Sunlight inactivation of human enteric viruses and fecal

Water Science and Technology Vol 46 No 11–12 pp 291–295 © IWA Publishing 2002
bacteria
R.S. Fujioka and B.S. Yoneyama
Water Resources Research Center, University of Hawaii, Honolulu, Hawaii 96822, USA

Abstract Three human enteric viruses (poliovirus, echovirus, coxsackievirus) suspended in seawater or
buffer were stable for 6 hr in the absence of sunlight but were inactivated at the same rate in the presence of
sunlight. Under summer sunlight conditions, at least 3 logs of these viruses were inactivated by one-hit
kinetics while under winter sunlight conditions only 1 log of these viruses was inactivated by two-hit kinetics.
Under these same conditions, 6 logs of E. coli were inactivated within 1 hr by one-hit kinetics under summer
and winter conditions. In comparison, E. faecalis was inactivated by two-hit kinetics and only 2.5 logs of
inactivation were observed after 4 hr of exposure to winter sunlight. Since human enteric viruses are
considerably more resistant to sunlight inactivation than E. coli and moderately more resistant than E.
faecalis, marine recreational water quality standards should be based on concentrations of enterococci and
not on coliform bacteria. Since the mechanism and rate of inactivation of coliphage and human enteric
viruses are similar, coliphages appear to be the best indicator for the presence of human enteric viruses in
recreational waters, especially coastal waters where abundant sunshine is available.
Keywords E. coli; E. faecalis; human enteric viruses; sunlight inactivation; water quality

Introduction
Recreational water quality standards are based on concentrations of fecal indicator bacteria
(E. coli, enterococci). However, the pathogens in water which cause diarrheal diseases
among swimmers are human enteric viruses (Cabelli, 1983). These sewage-borne viruses
are known to be unusually stable in marine waters and explain the fact that these viruses can
be recovered from marine water samples which were negative for fecal indicator bacteria
(Loh et al., 1979). Numerous studies (Davies-Colley et al., 1994; Fujioka et al., 1981;
Gameson and Saxon 1967; Kapuscinski and Mitchell 1981) have documented that sunlight
is the primary factor responsible for the rapid inactivation of fecal indicator bacteria in
marine recreational waters. More recently, coliphages or viruses, which infect coliform
bacteria, have been shown to be more resistant to sunlight inactivation than fecal indicator
bacteria (Fujioka and Siwak, 1985; Sinton et al., 1999) and have been proposed as the best
surrogate to monitor for the presence of human enteric viruses (Havelaar and Pot-
Hogeboom, 1988). However, sunlight inactivation of human enteric viruses has not been
sufficiently characterized. The objective of this study was to determine the ability of sum-
mer and winter sunlight conditions to inactivate human enteric viruses (poliovirus, cox-
sackievirus, echovirus) suspended in buffer or seawater and to compare their rates of
inactivation with those of E. coli and E. faecalis.

Methods
Prototype strains of Escherichia coli (ATCC 25922) and Enterococcus faecalis (UMRL
1053) were selected as representative fecal indicator bacteria. It should be noted that E. fae-
calis was previously classified as Streptococcus faecalis. Sabin Type 1 poliovirus (vaccine
strain) and sewage-isolated echovirus type 7 and coxsackievirus type B-5 were selected to
represent the major groups of human enteric viruses. Purified and washed cultures of these
microorganisms (108–109 CFU/ml or PFU ml) were diluted 1:10,000 in phosphate buffer or 291
 clear seawater. Aliquots (1.8 liter) of these mixtures were added to 2 litre borosilicate
beakers containing a sterilized magnetic bar. The exterior walls of these beakers were
covered with aluminium foil to prevent sunlight from entering the sides of the beaker. The
tops of the beaker were usually uncovered and this allowed sunlight exposure only through
the top of the beakers. In some experiments, the beaker was covered with a glass plate to
adsorb all wavelengths of sunlight <360 nm. The boxes were placed on a magnetic stirrer to
keep the water stirring slowly throughout the experiment. Iced water was added to the
R.S. Fujioka and B.S. Yoneyama

