United States
Environmental Protection
Agency
Water Engineering
Research Laboratory
Cincinnati, OH 45268
Research and Development
EPA/600/S2-85/142 Mar. 1986
v>EFV\ Project Summary
Survival of Parasite Eggs in
Stored Sludge
Edna S. Ka
eshiro and Gerald Stern
The inact
resistant pa
stored sludg
mine their
lagoons. Eg
can's, Toxoc,
tapeworm (i
to domestic
ginning and
anaerobic d
samples see
were stored
25°C, and i
sorted in th
conditions o
Nonsludge si
seeded with
digested si
similar cond
The total
time and te
number of vi
infectivity o
Ascaris egg!
storage tem|
months of si
ation rates of digestion-
site eggs in laboratory-
were measured to deter-
otential fate in sludge
s from roundworms (As-
a. and Trichuris) and a rat
rmenolepis) were added
sludges either at the be-
uring, or after aerobic or
estion. Digested sludge
This Project Summary was devel-
oped by EPA's Water Engineering 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).
ed with the parasite eggs Introduction
the laboratory at 4°C, at
a container that was in-
ground to simulate the
a sludge storage lagoon.
I samples (controls) were
fie same parasites as the
Iges and stored under
ons.
umber of eggs recovered
from the samples decreased as storage
perature increased. The
ble eggs and the potential
recovered Toxocara and
were related primarily to
rature and time. After25
rage at 4° C, the Toxocara
eggs and some Ascaris eggs remained
both viableand infective, whereas most
of the eggs stored at 25° C were non-
viable after' 0 to 16 months of storage
in sludge. Though storage temperature
and time were the most important
factors in the inactivation of these
eggs, minor effects were also associ-
ated with other factors— type of sludge
digestion, timing of egg addition (dur-
ing or after sludge digestion), type of
storage (in sludges versus in soil), pH,
and species of egg. These controlled
laboratory studies suggest that sludge
lagooning can be an effective method
for inactivating parasite eggs, par-
ticularly in warm geographic locations.
Enteric parasite eggs become concen-
trated in domestic wastewater sludge
after treatment of the raw sewage.
Sludge treatment by mesophilic ana-
erobic or aerobic digestion inactivates
some but not all of the parasites. Diges-
tion-resistant eggs of the enteric para-
sites Ascaris, Toxocara, Toxascaris, and
Trichuris can remain viable and capable
of infection. They must therefore be con-
sidered when sludge is disposed of, par-
ticularly on land.
Digested sludge is commonly used as
a fertilizer to replenish agricultural soil
and as an alternative to dumping, bury-
ing, or incineration. For example, approx-
imately 60% of all municipal sludge in
Ohio is applied to land. Once sludge
material is placed on land, however,
viable parasite eggs could become in-
fectious agents and pose a potential
health hazard to humans and domestic
animals.
A promising procedure for minimizing
the hazard from these parasite eggs is
additional treatment of the sludge in
storage lagoons. Sludge lagoons are
commonly used to store digested sludge
before further treatment or (and applica-
tion, or to provide permanent disposal.
Typically, sludge is stored in lagoons for
periods ranging from several months to
years. During this time, the solids settle
-------
to the bottom. Anaerobic decomposition
continues at the bottom of lagoons, and
the supernatant is periodically drawn off
and recycled to the sewage treatment
plant for further processing. Lagooning
has the advantage of being a simple
method that is economical in areas
where land is available. Long periods of
storage are known to cause substantial
inactivation of digestion-resistant para-
site eggs, but quantitative data are
sparse.
The purpose of this study was to in-
vestigate the inactivation rates of para-
site eggs stored in sludge under con-
trolled laboratory conditions to determine
their potential fate in storage lagoons.
