economic status. Interestingly, Chin, et al. (1967) were able to
demonstrate the presence of vaccine strain poliovirus in sewage when as few
as 0.3 percent of the local population had received the live-virus vaccine
shortly prior to the examination of the sewage. The Seattle study also
found children less than one year of age to have an average of 1.5 enteric
viral infections per year which dropped to 0.58 for those two to five years
of age and considerably lower for those over five.
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VIRUSES IN SEWAGE AND SEWAGE EFFLUENTS
Some enteric viruses enter surface waters directly from infected
individuals through swimming and other water activities. Viruses may also
enter surface water directly from waste discharges of individual dwellings
located along the water's edge. However, most human enteric viruses that
are present in surface water are introduced through the discharges of urban
sewerage systems. Sewage from these areas normally contains a wide variety
of enteric virus types that vary in concentration according to the time of
day and the season of the year. Wild viruses are shed in peak numbers in
the late summer or early fall. However, this seasonal peak becomes much
less pronounced in the semi-tropical region of the country. In recent
years, the predominant enteric viruses isolated from sewage have been the
vaccine-derived polioviruses and these viruses are found year-round without
a dominant summer-fall peak.
The number of enteric viruses isolated from sewage samples is very much
dependent upon the methodology used to recover the viruses. Concentration
methods, of which there are a large variety, are generally used and the
recovery efficiency may vary substantially. A few investigators have
attempted to quantitate virus in sewage by direct inoculation of the
unconcentrated sample into the cell culture assay system. Buras (1976)
used such a technique on sewage samples from Haifa, Israel, and reported
the surprisingly high number of 10° viral units/liter during midsummer.
The weekly average for eleven consecutive months dropped to 174,000
units/liter. Fannin, et al. (1977) reported up to 440 viral units/liter in
Chicago sewage with the direct inoculation procedure. EPA virologists
found 1,450 viral units/liter in a sample of Cincinnati sewage; however,
other samples were negative for virus by the same technique (Akin,
unpublished data). Grinstein, et al. (1970) found only two of 76 one-mi
samples positive for virus in Houston, Texas, sewage. Most investigators
in this country have concluded that viral concentration procedures are
required to quantify the virus level in sewage because of the relatively
low numbers that may be isolated during a particular sampling period.
Using various concentration procedures, virus levels in sewage from
U.S. urban areas have been reported up to approximately 6,000 units per
liter (Vaughn, 1977a). More common, however, are recoveries ranging from
near 100 to 400 virus units per liter (Safferman and Morris, 1976;
Grinstein, et al., 1970; Wellings, et al., 1974). Viral recoveries from
sewage in other countries (e.g. Israel, India, South Africa) have generally
been higher than those reported in U.S. studies (Buras, 1976; Rao, et al.,
1972; Nupen, 1970). This finding may reflect a less dilute sewage due to
water conservation practices and possibly to a higher prevalence of enteric
viral infections in these countries. As has been previously stated, a wide
variety of virus concentration/recovery methods are used. Since this is an
active area of research and experimentation, no standardized methodology is
utilized. Therefore, a true comparison of the currently available sewage
viral concentration data from one study area to another is not possible.
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Two factors have been identified that may further influence the
accuracy of the data; one factor may inflate the viral count and the other
may result in a substantial underestimation of the viral number. Most
quantitative virology is based on the counting of plaques produced in a
cell monolayer overlaid with semi-solid medium containing a vital stain.
The plaques are circumscribed areas produced by the death of a group of
cells within the monolayer. Several independent investigators, including
Brashear, Gerba and Riggs, have recently reported the inability to confirm
a viral causation for all of the plaques that appear on some cell types
when incubated with environmental samples (personal communications).
Therefore, the reporting of unconfirmed plaques as viral isolates may give
inflated numbers. On the other hand, it is known that viruses in the
aquatic environment are normally associated with particulate matter and
concentration procedures that remove particulates without prior virus
elution may result in an underestimation of the number of viruses present.
In many of the reports it is impossible to determine the consideration
given to these two sources of error. In addition, most studies have found
polioviruses to be the most frequent type of viruses isolated. The vast
majority of these have vaccine-like markers and are considered to be of
vaccine origin (Horstmann, et al., 1973). They are generally assumed to be
non-pathogenic. However, of the 142 cases of poliomyelitis reported in
this country in the last eight years, 44 have been "vaccine associated"
(MMWR, 1977). This observation suggests that the vaccine virus may be
pathogenic for a small susceptible portion of the population. Therefore,
the maximum (or average) number of pathogenic viruses that occur in raw
sewage from U.S. urban areas is not known. However, from the reports that
are available from field studies and with reasonable allowances for the
known variables, it would seem extremely unlikely that the total concentra-
tion of pathogenic viruses would ever exceed 10,000 virus units/liter of
raw sewage and would most often contain less than 1,000 virus units/liter.
Of possibly more importance to evaluating the drinking water hazard
than the amount of virus present in raw sewage are the following considera-
tions: (1) the efficiency of sewage treatment in virus removal, (2) the
portion of wastewaters that receive treatment, and finally, (3) the amount
of wastewater that constitutes the drinking water sources. A study
conducted in the early 1960's (currently being updated by EPA) indicated
that for the 155 U.S. cities studied, municipal wastewater constituted a
maximum of 18 percent of the surface water supplies for these cities
(FWPCA, 1966). Within the next few years, it is anticipated that legisla-
tion and public interest will result in at least secondary treatment for
practically all municipal wastewater entering a surface stream. By adding
a reasonable safety factor, we may assume that, in the near future, surface
water entering treatment plants will be composed of no more than 25 percent
wastewater and that this will have been subjected to at least secondary
treatment. Therefore, the efficiency of secondary treatment in the removal
of viruses from sewage becomes of primary importance in assessing the
waterborne transmission of viruses.
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The tera "secondary treatment" is generally applied to the treatment of
primary settled sewage by one of three biologically active processes: (1)
trickling filtration, (2) activated sludge digestion, or (3) oxidation pond
stabilization. The viral removal/inactivation potential of each treatment
has been studied at the laboratory and pilot scale level, as well as under
full-scale field conditions. Meaningful viral-removal-efficiency studies
can only be conducted by comparing the decrease in virus levels in the
effluent with time after adding a known level of viruses before treatment.