styrofoam boxes to bathe the beakers and to maintain the temperature of the water in the
beakers at 15–26°C. All experiments were started at 9–10 AM and samples were taken
before and during exposure to sunlight. In all experiments, the microbial populations dilut-
ed in phosphate buffer or in seawater and maintained at 23–26°C remained stable over 4–6
hr in the absence of sunlight (dark control). Inactivation of all microorganisms was deter-
mined based on concentrations (CFU/100 ml) of E. coli on mFC agar and of E. faecalis on
mEnterococcus agar using the membrane filtration method. Each group of human enteric
virus was added to separate beakers and their concentrations (PFU/ml) assayed by the
plaque assay method on Buffalo Green Monkey Kidney cell cultures as previously
described (Loh et al., 1979).
Results of numerous experiments conducted throughout the year were used to reach
basic conclusions. For this report, the results of a representative experiment conducted dur-
ing a typical sunny day during the summer and a comparative experiment conducted during
a typically sunny day during the winter are shown. The output of sunlight was measured by
using the LI-185S quantum meter to measure the intensity of the visible wavelengths
(400–800 nm) and UV wavelengths were measured by three radiometers manufactured by
UVP, inc. These meters measured UV which primarily peaked at 250 nm (UVX-25), 310
nm (UVX-31) and at 360 nm (UVX-36). The readings from all of these radiometers were
recorded throughout the experiments using LI-500 micrologger (Lamda Instrument Corp.).

Results and discussion

Figures 1 and 2 show the intensity of the various wavelengths of sunlight and show that
higher intensities of visible light (400–800 nm), and ultraviolet light at 360 nm, 310 nm and
at 250 nm were measured during a typical sunny day in the summer as compared to a typical

Figure 1 Measurements of sunlight using UVX-25, Figure 2 Measurement of sunlight using UVX-25,
UVX-31, UVX-36, and visible light radiometers UVX-31, UVX-36, and visible light radiometers
292 during summer conditions during winter conditions
sunny day during the winter. The results show that visible light and UV wavelengths, which
peaked at 360 nm, showed reduction close to 10% whereas UV wavelengths, which peaked
at 310 and 250 nm, showed a reduction of 12–15%.
Figure 3 summarizes the stability of all microorganisms suspended in seawater and
shows that 6 logs of E. coli were inactivated by one-hit kinetics after 1 hr exposure to
sunlight during the summer whereas E. faecalis was inactivated by two-hit kinetics with an
initial shoulder which lasted for 1 hr followed by 4 logs of inactivation during the next 3 hr

R.S. Fujioka and B.S. Yoneyama

of exposure to sunlight. A two-hit kinetic inactivation curve indicates that at least two tar-
gets in the cell must be hit before the cell is inactivated. Under the same sunlight conditions,
poliovirus was inactivated by one-hit kinetics and 3 logs of inactivation were observed after
4 hr of exposure to sunlight. Figure 4 shows that the rates of inactivation of poliovirus,
echovirus 7 and coxsackievirus B-5 in seawater under summer sunlight conditions were
similar and followed one-hit kinetics.
The same experiment was conducted during a typical sunny day during the winter when
the intensity of all wavelengths of sunlight were reduced (see Figures 1, 2). Figure 5 sum-
marizes the stability of all microorganisms and show that the kinetics of E. coli inactivation
was slightly retarded but was still characterized by one-hit kinetics and 6 logs of inactiva-
tion were observed after 1 hr exposure to sunlight. Thus, E. coli appears to be so sensitive to
sunlight inactivation that the rates of inactivation under summer and winter conditions
were similar. In contrast, the inactivation curve for E. faecalis was clearly slower during
this winter sunlight exposure and revealed an extended initial shoulder (2 hr) followed by
more than 2 logs of inactivation after 4 hr of exposure to sunlight. Under the same condi-
tions, the inactivation curve for poliovirus was considerably slower and resulted in only 1
log reduction after 4 hr of exposure to sunlight.
Figure 6 summarizes the stability of the three human enteric viruses and shows that the
inactivation curves for all three viruses were similar and were characterized by two-hit
kinetics. These results indicate that the major groups of human enteric viruses are inactivat-
ed to the same degree by sunlight and that poliovirus is a good model to predict the response
of other groups of human enteric viruses to sunlight inactivation.

