United States
 Environmental Protection
 Agency
 Municipal Environmental
 Research Laboratory
 Cincinnati OH 45268
 Research and Development
 EPA-600/S2-84-094  Sept. 1984
 Project  Summary

 Effect  of  Particulates  on
 Disinfection  of  Enteroviruses  in
Water  by  Chloramines
 Pasquale V. Scarpino
  The   inactivation  kinetics   of
chloramines (monochloramine and di-
chloramine) on an enterovirus, polio-
virus 1 (Mahoney), and on an enteric
indicator of fecal pollution, Escherichia
col/11229, were examined in laboratory
bench-scale studies using the kinetic
(stirred beaker) apparatus. The disinfect-
ing ability  of chloramines in the pres-
ence of viral aggregates  and organic
particulates was compared with viral in-
activation in pure buffer systems with
unassociated viruses and without added
particulates. In addition, comparisons of
chloramines, hypochlorous acid,  and
hypochlorite ion were made in a variety
of test situations involving, for example,
(1) several types of particulates (entero-
virus-associated animal cells, solids-
associated primary effluents, and fecal
suspensions), (2) aggregated and unas-
sociated single  viruses, (3) different
temperatures of reactivity, (4) different
pH's, and (5) different disinfectant com-
binations.
  Aggregated poliovirus was more re-
sistant to both monochloramine and di-
chloramine than were the unassociated
viruses. Almost doubling the mono-
chloramine dose from 12 to 22 mg/L at
5 C and pH 9 did not double the rate of
virus disinfection. Dichloramine inac-
tivated  poliovirus 1 less effectively than
did monochloramine. Monochloramine
formed at pH 9 and then adjusted to pH
7 gave a  stable solution of  mostly
monochloramine. Viral  disinfection
rates then examined at both pH 7 and 9
were similar, but monochloramine killed
the test bacterium E. coli 10 times more
rapidly  at pH 7 than at pH 9. Forming
monochloramine was about 1.2 times
 more effective as a disinfectant than
 newly made,  preformed monochlora-
 mine at 5 C and pH 9.
  Poliovirus 1 survivors that had been
 exposed eight times to monochloramine
 at 15 C and pH 9 were 2.3 times more
 resistant to monochloramine than both
 the initially used, unexposed virus and
 those viruses exposed fewer than eight
 times to monochloramine.
  Human epidermoid carcinoma (HEp-2)
 and Buffalo Green Monkey (BGM) cells
 were used to study the effects of cell-
 associated turbidity on the disinfection
 process. The object was to mimic the
 natural state  of  viruses as they are
 freshly discharged in feces. The rate of
 disinfection was influenced by both the
 disinfectant used and the cell-induced
 turbidity of the system. Both of the cell-
 associated viruses were more suscepti-
 ble to hypochlorous acid than to mono-
 chloramine. Increasing the turbidity
 increased the resistance of the cell-
 associated viruses to monochloramine.
  Total coliforms in fecal suspensions
 disinfected with hypochlorous acid
 showed  an initial rapid die-away  of
 greater than 99.9% during the first
 minute of interaction, followed by a pro-
tracted period of survival. The turbidity
of primary effluents also gave protection
to naturally occurring coliforms disin-
fected with monochloramine.
  This Project Summary was developed
 by EPA's Municipal Environmental Re-
 search Laboratory, Cincinnati, OH, to
 announce key findings of the research
project that is fully documented In a
 separate report of the same title (see
Project Report ordering information at
back).

