United States
Environmental Protection
Agency
Health Effects Research
Laboratory
Research Triangle Park NC 27711
Research and Development
EPA-600/S1-83-012 Sept. 1983
&EPA Project Summary
Enteric Virus Removal in
Wastewater Treatment
Lagoon Systems
Howard T. Bausum, Stephen A. Schaub, William E. Rose, and Paul H. Gibbs
This study assessed the removal of
indigenous enteric viruses from raw
wastewaters subjected to lagoon
treatment. Paired sites lacking mechan-
ical aeration (facultative pond systems)
were studied in the southeast.
southwest and north central regions of
the United States. Also included were
two systems that provide partial me-
chanical aeration of the wastewater in
the first pond. Facultative systems
were studied during summer, winter
and spring, 1979-1980; and aerated
systems during the late spring to early
fall season, 1979 with sampling
performed at all sites concurrently.
Intervals between sampling days
approximated the mean calculated
wastewater residence time for
individual cells. Thus, estimates of virus
reduction could be made in relation to a
common mass of water as well as on a
same sampling day basis. Samples were
obtained at three points in each system:
the raw wastewater and the effluent
from both the first and second pond in
series. Fecal coliform and chemical/
physical wastewater quality
measurements were performed
concurrently with virus sampling. The
adsorption-elution technique using
bentonite clay in the presence of added
calcium ion was used to concentrate
virus from large (10-30 I) wastewater
volumes. Virus samples were
reconcentrated in the laboratory, and
enteric virus levels were determined on
Buffalo green monkey kidney (BGM),
He La and human rhabdomyosarcoma
(RD) cell lines.
In facultative lagoon systems,
significant virus reduction occurred
throughout the year. Overall virus
removal in the first two ponds generally
exceeded 99 percent in summer, with
combined seasons averages above 95
percent. For all seasons, the first pond
of each system provided the greatest
contribution to virus removal. There
was a statistically significant (P< .05)
difference in virus removal on the basis
of season, with reduced virus
treatability during winter. However,
because virus levels in the raw
wastewater were lower in winter, the
level of virus leaving the lagoons was
fairly constant throughout the year.
Fecal coliform removals far exceeded
virus reductions and removal of other
wastewater constituents. Coliforms,
total organic carbon (TOC) and sus-
pended solids (SS) were, like virus,
removed primarily in the first pond of
the systems. Statistical analysis did
not, however, reveal any wastewater
parameters that were highly correlated
with virus removal.
In partially aerated lagoon systems,
the observed percent virus removal
during the spring to fall test period
was somewhat less than in the
facultative systems. However, virus
levels leaving both types of lagoon
systems were similar. In the aerated
systems the greatest removal occurred
in second downstream, non-aerated
ponds characterized by longer reten-
tion times.
This Project Summary was developed
by EPA's Health Effects Research
Laboratory. Research Triangle Park,
NC, to announce key findings of the
research project that is fully document-
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ed in a separate report of the same title
(see Project Report ordering
information at back).
Introduction
To the authors' knowledge this study is
the first quantitative evaluation of the
removal of human enteric viruses in oper-
ational, full-scale wastewater treatment
lagoon systems in which multiple sites,
regions and seasons were examined in
the United States Work reported by
others, using bench, pilot scale or single
full-scale lagoon systems has indicated
substantial virus reduction The principal
thrust of the present study was to deter-
mine if this observation is uniform and
reproducible, regardless of site, region or
season, in typically designed and operated
wastewater treatment lagoon systems. It
was not the goal of this study to identify
specific factors contributing to virus re-
moval, though tests were made for statis-
tical correlation in other water quality
parameters
The study consisted of two parts. (1) a
larger study encompassing three pairs of
wastewater treatment lagoon systems
representing widely separated
geographical regions within the United
States; and (2) a range-finding study,
limited to two systems, in which the first
one or two ponds in series received
partial mechanical aeration The goal of
this second study was to determine if
there were any differences in overall
virus reduction associated with the use of
artificial aeration
Site selection was preceded by identi-
fication of acceptable lagoon design and
performance criteria. These criteria were
then used in selecting systems with and
without mechanical aeration for the
various climatic regions. Included in the
larger study, using systems without arti-
ficial aeration were, two pond systems
in the upper midwest, at Beresford, SD,
and Lennox, SD; two in the Southeast, at
Jonestown, MS and Shelby, MS; and two
in the southwest, at El Paso, TX
(Northeast Ponds) and Kermit, TX. Study
sites using partial mechanical
aeration were located at Muskegon, Ml,
and Castle Rock, CO In the selection of
sites an effort was made to choose
systems similar in design, operation, size
and wastewater characteristics.
