ENTERIC VIRUSES IN GROUND AND
SURFACE WATERS:
A REVIEW OF
THEIR OCCURRENCE AND SURVIVAL
Elmer W. Akin, William H. Benton, and William F. Hill, Jr.
ENVIRONMENTAL PROTECTION AGENCY
Water Quality Office
Division of Water Hygiene
Gulf Coast Water Hygiene Laboratory
Dauphin Island, Alabama 36528
1971

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Copyright © 1971. Reprinted by permission of the Board of Trustees
of the University of Illinois from
PROCEEDINGS
THIRTEENTH WATER QUALITY CONFERENCE
February 1971
VIRUS AND WATER QUALITY: OCCURRENCE AND CONTROL
Edited by
Vernon L. Snoeyink
Department of Civil Engineering
University of Illinois
Environmental Protection Agency
of the State of Illinois

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ENTERIC VIRUSES IN GROUND AND SURFACE WATERS: A REVIEW
OF THEIR OCCURRENCE AND SURVIVAL*
ELMER W. AKIN, WILLIAM H. BENTON, AND WILLIAM F. HILL, JR.
Environmental Protection AgencyWater Quality Officei division of
Water Hygiene, Gulf Coast Water Hygiene Laboratory} Dauphin Island,
Alabama 36528.
ABSTRACT
Enteric viruses have been isolated from surface water samples
collected throughout the world. Investigations reviewed in this paper
indicate that enteric viruses were isolated from an average of 36% of
the surface water samples examined. These viruses are shed in the
feces of infected man and animals-and may enter water systems by way
of soil runoff and sewage, both treated and untreated. The increased
frequency of wa£er reuse for domestic purposes has increased the
probability of human contact vrith contaminated surface waters.
Laboratory studies indicate that viruses tend to adsorb to soil parti-
cles and would therefore be removed before they reach ground water or
soon after entering the underground system. However, epidemiological
studies have implicated both contaminated surface and ground water as
the transmission route in a limited number of infectious hepatitis
outbreaks. Remarkably, no w:despread waterborne epidemics of viral
disease other than infectious hepatitis have been substantiated. The
amount of endemic disease caused by contact with virus-contaminated
surface waters is completely unknown. The transmission route of
endemic viral disease is difficult to determine due to the large per-
centage of asymptomatic infections which occur with these viruses.
Numerous workers have studied the survival of enteric viruses in waters
and have found them to survive for a significant length of time to con-
sider water a potential route of viral disease transmission. They have
shown that 2 - 100 days are required for various members of the enteric
virus family to lose 99.9% of their initial infectivity when suspended
in different surface waters at 20-25°C. Our flow-through experimental
system has allowed us to study virus survival in estuarine water under
quasi-natural conditions. In these experiments, poliovirus 1 infec-
tivity was reduced by 99% in 4 hours at 30°C (summer conditions) and
5-15 hours at 22°C (autumn conditions). By yielding survival data
indicative of that occurring in the" natural setting, field studies of
this type may have a very practical application. With this information
and viral isolation data, water treatment requirements and recycling-
frequency guidelines may be realistically determined.
*Portions of this work will be submitted by the senior author in
partial fulfillment of the requirements for the degree of Doctor of
Public Health, University of Michigan, Ann Arbor, Michigan.

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INTRODUCTION
Trask et al. (1938) reported in the late thirties their finding
of the regular presence of poliovirus in the stools of poliomyelitis
patients. A few years later workers sought _and detected human
enteric viruses in domestic sewage. This occurred just as the
field of virology was coming into its own. The then new in vitro
cell-culture virus systems yielded an avalanche of virus data
including the isolation and identification of viruses not yet
associated with disease.
The isolation of viruses excreted through the gut of man has
increased to approximately 100 serotypes today, and more are sure
to be discovered. Increasingly, the enteric viruses are being
associated with diseases not even suspected of having a virus
etiology a few years ago; i.e., diabetes mellitus (Gamble et al.,
1969) and mongolism (Stoller and Collmann, 1965). These are in addi-
tion to a long list of conditions known for some years to be
caused by enteric viruses; i.e., meningitis, myocarditis, pericar-
ditis, hepatitis, paralysis, rashes, febrile illness, and gastro-
enteritis. The human health hazard of adenoviruses found to be
tumorigenic for experimental animals remains to be adequately
assessed.
Since the disposal of human waste inevitably involves water,
and since the recycling time for domestic and industrial water
usage has continually diminished with population and industrial
growth, and since every person regardless of age consumes some water

