United States
Environmental Protection
Agency
Risk Reduction
Engineering Laboratory
Cincinnati OH 45268
Research and Development
EPA/600/S2-89/015 Jan.1990
Project Summary
Persistence of Pathogens in
Lagoon-Stored Sludge
R. S. Reimers, M. D. Little, T. G. Akers, W. D. Henriques, R. C. Badeaux,
D. B. McDonnell, and K. K. Mbela
The project objective was to
investigate pathogen inactlvation in
lagoon-stored municipal sludges. The
in-field lagoons were located in
Louisiana (New Orleans) and in Texas
(Port Aransas), both semitropical
areas of the United States. Each
lagoon was filled with 7.56 m? of
anaerobically digested sludge to
which a spike containing a mixture of
Salmonella livingstone, poliovirus
Type 1, and Ascaris suum eggs was
added. The municipal sludge placed
in each lagoon was from the
respective local area. The field and
laboratory data demonstrated that 15
mo of storage was required for
pathogen inactivation to meet the
U.S. Environmental Protection Agen-
cy's (EPA) Process to Further Reduce
Pathogens (PFRP) criteria for lagoon-
stored sludges in a semitropical
climate. In this study, viable Ascaris
eggs were inactivated in 15 mo in the
New Orleans lagoon where the
temperature averaged about 25 "C
over a 5 mo period. Although a sim-
ilar temperature was observed for the
Texas (Port Aransas) lagoon, all
4scar/s eggs were dead after 12 mo
of storage, probably because of
petroleum organics in the Texas
sludge. Salmonella livingstone was
inactivated in 4 to 6 mo in both
lagoons at a log-reduction rate of 1.2
and 1.6 log Most Probable Number
(MPN)/mo/100 mL in New Orleans and
Port Aransas sediments, respectively.
Total conforms and fecal conforms
declined 2 to 6 logs within 12 mo.
Little, if any, die-off of fecal
streptococci, either on a volume or a
gram dry weight basis, was noted in
either lagoon. An increase of total
conforms was observed in both
lagoons after 10 mo. Poliovirus Type
1 was inactivated within 12 mo at
rates ranging from 0.01 to 0.02 log
PFU/mo/100 mL in the sediments of
both lagoons.
This Project Summary was
developed by EPA's Risk Reduction
Engineering 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 form at the back).
Introduction
Sludges from municipal wastewater
treatment plants often contain pathogens
that are hazardous to humans and
domestic animals. Partly because of this,
Congress passed the Clean Water Act of
1977 (P.L. 95-217). This act led to the
establishment of criteria for the control of
pathogens related to the land application
of sewage sludge (40 CFR 257). These
criteria specify the minimum level of
treatment needed before municipal
wastewater sludges can be applied to
land. In addition, EPA designated three
categories of municipal sludges destined
for agricultural use: "Not stabilized" (raw
sludge), "Processes to Significantly Re-
duce Pathogens" (PSRP), and "Proces-
ses to Further Reduce Pathogens"
(PFRP).
The criterion for a PSRP is that the
process reduces pathogenic viruses by 1
log or 90% and indicator bacteria (fecal
and total coliforms) by 2 logs or 99%. For
a PFRP process, pathogens are to be
reduced below detectable limits; i.e., 1
PFU for viruses, 3 MPN for pathogenic
bacteria, and 1 viable helminth egg per
100 mL of sludge. In November 1986, an
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EPA task force was established to review,
on a case-by-case basis, processes not
listed in the Federal Register that
nevertheless attain the required pathogen
reductions for either classification.
Lagoon storage, which is the subject of
this investigation, is one such process.
Lagoon storage of domestic waste
sludges as a means of inactivating
pathogens has received little attention.
Results from a recent EPA-funded
laboratory study indicated that when
small amounts of sludge containing
parasite eggs were stored at 25°C, the
eggs were destroyed after 10 to 16 mo
but that when sludge was stored at 4°C,
some Toxocara cants and Ascaris suum
eggs were still viable after 25 mo. These
results suggested that storing sludges in
lagoons in warm climates might be an
effective way of inactivating parasite eggs
and other pathogens and, in part, led to
the initiation of this project.