Eggs of roundworms (Ascaris, Toxocara,
and Trichuris) and those of a rat tape-
worm (Hymenolepis) were seeded into
the sludge before and during, or after
anaerobic or aerobic digestion. An earlier
study reported on the survival rates of
these eggs during the digestion pro-
cesses (M.I. Black et al., "Survival Rates
of Parasite Eggs in Sludge During
Aerobic and Anaerobic Digestion,"
Journal of Applied and Environmental
Microbiology, 1982, 44:5,1138-1143).
The present study examines the effects of
temperature, storage time, and type of
sludge digestion on the survival of eggs
under long-term storage conditions.
Procedures
Organisms and Digestion of
Sludge
Toxocara cam's, Trichuris vulpis,
Ascaris suum, Trichuris suis, and Hy-
menolepis diminuta eggs were collected
over a 5-month period beginning in
October 1979. Parasite eggs were added
to sludge that was processed by methods
simulating authentic high-rate municipal
digesters with constant removals and
additions (not digestion of batch sam-
ples).
Soil Controls
Topsoil was also spiked with eggs and
used as a nonsludge control. To duplicate
naturally occurring topsoil, the control
was made up of one part each of loam,
manure, and clay. The aerobically di-
gested sludge contained 2.0% to 2.9%
solids, and the anaerobically digested
sludge contained 2.9% to 3.4% solids.
Thus the topsoil control was made 3%
solids in water.
Storage Conditions
Each batch of digested sludge or soil
controls was divided into 100-g (wet
weight) aliquots and placed into presteril-
ized, 250-mL polypropylene bottles
sealed with screw caps (Nalge Co.,
Rochester, New York). Samples were
stored at 4°C, at 25°C, and in the ground.
The 4°C and 25°C samples were stored
in constant-temperature rooms. The pur-
pose of the sample placed in the ground
was to simulate conditions in a sludge
storage lagoon. In-ground samples were
placed in covered, 55-gal. galvanized
drums. These were buried with their rims
flush with the ground's surface in an area
where they were subjected to the normal
temperature fluctuations of the adjoining
earth. Digested sludge samples spiked
with all of the test species of helminth
eggs were prepared for storage in March
1980. Eight aliquots of each sample for
each of 15 different experimental condi-
tions were analyzed at approximately 3-
month intervals (a total of 120 samples
for each sampling period). The study was
terminated in 1983 after 33 months of
storage.
Temperatures inside the drums were
constantly monitored (Weathertronics
4120 thermograph*, Weathertronics,
Sacramento, California). Air tempera-
tures were obtained from the National
Weather Service of the National Oceanic
and Atmospheric Administration (Greater
Cincinnati Airport, Boone County, Ken-
tucky). As the seasons progressed, the
average monthly temperatures inside the
drums reflected those of the air with a
slight lag time, but they never got as cold
as the winter air. These records indicated
that the in-ground condition was a valid
simulation of a sludge lagoon, since
lakes, ponds, and other buffered environ-
ments typically exhibit this lag. Further-
more, the in-ground high and low tem-
peratures indicated that the samples
were insulated from the broad range of
air temperature fluctuations, again
properly simulating sludge lagoons.
Sampling of Spiked Sludges and
Spiked Soil Controls
The experimental setup for this study
consisted of four spiked, digested test
sludges plus one spiked soil control
sample for each of three different test
storage conditions (or a total of 15 dif-
ferent experimental conditions). To
determine the rate of inactivation of the
parasite eggs, eight samples for each of
the 15 experimental conditions (or a total
of 120 samples for each sampling period)
"Mention of trade names or commercial products
does not constitute endorsement or recommendation
for use.
were taken for examination approxi-
mately every 3 months over a total period
of 25 months. A modified sampling
schedule was maintained after 25
months of storage.
Recovery of Eggs from Sludge
and Soil
The pH of each sample was deter-
mined before eggs were isolated. The
entire content (100 g) of the sample in the
plastic bottles was filtered through a
U.S.A. standard testing sieve (No. 35
mesh) and washed with distilled water.
