Discussion by Pasquale V. Scarpino, Ph.D.
It is unfortunate, as emphasized by Nupen and Stander, that information presently available
concerning the inactivation of viruses in waste water by chlorine has too often been equivocal
because standard conditions for testing have not been used. In addition, there also has occurred in
many investigations undefined chemical conditions in the test systems, a lack of information concerning the types of titrable chlorine actually present in the chlorinated water, and the absence of
adequate quantitative techniques for detection of animal virus units.
It is unfortunate that no common method exists at this time for the detection of low levels of virus
in large volumes of relatively clean or finished water, since it is difficult to evaluate results based on
different concentration procedures. Nupen and Stander, for example, determined virus presence by
using two concentration techniques. One was a modification of the two-phase polymer separation
technique described by Shuval et al (1969), and the other was an ultrafiltration method that had the
advantage of monitoring 10 liters or more of clear or slightly turbid water in a time span measured in
hours. They reported on the average a 70% recovery of virus with the ultrafiltration method. Standard
Methods for the Examination of Water and Wastewater (1971), on the other hand, recommended the
use of Oliver's (1967) membrane filter technique as the most quantitative technique available for the
concentration of small quantities of virus from large volumes of water. Berg (1971), however, concluded based on his own studies that the insoluble polyelectrolyte method (Wallis et al., 1969) was the
most sensitive technique available at that time (1971) for large volumes of water despite its sometimes
low and eratic efficiency. More recently, interest has focused on a modified membrane filter method
described by Rao (1971), that was shown in parallel studies by Berg (1972) to be more efficient than
an aluminum hydroxide-protamine sulfate procedure. It would have been more informative if Nupen
and Stander also had been able to compare the overall recovery efficiencies of their concentration
methods to some of the more promising ones used by other investigators. However, this field of
concentration technology is advancing so rapidly that it sometimes seems that a recommended method
used today is replaced tomorrow by the development of a supposed more efficient and convenient
technique. Thus, one can be confused as to which method is "best". Perhaps there is really no best or
standard method, but several, depending upon the quality and quantity of the water to be examined,
and the recovery standard set.
It was concluded by Nupen and Stander that the absence of enterovirus in 10 liters of a water
sample using the ultrafiltration technique indicated from the virological point of view that the water
was safe for drinking" purposes. European Standards for Drinking-Water (1970) also recommended the
examination of a 10 liter sample of water, and stated that less than one plaque-forming unit (PFU) of
virus per liter of water provided a reasonable assumption that the water was safe for drinking. Melnick
(1971), on the other hand, proposed minimum standards of virus pollution of not more than one
detectable infectious virus unit per 10 gallons (37.85 liters) of recreational water used for bathing and
swimming, and not more than one detectable infectious virus unit per 100 gallons (378.5 liters) of
water used for drinking and food preparation. Berg (1971) considered 100 gallons (378.5 liters) of
water an absolute minimum for determiriing low ieyels of viruses in large quantities of water, and
emphatically stated that no virus should be allowed! in; a 1Ö0 gallon sample of disinfected, renovated,
and other potable waters. Thus, not only must a standard procedure or procedures be developed for
detection of low numbers of virus in large volumes of water, but experience must be gained concerning
the size of the sample that should be taken to provide the necessary confidence that the water is safe
for human use. Obviously, the choice of sample size will influence the concentration methods that can
be effectively used.
The importance of the HOC1 level for virus inactivation was stressed by the authors. Although both
HOC1 and OC1" are microbicidal, HOC1 has teen considered to be the most efficient disinfectant.
However, there are circumstances where chlorinated water at high pH can cause rapid inactivation of
the test viruses (Scarpino, In Manuscript).
Nupren and Standees paper Would have been more complete if more of their bacteriological data
could have been included. A wider margin of safety could also have been provided the water consumed
if the total coliform group had been tested for, instead of only Escherichia coli I. However, the
presence of fc. coli Ϊ (i.e. fecal coliforms) does constitute an extremely dangerous situation that
requires immediate remedial action (Public Health Service Drinking Water Standards, 1962; Geldreich,
1966; and Water Quality Criteria, 1968). Although fecal coliform determinations can be an important
adjunct test in our surveillance of raw waters entering a water treatment plant and potable waters, the
American usage has been not to recommend the fecal coliform test as a substitute for the total
coliform examination of potable waters (Standard Methods, 1971). Instead, the fecal coliform test has
been applied to studies of stream pollution, raw water sources, sewage treatment systems i bathing
143
It is unfortunate, as emphasized by Nupen and Stander, that information presently available
concerning the inactivation of viruses in waste water by chlorine has too often been equivocal
because standard conditions for testing have not been used. In addition, there also has occurred in
many investigations undefined chemical conditions in the test systems, a lack of information concerning the types of titrable chlorine actually present in the chlorinated water, and the absence of
adequate quantitative techniques for detection of animal virus units.