The overall objective of this study was
to evaluate the effectiveness of sludge
storage in lagoons as a method of
inactivating pathogens. A specific
objective was to determine whether the
storage of selected anaerobic sludges in
lagoons in areas where the mean ambient
temperatures approach 25°C could be an
effective way of inactivating selected
parasite eggs (Ascaris suum).
Salmonella, and enteroviruses (poliovirus
Type 1). An additional objective was to
detect changes in sludge characteristics
that could be correlated with pathogen
inactivation.
Procedure
A field lagoon was placed at the
Tulane University F. Edward Hebert
Research Center in Belle Chasse, LA,
and at the Nueces County Sewage
Treatment Plant No. 5 in Port Aransas,
TX. These locations were chosen for the
climatic and environmental conditions
associated with each. The size of each
sludge lagoon was about 10.3 m3, and
each was filled with 7.56 m3 (2,000 gal)
of anaerobically digested municipal
sludge from New Orleans, LA, and
Corpus Christi, TX, respectively. These
sludges were spiked with Salmonella
livingstone, Ascaris suum eggs, and
poliovirus Type 1. The parasites were run
through bench scale digesters so that the
spike would better simulate field
conditions. Samples were taken and
analyzed for bacteria and viruses at 24
hr, 1, 2, 4, 6, 9, 15, and 24 mo; parasite
samples were taken and analyzed every
3 mo. At each sampling period, abiotic
parameters were analyzed for oxygen,
temperature, oxidation-reduction potential
(ORP), pH, chemical oxygen demand
(COD), solids (total and volatile) (VSS),
ammonia, total Kjeldahl nitrogen,
alkalinity, hardness, nitrite, and nitrate.
Bottom and top samples were taken in
five different locations. All biotic and
abiotic analyses had the appropriate
quality control and assurance testing. In
addition, various knowns were also tested
to ensure the precision and accuracy of
the analytical test. To verify the lagoon
results in regard to Ascaris eggs, 20 L of
anaerobically digested municipal sludge
from the New Orleans and 20 L from the
Port Aransas area were each spiked with
Ascaris suum eggs and stored in large
plastic columns in the field in the New
Orleans area. Samples were taken
initially and every 3 mo thereafter.
Since there were no data concerning
the rate of settling for Ascaris suum
eggs, sludge settling tests were
conducted in a 1.8 m settling column with
sampling ports 0.3 apart. At 3, 6, 12, and
24 hr, 1 wk and 1, 2, and 3 mo, samples
for Ascaris suum eggs were taken and
suspended solids were analyzed .
All the sludge storage data were
analyzed for statistical significance of
pathogen die-off rate as a function of
such abiotic factors as temperature, VSS,
COD, pH, ORP, etc.
Results and Discussion
Bacterial Survival
The data pertaining to the inactivation
of indicator bacteria indicated that the
survival patterns for the coliforms and
fecal streptococci were of the order of 2
to 4 magnitudes of inactivation over 1 yr.
Although declines in indicator bacteria
(log MPN/gm suspended solids) were
preceded by a significant decrease in
Salmonella during the first 3 mo of
lagoon storage, the correlation of
decreases in indicator bacteria with
decreases in Salmonella appeared to be
nebulous. This becomes more obvious
when the increases in indicator organism
concentrations after the first year of
storage are examined - increases that
continued through the second year as a
possible result of outside contamination.
The importance of Salmonella being
reduced by more than 5 logs means that
lagoon storage under certain semitropical
conditions meets the criteria for PFRP in
relation to inactivation of pathogenic
bacteria (Figure 1).
Virus Survival
As can be seen in Figure 2, the
analyses of thermal control samples (1
mL vials of poliovirus in their original
medium) from both the New Orleans
the Port Aransas lagoons revealed ne
identical decreases in virus concen
tions after 180 days of storage in
lagoons' sediment fraction. Although
thermal control concentrations decrea;
by approximately 2 logs, concentrat
reductions approaching 6 logs wi
observed in the liquid and sedim
fraction samples collected over the sa
time period.