These studies should be based on actual detention times of the wastewater
in the treatment system. This information is difficult to obtain with
natural systems and such studies are usually done with high levels of
seeded virus using theoretical detention times based on plant design and
specification. Such studies have yielded highly variable viral removal
efficiencies, ranging from near zero to 99 percent (Berg, 1973a). This
variability may be due to: (1) the actual fluctuation in viral removal
efficiency of the treatment process, (2) a failure to collect time-phased
samples before, during and after the treatment process when there is
substantial variation in the viral load entering the plant, and (3) the
variation in efficiency of the virus recovery methods used. The addition
of a disinfection step to the treatment may also yield inconsistent
results. This is most likely due to the failure to add sufficient disin-
fectant to produce a viricidal residual for a desired contact time
(Olivieri, et al., 1971). In addition, the presence of protective protein
and particulate matter may physically interfere with the ability of a
disinfectant to produce a virus-free effluent.
The contribution of viruses to surface water by effluents frcm sewage
treatment plants can possibly best be determined by focusing on the number
of natural viruses found in these effluents rather than on a theoretical
percent removal by the treatment process. In a recent study conducted by
the Sanitation Districts of Los Angeles County (California), viruses were
recovered from 27 to 60 twenty-gallon samples of activated sludge effluent.
The positive samples contained frcm 0.4 to 136 viral units/liter (Miele,
1977). Sagik and Sorber (1977a) have studied the viral content of secon-
dary effluents from three systems in two Texas communities. Means of 5 and
179 viral units/liter were determined for two trickling filter systems and
a mean of 3 viral units/liter for the oxidation pond system. Heyward
(Personal communication) has recovered a mean of 165 and 269 units/liter
from activated sludge and trickling filter effluents respectively in a
community in Washington state.
The observations from these current studies have yielded virus levels
consistent with previously reported findings from unchlorinated secondary
effluents (Wellings, et al., 1974; Metcalf, et al., 1974). Therefore, it
would appear frcm the available data that the virus level in secondary
effluents in U.S. systems would not be expected to exceed 1,000 viral
units/liter. If a maximum of 10,000 units/liter were present in raw
sewage, as was suggested earlier in this report, then the typical viral
reduction by secondary treatment may be considered to be on the order of 90
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8
percent. Disinfection and/or tertiary treatment may reduce the virus level
by one to four additional orders of magnitude. However, these additional
treatment steps will most likely not be routinely applied to wastewaters
that are discharged directly into surface water streams.
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VIRUSES IN SURFACE WATERS AND TREATMENT PLANT INTAKES
There are no known specific viricides in fresh surface waters, and at
cool temperatures, viruses have been found to survive for many days and
perhaps months before their infectivity is destroyed by oxidative and
other catabolic forces (Akin, et al., 1971; Herrmann, et al., 1974). To
further extend the maximum viral pollution concept, we will assume that no
viral inactivation occurs in wastewater from the point of discharge into a
stream until it enters the intake of a downstream treatment plant. Based
on data from an earlier study (FWPCA, 1966), an estimate was made that
water treatment plants that use the worst polluted surface waters would
have no more than 25 percent of their water composed of wastewater
effluents. By using this figure, the maximum virus level at a treatment
plant intake would be on the order of 250 viral units/liter (or 95,000
units/380 liters, a typical sample volume equivalent to 100 gallons). This
"worst case" number is truly hypothetical in that it represents the
combination of extreme conditions at each step along the way from raw
sewage to a surface water treatment plant intake. The few studies that
have been conducted to determine virus levels at intakes have found
considerably fewer viruses than this estimated maximum number. Berg
(1973b) reported the isolation of 6 and 38 viral units/380 liters at two
intakes in the Midwest. Brashear (unpublished data) found two of fourteen
380-liter samples positive for virus at an intake on the Ohio River. The
two positive samples yielded a total of only one and two viral units. A
current EPA-funded study has reported the isolation of a maximum of 38
viral units/380 liters at an intake on the Missouri River. Viruses were
isolated from 27 of 42 190-liter samples (O'Conner, et al., 1977). A
preliminary joint study conducted by EPA and a contractor in December,
1975, at the same site had yielded between 9 and 86 viral units/380 liters
(Akin, unpublished data). Even though these data do not represent
extensive sampling, they do show the viral concentrations recovered from
relatively heavily polluted surface waters. The maximum viral
concentration observed was about three orders of magnitude lower than the
predicted "worst case" number. More virus sampling of surface water should
be conducted at plant intakes in order to better quantitate the true
maximum virus challenge to the water treatment technology employed by the
typical plant.
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10
THEORETICAL CONCENTRATIONS OF VIRUSES IN TREATED DRINKING WATER
Few persons knowledgeable in public health and sanitation would deny
the occurrence of waterborne transmission viral disease. Waterborne
outbreaks of infectious hepatitis and gastroenteritis have been reported.
Such outbreaks are reported to the USPHS, Center for Disease Control, by
state or local health personnel and are formally investigated by a joint
effort of CDC and EPA at the request of the appropriate state health
department. Most of these outbreaks have occurred with untreated water
supplies and with treated water supplies that have had obvious deficiencies
in the system (Craun and McCabe, 1973; CDC, 1976). The answer to these
problems lies in the application of established sanitary engineering
principles using "fail-safe" systems that insure uniform and uninterrupted
water treatment. Also, a vigorous effort to encourage compliance with
sanitary codes designed to prevent recontamination of treated water in the
distribution system could substantially reduce the number of outbreaks. An
important question that remains is whether drinking waters are virologi-
cally safe from the endemic spread of infectious disease when they are
derived from unprotected surface water sources that have been subjected to
"good" conventional water treatment processes.
Numerous studies conducted over the past 15 to 20 years have shown that
conventional water treatment can typically remove or inactivate six to
eight orders of magnitude of virus (Robeck, et al., 1962; Chang, 1968;
Sobsey, 1975). This overall level of reduction represents a summarization
of the reductions derived from studies of the individual treatment steps
using laboratory cultured virus inoculum. Applying the larger reduction
figure (108), Sproul (1976) has calculated that the treatment of a source
water containing 300 viral units/380 liters would result in a finished
water that contained one infectious unit of virus per 120 million liters.