Communities without substantial
industrial discharge were selected, with
the exception of Muskegon, where about
60 percent of the wastewater is of
industrial origin, coming largely from
paper mills. All systems receive raw,
unsettled sewage. The systems serving
Muskegon and El Paso serve substan-
tially larger populations than the remain-
ing systems All systems have at least two
ponds operated in series; all have been in
service at least 5 years, and all meet EPA
discharge standards for biochemical
oxygen demand and SS. Calculated
combined water retention time for the
two ponds in the non-aerated systems
varies from 24 days (Kermit) to 1 38 days
(Jonestown) with little difference in resi-
dence time between the first and second
pond in each system and all operate with
a continuous effluent discharge. At
Muskegon and Castle Rock, residence
time in the first or partially aerated
portion is much shorter (Muskegon,~2
days, Castle Rock, 6.7 days), while
residence in the subsequent non-aerated
cells is relatively long (Muskegon,— 150
days; Castle Rock, —63 days) At
Muskegon, the large non-aerated
lagoons are subject to seasonal
drawdown, with the water applied to
cropland At Castle Rock, year-round
discharge occurs with the final pond in
series subject also to additional
drawdown for irrigation.
The six paired sites without mechanical
aeration were studied m each of three
seasonal test periods late Julyto October
1979; January to March 1980, and late
March to May 1 980. During each season
concurrent sampling was performed at
three periods m time These sampling
times were on approximately 25 day
intervals, thereby spanning a period of
about 50 days m each season. The two
systems using mechanical aeration were
studied at only three points in time,
representing approximately the begin-
ning, midpoint and end of the summer
drawdown season Sampling was approx-
imately simultaneous at the two sites.
On each sampling day, samples for
virus concentration and analysis and for
physical, chemical and coliform
determinations were obtained at three
points in each lagoon system. In non-
aerated systems these were- (1) the
influent wastewater (raw sewage) before
entering the first lagoon, (2) the effluent
from the first lagoon, or the first pair of
lagoons if operated in parallel (Lennox
and El Paso); and (3) the effluent from the
second lagoon. In systems with
mechanical aeration, sampling points
were (1) raw sewage; (2) the effluent
from the mechanically aerated portion
whether one pond (Castle Rock) or two in
series (Muskegon); and (3) the effluent
from the final pond(s), which were
subjected to seasonal drawdown. At the
sites with aeration, duplicate samples
were taken for virus concentration an<
evaluation.
The following waterquality parameters
other than virus, were determined: SS
TOC electrical conductivity, feca
coliforms and total dissolved solids (TDS)
Also measurements of watei
temperature, pH and dissolved oxyger
(DO) were made both morning and, when
possible, afternoon
Preparation of virus concentrate
samples in the field as well as measure-
ment of fecal coliforms and most
physical/chemical water quality
parameters were performed by local
contractors.
For virus concentration m the field the
sample volume was 20-35 gal. (76-132 I)
for pond effluents and 10-20 gal. (38-761)
for raw influent wastewater. Pond
effluent samples were simple grab
samples.Raw wastewater samples were
composites formed by collecting portions
of the sample in the early morning, at
noon, and the previous evening. Samples
were batch-concentrated during
the technique of adsorption to bentonite
clay in the presence of calcium ion The
clay particles were trapped on high solids
capacity fiberglass filters, and the loaded
filters were shipped under ice in the
presence of 2 percent beef extract, pH 9.
In the laboratory, virus was eluted from
the filters at pH 9 and further concentra-
ted by the organic flocculation method at
pH 3.5. The floe material was centrifuged,
and the centrifuge pellet was then dis-
solved with 0.15 M Na2HPO4. Total
concentration factors were —4000-fold
for pond samples Virus levels in the
samples were determined by standard
plaque assay procedures using three
heteroploid continuous cell lines- Buffalo
green monkey kidney (BGM), HeLa, and
human rhabdosarcoma (RD).