For both lagoons, poliovirus Type
survival was significantly greater in
sediment than the liquid fractions. Initi;
it was suspected that survival in the liq
phase was greater in the New Orlea
than in the Port Aransas lagoon, but <
weight conversions showed that the s
vival pattern differences could
attributed to differences in initial cona
trations following spiking. As can be se
in Figure 2, which compares dry weu
titration results for both the New Orlea
and Port Aransas lagoon virus samples
adjustments are made for the differenc
in initial spiking concentrations (apprc
imately 1 log), the inactivation curv
over storage time are nearly identic
Thus, the titration differences could
attributed to one of two facts: (1) t
initially lower virus concentrations in t
Port Aransas lagoon could have bei
from poor mixing and sampling, or (2) tl
petroleum constituents in the Port Ara
sas lagoon sludges reacted immediate
to inactivate the spiked virus durir
mixing, but after mixing and lago<
storage, the chemical constituents hi
little or no effect on virus survival.
Parasite Survival
Lagoon-Stored Sludge
The results of parasite analyses >
samples from the two lagoons are show
in Figures 3 and 4 (Sludge Lagooi
where the percent inactivation of Ascar,
eggs over time is shown. In the Ne
Orleans lagoon (Figure 3), a die-off of th
eggs began to occur after 3 mo and by
mo 33% of the viable eggs had bee
inactivated. By 9.3 mo, 74% of the egg
had been inactivated, and by 12.2 m<
98% of the eggs had been inactivatec
No viable eggs were recovered subse
quently.
In the case of the Port Aransas lagoor
the die-off of Ascaris eggs occurre
much more rapidly. After 3 mo, 89% c
the viable eggs in the original spike ha
been inactivated, and by 6.5 mo, nearl
total inactivation (99.9%) had occurred
The difference in the die-off of eggs i
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-2
New Orleans
10 15
Months
Port Aransas
Salmonella Detection Limit - None Detected
After Initial Sampling 24 Hours Post-spike.
None Detected
I I I I I I I I I I I
10
15
20
Months
O Aqueous Fraction
D Sediment Fraction
Figure 1. Graph of Salmonella organisms per gram dry weight versus time m months for
New Orleans and Port Aransas sludge lagoons.
the two lagoons was significant
(p = 0.004).
Small PVC tubes were placed in each
lagoon and filled with sludge spiked with
a higher concentration of Ascaris eggs so
that the die-off of eggs could be
monitored more accurately. In the sludge
in the tube placed in the New Orleans
lagoon, the eggs died off at about the
same rate as the eggs in the lagoon
sludge (Figure 3). The die-off in the tube
placed in the Port Aransas lagoon was
also similar to that in the sludge in the
same lagoon (Figure 4). The Ascaris
eggs in distilled water in vials placed in
each lagoon died off within about 6 mo
(Figure 5).
Sludge Stored in Large Columns
This part of the project was initiated to
verify certain results obtained in the
study of the survival of Ascaris eggs in
sludge stored in the lagoons- the more
rapid die-off of eggs in the Port Aransas
lagoon sludge as compared with that in
the New Orleans sludge, and the rapid
die-off of eggs in the distilled water
controls in both lagoons. In addition, the
Ascaris eggs used in spiking the sludge
in each lagoon had a relatively low level
of viability, 13.7%, and it was desirable to
determine if another batch of Ascaris
eggs with a higher initial rate of viability
could survive longer in stored sludge.
The mactivation of Ascaris eggs m
sludge stored in the two large plastic
columns is shown m Figures 3 and 4. The
viability of the Ascaris eggs in the
digested sludge used to spike these
columns was 90.6%. In the column with
New Orleans sludge, significant die-off
did not occur until after 9 mo of storage.
At 9 mo, only 11% of the viable eggs in
the initial samples had been inactivated,
but after that, the die-off was more rapid.