If the same treatment conditions are uniformly applied to a source water
containing the maximum theoretical number of 95,000 viral units/380 liters,
then one infectious unit would be present in about 0.4 million liters of
finished drinking water.
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11
DISINFECTION OF DRINKING WATER
Studies conducted in the 1950's demonstrated that poliovirus and other
enteroviruses were inherently much more resistant to chlorine than the
coliform bacteria used to indicate the sanitary quality of drinking water
(Clarke and Kabler, 1954; Clarke, et al., 1956). Investigations conducted
during this period also demonstrated considerable differences in chlorine
inactivation rates among various enteric virus strains and types (Kelly and
Sanderson, 1958). Other observations discussed elsewhere in this report
have shown their generally high survival under certain environmental
conditions including sewage treatment processes and their apparently low
minimum infectious dose level (about 1 viable unit). These findings have
raised the level of interest and concern about the possibility of hazards
posed by the viruses in drinking water. Since disinfection is considered
the last line of defense against disease transmission by drinking water,
much effort has been expended in assessing the efficiency of disinfection
and factors which may interfere with efficient disinfection of drinking
water. The main interfering factors include the inherent or native
differences in viral resistance to disinfection and those conferred on
viruses by their association with particulate matter.
Inherent Virus Disinfection Resistance
Liu, et al. (1971, 1973) conducted a broad investigation of the
free-chlorine resistance of 25 human enterovirus types in chlorine
demand-free water and in Potomac River water at a pH of 7.8. Included were
three reoviruses, three adenoviruses, three polioviruses, eight coxsackie-
viruses, and eight echoviruses. They found that the time required for
99.99 percent inactivation at 2° C and 0.5 mg/1 free residual chlorine
ranged from 2.7 minutes for reovirus 1 to more than 120 minutes for
coxsackie A6 virus.
More recently EPA has funded research to intensively examine the
disinfection resistance of six of the 25 enteroviruses used in Liu's
investigation, including two of which were very sensitive, two which were
very resistant and two which were intermediate in resistance (Engelbrecht,
et al., 1978). The resistance of all six of these viruses has been
examined under chlorine demand-free conditions at pH 6.0 and 10.0 and three
of these viruses have been studied at the pH used by Liu, et al. (1973),
i.e., 7.8. The disinfection criteria and experimental conditions were
similar, consisting of the time required for 99.99 percent inactivation at
free chlorine levels of 0.5 mg/1.
The results of the studies conducted at pH 7.8 indicated that the time
required for 99.99 percent inactivation (4.5 to 6.7 minutes) was much
shorter than indicated by the results of the Liu et al. (1973) study (8.0 -
39.5 minutes). The resistance of the six enteroviruses to hypochlorous
acid (HOC1) at pH 6 was found to vary by a factor of five in the time
required for a 99.99 percent reduction (1.4 to 6.8 minutes). The time
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12
required for a 99.99 percent reduction of these same viruses by the
hypochlorite ion (OC1~) at pH 10.0 ranged from 23.6-193 minutes, or 10 to
150 times that required for equivalent reduction at pH 6.0. Interestingly,
the virus type (echo 1) that showed the greatest sensitivity at pH 6.0 was
the one most resistant at pH 10.0. This result indicated that changing the
pH had different effects on the chlorine resistance of different viruses in
addition to the alteration of the chlorine species present. Further
indication of this phenomenon has been shown in chlorine dioxide (CIC^)
disinfection studies. In contrast to chlorine, C102 does not react with
water to hydrolyze to form other compounds. The C102 molecule remains
intact in the same form over a wide pH range (Benarde, et al., 1965). Yet
the rate at which C1C>2 inactivates polio virus 1 increases as the pH is
increased fron 4.5 to 9.0. Poliovirus 1 was inactivated over three times
faster at pH 9.0 as at pH 7.0 (Oonier, et al., 1977). Previously,
Benarde, et al. (1965) had shown a similar effect in E. coli disinfection
studies using chlorine dioxide. Thus it appears that both 12. coli and
poliovirus 1 are much more sensitive to ClOp at slightly alkaline pH's
than at neutral or acid pH's. Morris (1970; pointed out the possibility
that pH changes might alter the ionic charge and interfacial potential at
the microbial surface, resulting in changes in sensitivity of the microor-
ganism to germicidal action. He pointed out the need for such information
in attempting to develop systematic knowledge of disinfection and
disinfection mechanisms.
Scarpino, et al. (1972) obtained unusual disinfection results which may
have been caused by changes of this nature. They reported a reversal in
the viricidal efficiency of hypochlorous acid and hypochlorite ion. Their
results showed that hypochlorite ion was seven times as effective as
hypochlorous acid against poliovirus 1 although companion studies conducted
with E_. coli gave conventional results; hypochlorous acid was 50 times as
effective as hypochlorite ion (hypochlorous acid has been well established
in the literature as a much stronger disinfectant than hypochlorite ion).
In discussing these results, they speculated that components of a borate
buffer system used in one of their studies may have had a major effect on
HOCL^±OC1~ equilibrium, may have suppressed ionization, or may have
resulted in the formation of previously undescribed viricidal forms.
Subsequent studies indicated that the presence of potassium chloride in the
pH 10.0 buffer was responsible for the enhanced disinfection rate shown by
hypochlorite (Scarpino, unpublished data). More recently, Engelbrecht, et
al. (1978) have confirmed these results and also have shown that the
presence of potassium chloride also accelerated virus inactivation by
hypochlorous acid.
The results of two recent studies indicate that viruses may develop
increased resistance to chlorine. Studies by Bates, et al. (1977) showed
that progeny of poliovirus 1 repeatedly cycled through chlorine disinfec-
tion were somewhat more resistant to chlorine than the original parent
stock virus. The resistant progeny required about 2.5 times the length of
exposure required by the parent stock for inactivation to the same level.