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each day of his life, it is imperative that we know the fate of
infectious and dangerous substances that enter our water systems.
Enteric viruses can be readily isolated from domestic sewage
and present treatment methods do not produce virus-free effluents
(Kollins, 1966; Grabow, 1968). In addition, sewage from approxi-
mately 60% of the population of the United States receives no
significant treatment before it enters a disposal area, usually
a body of water (Holcomb, 1970). Human enteric viruses have also
been isolated from rectal samples of domestic and wild animals,
identifying soil runoff as another potential source of water contami-
nation (Kalter, 1967; Lundgren et a!., 1968; Grew et al., 1970).
With this empirical knowledge, we can deduce that human enteric
viruses are present to some degree in most natural waters.
GROUND WATERS
The possibility of ground water viral pollution has received
very little concern. However, laboratory studies have shown that
viruses readily adsorb to a wide variety of substances including
natural clays and silts. Carlson et al. (1968) have reported the
effective adsorption of bacteriophage T2 and poliovirus to kaolinite
4, montmorillonite 23, and illite 35 in the presence of electrolytes.
Jakubowski (1969) confirmed these results with poliovirus 1 and
extended his observations with clays and marine silt. This adsorp-
tion was found to be reversible. Free infectious virus could be
eluted from the particles in the presence of a proteinaceous

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solution. Drewry and Eliassen (1968) have found bacteriophages
Tl, T2, and fZ to effectively adsorb to nine different soil types
taken from California and Arkansas. Most workers emphasize the
influence of pH and cation concentration on virus adsorption.
Increasing adsorption occurs with the reduction of pH below 8.0 and
with the addition of cations, especially the divalent species.
Romero (1970) has recently reviewed much of the literature on the
movement of bacteria and viruses through porous media. He reported
that soil uniformly composed of fine sand with a high clay content
is the best aquifer material for removing biological contaminants
and concludes that these pollutants are able to travel a maximum
of 100 feet in this environment. Robeck et al. (1962) found that
two feet of packed sand removed poliovirus 1 from slow moving
water but penetration of near 100% of the virus occurred as the
flow rate was increased. Hori et al. (1970) recently reported that
poliovirus 2 was able to penetrate a 6-inch column of three types
of Oahu (Hawaii) soil rather rapidly, even though a significant
reduction in virus titer was found in the filtrate. Considering
these findings, one would expect ground water to be cleansed of
viruses by entrapment and adsorption after traveling a short
distance. Bound viruses would thereby be broken down by soil
microorganisms and environmental conditions as is other organic
matter. However, sensitive methods are lacking to readily detect
viruses at the levels they would probably occur in ground water.

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Therefore, confirmation or rejection of laboratory findings as they
apply to the natural setting must be sought by other methods. Indeed,
epidemiological findings have helped in this regard. Taylor et at.
(1966) have reviewed the data oil 48 outbreaks of suspected waterborne
infectious hepatitis involving approximately 31,000 people. He
found well or spring water to be the suspected source of the virus
iti 21 of the 48 outbreaks.
Perhaps the early work of Neefe and Stokes (1945) provides the
best quantitative data- They convincingly showed that a covered
well providing drinking water at a summer camp was contaminated by
the infectious hepatitis agent(s) fro® a cesspool 75 feet away.
Tucker et al. (1954) similarly found that a spring supplying water
to a campground was contaminated by faulty sever lines "near" the
spring. Shuval (1970) has recently isolated enteric viruses from,
a municipal well which was under surveillance as a possible common
source for localized infectious hepatitis cases. These data would
indicate that some enteric viruses are able to travel a finite
distance through soil and enter a water supply. Of course, channels
and breaks in subsurface rock formations may have been followed
by the contaminated water rather than an actual soil percolation,
thus allowing the viruses to travel much farther than laboratory
data would indicate.
Epidemiological findings seem to be at odds with laboratory
data concerning the cleansing of ground water by filtration and