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Introduction

Background
  Knowledge about  virus inactivation  in
water is  assuming greater importance  as
streams, rivers, and lakes that serve as drink-
ing water sources for many cities become
more and more contaminated with sewage.
Enteric viruses infective for man are the most
important viral agents known to be present
in water and wastewater, and more than 100
different  types may be present  in human
feces. Enteric viruses include the entero-
viruses (primarily polioviruses, coxsackie-
viruses, and echoviruses), hepatitis type  A,
Norwalk type agents, rotaviruses, reoviruses,
adenoviruses, and parvoviruses.  Since en-
teric viruses are found  in the feces of infected
persons and are readily isolated from  urban
sewage, they may enter water supplies and
present health hazards to humans. Virolo-
gists in several countries have reported the
presence of enteroviruses in drinking  water
samples obtained from public water supply
systems that use conventional  treatment
methods  of filtration  followed by disinfec-
tion. These studies all involve water that is
bacteriologically safe and contains a chlorine
residual considered  to be virucidal. The
passage of viruses through a water treatment
plant's treatment train could  be due  to  an
enhanced viral resistance to  chlorine, the
presence of natural paniculate matter, the
association of the viruses with  the alum used
for flocculation,  or virus association with
organic matter.
  Most viruses in the natural environment
are associated with solids and do not  occur
in  a free  state. The association of viruses
with solids  does not necessarily mean virus
inactivation. In fact, clay solids do not ap-
pear to have any deleterious effect on the
viruses. Concern exists that particulates (tur-
bidity) in  drinking water may  interfere with
disinfection. Thus, turbidity in drinking water
may alter the virus minimal infectious dose
by  protecting the viruses. This  possibility
resulted  in the  National Interim Primary
Drinking Water Regulations (NIPDR),  which
allow a  maximum  contaminant  level of 1
Nephelometric Turbidity Unit (NTU),  or  up
to 5 NTU if that level does not interfere with
achieving and maintaining disinfection.
   Studies  suggest the occurrence  of  an
evolutionary or adaptive alteration that  in-
creases the resistance of the virus popula-
tion after repeated sublethal exposures to
free chlorine. Virus inactivation in  water ap-
pears to be favored by acid conditions.

Objectives
   Because chloramine (combined chlorine)
is  commonly used in place of chlorine  to
disinfect waters with high levels of trihalo-
methane precursors (organics), more precise
data are needed on the efficiency of chlora-
mine disinfection alone and in the presence
of particulate matter (turbidity) in drinking
water. The main objective of this study was,
therefore, to investigate the effects of par-
ticulates in water on the disinfection  of
enteroviruses by chloramines. The complete
study objectives are outlined below:
  1. To determine the effect of turbid water
on the disinfection of test microbes (primarily
poliovirus 1 and the reference bacterium E.
co//) using combined available chlorine (the
chloramines). These results were then com-
pared with those of free  chlorine (hypo-
chlorous acid and hypochlorite ion). The
relationship  of   particulate  material  to
disinfection efficiency was then examined.
Particulates included human fecal solids,
sewage-primary effluent solids, and animal-
cell-associated poliovirus 1.
  2. The disinfection ability of chloramines
(both monochloramine and  dichloramine)
was studied at various chloramine concen-
trations, temperatures, contact times, and
pH values; at various concentrations and
types of particulates; and with single versus
aggregated preparations of test virions.
Comparisons were also made of disinfection
efficiencies for monochloramine used as a
preformed dose and as forming doses. Also
studied were the virus inactivation with dou-
ble monochloramine doses and the addition
of multiple doses of poliovirus 1 during the
progress of the experiment.
  3. To select a monochloramine-resistant
poliovirus  1 mutant.
Methods
  All of the disinfection studies were per-
formed  using  the  kinetic  apparatus. The
poliovirus 1 (Mahoney strain) stocks used in
these studies were prepared as either ag-
gregates or singles. Enterovirus-associated
animal cells were prepared to simulate natu-
rally found cell-associated  viruses that can
be  excreted from  the intestinal tract  of
humans. Two cell lines were used — human
epidermoid carcinoma (HEp-2) and  Buffalo
Green Monkey (BGM) cells. Primary effluent
from a municipal sewage treatment plant and
human feces were used to prepare solids-
associated,  naturally occurring  coliforms.
Animal viruses were titered by the plaque
technique in BGM continuous cell lines. £.
co//  survivors  were recovered  and
enumerated with surface-inoculated tryp-
ticase soy agar plates, whereas primary
effluent  studies  used  the most probable
number multiple-tube fermentation method
through the confirmed test.
Results and  Discussion

Monochloramine and
Dichloramine Disinfection of
Poliovirus 1 Singles and
Aggregates
  A number of studies have implicated ag-
gregates in the viral inocula as the cause of
aberrations in survival curves when viruses
are exposed to  destructive chemical and
physical agents such as disinfectants. This
study showed aggregated poliovirus 1 to be
1.7 times more resistant to the disinfectant
than the  singles preparation  (Figure  1).
Similar results were found in the dichlora-
mine studies. The disinfectant apparently
penetrates slowly into the aggregated viral
mass, thus enabling some viruses to survive
and develop in the tissue culture recovery
system.