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13
The existence of extremely chlorine resistant polio 1 viruses in the
environment has also been reported recently (Shaffer, et al., 1977). -At
free chlorine levels of about 0.5 mg/1 and pH 7.1, these isolates showed an
initial decline in number of about 90 percent in 2 minutes with virtually
no decrease in titer after 30 minutes exposure. These findings are
contrary to the results of many previous studies by others. Isolates of
these viruses have been obtained for confirmatory studies and further
investigation in two extramural research projects currently being funded by
EPA.
Concern has also been expressed that the inherent disinfectant
resistance of viruses grown from a human host (usually in epithelial cells
in the digestive tract) may differ from that of viruses adapted to
replication in laboratory cell culture systems. Use of cell-culture-grown
viruses in "seeded" field studies to determine the efficacy of virus
removal and inactivation by water treatment processes has also been
criticized (Sproul, et al., 1969; see discussion by Shuval at the end of
Sproul's article). In efforts to provide information on both of these
points of controversy, EPA has recently funded a laboratory study designed
to compare disinfection resistance of naturally-occurring and cell-culture-
grown viruses and a field study designed to determine the efficiency of
water treatment processes for removal and inactivation of naturally-
occurring enteroviruses.
Aggregation or clumping of viruses has long been considered to be a
factor involved in increased viral resistance to disinfection. Until
recently, however, no evidence of this has been available. Through a
series of EPA research grants at the University of North Carolina, evidence
of this phenomenon and information on the factors involved in virus aggre-
gation and deaggregation have been obtained (Sharp, et al., 1976; Floyd, et
al., 1976; Floyd and Sharp, 1977; Young and Sharp, 1977). Although the
effects are complex and appear to be different with different viruses, some
general information has been developed from these studies as follows:
1. Although differences in halogen resistance of up to 300-fold between
aggregated and dispersed viruses have been shown, the differences
thus far shown are differences in rates of inactivation. No
evidence of complete resistance by the aggregates to inactivation
has been found.
2. The type and concentration of metallic cations and pH are important
determinants with regard to aggregation. Aggregation does not
appear to be related to virus isoelectric point characteristics.
3. Induced aggregates of some viruses are quite stable although those
of other viruses can be dispersed easily. Natural virus aggregates
appear to be more stable than induced aggregates.
Additional studies to improve the state of our knowledge in this area are
currently in progress.
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14
While disinfection information on a large number of enteroviruses is
available, data on several viruses including infectious hepatitis A virus
and gastroenteritis virus(es), each of which have been implicated in
waterborne disease outbreaks, are not available because of technological
problems associated with conducting such research on these particular
viruses. Disinfection studies on infectious hepatitis A 'virus under EPA
funding are now in progress.
Protective Effects of Particulate Matter
For disinfection to be effective, contact must occur between the
disinfectant and the microorganism. Because source waters may contain a
variety of inorganic and organic particulates, some of which may originate
as fecal wastes, and because of the tendency of microorganisms, particular-
ly viruses, to adsorb to various kinds of particles, removal of particulate
matter (usually measured by light scattering) has been considered very
important during water treatment. Although the major reason for removal of
particulate matter (turbidity) has been the concern that it may interfere
with disinfection, until recently little direct evidence of such interfer-
ence had been found. The reduction of the Public Health Service standard
for turbidity from 5 Nephelometrlc Turbidity Units (NTU) to 1 NTU specified
in the National Interim Primary Drinking Water Regulations has brought the
need for information in this area into sharp focus.
Previously, laboratory studies had established that enteric bacteria
and enteroviruses ingested by aquatic nematodes found in some water
supplies were protected against very high doses of chlorine (Chang, et al.,
1960). In another instance, persistance of coliforms in a water supply
system containing a substantial level of residual chlorine was attributed
to ingestion and survival of the coliforms in small crustaceans present in
the water (Tracy, et al., 1966). More recent studies indicate that virus
adsorbed onto surfaces of particles, such as clay, remained exposed to the
disinfectant and inactivation rates were affected only slightly, if at all,
when compared to the inactivation rates of free virus particles (Syraons and
Hoff, 1975; Stagg, et al., 1977). Viruses precipitated with aluminum
phosphate also showed inactivation rates similar to those of free viruses
(Hoff, 1977). Boardman (1976) reported that poliovirus associated with
kaolin, calcium carbonate and alum was inactivated at a slower rate than
free virus particles. The effect was greatest when the particles (calcium
carbonate and alum) were formed in the presence of virus.
Disinfection studies on viruses associated with organic material
presents a different picture. Moffa and Smith (1974) showed that
cell-associated poliovirus was more resistant to inactivation by chlorine
dioxide than freely suspended virus. Other research in this area also
indicates that this type of virus-particulate complex is much more
resistant to inactivation than freely suspended virus (Hoff, 1977). Virus
associated with cell debris could be detected after exposure to more than
1.5 mg/1 of HOC1 for nearly an hour. Under similar conditions, free virus
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15
was not detectable after two minutes exposure. While it is not known how
well such complexes simulate viruses as they exist in natural waters, it is
likely that this model is more representative of natural conditions than a
model employing freely suspended virus. Studies showing that coliforras
associated with the solids in sewage effluents are very well protected from
inactivation (Hoff, 1977) provide indirect evidence that viruses present in
such solids also would be protected. Additional studies of this type using
chlorine dioxide and ozone as disinfectants are in progress. It is
significant that cell-associated viruses, while extremely well protected
against inactivation, are still able to initiate infection of cell cultures
(Hoff, 1977). Other recent studies have also shown that enteroviruses
associated with both inorganic and organic solids remain infective for cell
cultures (Moore, et al., 1974) and animals (Schaub and Sagik, 1975).
As indicated above, it is not known at present the state in which
enteroviruses exist in natural waters. Data indicate that viruses
intimately associated with certain types of participates are much more
likely to survive disinfection than free viruses. Additional research to
further refine our knowledge in this area is needed. The data also
indicate that in addition to the final barrier, disinfection, water
treatment unit processes which remove participates constitute important
barriers for preventing virus passage through these water treatment
processes.
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16
TESTING OF DRINKING WATER FOR VIRUSES
Even though the development of methods for recovering viruses from
water has advanced markedly during the past few years, no practical method
exists for the sampling of extremely large volumes of drinking water.