Figure 3 Comparative stability of poliovirus vs. Figure 4 Comparative stability of poliovirus,

E. coli and S. faecalis to sunlight exposure during echovirus, and coxsackievirus to sunlight exposure
summer conditions during summer conditions 293
R.S. Fujioka and B.S. Yoneyama

Figure 5 Comparative stability of poliovirus vs. Figure 6 Comparative stability of poliovirus,

E. coli and S. faecalis to sunlight exposure during echovirus, and coxsackievirus to sunlight exposure
winter conditions during winter conditions

We previously reported (Fujioka and Siwak, 1985) that wavelengths of sunlight that
penetrate glass were effective in inactivating fecal indicator bacteria but not coliphage. To
determine if this observation applies to human enteric viruses, poliovirus, suspended in
seawater, was exposed to sunlight with and without a plate glass cover under summer sun-
light conditions. Without the glass cover, more than 3 logs of poliovirus were inactivated
after 4 hr of exposure to sunlight. With the glass cover, the virus sample was exposed to
sunlight consisting of those wavelengths greater than 360 nm for 4 hr and no inactivation
was observed. Based on this observation, we conclude that sunlight inactivation of
poliovirus and coliphage occur by the same mechanism and differ from the mechanism by
which bacteria are inactivated. This conclusion is not surprising because the structure of the
virus is very different from that of bacteria. A unique characteristic of human enteric virus
is that its structure become stabilized to inactivation by heat and by denaturating chemicals
in the presence of calcium and magnesium (Fujioka et al., 1969). Thus, poliovirus was sus-
pended in phosphate buffer with and without 0.1 M of MgCl2 and exposed to sunlight under
summer conditions. The presence of Mg++ did not inhibit the rate of sunlight inactivation of
poliovirus indicating that the mechanism of sunlight inactivation differs from the mecha-
nism by which poliovirus is inactivated by heat and denaturating agents.

Conclusions
In this study, the inactivating effects of sunlight were assessed using purified and washed
cultures of fecal indicator bacteria and human enteric viruses, which were suspended in
clean buffer or seawater. These conditions simplify the characterization of sunlight inacti-
vation of microorganisms because many of the interfering factors in natural samples such
as turbidity, adsorption of microorganisms to particles and presence of chemicals, which
can enhance or inhibit the reaction with sunlight, were eliminated. Based on the results of
this study, the following conclusions are made. First, three human enteric viruses
(poliovirus, echovirus, coxsackievirus) were inactivated to the same extent in the presence
of sunlight and were more resistant to sunlight inactivation than fecal indicator bacteria.
Second, evidence was presented to show that the mechanism of sunlight inactivation of
coliphage and human enteric virus is similar. Moreover, since both are more resistant
to sunlight inactivation than fecal indicator bacteria and since the method to assay for
294 coliphage is feasible, our results support the proposal that coliphages are the best surrogate
for the presence of human enteric viruses in environmental waters (Havelaar and
Pot-Hogeboom, 1988). Third, E. coli is so sensitive to sunlight inactivation that it should
not be used to monitor recreational waters for the presence of human enteric viruses.
Fourth, E. faecalis was moderately resistant to sunlight inactivation and can be correlated
to the presence of human enteric viruses because: a) this bacteria is always present in
sewage at much higher concentrations than viruses, b) the method of assay is simple, rapid
and inexpensive and c) large volumes of water can be assayed. This conclusion is supported

R.S. Fujioka and B.S. Yoneyama

by the report by Cabelli (1983) that concentrations of enterococci in marine water are better
predictors of diarrheal diseases among swimmers at marine beaches than E. coli.

Acknowledgements
This study was funded by the Department of Health, State of Hawaii under contract number
88-465. This is contributed paper CP-2000-04 of the Water Resources Research Center,
University of Hawaii at Manoa, Honolulu.

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