Conclusions and
Recommendations
At the six paired non-aerated sites,
virus levels in influent sewage were
highest in summer (mean = 348 plaque-
forming units (pfu/l) and lowest in spring
(mean = 54.6 pfu/l). This was not
reflected in the virus levels leaving the
second pond in these systems. These
were generally in the range 0.2 to 1.1
pfu/l, though somewhat higher at the
Kermit site (1.3 to 5.6 pfu/l) and at the
South Dakota sites in winter (2.2 to 10.6
pfu/l). Mean percent removal for the
various sites ranged from 99.3 to 99.9 in
summer, from 93 9 to 99.1 m winter and
from 89.3 to 99.6 m spring. Combined
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season site means varied from 93.3 at
Beresford to 99 6 at Jonestown Results
obtained from the various cell lines used
were in substantial agreement. Removal
of human enteric virus in these systems
was consistently good regardless of site,
region or season and was comparable to
or better than that typically achieved in
conventional secondary sewage
treatment (without disinfection)
Virus removal occurred preponder-
antly in the first pond of each system (88
to 98 5 percent as compared to 62 to 69
percent for second ponds) This may be
related to the higher levels of particulates
available for virus adsorption and
settling or to the high levels of bacteria
and algae which may have antagonistic
interactions with the viruses Similar
results occurred for SS, TOC, and fecal
coliforms
The effect of season, region, site snd
season x region on virus removal was
studied by analysis of variance (ANOVA)
A seasonal difference for the first pond
and for the combined ponds were the only
effects found significant at the 5 percent
level The seasonal difference did not
depend on region The better virus
removals encountered in summer are in
keeping with other work reporting the
importance of temperature in virus
removal, although in the present study a
significant correlation was not obtained.
One objective of this study was to
measure change in various water quality
parameters as the water passed through
the lagoons to determine if any were
correlated with virus removal No
correlations could be detected either
within or across seasons Suspended
solids and TOC removals were generally
good (above 70 percent) only during
summer, while fecal coliform removals
generally exceeded 99 percent through-
out the year
Results from the limited study on
systems receiving partial aeration in the
first ponds indicate no significant differ-
ence in the virus content of effluent water
between them and the non-aerated
systems. Mean effluent values were 0 56
pfu/l for Muskegon and 0 57 for Castle
Rock In contrast to the non-aerated
systems, virus removal occurred
preponderantly in the downstream, non-
aerated pond(s). This is attributable tothe
very short retention times in the first
(partially aerated) portion and the long
retention times in the non-aerated
portion Virus reduction within the non-
aerated (or second stage) ponds at Castle
Rock was 97 percent and thus within the
range observed for the non-aerated
systems. Assessment of percent virus
reduction in the Muskegon system was
not totally satisfactory because of very
low apparent virus levels in the influent
sewage. This may have resulted by the
presence of materials that interfered with
the efficiency of sample concentration or
that were toxic to viruses or to the cell
monolayers used in their enumeration
The presence of 0.2 to 10 pfu/l of
enteric virus in pond effluents could
represent a low level health hazard if
released undismfected to a receiving
stream used for recreation or as a
drinking water source Disinfection, land
wastewater application or the addition of
more than two ponds in series with a
longer retention time would provide
further virus reduction.
While the present study provides a
general verification of the year-round
capacity of wastewater treatment lagoon
systems to remove enteric viruses, under
typical conditions in the United States,
the following should be borne in mind. (1)
optimization of this virus removal
capability will require a better
understanding of the mechanisms
involved and their interaction; and (2) the
impact of adverse conditions such as
excessive loading, extended freezing or
toxic chemical inputs has not been
evaluated.
Studies should be performed to assess
the role of virus adsorption to wastewater
particulates or to algae and other
microorganisms. Other areas which
should be addressed in virus removal are
the kinetics of virus adsorption, sedimen-
tation rates, the effects of thermal
turnover and excessive hydraulic loading,
the rate of mactivation or irreversible
solids association, and the potential for
enhanced virus removal by association
with settleable solids through addition of
clay or cation, reduction in pH or use of
coagulant aids.
Howart T. Bausum, Stephen A. Schaub. William E. Rose, and Paul H. Gibbs are
with the U.S. Army Medical Bioengineering Research and Development
Laboratory, Fort Detrick, Frederick, MD 21701.
Elmer W. Akin is the EPA Project Officer (see below).
The complete report, entitled "Enteric Virus Removal in Wastewater Treatment
L a goon Systems," (Order No. PB 83 -234 914; Cost: $10.00, 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:
Health Effects Research Laboratory
U.S. Environmental Protection Agency
Research Triangle Park. NC 27711
ftUS GOVERNMENT PRINTING OFFICE 1983-659-017/7170
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