Farrah, et al., (1977) have speculated that the sampling of 100,000 liters
of water was possible with a viral recovery procedure tested in their
laboratory. However, investigators who have routinely conducted environ-
mental virus testing have not exceeded sample volumes of 1,900 liters (500
gal). Obviously, these sample volumes are insufficient to recover viruses
if the observed viral level in source waters and the predicted reductions
through treatment accurately reflect the real world situation. Concern for
the validity of this conclusion has been a major force in the initiation of
field studies and surveys for human viruses in treated waters from
relatively small sample volumes of 380 to 1,900 liters.
The EPA began studies in July, 1972, using virus concentration methods
tested in its laboratories. These studies have continued to the present
time and have incorporated improvements in the methodology as they have
been developed. The EPA results obtained with the methods that were used
prior to the development (by numerous investigators), testing (Hill, et
al., 1976), and publication (APHA, 1976a) of a tentative standard method
have been reported (Clarke, et al., 1975; Akin, et al., 1975). Currently,
this standardized method is being used by EPA to test up to 1,900-liter
samples of drinking water from sites throughout the country.
In the current EPA study, 119 drinking water samples have been
collected for viral analysis from 42 communities. A listing of the sites
is shown in Table 1. Also shown are the total number of samples collected
at each site, the mean sample volume and the type of treatment the drinking
water received. Data on the fecal coliform content of the source water are
given so as to provide some indication as to the degree of domestic
pollution of the raw water supply. The sampling sites fell into two major
categories: (1) sites that utilized surface waters that contained signifi-
cant domestic wastewater and invariably treated the water with the full
conventional processes, and (2) sites that used surface waters from
supposedly protected watersheds that were marginally treated, if treated at
all. Arrangements were made through state health departments and the local
water utilities for the sampling. A mobile laboratory was taken to each
site; the sample was collected and partially processed on site before
returning to the Cincinnati laboratory.
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17
Table 1. Community Drinking Water Systems Sampled for Viruses During the
Current EPA Field Study*
No.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
Site
Chester, IL
Indianapolis, IN
Cape Giardeau, MO
Glasgow, MO
Kansas City, MO
Lexington, MO
St. Joseph, MO
Hueston Woods, OH
Rocky Fork, OH
Stonelick, OH
Altoona
(Blair Gap) , PA
Bradford, PA
Central City, PA
Curwensville , PA
Derry, PA
Emporium, PA
Johnstown, PA
Lock Haven, PA
Nanty Glo, PA
Reynoldsville, PA
Sheffield, PA
Shomokin, PA
Unionville, PA
Williamsport, PA
Wilpen, PA
Alburg, VT
Barre, VT
Coventry, VT
Island Pond, VT
Montgomery Center,
Montpelier, VT
Orleans, VT
Rutland, VT
Troy, VT
Fairfax, VA
No.
Collected
10
16
8
10
3
12
4
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
VT 1
1
1
1
1
8
Mean
Sample
Volume
(liter)
1851
1832
1522
1885
1893
1904
1893
984
1249
1893
1094
307
613
1325
606
257
678
632
428
363
212
379
1893
424
602
1718
791
1893
1893
1893
1211
1893
844
1893
379
Fecal Coliforms
in Source Water
Counts per 100 ml
Treatment**
C
C
C
C
C
C
C
C
D
N
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
N
D
D
D
D
D
N
C
Range
180-13000
1-2000
430-8000
400-5100
240-490
2300-16000
670-2600
13
240
ND
60
2
2
0
6
0
4
1
5
25
28
ND
3
6
32
9
11
0
0
26
100
19
8
4
0-5
Geo. Mean
1429
90
1345
1154
343
5980
1434
13
240
ND
60
2
2
0
6
0
4
1
5
25
28
ND
3
6
32
9
11
0
0
26
100
19
8
4
1
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18
Mo.
36
37
38
39
40
41
42
Site
Cedar Grove, WV
Clarksburg, WV
Fayetteville, WV
Keyser, WV
Milton, WV
Ripley, WV
Teas Valley, WV
No.
Collected
4
2
2
1
2
5
5
Mean
Sample
Volume
(liter)
954
1578
1730
939
1578
1673
1893
Fecal Coliforms
in Source Water
Counts per 100 ml
Treatment**
C
C
C
C
C
C
C
Range
290-870
0-48
62-160
19
63-690
170-3400
0-4800
Geo. Mean
522
7
99
19
209
409
37
* No viruses were isolated from any of these samples
** C = Conventional treatment (coagulation/sedimentation, filtration and disinfection;
except at Hueston Woods, Ohio, which used slow sand filtration
and disinfection)
D = Disinfection only
N = No treatment
ND « Not Done
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19
The viral recovery procedure used was essentially that of the tentative
standard method (APHA, 19?6a) and is outlined in Figure 1. As can be seen
in Table 1, the 1,900-liter sample volume was not always achieved due to
the plugging of the viral adsorbents by the particulates present in the
water. In order to evaluate the recovery efficiency of the method with
samples from various water supplies, an extremely low level of poliovirus
vaccine was seeded periodically into a water sample and the concentrate
assayed in cell cultures by the plaque technique. The results of the
positive-control tests are shown in Table 2. The recoveries ranged from 0
to 70 percent and reflected the limitations of the method. Ill-defined
changes in water quality that interfere with viral adsorption and elution
are thought to be responsible for the low and variable recoveries
experienced within systems as well as between systems (Sobsey, 1978).
The field studies conducted by EPA comprise the most extensive sampling
of drinking water supplies that has been reported. In addition to these
studies, a joint EPA-grantee longitudinal study is currently examining
1,900 liters of finished water per week from a plant that utilizes the
Missouri River as its source water. The incoming water is highly polluted
with domestic waste, as indicated by the fecal coliform densities and the
isolation of human enteric viruses (O'Conner, et al., 1977). The Carborun-
dum Company, through its leasing of a viral sampling service, has also
claimed wide experience in the testing of various types of waters including
finished waters (Shaffer, et al., 1977). However, their specific findings
are considered confidential by the lessee and are not reported in detail in
the literature. Some yet unpublished studies are also being conducted by
the Epidemiology Research Center of the State of Florida (EPA funded), the
Fairfax County, Virginia, Water Authority and the University of Maryland.