Effects of Temperature On
Inactivation  of Poliovirus  1
Singles by Monochloramine and
Dichloramine
  Since chemical disinfection is a rate pro-
cess,  the  chemical reaction rate increases
with increasing temperatures. The empirical
rule of thumb is that the rate of the reaction
increases  by a factor of 2 to 3 for each
10-degree rise in temperature. The tempera-
ture coefficient for a 10-degree change (Q10)
when destroying virus by free chlorine has
been observed to increase the rate of virus
inactivation by a factor of 2 to 3 (200 to 300
times).  Studies  for  99% inactivation  of
poliovirus 1 singles by monochloramine at
pH 9 and  temperatures of 5, 15, and 25 C
yielded  an  average  Q10 value  of 2.75,
whereas at the 90% level, the average value
was 1.95.  The 90%  inactivation kinetics of
poliovirus  1 singles by dichloramine at 5 and
15 C yielded a Q10 of 2.5, which was within
the 2  to 3 factor increase noted earlier.

Effects of pH on Inactivation of
Poliovirus 1  Singles  and E. coli
by Monochloramine
  Most finished  drinking waters  in  the
United States are maintained below pH 9 —
usually between 7 and 9. At pH 9 and above,
the chloramine that is formed when hypo-
chlorous  acid  reacts  with  ammonia  is
predominantly monochloramine. Thus many
research studies are done at pH 9 or above.
However,  pH values lower than 9 can be en-
countered in drinking water treatment.  To
determine the disinfectant quality of a still
predominantly monochloramine system at a
pH below 9, monochloramine levels were
first preformed at pH 9, and the pH was im-
mediately adjusted to 7. Initial concentrations

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of monochloramine were stable at this lat-
ter pH for more than 4 hr. The change in pH
had no apparent effect on the  disinfection
of the poliovirus 1 singles, but  it increased
the rate of monochloramine disinfection of
E co//about 10 times (Figure 2). The effect
of pH on transport mechanisms across the
bacterial cell membrane may have influenced
the greater monochloramine destructive ef-
fect at pH 7. Earlier work has demonstrated
that Entamoeba histolytica cysts  take up
more chlorine and have lower survival rates
at low pH. Other research using  the sur-
rogate  animal  virus,  bacteriophage f2  (in-
stead of an animal virus like poliovirus 1)
produced greater virus inactivation at lower
pH. The fact that the present study did not
produce a similar effect points up the need
for caution when using a surrogate animal
virus. These studies  should be continued
using other animal viruses to determine
whether inactivation by monochloramine is
greater at pH 7 than  at pH 9.

Disinfection ofE. coli Using
Preformed and Forming
Monochloramines
   Chloramine research  studies  usually  use
preformed monochloramines as the disinfec-
tant. For many years, ammonia (NH3)  has
been combined with  chlorine (CI2) to form
chloramines for the treatment  of  drinking
water. Ammonia is still deliberately added to
some chlorinated public water  supplies to
provide  a combined  available   chlorine
residual (i.e., chloramines). Monochloramine
is the principal  chloramine that is  en-
countered in drinking water treatment, but
in recent years, chloramines have not been
recommended as a primary disinfectant be-
cause of their perceived low germicidal effi-
ciency. Another concern is whether forming
monochloramines are better disinfectants
than preformed monochloramines. Thus the
present study attempts to cast more  light
upon the  real-world situation in which
monochloramines are formed during the pro-
cess of disinfection and are not added in the
preformed state.
   The disinfection efficiencies of preformed
monochloramine (NH2CI)  and   forming
monochloramine (free  chlorine and NH3)
against the test bacterium £ coli were com-
pared along with reference to the disinfec-
tion  ability  of free  chlorine  alone.  The
forming  monochloramine  (Figure 3)  was
about 1.2 times more  effective than  the
preformed  monochloramine. Split-second
exposure  of the E coli inoculum to  the
hypochlorite-hypochlorous acid mixture that
existed at pH 9 in the forming monochlora-
mine study may have been responsible for
the initial faster kill of the test  bacteria.
      10
i
                                                Poliovirus 1 "Singles"

