X-/EPA
United States
Environmental Protection
Agency
Municipal Environmental Research
Laboratory
Cincinnati OH 45268
Research and Development
EPA-600/S2-81-088 July 1981
Project Summary
Virus Inactivation in
Wastewater Effluents by
Chlorine, Ozone, and
Ultraviolet Light
R. A. Fluegge, T. G. Metcalf, and C. Wallis
Virus levels before and after disin-
fection were studied in four full-scale
wastewater treatment plants: two
used chlorine as the disinfectant, one
used ozone, and one used ultraviolet
light. A second ozone study was con-
ducted on a pilot scale at one of the
treatment plants.
Results for the viral content in
wastewater effluents indicated no
consistent correlation between virus
concentrations and any of the "tradi-
tionally" measured indicators of pol-
lution. No seasonal variation of virus
concentration was detected in this
four-season study. A diurnal variation,
however, was noted in one of the
plants during two separate samplings:
one in December and one in July.
Maximum virus concentrations oc-
curred in this effluent between 2:00
and 4:00 a.m. for each of these two
sampling periods.
Ten different virus types were iso-
lated. Poliovirus 1 was the predomi-
nant virus type found in both the
treated and treated-plus disinfected
effluents.
Experiments with pure strains and
known quantities of attenuated viruses
to determine recovery efficiencies
indicated that variability in viral seed
recovery data, although fairly small in
a controlled laboratory environment,
is more marked in a field situation.
This Project Summary was devel-
oped by EPA's Municipal Environmen-
tal Research Laboratory, Cincinnati,
OH, to announce key findings of the
research project that is fully docu-
mented in a separate report of the
same title (see Project Report ordering
information at back).
Introduction
Wastewater Treatment Plants
Four treatment plants were studied
during this program: one each in Cin-
cinnati (Ohio), Estes Park (Colorado),
Marlborough (Massachusetts), and
Waldwick (New Jersey).
The Muddy Creek Works (Cincinnati),
studied in December 1976, is a second-
ary treatment facility using a conven-
tional activated sludge system followed
by disinfection with chlorine. Effluent
samples were collected both before and
after applying the disinfectant. Virus
content as well as the "traditional"
water quality parameters were mea-
sured. The chlorine dose produced
average total chlorine residual of 1.2
mg/L, after a contact time of 50 min. In
July 1977? ozone was applied as an
alternative disinfectant for this same
effluent. The effluent was transported
by tank truck to the Robert A. Taft
Laboratory (Cincinnati), the location of
EPA's pilot ozone test facility. The
packed-column ozone contactor used
an applied ozone dose of 8 mg/L with a
contact time of 30 sec.
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The Estes Park Upper Thompson
Sanitation District Treatment Plant
(UTSD), sampled during the spring of
1977, consists of a conventional acti-
vated sludge plant followed by second
stage attached growth nitrification, tri-
media filtration, and disinfection with
ozone. Ozone was produced from air
using a corona discharge generator. The
contactor was a baffled, closed, rectan-
gular tank in which effluent flowed both
concurrent and countercurrent with
ozone, which was injected through
porous stone diffusers placed at the
bottom of the tank. The contact time at
average flow was 37 min. Changes in
operating conditions permitted a study
to be completed that indicates dosage
rates of approximately 5 mg/L are
required to ensure virus die-off.
The Marlborough Easterly Wastewater
Plant has a two-stage activated sludge
system for nitrification followed by dis-
infection with chlorine. The effluents
collected and studied from this plant in
October 1977 had an average total
chlorine residual of 1.3 mg/L after a
contact time of 50 min.
The Northwest Bergen County Treat-
ment Plant (Waldwick), sampled during
April and May 1978, is a conventional
and/or step aeration activated sludge
system followed by disinfection with
ultraviolet radiation. Although this plant
has a design flow of 32,100 mVd (8.5
mgd), during these studies the flow was
only 19,000 mVd (5 mgd). Clarified
secondary effluent flowed through an
ultraviolet disinfection unit manufac-
tured by Pure Water Systems, Inc. This
prototype unit consisted of a stainless
steel rectangular compartment housing
400 ultraviolet lamps that were protected
from the effluent by 23-mm (0.9-in.
O.D.) quartz jackets spaced 12.7 mm
(0.5 in.) apart. Contact time was approx-
imately 3.6 sec.
Experimental Procedures
An Aquella* virus concentrator (Car-
borundum Company) mounted inside a
mobile field laboratory concentrated
waterborne viruses from large samples
on a continuous basis. To ease handling
and shipping problems, samples of
approximately 380 L(100 gal) of effluent
were concentrated to as small a volume
of liquid as practicable for analysis.
Sodium thiosulfate, dilute HCI, and
AICI3 were added to the effluent sample.
'Mention of trade names or commercial products
does not constitute endorsement for use by the U.S.
Environmental Protection Agency.
and the solution passed through virus-
adsorbing filters. These filters were
removed and placed into 1 to 3 L of
glycine solutions; the solution was
further concentrated and stored in fetal
calf serum at dry ice temperatures
before shipment to the virus assay
laboratory.
Analysis of samples for natural virus
was carried out on two types of cell
cultures: a continuous cell line (Buffalo
Green Monkey, BGM) maintained at the
University of New Hampshire virus
laboratory and a primary cell line (Afri-
can Green Monkey Kidney, PMK), which
was prepared fresh for each sample.