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20
Figure 1. Recovery Procedure Used to Isolate Viruses From Drinking
Water Samples
Procedure
Sample Volume
Water Tap
1
Virus Concentrator
(Balston Virus Adsorbing Filter)
i
Elution of Virus Adsorbent
(Glycine Buffer pH 11.5)
Return of Eluate to Central Lab
(on Wet Ice)
Reconcentration of Eluate
by Second Filter Adsorption
\
Elution of 2nd Adsorbent
(Glycine Buffer pH 11.5)
I
Frozen Storage of Eluate
(-80° O
I
Assay for Virus in 4 Cell Culture
Types (Observed 28 days for Cyto-
pathic Effect)
I
Confirm and Identify Isolates
1,900 liters
1,400 ml
20-80 ml
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21
Table 2. Results of Positive-Control Seed-Study with Poliovirus Vaccine
and Treated Drinking Water from Six Communities
SITE
SAMPLE VOLUME
TOTAL VIRUS INPUT
VIRUS RECOVERED
Indianapolis, IN
Indianapolis, IN
Indianapolis, IN
Indianapolis, IN
Indianapolis, IN
Lexington, MO
Lexington, MO
Glasgow, MO
Chester, IL
Ripley, WV
Cedar Grove, WV
(Liters)
3,, 900
1,900
1,900
1,900
1,900
1,900
1,900
1,817
1,900
1,900
1,518
(PFU)
37
78
43
41
61
20
10
82
75
106
106
PFU
3
0
1
4
1
5
7
6
25
3
0
%
8
0
2
10
2
25
70
8
33
3
0
PFU s plaque forming unit
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22
RESULTS OF VIRAL TESTING OF DRINKING WATER SYSTEMS
Since 1972, EPA has collected 255 drinking water samples from water
supply systems in 56 communities. An additional 50 samples have been
collected by an EPA-grantee in the ongoing longitudinal study. No
confirmed virus isolations have been made from any of these samples and the
Agency is aware of only two studies in which viral isolations from a
treated water supply have been claimed. An echovirus was reported to have
been isolated from a chlorinated supply in Dade County, Florida (Wellings,
et al., 1975). In an EPA-funded survey, poliovirus was reported to have
been isolated from four water samples collected in the summer of 1975 from
'a water supply in Fairfax, Virginia, that utilized full conventional
treatment (Hoehn, et al., 1977). The latter finding created considerable
concern because the source water quality and the extent of treatment
applied would have suggested the great improbability of virus recovery in
the volumes of water tested. The authenticity of the isolations, however,
has been questioned (Akin and Jakubowski, 1977), but no obvious source of
sample contamination was evident. A subsequent test of the same water
supply a year later was conducted by EPA (in-house) and the laboratory that
reported the positive finding (Carborundum Company). Eight 380-liter
finished water samples were concurrently collected by each laboratory
during July, 1976. The concentrated samples were split into equal volumes
and half of each sample was assayed for viruses by each laboratory. An
additional 34 samples were collected between June and September, 1976, and
assayed only by the Carborundum laboratory.
A single viral isolate (identified as poliovirus 1) was made from a
sample collected on September 2nd by the Carborundom team and assayed in
their laboratory (Hoehn and Randall, 1977). No other viruses were isolated
from the 50 380-liter finished water samples. However, six units of
poliovirus 1 were isolated from a rectal swab taken from a Carborundum
field-team member on the day that the positive water sample was collected.
The periodic collection of rectal specimens from all personnel directly
involved with the study had been initiated during the 1976 sampling to
identify possible viral contamination sources. The possibility that the
water sample could have been contaminated by the worker cannot be ruled
out; however, no direct contact or mechanism of transmission was evident.
The previously mentioned findings raised serious questions as to the
differences in viral recovery sensitivity of methodologies used by the two
laboratories and to the environmental quality controls of the Carborundum
procedures. Therefore, an intensive double-blind seeded study involving
both laboratories and sponsored by the EPA Office of Drinking Water was
planned and is currently underway.
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23
VIRUSES IN GROUND WATER
Once considered a reliable source of microbiologically-saie drinking
water that did not require treatment, the purity of ground water is now
being questioned. The rapidly moving trend to the land application of
domestic wastewaters has made the penetration of viruses through soil into
aquifers a distinct health concern. Studies have shown that viruses can be
isolated from ground waters when domestic waste is applied to or released
into certain types of overlying soil (Mack, et al., 1972; Wellings, et al.,
1972; Vaughn and Landry, 1977). In an effort to better quantitate and
define this potential hazard, EPA has funded studies in this area. Vaughn
(1977b) has shown that viruses in a tertiary-treated effluent can penetrate
5.3 meters of a sandy Long Island, New York, soil from a recharge basin and
can be isolated from the underlying ground water. A seeded study conducted
with high concentrations of poliovirus introduced into the recharge basin
has shown that the virus can penetrate the soil and enter the upper aquifer
(7.6 meters below the surface) within 24 hours. Studies by Sagik and
Sorber (1977b) in Texas have shown that the spray irrigation of secondary-
treated wastewater may transmit viruses at least through 1.4 meters of clay
soil. Gilbert, et al. (1976) conducted similar studies at a recharge site
in Arizona and were unable to recover viruses from about 50 water samples
collected 6 to 9 meters below the surface at a seven-year-old recharge
site. The findings of these two studies indicate that the virus-soil
interactions are complex and many variables may be involved in the viral
contamination of ground water from wastewater recharge sites.
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24
RISK OF VIRAL EXPOSURE FROM DRINKING WATER
Assumptions have been made in this report that suggest that one
tissue-culture-infectious unit (TCIU)/O.4 million liters would be the
maximum number of viruses that most likely could be transmitted through
drinking water under currently accepted treatment and sanitary standards.
At this concentration, the residents of a typical city of 100,000 popula-
tion would be exposed to about 110 TCIU of viruses each day in the water
used for domestic purposes based on U.S. average community use of 640
liters/person/day and 69 percent residential use (Murray and Reeves, 1977).
