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-

-------
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

-------
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

-------
United States
Environmental Protection
Agency
Center for Environmental Research
Information
Cincinnati OH 45268
Postage and
Fees Paid
Environmental
Protection
Agency
EPA 335
Official Business
Penalty for Private Use $300

-------