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adsorption. However, it must be kept in mind that the epidemiological
findings concern the infectious hepatitis virus only and the laboratory
data were obtained with lab-strains of poliovirus and bacteriophage.
In addition, the data are so limited and the factors so variable
in the natural settings that it remains impossible to draw general
conclusions as to the health hazard associated with the utilization
of ground water near a pollution source.
SURFACE WATER
Considerable data are available concerning the viral contamination
of surface waters. Using recovery methods realized to be quantita-
tively questionable, field surveys have shown human enteric viruses
to be present in rivers and estuaries around the world. Coin et at.
(1964) using a swab method found 21% of 1156 samples of river water
around Paris to be positive for enteric viruses. Foliquet et at.
(1966) found viruses in river and drinking water (9 and 8% of samples
positive, respectively) from French communities along the Meurthe
and Moselle Rivers. Bagdasar'yan (1968) reported 34% of 164 river
water samples collected within the city limits of Moscow contained
enteric viruses. Farrohi (1966) observed that 38 - 63% of his samples
from two Swiss rivers emptying into Lake Geneva contained enteric
viruses. Shuval (1970) isolated enteric viruses from 3 of 34
Jordan River samples collected up to 15.5 miles from the primary
contamination source. The Metropolitan Water Board of London has
determined that 56£ of 15 intake water samples (Thames River) were

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positive for enteric viruses (Taylor, 1969). In this country,
Metcalf and Stiles (1968) have found 27 - 52% of their water samples
taken from polluted tidal rivers contained enteric viruses. Twenty-
seven percent of the water samples from the receiving bay were virus
positive, Lamb et at, (1964) isolated enteric viruses from 27%
of 71 upper-Illinois River samples one mile from a sewage outfall.
Grinstein et at. (1970) have used a newly developed concentration
method for sampling river water in the Houston (Texas) area. Nearly
100% of the samples collected 5 miles downstream from the nearest
sewage outfall were positive for enteric viruses. These selected
reports unquestionably document the fact that enteric viruses shed
from their natural host by the fecal route survive for a significant
length of time in an aqueous environment to become potential health
hazards.
Laboratory studies have been conducted which were aimed at
elucidating major factors affecting virus survival, Prier and
Riley (1967) have attempted to single out some of the factors
exhibiting an effect on virus survival in water (Table 1). Of the
factors listed, temperature is the only one which is well defined
and consistent. Under otherwise identical conditions, virus
survival is inversely related to temperature. This relationship
is seemingly true throughout the entire temperature range. Figure 1
exemplifies the effect of temperature upon the inactivation curve
of two poxviruses. Note the two types of curves observed; i.e.,

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TABLE 1
FACTORS THAT MAY AFFECT VIRUS SURVIVAL IN WATER (PRIER AND RILEY, 1967)
1.	Time virus is in water
\
2.	Nature of water (lake, stream, well)
3.	Rate of water flow
4.	Temperatures of water
5.	Chemical content of water
6.	Organic content of water

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o
40
2
\
K
2
50
45

55
20
40
60
MINUTES
50
52.5
60'
20
40
60
MINUTES
FIGURE 1. Thermal inactivation in aqueous environments of (A)
variola virus (Hahon and Kozikowski, 1961) and (B)
vaccinia virus (Kaplan, 1958).

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one component curves with variola virus (A) and two component curves
with vaccinia virus (B).
Factors other than temperature have been less precisely defined.
Purity of water seems to have a significant effect on survival time.
In general, enteric viruses have been found to survive longer in
distilled water than in polluted water and interestingly, longer
in "grossly" sewage-polluted water than in "moderately" polluted
water (Table 2). Cioglia and Loddo (1962) reported that the addition
of sewage to river and seawater increased the survival time of selected
enteric viruses. It has been suggested that gross pollution provides
surface water an ample supply of proteinaceous material which
protects the virus and reduces available oxygen thereby retarding
oxidation of the virion. These processes may be Involved but do
not explain the shortest survival time in the moderately polluted
water.
A number of workers have studed the survival of enteric
viruses under laboratory and field conditions. Table 3 Is an
attempt to present some of these findings in a form permitting as
much comparison as possible. Data are arranged from the shortest
to the longest survival time found to be required for a 3 log (99.9%)
inactivation at 20 - 25°C without regard to the virus type. Much
pertinent information is of necessity omitted from the table; e.g.,
method of titration, characterization of suspending waters,
experimental conditions. Nevertheless, a general pattern of