                                                Poliovirus 1 Aggregates
                  J	I	1   I   I  I  I  I
                                              J	I	I	I   I   I  I  I
        10
                                            100

                                         Minutes
                                                                     1000
Figure 1.
   Concentration-time relationship for 99% activation of poliovirus 1  singles and
   aggregates by monochloramine at pH 9 and 15 C.
 100
           NH2CL Level
              (mg/L)

• Poliovirus 1
A Poliovirus 1
&E. coli
0 E. coli
                        8.5
                        8.7
                        2.0
                        2.2
 PH


7.0
9.0
7.0
9.0
  10
  1.0
 0.1
 .01
                                            100
Preformed NH2CI
                                             1.0
                                             .01
                                                   A  1.92 mg/L NH2CI, Preformed
                                                   •  1.86 mg/L NH2CI. Forming
                                                   A 0.5 mg/L (HOCL + OCI-)
          60    120   180   240  300

                  Minutes

Figure  2.    The inactivation of poliovirus 1
            singles and Escherichia coli at 5
            C by monochloramine at pH 9
            and 7 (preformed at pH 9).
                                           10   20  30  40  50  60  70

                                                     Minutes


                                  Figure 3.   Disinfection of E. col i / 7 223 at 5
                                             C and pH 9 by  forming and
                                             preformed   monochloramines
                                             compared  with   a 0.5-mg/L
                                             mixture  of hypochlorous acid
                                             (HOCIj  and  hypochlorite  ion
                                             roc/-/

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  The disinfection rates for the forming and
preformed monochloramines were the same
after the first 15 min of the study; the ob-
served differences in the positioning of the
lines can be attributed to the differences in
bacterial numbers after the first 3 min of the
study. Although the original bacterial inocula
were similar, there was a  greater initial kill
of the bacteria in the forming monochlora-
mine study than in the preformed mono-
chloramine.  After the first 3 min of the
forming monochloramine  experiment, the
ability of the newly formed  monochloramine
to kill the remaining bacteria was the same
as that encountered in the preformed study,
but there was more bacteria to  disinfect in
the preformed study.  The brief' initial ex-
posure of the bacteria in the inoculum to the
free chlorine present  before the mono-
chloramine was completely formed appears
to account for the differences between the
two monochloramine survival curves.
Sequential Addition of
Poliovirus 1 to  Determine the
Extent of Monochloramine
Disinfecting Efficiency
  Survival  curves in these studies  often
show retardant die-away and inactivation
patterns. Although the disinfectant level was
never depleted during the course of the ex-
perimentation, the  question arose as to
whether changes had nevertheless occurred
in the disinfectant's efficiency to account for
retardant curves. Thus a second inoculum
of poliovirus 1 was added 2 hr after the first
virus administration  to determine whether
the inactivation rate of this subsequent virus
inoculum would mimic the first portion of the
curve. The rapid initial inactivation rate reap-
peared, indicating that the disinfection effi-
ciency of the original monochloramine had
not been affected or altered and that this
monochloramine was still capable of inac-
tivating the additional inoculum.

Effect of Increasing
Concentrations of
Monochloramines on the
Inactivation of Poliovirus
1 Singles
  During this research, it was noted that in-
creasing the monochloramine concentration
did  not proportionally increase the rate of in-
activation of poliovirus 1 singles. Though 12
mg/L of monochloramine was about four
times more effective at the 99% inactivation
point than the lower concentration of 5.4
mg/L, 22 mg/L was as effective as 12 mg/L.
This result  is contrary to that of some re-
searchers  who used E. coli as  the test
organism and consistent with results of other
investigators who used viruses.