The filtrate was returned to the same
bottle and centrifuged at 400 X g for 5
min at 4°C. The supernatant was
decanted, and 100 mL of 3° (w/v) lactal-
bumin hydrolysate (Sigma, St. Louis,
Missouri) was added to decrease ad-
herence of the eggs to the sludge. The
mixture was centrifuged as above, the
supernatant was discarded, and the
pellet was washed free of lactalbumin
hydrolysate with distilled water. After
centrifugation, the pellet was resus-
pended in 50 mL (final volume) distilled
water and layered onto a 150-mL con-
tinuous sucrose density gradient (spe-
cific gravity 1.26 to 1.00); then it was
centrifuged at 800 X g for 15 min. The
pellet formed at the bottom of the
gradient was discarded, and the material
within the gradient was collected, diluted
with an equal volume of distilled water,
and centrifuged at 800 X g for 5 min. The
pellet material was washed three times
with distilled water.
At the onset of storage, recoveries of
eggs from sludge were determined for
tests involving eggs added at the be-
ginning and during aerobic or anaerobic
digestion. The recoveries are listed in
Table 1.
Similar recoveries were not deter-
mined for eggs added to soil controls
because of lack of manpower at this
labor-intensive time when hundreds of
samples were being prepared for this
study. Estimates of the average number
of eggs in each 100-g, wet-weight soil
sample were based on the number initi-
ally added. These averages were as
follow:
Ascaris 10,400
Hymenolepis 12,500
Toxocara 2,100
Trichuris 900
Eggs recovered from all samples were
incubated at 25°C in the dark for 30 days
to cause embryonation.
-------
Data Analysis
The data were analyzed to determine
whether each of the various storage
conditions had an influence on the
'• recoverability and viability of the test
parasite eggs. Two-factorial analysis of
variance (ANOVA) with repeated mea-
sures was used (BMDP Statistical Soft-
ware, Inc., University of California). The
first ANOVA used an abridged set of data.
The data eliminated from these analyses
were those of months 0, 19, 30, and 33
because complete data for the parasite-
spiked sludge samples were not avail-
able. Also, data for the parasite-spiked
soil samples were not included in this
ANOVA because they might not be com-
parable with those of sludge samples.
The soil samples were not treated
aerobically or anaerobically, and eggs
were not added to the soils over a period
of 15 consecutive days (as would be re-
quired for a strict statistical analysis of
variance evaluation).
Nevertheless, in the real world, soils
are not normally digested aerobically or
anaerobically to stabilize against putre-
faction or to reduce vector attraction.
Also, the test parasites were added di-
rectly to the soil samples in a manner
similar to that used with the test para-
sites added to the sludges after digestion,
and they were exposed to the same
temperatures and storage time condi-
tions. We therefore decided that useful
information would be provided (1) by
performing another ANOVA that in-
cluded the test parasite soil samples and
(2) thereby enabling comparisons with
the ANOVA that excluded the soil sam-
ples. Again, data for months 0, 19, 30,
and 33 were not included because com-
plete data sets were not available.
Infectivity of Recovered Eggs
The infective potential of Ascaris and
Toxocara eggs was analyzed. Samples
containing viable and nonviable eggs
were concentrated to 2 mL and intubated
into male Holtzman albino rats (Charles
River Breeding Lab., Inc., Wilmington,
Massachusetts). After 8 days, the in-
fected rats were sacrified by anaesthesia
with diethyl ether, and their lungs, livers,
kidneys, and brains were removed. Each
organ was placed into 50 to 100 mL of
physiological saline solution and cut into
small pieces. One Baermann apparatus
was set up for each organ. The funnel
was filled with saline at 37°C, and two
layers of cheesecloth were used to sus-
pend the minced organ within the saline
in each funnel. Lamps with 60-W
tungsten bulbs were placed approxi-
mately 2 in. from the junction of the head
and stem of each funnal to create a
thermal gradient. After 50 to 60 min, the
suspended organ was removed and dis-
carded. The contents of each funnel were
placed into 50-mL, glass, conical tubes,
and 10 mL of 0.2% (w/v) saponin was
added to each tube to lyse red blood cells.