It is unfortunate that no common method exists at this time for the detection of low levels of virus
in large volumes of relatively clean or finished water, since it is difficult to evaluate results based on
different concentration procedures. Nupen and Stander, for example, determined virus presence by
using two concentration techniques. One was a modification of the two-phase polymer separation
technique described by Shuval et al (1969), and the other was an ultrafiltration method that had the
advantage of monitoring 10 liters or more of clear or slightly turbid water in a time span measured in
hours. They reported on the average a 70% recovery of virus with the ultrafiltration method. Standard
Methods for the Examination of Water and Wastewater (1971), on the other hand, recommended the
use of Oliver's (1967) membrane filter technique as the most quantitative technique available for the
concentration of small quantities of virus from large volumes of water. Berg (1971), however, concluded based on his own studies that the insoluble polyelectrolyte method (Wallis et al., 1969) was the
most sensitive technique available at that time (1971) for large volumes of water despite its sometimes
low and eratic efficiency. More recently, interest has focused on a modified membrane filter method
described by Rao (1971), that was shown in parallel studies by Berg (1972) to be more efficient than
an aluminum hydroxide-protamine sulfate procedure. It would have been more informative if Nupen
and Stander also had been able to compare the overall recovery efficiencies of their concentration
methods to some of the more promising ones used by other investigators. However, this field of
concentration technology is advancing so rapidly that it sometimes seems that a recommended method
used today is replaced tomorrow by the development of a supposed more efficient and convenient
technique. Thus, one can be confused as to which method is "best". Perhaps there is really no best or
standard method, but several, depending upon the quality and quantity of the water to be examined,
and the recovery standard set.
It was concluded by Nupen and Stander that the absence of enterovirus in 10 liters of a water
sample using the ultrafiltration technique indicated from the virological point of view that the water
was safe for drinking" purposes. European Standards for Drinking-Water (1970) also recommended the
examination of a 10 liter sample of water, and stated that less than one plaque-forming unit (PFU) of
virus per liter of water provided a reasonable assumption that the water was safe for drinking. Melnick
(1971), on the other hand, proposed minimum standards of virus pollution of not more than one
detectable infectious virus unit per 10 gallons (37.85 liters) of recreational water used for bathing and
swimming, and not more than one detectable infectious virus unit per 100 gallons (378.5 liters) of
water used for drinking and food preparation. Berg (1971) considered 100 gallons (378.5 liters) of
water an absolute minimum for determiriing low ieyels of viruses in large quantities of water, and
emphatically stated that no virus should be allowed! in; a 1Ö0 gallon sample of disinfected, renovated,
and other potable waters. Thus, not only must a standard procedure or procedures be developed for
detection of low numbers of virus in large volumes of water, but experience must be gained concerning
the size of the sample that should be taken to provide the necessary confidence that the water is safe
for human use. Obviously, the choice of sample size will influence the concentration methods that can
be effectively used.
The importance of the HOC1 level for virus inactivation was stressed by the authors. Although both
HOC1 and OC1" are microbicidal, HOC1 has teen considered to be the most efficient disinfectant.
However, there are circumstances where chlorinated water at high pH can cause rapid inactivation of
the test viruses (Scarpino, In Manuscript).
Nupren and Standees paper Would have been more complete if more of their bacteriological data
could have been included. A wider margin of safety could also have been provided the water consumed
if the total coliform group had been tested for, instead of only Escherichia coli I. However, the
presence of fc. coli Ϊ (i.e. fecal coliforms) does constitute an extremely dangerous situation that
requires immediate remedial action (Public Health Service Drinking Water Standards, 1962; Geldreich,
1966; and Water Quality Criteria, 1968). Although fecal coliform determinations can be an important
adjunct test in our surveillance of raw waters entering a water treatment plant and potable waters, the
American usage has been not to recommend the fecal coliform test as a substitute for the total
coliform examination of potable waters (Standard Methods, 1971). Instead, the fecal coliform test has
been applied to studies of stream pollution, raw water sources, sewage treatment systems i bathing
143