Effect of Chloride  Ions on
Monochloramine Disinfection of
Poliovirus 1 Singles and
E. coli
  Poliovirus 1 has been found to be inac-
tivated more rapidly by chlorine in the form
of hypochlorite ion (OCI') at pH 10 than by
hypochlorous acid (HOCI) at pH 6. The
borate buffer system (containing KCI) is
believed to have an  influence on the
hypochlorite ion  and hypochlorous  acid
virucidal relationships. Since 0.2 N HCI was
used  in this study to prepare preformed
monochloramine at pH 9, the effect of the
chloride ion on the disinfection process was
investigated. The addition of 0.2 N HCI made
the 0.05 M borate buffer system (without
KCI) about 0.02 M with respect to chloride
ions.  In a similar study with £ coli at 5 C,
3.2  mg/L  monochloramine was formed at
pH 7, 0.02 M chloride ions were added as
the sodium salt, and disinfection was com-
pared  with  that at the  same  level of
monochloramine at pH 7, but without the
added chloride ions.  No effect of the added
chloride ions was observed. Thus the ob-
served difference in disinfection at pH 9 and
pH 7 for E. coli (Figure 2) was due to the pH
change to 7 and not to the addition of chlo-
ride ions when the 0.2 N  HCI was added to
the buffer system.

Selection for Monochloramine-
Resistant Poliovirus 1
  Studies have  suggested  that viruses
become resistant to free  chlorine after
repeated sublethal exposures. Thus a test
was undertaken to determine whether suc-
cessive  exposures of virus to monochlora-
mine  would have the same  effect.  Two
procedures were used  to  prepare virus
singles  (aggregates  could  increase virus
resistance). Polioviruses  prepared by both
procedures were exposed separately under
the same test conditions for similar time
periods. After exposure to monochloramine,
the more resistant plaques representing sur-
vivors were  isolated, regrown,  and re-
exposed to monochloramine. Eight repetitive
monochloramine exposure cycles were per-
formed  for viruses prepared by both pro-
cedures.  The viruses  prepared by one
method developed no resistance to mono-
chloramine, whereas those prepared by the
other method gradually did. The poliovirus
prepared by the latter method and exposed
eight times were 2.3 times more resistant to
monochloramine  than either  unexposed
poliovirus or virus exposed seven times to
monochloramine (Figure 4).
 200
 , Initial Singles Poliovirus
  exposed to 9.15 mg/L NH2C/

  Poliovirus 1 Singles
  survivors of 7 exposures
  now exposed to 10.5mg/L NH,CI

• Poliovirus 1 Singles
  survivors of 8 exposures now
  exposed to 8.95 mg/L NH2CI
  ,0/L.
             7234
                   Hours

Figure 4.    Inactivation  of prepared polio-
            virus at pH 9 and 15 C before
            and after repeated exposure to
            monochloramine.
Disinfection of HEp-2-Cell-
Associated Poliovirus  1
With Monochloramine
  Disinfection  studies  with  animal-cell-
associated poliovirus 1 were performed using
two continuous cell lines, HEp-2 and BGM
kidney cells. The cell-associated virus system
approximates the state of viruses as they are
excreted from the body into domestic sew-
age. The viruses are protected by their cell
association (Figure 5). This effect is more
dramatic  when the viruses  are 99.9%
through 99.99% inactivated.
  The effect of organic turbidity on the
disinfection of HEp-2 cell-associated polio-
virus was studied with monochloramine con-
centrations ranging from 4.15 to 21.0 mg/L
at 5 C and pH 7 and 9 (Figure 6). Nearly
doubling the monochloramine dosage (from
12.2 to 21.0  mg/L) at pH 9 in the presence
of almost the  same turbidity  reduced the
time required for 90% virus inactivation from
50 to 30 min. Increasing the turbidity from
0.8 NTU to  2.0 NTU at monochloramine
levels of 10.35 and 11.3 mg/L, respectively,
and pH 7 significantly decreased disinfection
efficiency. Turbidity caused by the presence
of animal cells interfered with the disinfec-

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 tion process. The pH change from 9 to 7 had
 no apparent effect on the rate of poliovirus
 1 inactivation whether or not the virus was
 associated with animal cells.
  700
^ 70
2
5
k.
3
  1.0
   10
       O 2.2  mg/L; Cell-Assoc. (2.45 NTU)
       A 2 28 mg/L. Cell-Assoc. (1.25 NTU)
       • 2.98 mg/L. Aggregated
     _ ±3,04 mg/L, Aggregated
            8 '
                      HEp-2
                      Cell-associated
                  •*—  Poliovirus 1
    Aggregated
    Poliovirus 1
   Oil
            12345