The tubes were centrifuged for 2 min at
500 X g, and the supernatant was dis-
carded. The volume of the pellet remain-
ing in each tube was examined. A small
pellet (0.5 mL) required no further treat-
ment and was resuspended in 20 mL of a
solution containing 0.1% Tween-80 and
10% formalin-saline (5.4 mL of 37%
formalin in 14.6 mLof0.1%(v/v)Tween-
80 physiological saline solution) to fix
and preserve the larvae. Fixed samples
were stored at 4°C until examined for
larvae.
Tubes containing 0.5 mL or more of
tissue pellet required further reduction
before they could be analyzed. These
pellets were suspended in 30 mL of a
pepsin digestion solution (2.5 g of pepsin
in 500 mL of 0.85% (w/v) sodium
chloride plus 3.5 mL cone. HCI) and in-
cubated at 30°C for 24 hr on a rotating
platform at 200 rpm. The digested tissue
was rinsed free of the pepsin solution
with distilled water and fixed and storage
as above. The number of Ascaris and
Toxocara larvae in each sample were
counted under 125X magnification using
a phase-contrast microscope.
Table 1. Egg Recoveries from Sludge
Eggs/100 g of Sludge (wet weight)
± Standard Error of Measurement (n=3)
E99 Type
Aerobic Digestion
Anaerobic Digestion
Ascaris
Hymenolepis
Toxocara
Trichuris
4,800± 176
4.8OO + 199
1.700± 127
400 ± 40
5,300 ±475
7.500 ± 535
1 .500+ 168
900 ± 63
Results and Discussion
Effects of Sludge Digestion
During the 15-day period of meso-
philic anaerobic digestion approximately
23% of the A, suum eggs were inacti-
vated. T. cam's and Trichuris spp. eggs
were not affected. Aerobic digestion
inactivated 38% of the Ascaris eggs, 11%
of the Trichuris eggs, but none of the
Toxocara eggs. Therefore, it appears that
laboratory aerobic digestion inactivated
the parasite eggs more effectively than
laboratory anaerobic digestion.
pH
The pH of soil and sludge samples
changed with storage time. Large initial
increases occurred in the pH values of all
soil samples (from pH 6 to pH 7 to 8) and
anaerobically digested sludge samples
(from pH 7.2 to pH 8.5 to 9), whereas
aerobically digested sludge samples
showed an initial decrease in pH (from pH
7.2 to pH 6 to 7). After long-term storage,
the largest changes in pH were seen in
soil stored at 25°C, aerobically digested
sludge stored at 4°C, and anaerobically
digested sludge stored at 25°C, and in the
ground. No obvious correlations were
noted between the pH of samples and the
recovery, viability, or infectivity of para-
site eggs.
Ascaris
A substantial reduction occurred in
the recovery of Ascaris eggs, especially
within the first 3 months of storage; but
more eggs were recovered from sludges
than from the soil samples. The tempera-
ture at which Ascaris eggs were stored
had little effect on the fraction recovered.
Viability, however, was dramatically
affected by the storage temperature and
time. Viability decreased very slowly with
storage at 4°C. After 25 months, more
than 50% of the Ascaris eggs recovered
from samples stored at 4°C were still
viable; but 10 to 16 months of storage at
25°C rendered most of the recovered
eggs nonviable. Ascaris eggs stored in
the ground in anaerobically digested
sludge lost their viability much earlier
than those in aerobically digested sludge.
However, after 30 months of storage, low
levels of viable eggs were still recovered
from the digested sludge stored in the
ground. Viable Ascaris eggs were re-
covered from the soil controls from each
storage condition over the 33-month test
period. Overall, anaerobically digested
sludge appeared to have a greater effect
on reducing Ascaris egg viability than did
-------
aerobically digested sludge or the soil
control.