Only a portion of the total volume used would be ingested in an "uncooked"
condition whereby .the virus could remain in an infectious form. Eased on
information compiled by Lippy (unpublished data) it is estimated that an
"average" person drinks about one liter of tap water/day. At this
consumption rate and under the conditions outlined above, one TCIU of virus
would be ingested with water by a single individual in the typical city
about every four days. The number of cases of clinical disease that would
result from the infections produced by this level of ingestion is unknown,
but would most likely be several orders of magnitude lower than the
ingestion frequency. Factors important in the manifestation of clinical
disease from viral ingestion include: (1) the immune state of the consumer
as a result of previous exposure to the virus, (2) the number of viral
particles required to produce infection, and (3) the virulence of the virus
(i.e., the disease:infection ratio).
The hypothetical level of exposure to viruses in drinking water would
appear to be low. However, this assessment has not been based on rigorous
data in many areas. Even though the model used the maximum detected virus
levels and allowed the virus environmental survival advantages (to approxi-
mate a "worst case" situation), the actual numbers of viruses present most
assuredly are underestimated due to the inefficiency of available virus
recovery methodology. We experienced a rather low mean recovery of 15
percent from cur positive controls using poliovirus (Table 2). Most
method-development studies have been conducted with laboratory-cultured
vaccine poliovirus because it is a hardy virus that is safely and easily
enumerated in cell culture. Very little recovery-efficiency data are
available for the approximately 100 enteric viruses remaining to be
studied. However, limitations in the concentration procedure exist with
some of the adenoviruses, and limitations in cell culture assay systems for
recovering some of the coxsackie A viruses, the hepatitis A virus and the
gastroenteritis agents have long been recognized. The efficiency of the
entire recovery system for the complete spectrum of the wild enteric
viruses in the state that they exist in nature is not known. Therefore, it
seems quite possible that no more than one percent of the total number of
viral units present in environmental samples is actually enumerated with
currently available methods. Also, the viral removal efficiencies by
sewage and water treatment processes are influenced by meterological
conditions and are subject to human, equipment and process failure and,
therefore, optimum treatment conditions are not always maintained. The
awareness of these limitations coupled with the unexpected and as yet
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25
unexplained finding of poliovirus in relatively small volumes of treated
finished water in Virginia (Hoehn, et al., 1977) warn against the drawing
of premature conclusions as to the magnitude of the risk.
Basic to determining the true risk of infection from ingested viruses
is an understanding of the minimum number of TCIU required to produce
infection in a susceptible person. A widely quoted study has shown that
only one TCIU could be required (Katz and Plotkin, 1967). However, this
study was conducted in premature infants who were exposed to poliovaccine
virus via gavage and may not reflect the actual risk in nature. More
extensive studies are currently being funded by EPA. Investigators at the
University of Wisconsin are using a wild porcine enterovirus and piglets to
study the minimum infectious and pathogenic dose of viruses in this animal
model system. It has been assumed that a low dose of wild virus may result
in asymptomatic infection in a susceptible host, whereas, a larger dose may
result in overt disease. However, such a dose-response relationship has
not been quantitated.
Other investigators at the University of Wisconsin are conducting a
study in humans under DHEW safeguards established for the protection of
human subjects. The study involves the feeding of low doses of oral
(Sabin) poliovaccine to 3- to 6-month old infants in order to determine the
minimum number of particles that must be ingested to produce an immunologic
response with the protective vaccine. The full dose of vaccine is given 10
days later so that infants not responding to the low dose will be protected
against any subsequent natural exposure to the wild pathogenic poliovir-
uses. These virus feeding studies will hopefully provide definitive
evidence as to the likelihood of producing an immunologic response by the
natural ingestion of extremely low numbers of waterborne virus particles.
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26
CONCLUSIONS AND RECOMMENDATIONS FOR FUTURE WORK
The existence of an appreciable health risk from the waterborne
transmission of enteric viruses through drinking waters in this country is
not apparent. This may be because the risk is truly minimal or it may only
reflect the difficulty in documenting the endemic spread of low levels of
infectious viruses through water supply systems. The finding of viruses at
intakes to water treatment plants with rather inefficient virus recovery
methods coupled with the likelihood that water treatment processes are not
uniformly applied and are not as efficient in removing naturally-occurring
viruses as has been indicated with seeded studies suggest that the latter
may be true. Many questions that bear directly on the subject remain
unanswered. It has long been hypothesized that low levels of viruses are
transmitted through drinking water and thereby give rise to sporadic
asymptomatic infections in those people ingesting this water. These cases
in turn give rise to person-to-person spread of large numbers of viruses
that result in cases of overt disease. An adequate approach for testing
this hypothesis has not been devised.
Virus recovery methods have been improved but remain relatively
inefficient in recovering all viruses from wastewaters and natural waters
(Sobsey, 1978; Williams and Jakubowski, 1978). Reported recoveries with
laboratory-cultured seeded viruses typically range from 0 to 70 percent.
In addition, some investigators believe that a major portion of the natural
viral population is firmly attached to or deeply embedded in particulates
and is not recovered at all by currently available methods. This methodol-
ogy question must continue to be addressed. The embedded particles may be
protected from water disinfection. Therefore, laboratory studies with
seeded viruses that do not represent the physical state of natural viruses
may yield a false viricidal efficiency of such disinfectants. In addition,
recent findings indicate that some viruses may not be removed by
adsorption-sedimentation-treatment procedures nearly as readily as the
standard testing virus, poliovirus 1 (Farrah, et al., 1978):
The viral agents of infectious hepatitis and acute gastroenteritis
(diseases that can be spread by water) cannot be recovered from
contaminated water due to lack of laboratory culture systems. This may
also be true of other viruses. Therefore, significantly more viruses may
be present in all types of surface waters than have been reported. Ground
water may also be contaminated with viruses; factors important in the
prevention of viral penetration through soil into the aquifer at wastewater
recharge sites must be defined. More basic studies that deal with the
molecular and the physico-chemical levels of virus interactions need to be
conducted so as to better understand the mechanism of viral attachment and
adsorption to particulates as well as the viral-inactivating factors
present in soils and the aquatic environment. The minimum infectious dose
of enteric viruses when ingested with food and water has not been satisfac-
torily evaluated and, therefore, needs further work.