At 12 mo, 76% of the eggs had been
inactivated, and complete mactivation
was observed at 15 2 mo
In the case of the eggs in the sludge
of the Port Aransas column, i.e., sludge
from Corpus Christi, TX, the rate of
mactivation of Ascaris eggs was less than
that observed in the New Orleans
column. In the Port Aransas column, the
mactivation of the eggs occurred at a
fairly steady rate with 7% mactivation
observed at 1 mo, 23% at 3 3 mo, 54%
at 6.4 mo, 90% at 9 mo, and nearly
complete mactivation (99.9%) at 12 mo
Although the time it took for complete
mactivation of eggs to occur in the sludge
in the two columns was not statistically
different, the inactivation that had
occurred in the Port Aransas column at 6
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Thermal Control New Orleans
(PFU's MOO ML)
Thermal Control
Port Aransas
Sediment Fraction
New Orleans
v Liquid Fraction
\ New Orleans
Sediment Fraction
"* njPort Aransas
Liquid Fraction*^
60 90
Days After Spiking
Figure 2. Composite of Poliovirus survival New Orleans and Port Aransas lagoons.
The solids in the sludge settled at ab
the same rales as the Ascaris eggs.
When these results are compared v
those in previous studies, it would app
that Ascaris eggs settle more slowly in
anaerobic sludge than in raw sewat
This is to be expected since anaero
sludge is thicker.
For the New Orleans municipal sludi
changes in abiotic parameters tt
correlated with Ascaris egg inactivati
included only volatile solids. Fee
streptococcus mactivation correlated vv
the fluctuation of volatile solids a
temperature. The inactivation of otr
indicator organisms and Salmonella w
not observed to correlate with any abio
changes. For the Corpus Chnsti mui
cipal sludge, no abiotic parameters c<
related with Ascaris egg inactivatic
Poliovirus inactivation correlated wi
changes in pH, volatile solids, and to
solids. Fecal streptococcus mactivatit
correlated only with conductivity. Tl
abiotic variables could not be used
indicators of inactivation, but the;
abiotic parameters were related to tl
mechanism by which the die-o
occurred.
and 9 mo was significantly greater than
the inactivation that had occurred at
these times in the New Orleans column
(p < 0.05).
Eggs in the distilled water controls
placed in the two columns died at similar
rates, although there was considerable
fluctuation in the viability of the eggs
from individual vials examined between 3
and 12 mo of storage. In the vials
examined from each column at 12 mo,
nearly all of the eggs had died (96% and
98%), and at 15.2 mo no viable eggs
were observed in vials taken from the two
columns. The inactivation of eggs in
these distilled water controls was not
significantly different from the inactivation
of eggs in the column with the New
Orleans sludge.
The storage of Ascaris eggs in
sludges in lagoons and in large columns
produced several interesting results. First
of all, the eggs survived for a significantly
shorter time in the Port Aransas lagoon
than in the New Orleans lagoon. This
difference in the survival of eggs in the
two lagoons was probably due to the
presence of petroleum by-products in the
sludge in the Port Aransas lagoon, which
affected the viability of the eggs. Since
the temperatures observed in the two
lagoons varied little from each other,
temperature was unlikely to have caused
the observed difference.
While the Ascaris eggs stored in
Corpus Christi sludge in the column
survived longer than they did in the
sludge stored in the Port Aransas lagoon,
they were inactivated at a more rapid rate
than were the eggs in the New Orleans
sludge column. This again was attributed
to the presence of petroleum by-products
in the Texas sludge. The Ascaris eggs in
the New Orleans sludge column survived
longer and had a later onset of
inactivation than did the eggs in the New
Orleans sludge lagoon. These reactions
are partly attributable to the 90.6% rate of
egg viability in the column spike as
compared with the 13.7% for the lagoon
spike.
The results of the settling experiment
showed that when the anaerobic sludge
containing Ascaris eggs was allowed to
settle under quiescent conditions, some
eggs still remained in the upper 30 cm of
the sludge for at least 7 days. At 24 hr,
only approximately 20% of the original
number of eggs remained in the upper
one-half (1 meter) of the settled sludge
and, after 1 wk, less than 1% of the eggs
were in the upper one-half of the sludge.