                   Minutes

 Figure 5.   Inactivation of aggregated and
            HEp-2 cell-associated poliovirus
            1 with hypochlorous acid at pH 6
            and 5 C
                                                20
                                                10
                                            l
                                                                                   O
                                                        NH-,CI Level Turbidity   pH
                                                                                                    O
                                                        O  Poliovirus 1 Singles
                                                               J	I—I  I  I  I I
                                                                                           i    l   l  I  I l  l
                                                                              10
                                                                                                         100
                                                                                   Minutes
                                            Figure 6.
                                                       Concentration-time relationship for 90% inactivation of poliovirus 1 singles and
                                                       HEp-2 cell-associated poliovirus 1 at different turbidity levels and concentrations of
                                                       monochloramine at 5 C and pH 7 and 9
Disinfection of BGM
Cell-Associated Poliovirus 1
With Hypochlorous Acid,
Monochloramine, and
Dichloramine
  The inactivation of BGM-cell-associated
poliovirus 1 was studied using three disinfec-
tants — hypochlorous acid at 15 C and pH
6.0, monochloramine at 15 C and pH 9, and
dichloramine at 5 C and pH 4.5. All the sur-
vival curves showed extended tailings caused
by the association of the poliovirus 1 to the
cells and to themselves (aggregation) dur-
ing the disinfection  process. Disinfection
rates of BGM-cell-associated  poliovirus 1 by
monochloramine and hypochlorous acid at
similar turbidities and concentrations were
compared. Whereas only 15 sec was needed
for 90% of the cell-associated viruses to be
inactivated by the hypochlorous acid, 95 min
was required to reach 90% inactivation with
the monochloramine. Even under these dif-
ficult conditions for disinfecting viruses,
hypochlorous acid was about 380 times as
effective  as the monochloramine. Figure 7
summarizes the monochloramine concen-
                                           S
                                           O
                                           \

                                               20
                                               10
 D
 A
 a
                                                        *
                                                        o
Turbidity
 (NTU)

 0.7
  1 0
  1.98
 20
   .65
   90
  1.6
NH2CI Level
  (mg/L)

   332
   3.16
   5.47
   6.0
   7.0
   7.0
   1 16
                                                             Poliovirus 1 Singles
                                                                                               •  •¥•
                                                                     I

                                                                     I
                                                                            70
                                                                                                       700
                                                                                  Minutes
                                           Figure 7.
Concentration-time relationship for 90% inactivation of BGM-cell-associated and
unassociated poliovirus 1 by various concentrations of monochloramine at15C and
pH9.

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tration-time plot for the 90% inactivation of
BGM-cell-associated  and  unassociated
viruses. The BGM-cell-associated poliovirus
1 points are  represented by bold symbols.
Most of these associated points were above
the poliovirus 1 unassociated singles curve,
indicating that the cell-associated  viruses
were being protected from inactivation.
  A final study was done comparing the in-
activation of BGM cell-associated polio-
viruses with the survival  of unassociated
poliovirus 1 singles using dichloramine as the
disinfectant  at 5  C and  pH 4.5.  No dif-
ferences were observed  in the  disinfection
rates of  the  two  poliovirus preparations,
though the  dichloramine concentrations
were similar (17.0 mg/Lforthe unassociated
versus 17.35 mg/L for the associated polio-
virus). The lack of  protection could be due
to rapid penetration of the cell mass by the
dichloramine.

Coliform  Disinfection Studies
  Disinfection studies using coliforms were
divided into two groups: (a) disinfection of
naturally  occurring coliforms  from fecal
suspensions, and (b) disinfection of fecal col-
iforms associated with  primary  effluent
solids. The turbidity associated with either
system was found to interfere with disinfec-
tion efficiency.