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27
Two recent reports have recommended areas that need further research
(APHA, 1976b; NAS, 1977). Based upon the information presently available,
it is recommended that research should be initiated or continued in nine
areas in order to more fully evaluate the possible health risk from viruses
which might be in drinking water. These recommendations are:
1. The development of improved recovery methods with emphasis on
increased sensitivity for the total number and types of viruses that
may be present in water and wastewaters.
2. The further evaluation of the disinfection capability of chlorine
and other disinfectants on natural viruses under field conditions
and new viruses implicated in waterborne disease outbreaks (e.g.,
gastroenteritis virus).
3. The development of practical methods to remove/inactivate all
detectable viruses from treated sewage and sludge.
4. The further evaluation of the viral contamination of ground water as
one of the factors to be considered in the land application of
wastewater and sludge.
5. The development of a broader data base for estimating the minimum
infective dose for ingested viruses.
6. The development of methods for the laboratory cultivation of
hepatitis A virus and agents of acute oral gastroenteritis.
7. The evaluation of the role of lower animals as reservoirs of viruses
that may infect humans.
8. The development of epidemiological approaches to determine the
extent of endemic waterborne viral transmissions.
9. The elucidation of specific factors and mechanisms responsible for
viral inactivation and destruction in natural waters and soils.
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28
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40. Hoehn, R.C., C.W. Randall, E.A. Bell, Jr. and P.T.B. Shaffer. 1977.
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in Sewage. J. San. Eng. Div., ASCE, 97:661-673.
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64. Safferman, R.S. and M-E. Morris. 1976. Assessment of Virus Removal
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APPENDIX I
Virus-in-Water Related Research Projects Funded by the Environmental
Research Center-Cincinnati During FY 77 & 78
Title
Occurrence of Viruses in Drinking Water
Supplies.
Evaluate Methods for Concentrating and
Recovering Viruses from Drinking Water.
Improve Methods for Isolation, Identification
and Recovery of Human and Other Animal Viruses
from Surface Waters.
Increase Isolation Sensitivity of In Vitro
Systems for Detecting Enteric Viruses.
Evaluate Tentative Standard Method for
Selected Enteric Viruses.
Identification, Isolation and Characterization
Hepatitis A Agent.
Isolate, Cultivate, Characterize Etiologic
Agent(s) of Non-Bacterial Gastroenteritis
(Norwalk, Hawaii Agents).
Determine Minimal Oral Infectious Dose and Oral
Pathogenic Dose of Enteroviruses in a Natural
Animal Host.
Human Health Hazards of Viruses in Drinking
and Recreational Waters.
Longitudinal Study of Viruses and Coliforms in
Raw and Treated Water.
Factors Affecting the Adsorption, Transport and
Infectivity of Animal Virus in Soil-Water
Systems.
Investigation of Potential Virus Survival and
Movement at a Land Reclamation Site Utilizing
Sewage Sludge.
Fate of Human Viruses in Groundwater Recharge
Systems.
Human Enteric Virus Survival in Soil Following
Irrigation with Sewage Plant Effluents.
Activity Type
In-house
In-house
In-house
In-house
Grant
Grant
Grant
Grant
Grant
Grant
Grant
Contract
Grant
Grant
Supporting Lab*
HERL
HERL
HERL
HERL
HERL
HERL
HERL
HERL
HERL
HERL
HERL
HERL
HERL
HERL
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36
Title
Effect of Virus Particle Aggregation on the
Disinfection of Water Supplies.
Virus Sensitivity to Chlorine Disinfection of
Water Supplies.
Inactivation of Naturally Occurring Entero-
viruses.
Effect of Particulates on Disinfection of
Enteroviruses in Water by Chlorine Dioxide.
Effect of Particulates on Ozone Disinfection
of Bacteria and Viruses in Water.
Removal of Virus from Public Water Supplies.
Effect of Turbidity on Disinfection by Chlorine.
Methodology for Concentration, Recovery, and
Identification of Viruses from Ambient Waters
and Wastewaters.
Development of Methods for Quantitation of
Adsorbed Viruses in Waste and Other Water.
Quantitative Methods for Virus in Water.
Identification and Detection of Water-borne
Viruses by Immunoenzymatic Methods.
Development of Field Virus Concentration
Technology.
Development 'of Methods for the Detection and
Inactivation of Viruses in Various Waters.
Activity Type
Grant
Grant
Grant
Grant
Grant
Grant
In-house
In-house
Grant
Grant
Grant
Grant
Grant
Supporting Lab*
MERL
MERL
MERL
MERL
MERL
MERL
MERL
EMSL
EMSL
EMSL
EMSL
EMSL
EMSL
* HERL - Health Effects Research Laboratory
MERL - Municipal Environmental Research Laboratory
EMSL -.Environmental Monitoring fi Support Laboratory
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APPENDIX II
GLOSSARY
Asymptomatic - Presenting no subjective evidence of disease.
Double Blind Experiment - As used in this report, a study in which EPA
(Cincinnati) and the Carborundum Company exchanged and analyzed viral
samples, the virus identity and concentrations of which were only known to
a third party.
Elution - The removal of virus from material by washing with a liquid.
Enterovirus - A virus that infects cells of the intestinal tract.
Etiology - The causal relationship between a virus and the specific
disease.
Gavage - The introduction of material into the stomach by a tube.
Infectious Units - Either a single virus particle or a stable viral clump
that is infectious for a living host system.
Longitudinal Study - Research occurring over a period of time.
Plaques - Small, clear, circular areas on a lawn of growing cells
(monolayer) which result from the virus-induced deaths of groups of cells.
The number of plaques indicates the concentration of virus particles.
Progeny - Viral descendents.
Recharge Basin - An underground basin in which water is deliberately added
to restore water capacity.
Seeded Virus Study - An experiment in which a known concentration of virus
is added to some medium such as water.
liter - The concentration of viruses in a given volume of liquid.
Vaccine-like Markers - As used in this report, proteins on the coat of a
strain of poliovirus which react with various types of antibodies in a
manner similar to those of the specific poliovirus used in the preparation
of live (Sabin) vaccines. The markers could also pertain to growth at a
specific temperature.
Wild Viruses - Viruses recovered from the environment or from an infected
individual.
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