After 1 mo, all of the eggs were found in
the bottom 1 m of the sludge column.
Recommendations
Study results indicate that lagoc
storage of municipal sludges undi
certain conditions in semitropical climate
can be used to inactivate pathogens.
was also observed that Ascaris egc
were far more resistant to inactivatic
than were bacteria and viruses. Based c
these findings the following i
recommended:
1) Initiate drying bed studies, with th
use of raw sludge and aerobically an
anaerobically treated sludges, t
determine the exact stabilizatio
conditions needed for producm
sludges that meet PFRP criteria.
2) Investigate the applicability of usm
combined treatment processe
(digestion followed by lagoon and/c
drying bed storage) to mactivat
enteropathogens in sludges beini
processed in rural and/or smai
Publicly Owned Work Treatment
(POTW).
3) Determine whether petroleum hydro
carbons would inactivate enteropatho
gens in non-hazardous petroleun
sludges, petroleum-contaminated mu
nicipal sludges, and pit muds beint
co-disposed with municipal sludges.
4) Determine the appropriate controls fo
studies of the survival of Ascaris egg!
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-25-
100
Sludge/Tube/Lagoon
Sludge Column
Sludge Lagoon
6 9 12
Months of Storage
15
18
Figure 3. Inactivation of Assarts eggs stored in New Orleans sludge in lagoon, tube in the
lagoon, and large column.
Sludge/Tube/Lagoon
Sludge Column
Sludge Lagoon
6 9 12
Months of Storage
15
18
Figure 4. Inactivation of Ascans eggs stored in Port Aransas sludge in lagoon, tube in the
lagoon, and large column.
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New Orleans Lagoon
Port Aransas Column
New Orleans Column
100
Months of Storage
Figure 5. Inactivation of Ascans eggs stored in distilled water in vials stored in New Orleans
and Port Aransas lagoons and large columns (controls).
in different sludges or sludi
products.
Conclusions
After 15 mo of storage in the N«
Orleans, Louisiana, and Port Aranse
Texas, lagoons, Ascaris suum eggs we
inactivated. Both localities are
semitropical zones. In the Texas lagoo
Ascaris inactivation was at a faster ral
perhaps because of petroleu
contaminants in the sludge. Salmonei
livingstone and poliovirus Type 1 we
inactivated within 6 mo of storage, ar
total and fecal coliforms dropped 2 to
logs. The fecal streptococci, howeve
decreased very little.
With the New Orleans municip
sludge, die-off of pathogens appeared
be a result of temperature, where 2
Ascans egg die-off in the Texa
petroleum-contaminated sludge wa
related more to petroleum residues thi
were estimated to be around 15% to 20C
by volume.
Finally, the die-off data for both lagoc
sites not only indicated pathoge
reductions within 15 to 18 mo but was i
accordance with published processes t
further reduce pathogens (PFRP) i
sludge treatment processes or proces
schemes.
The full report was submitted i
fulfillment of Cooperative Agreement N(
CR 810289 by Tulane University unde
the sponsorship of the U.S
Environmental Protection Agency.
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R.S. Reimers, M.D. Little, T.G. Akers, W.D. Henriques, R.C. Badeaux, D.B.
McDonnell, and K.K. Mbela are with Tulane University, New Orleans, LA
70112.
Albert D. Venosa is the EPA Project Officer (see below).
The complete report, entitled "Persistence of Pathogens in Lagoon-Stored
Sludge," (Order No. PB 89-190 359/AS; Cost: $28.95, 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:
Risk Reduction Engineering Laboratory
U.S. Environmental Protection Agency
Cincinnati, OH 45268
United States
Environmental Protection
Agency
Center for Environmental Research
Information
Cincinnati OH 45268
Official Business
Penalty for Private Use $300
EPA/600/S2-89/015
8°§°mi>
MUCI
CHICAGO
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