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TABLE 2
AVERAGE TIME IN DAYS OF 99.9% REDUCTION IN TITER OF INDICATED ENTERIC VIRUSES
WHEN SUSPENDED IN WATER WITH THREE LEVELS OF DOMESTIC POLLUTION
AT 3 TEMPERATURES (CLARKE, et at., 1964)
Amount of
pollution
Phldovirus 1 Echovirus 7
Echovirus 12
Coxsackievirus A-9
2B°C 20°C 4°C
28°C 20°C 4"C
28°C 2Q°C 4eC
28°C 20°C 4°C
Low
(river water)
Moderate
(river water)
Heavy
(sewage)
17 20 27
11 13 19
17 23 110
12 16 26
5 7 15
28 41 130
5 12 33
3 5 19
20 32 60
<8 <8 10
5	8 20
6	N.D* 12
* N .D. - Hot done.

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TABLE 3
SS OF INFECTIVITY OF ENTERIC VIRUSES WHEN SUSPENDED IN VARIOUS AQUEOUS ENVIRONMENTS
'pe of water
References
Virus
Temperature (°C)
4-6
15-16 20-25
ja or
ftuarine water
Cioglia & Loddo (1962)
Shuval (1970)
Akin, et at. (UP)*
Matussian &
Garabedian, (1967)
Lycke, et at. (1965)
V
Akin, et at, (UP)
Akin, et at, (UP)
Akin, et at. (UP)
McLean & Brown (1968)
Shuval, et at. (1971)
Cioglia & Loddo (1962)
Cioglia & Loddo (1962)
Cioglia & Loddo (1962)
Cioglia & Loddo (1962)
Metcalf & Stiles (1967)
Metcalf & Stiles (1967)
Metcalf & Stiles (1967)
Coxsackie B-3
Polio 1
Coxsackie B-l
Polio 1
Polio 3
Reo 1
Echo 6
Polio 1
Polio 2
Polio 1
Polio 1
Polio 2
Polio 3
Echo 6
Coxsackie B-3
Echo 6
Polio 1
Daye/tog titer reduction
90/3
9/2
45/3
60/3
30/3
30/3
88/3
130/3
8/3
9/3
15/3
15/3
8/3
15/3
14/3
16/3
15/3
2/3
2-6/3
3/3
3-6/3
4/3
4/3
4/3
5/3
5/3
8/3
8/3
8/3
8/3
15/3
28/3
fcble 3 continued.

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Page 2 of Table 3
BLE 3. LOSS OF INFECTIVIXY OF ENTERIC VIRUSES WHEN SUSPENDED IN VARIOUS AQUEOUS
ENVIRONMENTS - CONT'D.
ype of water
References
Virus
Temperature (°C)
4-6
15-16 20-25
«Jal river water
Akin, et al. (UP)
Akin, et al.	(UP)
Akin, et al.	(UP)
Akin, et al.	(UP)
Coxsackie B-l
Reo 1
Echo 6
Polio 1
Days/log titer reduction
3/3
1 5/3
6/3
6/3
Cioglia & Loddo (1962)
Coxsackie B-3
75/3
8/3
2/3
Prier & Riley (1967)
Echo 6
7/0.5
-
3/3
Prier & Riley (1967)
Polio 1
7/1
-
3/3
Prier & Riley (1967)
Coxsackie B-3
7/1.7
-
3/3
Clarke, et al. (1964)
Echo 12
19/3
-
5/3
Clarke, et al. (1964)
Echo 7
15/3
-
7/3
Clarke, et al. (1964)
Coxsackie A-9
10/3
-
<8/3
Cioglia & loddo (1962)
Polio 2
75/3
15/3
8/3
Cioglia & Loddo (1962)
Polio 3
30/3
8/3
8/3
Cioglia & Loddo (1962)
Echo 6
60/3
15/3
8/3
Clarke, et al. (1964)
Coxsackie A-9
20/3
-
8/3
Clarke, et al. (1964)
Echo 12
33/3
-
12/3
Clarke, et al. (1964)
Polio 1
6/3
-
13/3
Cioglia & Loddo (1962)
Polio 1
60/3
45/3
15/3
Clarke, et al. (1964)
Echo 7
26/3
-
16/3
table 3 continued