The infectivity of recovered Ascaris
eggs incubated under conditions sup-
porting embryonation reflected their
viability. Eggs stored at higher tempera-
tures decreased in infectivity more
rapidly than those stored at lower tem-
peratures. Ascaris eggs aerobically
digested along with the sludge (Phase II)
were noninfective (did not cause infec-
tions in test rats) after 25 months when
stored at 25°C, in the ground, and at4°C.
In the case of Ascaris eggs added after
aerobic digestion (Phase I), a small
number of eggs from samples stored in
the ground for 33 months still appeared
capable of causing low levels of in-
fectivity, even though there was no visual
evidence of viability. Thus even when the
egg population appeared to be nonviable,
a few were occasionally capable of
causing infections in test animals. Also,
some eggs that appeared to be viable did
not produce infection in the test animals.
Thus tests for egg viability alone may not
be sufficient to ensure noninfectivity.
Joxocara
The type of sludge (aerobic or ana-
erobic) had no affect on the recovery of
Toxocara eggs; however, fewer eggs
were recovered from the soil samples
stored at 25°C and in the ground. The
data .on Toxocara were more scattered
than those obtained lor Ascaris, probably
because of the smaller numbers of
Toxocara eggs initially seeded in the
samples. Storage temperature and time
affected the viability of Toxocara eggs in a
manner similar to Ascaris eggs. The
viability of eggs decreased as the average
storage temperature increased.
The viability of eggs stored in aerobi-
cally digested sludges decreased more
slowly than in eggs stored in anaerobi-
cally digested sludges. The eggs re-
covered from samples stored at 25°Cand
in the ground were essentially nonviable
after 13 months of storage in sludge.
After 2 years in storage at 4°C, a signifi-
cant number of the recovered Toxocara
eggs were still viable. The viability of
Toxocara eggs in the soil controls was not
affected by storage at any temperature.
The difference in the viability of Toxocara
eggs stored in sludge and in soil was the
most dramatic among all the parasite
species tested. Thus it appears that the
sludges also had some detrimental effect
on Toxocara egg viability.
Though fewer Toxocara eggs were
recovered than Ascaris (and therefore
fewer total eggs were intubated into
rats), these eggs were just as capable of
causing infections. Higher storage
temperatures decreased the infectivity of
the eggs more effectively than did lower
temperatures. Also, sludge storage of
eggs decreased their infectivity more
effectively than did soil storage.
Tr/churis
Recoveries of Trichuris eggs from all
samples were similar: all were lowafter 3
months of storage. The total number of
Trichuris eggs seeded into each sample
was smaller than for any other species.
Because a low number of Trichuris eggs
were recovered, the viability data on this
species were very scattered. But eggs
stored at lower temperatures generally
had greater viabilities, and eggs stored in
soil maintained their viabilities longer
than the eggs stored in the sludges.
Hymenolepis
Though these eggs were not viable at
the onset of sludge digestion and sample
storage, they could still be recovered
from the samples, especially those stored
at 4°C. Temperature also affected the
recovery of Hymenolepis eggs, which
decreased more rapidly in the samples
stored at higher temperatures. As with
Toxocara, fewer Hymenolepis eggs were
recovered from soil stored at 25°C and in
the ground than were recovered from
sludges.
Conclusions and
Recommendations
The destruction of roundworm eggs
was minimal during the 15-day treat-
ment period of mesophilic anaerobic or
aerobic digestion. After 3 months of
storage at 25°C, at 4°C, and in the
ground, the roundworm eggs recovered
from both types of sludges were viable.
Ascaris and Toxocara eggs were infective
in rats.
After 16 months in storage at 25°C,
very few of the recovered A scan's eggs in
the digested sludges appeared to be
viable; nevertheless, a low level of in-
fection in rats was observed after 22
months of storage at 25°C. At least 2
years of storage in sludge at 25°C was
needed to inactivate most of the Ascaris
eggs. When Ascaris eggs added after
aerobic digestion (Phase I) were stored in
the ground and exposed to normal tem-
perature fluctuations, inactivation (both
nonviable and noninfective) took at least
33 months. Storage at 4°C had little
effect on decreasing Ascaris viability and
infectivity. Thus the temperature and
length of time at which digested sludge
was stored appeared to be the most im-
portant factors for inactivating Ascaris.