Conclusions
  Virus aggregates are, along with  organic
particulates, a major part of the mechanism
for the maintenance of  virus infectivity in
water. In these studies, aggregated polio-
virus 1 (at the 99% inactivation point) at 15
C and pH 9 was about 1.7 times more resis-
tant to disinfection  by monochloramine than
unassociated virus  singles. The singles virus
preparation disinfected by dichloramine at 15
C and pH 4.5 was inactivated (at the 90%
inactivation point)  about 8 times as rapidly
as the aggregated virus.
  The average temperature coefficient for a
10-degree change  (Q10) was 2.75 in mono-
chloramine-temperature reactivity  studies
with  poliovirus  1  singles at   pH   9 and
temperatures  of  5,  15,  and  25  C. For
dichloramine, a 10-degree change  in tem-
perature gave a Q10 of 2.5 for poliovirus  1
singles. Both Q10 values are within  the 2 to
3 factor increase used as the rule of thumb
for each 10-degree rise in temperature.
   Monochloramine formed at pH 9 and then
adjusted to pH 7 was a better disinfectant
for bacteria but not for the  test virus. Lower-
ing the pH  from 9 to 7 increased mono-
chloramine disinfection efficiency about 10
times for the bacteria.
   Forming monochloramine was about 1.2
times  more effective than the preformed
monochloramine for disinfecting E coli. The
faster disinfection rate could be due to the
initial presence of hypochlorous acid before
the  monochloramine  was  completely
formed.
  Resistance to monochloramine developed
gradually in single viruses prepared by one
of the two  methods used. Poliovirus 1 sur-
vivors exposed  eight times to monochlora-
mine and then  disinfected with 8.95 mg/L
monochloramine were 2.3 times more resis-
tant to monochloramine than either the virus
never exposed  to monochloramine or the
virus previously exposed  seven times to
monochloramine.
  The presence of HEp-2  and BGM cell-
associated  turbidity interfered with the cell-
associated  virus by hypochlorous acid and
monochloramine but not by dichloramine.
  The solids in human feces and primary ef-
fluents protect naturally occurring coliforms
from disinfection.
 Recommendations
  A dramatic increase in monochloramine
 disinfection efficiency was produced for E.
 coli by lowering the pH of monochloramine
 from 9 to 7.  Since most finished drinking
 waters are maintained in the United States
 at a pH below 9, the mechanism of action
 should be further investigated. In addition,
 other  animal viruses  besides  poliovirus
 should be studied to determine whether they
 are affected  by the pH  change.
  Additional  studies are required with dif-
 ferent cell lines to determine whether they
have similar viral protective effects during
disinfection.
  Studies should be conducted to determine
the cost effectiveness of reducing  the tur-
bidity levels from 5 to 1 NTU in drinking
water treatment. A reduced turbidity level of
1 NTU  is recommended because  of our
studies and  those of others  on coliforms
associated with primary effluent solids, fecal
solids,  and cell-associated viruses.
  In future turbidity studies,  methods for
determining the organic or inorganic nature
of the  particulates must be developed to
ascertain  their potential  for inhibiting
disinfection.
  Methods should be established for stan-
dardizing the apparatus and the methods for
disinfection research and for  reporting the
physical state of the test organisms — that
is,  viral-associated  (aggregates  or  cell-
associated) or  unassociated  (singles)  pre-
parations.
  The usefulness of chloramines, especially
the monochloramines,  in field situations
should be more carefully evaluated. Under
certain conditions such as forming situa-
tions, they may be useful.

  Dichloramine's ability to penetrate organic
masses such as cells should be more thor-
oughly investigated.
  The  development  of resistant strains of
viruses  in  nature should  be  thoroughly
studied.  Laboratory studies can only point
out a potential problem; field studies are re-
quired to pinpoint possible health risks that
might exist in  the  natural environment.
   Pasquale Scar pi no is with University of Cincinnati, Cincinnati, OH 45221.
   John C.  Hoffis the EPA Project Officer (see below).
   The  complete report, entitled  "Effect of Particulates  on Disinfection of
     Enteroviruses in Water by Chloramines," (Order No. PB 84-190 693; Cost:
     $ 11.50, subject to change) will be available only from:
           National Technical Information Service
           5285 Port Royal Road
           Springfield, VA 22161
            Telephone: 703-487-4650
   The EPA Project Officer can be contacted at:
           Municipal Environmental Research Laboratory
            U.S. Environmental Protection Agency
            Cincinnati, OH 45268
 *USGPO:   1984-759-102-10681

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