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Page 3 of Table 3
fe 3. LOSS OF INFECTIVITY OF ENTERIC VIRUSES WHEN SUSPENDED IN VARIOUS AQUEOUS
ENVIRONMENTS - CONT'D.
|>e of water
Temperature (eC)
References
Virus
4-6
15-16
20-25


Day8/log titer reduction
Clarke, et al. (196A)
Polio 1
27/3
-
20/3
Poynter (1968)
Polio 3
50/3
18/3
-
Taylor (1967)
Polio 3
67/3
7/1.3
7/2
Clarke & Chang (1959)
Coxsackie A-2
-
-
5/2
Clarke & Chang (1959)
Coxsackie A-2
-
-
47/2
Taylor (1967)
Coxsackie B-5
-
24/1
-
Poynter (1968)
Coxsackie B?**
18/2
-
-
Prler & Riley (1967)
Coxsackie B-3
7/1.7
-
3/3
Prier & Riley (1967)
Polio 1
7/1.5
-
3/3
Clarke, N. A. (UP)
Echo 7
22/3
-
4/3
Prier & Riley (1967)
Echo 6
5/3
-
5/3
Clarke, N. A. (UP)
Coxsackie A-9
6/3
-
<6/3
Clarke, N. A. (UP)
Polio 1
27/3
-
6/3
Joyce & Weiser (1967)
Coxsackie ?
18/3
-
6/3
Clarke, N. A. (UP)
Echo 12
14/3
¦m*
<6/3
Joyce & Weiser (1967)
Polio ?
21/3
-
10/3
Joyce & Weiser (1967)
Echo ?
23/3
-
12/3
Joyce & Weiser (1967)
Echo ?
21/3
-
20/3
ir water
it'd.)
t
unded fresh
ater
3 continued

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rage 4 of Table 3
E 3. LOSS OF INFECTIVITY OF ENTERIC VIRUSES WHEN SUSPENDED IN VARIOUS AQUEOUS
ENVIRONMENTS - CONT'D.
te of water
References
Virus
Temperature (°C)
4-6
15-16 20-25
funded fresh
*r - Cont'd.
Joyce & Welser (1967) Polio ?
Joyce & Weiser (1967) Polio ?
Joyce & Weiser (1967) Echo ?
Day $/log titer reduction
52/3
52/3
42/3
21/3
22/3
24/3
Clarke, N. A. (UP)
Echo 7
85/3
10/3
Clarke, N. A. (UP)
Echo 12
130/3
11/3
Clarke, N. A. (UP)
Coxsackie A-9
98/3
15/3
Clarke, N. A. (UP)
Polio 1
140/3
95/3
McLean & Brown (1968)
Polio 2
12/No loss
5/1
Clarke & Chang (1959)
Coxsackie A-2
<•* «
100/2
Taylor, (1967)
Polio 3
167/3
-
Poynter, (1968)
Polio 3
168/3
-
onized or
tilled water
Akin, et al. (UP)
Akin, et al. (UP)
Akin, et al. (UP)
Akin, et at. (UP)
Poynter, (1968)
Poynter, (1968)
Reo 1
Coxsackie B-l
Echo 6
Polio 1
Coxsackie B?
Polio 3
95/2
180/1
3/3
5/3
14/3
11/1
56/1
Unpublished data
Virus serotype not reported

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-8
inactivation as well as variability within and between enteric
virus survival times may be gleaned from the table.
ESTUARINE FIELD STUDIES
Most of the survival data herein reported were obtained under
laboratory or artificial conditions which may not yield results
representative of the natural setting. This fact has concerned
us at the Gulf Coast Water Hygiene Laboratory and we have therefore
initiated survival studies under more natural conditions. A
diagrammatic representation of our experimental system is shown in
Figure 2. "Fresh" estuarine water was continuously pumped from
Little Dauphin Island Bay into a tank in our wet laboratory with
the overflow being returned to the bay. Water for these experiments
was siphoned from the wet laboratory to the constant head tank
located just outside the laboratory. From there, the estuarine
water was siphon-fed into the 1000-gallon fiber glass tank at a
rate of 7 liters/min, Water passing through the distribution
manifold was mixed with the 1000 gallons of estuarine water in the
tank by electric stirrers. Excess water then flowed out the central
standpipe and thence through a flow meter. In this flow-through
system, marine life and biological metabolites are not allowed to
accumulate to a level alien to that found In the natural setting.
Therefore, this system should yield virus survival patterns more
indicative of those which actually occur in the complex estuarine
environment. Temperature was recorded constantly. Salinity,