The virus assays were made by plaquing
methods that permitted separation and
enumeration of viruses present in the
samples. Virus isolates were identified
by serum neutralization tests using
eight Lim-Benyesh-Melnick (LBM) anti-
serum pools.
Results
Virus isolation rates were determined
for each of the samples by equally
weighting each sample that contained
one or more virions. The data from each
sample were then tabulated to form the
reported average. Based on this weight-
ing, 38% of all samplings, before disin-
fection, contained one or more virions.
The actual virus isolation rates for the
effluent before disinfection were: UTSD
Treatment Plant, 69%; Muddy Creek
Works, 75%; Taft Pilot Plant, 57%;
Marlborough Easterly Plant, 38%; North-
west Bergen County Plant, 56%.
There were no major differences in
seasonal isolation rates, particularly for
the two sampling periods completed in
July and December at the Muddy Creek
Works. The latter two rates for non-
disinfected effluent were 75% and 60%,
respectively.
Significantly lower isolation rates
were encountered after disinfection.
Reductions occurred for each of the
three disinfectants (ultraviolet, chlorine,
and ozone) studied. The actual virus
isolation rates for the effluent after
disinfection were: UTSD Treatment
Plant (all sample days), 50%; UTSD
(eliminating 4 days of deliberately
decreased ozone dosage), 33%; Muddy
Creek Works, 38%; Taft Pilot Plant, 7%;
Marlborough Easterly Plant, 0%; North-
west Bergen County Plant, 13%.
Virus isolation rates were reduced
75% when ultraviolet light was used as
a disinfectant; 50% when ozone was
used at UTSD; 88% when ozone was
used for Muddy Creek at Taft; and 50%
and 100% (none detected) for the two
chlorine samplings. Similar results
were obtained by analyzing the same
data using virus titer rather than isola-
tion rates.
The relative distribution of viruses by
classification, based on the total number
of viruses in all nondisinfected effluents,
was measured. The polio and coxsack-
ievirus types were isolated and individ-
ually identified. All three types of polio-
viruses (1, 2, and 3) were shown to be
present in the effluents. These viruses
were not assayed to determine their
virulence. Coxsackie A9, B1, B2, B3, B4,
B5, and B6 were also isolated and
identified during this program. Except
for the coxsackie B5 virus, all virustypes
were found in the treated as well as the
treated plus disinfected effluent.
Virus levels in treated effluent varied
diuirnally in one of the plants—the
Muddy Creek Works. Virus concentra-
tions were greater (as high as 40 viruses
per 380 L (100 gal)) between 2:00 and
4:00 a.m. in both December and July.
An increase of almost an order of
magnitude was found for virus liters
between 2:00 and 4:00 a.m. compared
with measurements during the remain-
der of the day. This increase remains
unexplained.
The diurnal variations demonstrated
at the Muddy Creek Treatment Plant
provided a unique opportunity for com-
parisons with the traditionally measured
water quality indicators. Linear regres-
sion analyses performed on the July
data determined indicators that might
correlate with virus levels. Total coliform,
fecal coliform, chemical oxygen demand,
and total organic carbon had a positive
correlation coefficient. No correlation
was evident for total suspended solids
and turbidity.
A similar analysis was performed of
effluent data collected in December. In
this instance, no correlation was found
with total coliforms and fecal coliforrns
whereas a positive correlation with total
suspended solids and turbidity occurred.
Both COD and TOC exhibited a positive
correlation with virus tilers in December
as well as July.
Conclusions
In general, the present study provides
a framework for analyzing the effective-
ness of the various disinfectanls to
inactivate viruses. Questions in many
areas, however, remain unanswered.
The amount of data required to provide a
statistically accurate profile on the
relative disinfection abilities of chlorine,
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izone, and ultraviolet radiation are
attainable only if further sampling
studies are undertaken as part of a
coordinated research program.
Because virus levels encountered in
each of the tested effluents before dis-
infection were extremely low, detection
of significant reductions after disinfec-
tion was very difficult. In all cases,
reductions were noted, but the param-
eter used to measure these reductions
was virus isolation rate (i.e., the number
of samples positive for virus divided by
the total number of samples tested per
treatment plant), not virus liter. Thus,
the trend was there, but only qualitatively.
The low levels of indigenous viruses
in the undisinfected effluents were not
anticipated. There was also a further
complication. Seeding experiments
with attenuated poliovirus strains at
each of the treatment plants indicated
that the method of concentrating large
volumes of effluent (380 L) down to 1 to
2 L was quite inefficient (approximately
10%) and exceedingly variable. This
made quantitative analysis and inter-
pretation even more difficult.
There is still a need to determine how
well disinfectants inactivate indigenous
viruses under actual operating condi-
tions. This study, however, clearly
demonstrated that virus recovery tech-
niques need considerable improvement.
Field studies should be undertaken only
after a viable measurement technique
exists.
The full report was submitted in
fulfillment of Grant No. R804991 by the
Carborundum Company, Niagara Falls,
New York, under the sponsorship of the
U.S. Environmental Protection Agency.
R. A. Fluegge is with Radian Corporation. Austin. TX 78766; T. G. Metcalfis with
the University of New Hampshire. Durham, NH 03824; and C. Wa/lis is with
Baylor University, Houston, TX 77025.
Albert D. Venosa is the EPA Project Officer (see below).
The complete report, entitled "Virus Inactivation in Wastewater Effluents by
Chlorine, Ozone, and Ultraviolet Light," (Order No. PB 81-208 183; Cost:
$9.50, 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
> US GOVERNMENTPWNT1NQOFFICE 1«1 -757-012/7210
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