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CONSTANT-HEAD TANK
FLOW
TEMPERATURE
RECORDING PROBE
ELECTRIC STIRRER
OVERFLOW
2M0'
DISTRIBUTION
MANIFOLD
1,000 GALLON FIBERGLAS TANK
FLOWMETER
FIGURE 2, Flow-through system for studying the survival of virus
in estuarine water.

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turbidity, and flow rate measurements were made Intermittently
throughout the experimental period. The fiber glass tank was filled
with estuarine water from the wet laboratory on the day before an
experiment was run. A Plexiglas cylinder 5 cm x 94 cm was inserted
vertically into the tank and filled with distilled water (approxi-
mately 1700 ml). Since laboratory data have previously been
collected on the inactivation curve of poliovirus suspended in
distilled water at 23°C, this Plexiglas unit when dosed with virus
provided a virus control and a comparison for the inactivation
patterns obtained in the flow-through system. On the morning of
the experiment a predetermined multiplicity of poliovirus 1 was
added to the tank and cylinder. After 30 minutes of mixing, the
initial samples were taken and the incoming siphon begun.
At each sampling interval, a pool of 100 ml was obtained by
collecting 10 ml from 10 locations within the tank. From this
pool, 5 ml were removed in duplicate and placed into a test tube
containing lyophilized nutrient broth which provided protective
proteins for the virus without significantly diluting the sample.
Ten-ml portions were also removed from the distilled water cylinder
and handled in the same manner as the tank samples. These samples
were immediately placed at -80°C and held at that temperature until
assayed for virus by the plaque technique using HEp-2 cell monolayers.
Three ml of each sample were assayed with results reported as the
mean PFU/ml of sample.

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-.10
The reduction in plaque counts at each sampling interval
represented a virus loss due to dilution as well as inactivation.
To differentiate loss by dilution from loss by inactivation, a
correction factor (CF) was obtained in the following manner.
Either immediately prior to or following each experiment, the
1000-gallon tank was filled with high salinity estuarine water and
the salinity determined hydrometrically. The mixers were engaged
and tap water having a negligible salinity (0.2 - 0.4 ppt) was
introduced into the tank through the distribution manifold.
Hourly salinity determinations were made from samples collected at
the tank effluent. Flow rate determinations were made intermittently
throughout the sampling period. The reduction in salinity occurred
geometrically and proceeded as a linear first order react* ->n.
Therefore, the rate of salinity reduction (dilution) could be
— (Qrhbt)
calculated from the equation, N^/N = e	, where is the
initial salinity at time zero; is the salinity at time t\ a is
the intercept and b is the reduction rate (slope function). A curve
can be drawn of the form Y - a + bx which produces a straight line
of best fit to the points observed. Since the kinetics of salinity
reduction in this system should be practically identical to virus
dilution in the experimental system, the slope of the line should
be the same in both cases. Under the conditions employed, the
Initial tank contents were diluted in half every 7-8 hours. The
correction factor for each time interval during an experiment was

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calculated from the slope function and intercept of this line. The
actual plaque count at each time interval was multiplied by the
appropriate correction factor to obtain the calculated plaque count
If no dilution had occurred.
Four experiments have been conducted to date; two under summer-
conditions with a temperature range of 28 - 33°C [salinity, 21 - 27
ppt; turbidity, 28 - 36 Jackson Turbidity Units (JTU)] and two under
autum conditions with a temperature range of 20 - 24°C (salinity,
18 - 21 ppt; turbidity, 32 - 44 JTU). The results of these four
experiments are shown in Figure 3. The two summer experiments
are represented by a single inactivation curve since the calculated
least squares curves were practically identical. As can be seen,
lose in poliovirus infectivity in the summer experiments occurred
exponentially with a 4 log (99.99%) reduction occurring in seven
hours. The inactivation curve of the autumn experiments, however,
began to "tail" between 4 and 8 hours resulting in a 2.5 and 3.5 log
reduction in 24 hours for autumn experiments one and two, respectively.
Even though temperature, salinity, turbidity, and flow rate measure-
ments were very similar for the two autumn experiments, a one log
difference in virus reduction occurred in the 24-hour samples. The
loss of virus Infectivity in the distilled water cylinders did not
exceed 0.5 log in six hours under summer conditions and did not
exceed 0.5 log in 24 hours under autumn conditions*

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4 8 12 16 20 24
HOURS
FIGURE 3, Inactivation of poliovirus 1 in estuarine water under field
conditions during summer (0, A) and autumn (0* A) months.

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-12
DISCUSSION AND CONCLUSIONS
The exact method of viral inactivation in nature is not
clearly understood. Pohjanpelto (1962) and Lund (1963) have
reported that oxidation is important in the Inactivation of
poliovirus above 50°C but plays a very small role in spontaneous
inactivation at temperatures below 37°C, It still remains obscure
as to which part of the virion participates in the oxidative
inactivation at the higher temperature and the ill-defined inactiva-
tion process at lower temperatures. Dinnaock (1967) has suggested
that loss' of iufectivity can occur by nucleic acid or protein
damage and that the stability of each moiety varies with temperature.
Inactivation at a given temperature would therefore take place
through whichever component is the least stable at that temperature.
Regardless of the method of inactivation, the reviewed data clearly
show that iriactivation time is inversely related to temperature
and that a matter of days to weeks are required for a 99,9%
inactivation of various enteric virus types at prevailing surface
water temperatures.
The wide time-range required for a 3 log (99.9%) reduction
indicates that significantly different inactivation rates occur
within the enteric virus family and that major factors other than
temperature are important in inactivation (or survival) of these
particles in the aqueous environment* These other factors have not
been clearly defined.

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Enteric viruses shed from the gut of animal hosts are able
nevertheless to survive for a significant length of time in natural
bodies of water to spread disease by this route. Of the reports
reviewed, an average of 36% of the water samples collected contained
one or more viruses. In addition, this transmission route has been'
well documented in a limited number of explosive outbreaks of
infectious hepatitis. The most recent outbreak involved 3.8% of
the population of a French town with evidence pointing to an
extensively contaminated municipal water distribution system as
the culprit (Gavan and Nutt, 1970). These outbreaks are rare and
it seems safe to say that widespread viral morbidity and mortality
are not occurring in this country as a result of contact with surface
waters. The extent of endemic disease spread by enteric-virus-
contaminated water, however, is anyone's guess. Epidemiological
studies designed to answer this question are extremely costly and
to date have not obtained sufficient support to initiate adequate
investigations. Many enteric viruses produce an asymptomatic
response in 90% or more of the persons infected, thereby producing
a confusing transmission pattern when overt disease cases alone
are considered. Costly and time consuming antibody studies conducted
on a latfge segment of the population are required to determine
infectivity patterns and therefore an accurate transmission
picture. Less costly viral survival studies yield data that can

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provide an indirect evaluation of the health hazard.of human
contact with water known to have received viral contaminated
materials.
It should be emphasized that our present water supply is being
recycled for domestic and Industrial usages at an increasing
frequency. This, of course, allows less time for nature's purifi-
cation process to accomplish its task. Knowledge of viral
survival patterns in natural water systems could be put into
immediate practical use. These data coupled with the results of
virus monitoring (assuming that a satisfactory concentration-
isolation procedure is forthcoming) would provide a sound basis
for determining water treatment needs and safe reuse frequency.
The fate of enteric viruses in estuarine as well as fresh
water has health importance. The increased coastal population
growth has been accompanied by Increased exposure of recreational
and seafood-producing coastal waters to domestic waste. Several
workers have studied virus survival in seawater and have found It
to possess a virucidal property (Magnusson et at., 1966; Matossian
and Garabedian 1967; Shuval et al.t (1971). The work of Magnusson
et al., (1967) and Shuval et al., (1971) indicates that a viable
marine bacterium is responsible and that filtration or/heating the
seawater to 90°C removes the virucidal property for poliovlrus.
Conversely, Matossian and Garabedian (1967) using poliovlrus 1
found filtration or boiling had little or no effect upon the

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virucidal properties of Mediterranean Sea samples. Likewise, our
studies show that filtration and autoclaving does not eliminate
the virucidal property of Gulf Coast estuarine water upon poliovirus
1 (unpublished data). Mitchell and Jannasch (1969) studying 3>X 174
bacteriophage have concluded that three major factors are working
in seawater to influence phage survival: (1) inactivating micro-
organisms, (2) inactivating chemicals, and (3) protective organic
matter. Considering the number of possibilities within each of
the three factors, there can be an almost infinite number of
combinations exerting an effect on virus survival. It may be
practically impossible to isolate each combination of factors which
significantly lengthens survival time or hastens inactivation of
a virus particle in the various aqueous environments. Even if the
combination of factors were elucidated, the result of their
interactions may be so complex that virus survival predictions
without actual field testing would be ill-advised.
For the above reasons, we have begun conducting our survival
studies under conditions more representative of nature but yet
controlled to the extent that results lend themselves to interpre-
tation. From the four experiments we have conducted in our flow-
through system, the effect of temperature is clearly seen. A
temperature reduction of a mean of 9°C from 31°C extended the
99.9% inactivation time three-fold (to a mean of 18 hours). It is
interesting to note that the. inactivation curve of the two summer

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experiments, conducted one month apart, are practically identical.
The two autumn experiments, conducted 3 weeks apart, gave similar
inactivation patterns but at a significantly different order of
magnitude; i.e., approximately one log difference after 24 hours
(Figure 3). This difference may reflect the occurrence of virucidal
biological shifts in the marine environment during the change in
seasons which were not apparent from the monitored environmental
parameters. Also of interest is the change in inactivation rate
occurring in the fall experiments between 4-8 hours. We may
speculate that the initial rapid drop in virus titer does not
represent inactivation at all but rather aggregation of virus
particles to each other and to "marine silt." This could occur
by the adsorption of virions in the presence of divalent cations
in the seawater. Therefore, the flatter slope may represent the
inactivatioii of stable configurations of virus aggregates. Chang
(1967) has strongly emphasized the influence of clumping on viral
survival curves. However, we do not rule out the possibility of
the presence of multiple inactivation forces which may dominate
during different phases of the inactivation period thereby changing
the inactivation rate. Laboratory studies are being planned to
clarify the aggregation possibility. Field studies Under winter
and spring conditions are also being planned in an effort to
determine the seasonal variability in virus survival time as well
as the survival patterns of poliovirus 1.

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We have found a significantly different inactivation rate of
poliovirus 1 under laboratory conditions versus our flow-through
system when fresh estuarine water was used in both cases. A reduction
in infectivity of 99.9% occurred in 72 hours in the laboratory
studies contrasted to a mean of 18 hours in the flow-through system-
at 23°C (unpublished data). Likewise, Metcalf and Stiles (1967)
found that poliovirus 1, echovirus 6, and coxsackievirus B-3
survived longer under laboratory conditions contrasted to suspension
of these viruses in an estuary within cellulose dialysis tubes.
This phenomenon still remains to be explained, and when all published
findings are considered, major factors affecting the inactivation
of enteric viruses in the natural marine environment are yet to
be elucidated.
If our flow-through experimental system is at all representa-
tive of a "real" estuarine environment, then it would seem that at
least one enteric virus (poliovirus 1) is inactivated at a faster
rate in nature than in the laboratory setting. Assuming that this
is true for all enteric viruses, then laboratory survival studies
have a built-in margin of error on the side of safety. This would
be welcome from a public health standpoint by allowing some added
tolerance in making viral hazard predictions based on laboratory
survival studies of highly pathogenic viruses. Of course, the
elusive infectious hepatitis agent(s) still remains the villain a
8upeviov of water transmissible Infectious agents. The limited data

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available Indicate it may be many times more resistant than any
known virus to nature's purification process as well as to chemical
disinfectants. However, until it is isolated and adequately
studied, viral hazard predictions based on survival studies must
be obtained with the most resistant enteric virus readily available
for study. This determination is yet to be made.
ACKNOWLEDGMENTS
We thank William Zirlott and Clinton Collier for their
assistance in setting up and maintaining the flow-through experimental
system.
DISCLAIMER CLAUSE
Mention of any commercial products in this paper does not imply
endorsement by the Federal Government.

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