The higher the storage temperature and
the longer the storage time at this high A
temperature, the more rapidly the round- •"
worm eggs were rendered harmless.
Although the eggs of Toxocara were
more resistant to digestion than those of
Ascaris, they were more quickly de-
stroyed during storage. After only 10
months in storage at 25°C, none of the
Toxocara eggs recovered from the sludge
were capable of producing an infection in
rats. Toxocara eggs stored in the ground
for 25 months could not produce an in-
fection in rats even though they appeared
to be viable. Storage at 4°C for more than
2 years did not effect their infectivity. As
with Ascaris, Toxocara eggs were more
quickly inactivated in sludges at higher
storage temperatures with longer storage
time.
Comments are difficult to make on the
viability of Trichuris eggs recovered. As
mentioned earlier, the recovery rates for
these eggs were low and erratic, and
large standard deviations were observed.
Therefore only a most general statement
can be made about the viability of these
eggs. After 13 months of storage in
sludges at 4°C, at 25°C, or in the ground,
most of the Trichuris eggs were no longer
viable. Eggs stored in soil, however, ap-
peared to retain their viabilities for at
least 25 months.
A summary of specific conclusions
follows:
1) During the 15-day period of meso-
philic anaerobic digestion, approxi-
mately 23% of the A. suum eggs
were inactivated; T. cam's and
Trichuris spp. eggs were not af-
fected.
2) Aerobic digestion inactivated 38% of
the Ascaris eggs, 11 % of the Tri-
churis eggs, but none of the Toxo-
cara eggs.
3) The total number of eggs recovered
from the stored samples decreased
with time.
4) Eggs of all species studied were
mostly inactivated faster (16 months
in anaerobic digested sludges and
about 25 months in aerobic digested
sludges) when stored at higher
(25°C) rather than at lower tempera-
tures (4°C or in the ground).
5) Ascaris eggs aerobically digested
along with sludge (Phase II) were
noninfective after 25 months when
stored at 25°C, in the ground, and at
4°C.
6) Ascaris eggs anaerobically digested
with sludge (Phase II) were non-
-------
infective by 16 months when stored
at 25°C, but were still infective after
22 months when stored at 4°C or in
the ground.
7) Either aerobic or anaerobic digestion
makes T. can/s eggs more suscepti-
ble to inactivation when compared
with eggs stored in soil controls.
8) Trichuris spp. eggs in the digested
sludges (Phase II) appeared to be in-
activated by storage at 25°C and in
the ground after 16 months, but
could be viable even after 25 months
when stored at4°C. However, a con-
clusive statement on Trichuris vi-
ability is difficult to make because of
the low initial innoculum levels and
subsequent low recovery of these
eggs.
9) H. diminuata proved very sensitive,
since storage of the eggs at 4°C
alone before seeding into the sludges
rendered them nonviable and non-
infective.
10) Viability of the eggs did not always
predict infectivity; that is, eggs that
did not look viable were sometimes
infective and vice versa.
This 3-year investigation into the
persistence of certain roundworm eggs
during storage after anaerobic or aerobic
digestion indicates the need for addi-
tional research. The following studies
should be done on the survival of para-
sitic eggs in sludges.
1) An investigation of the types and
numbers of microbial agents present
in stored sludges and their contribu-
tions to the destruction of parasite
eggs.
2) An investigation of the factors in the
digestion processes that apparently
increase the resistance of A. suum
eggs to inactivation when they are
subsequently placed in storage.
3) A thorough measurement of the
redox potential of sludges during
storage and the relation of the
electrochemical potentials to the
organic and inorganic chemical
reactions that may affect parasite
destruction.
4) A larger-scale investigation into the
effects of long-term storage on the
destruction of eggs from certain
parasites (it should more closely
duplicate existing lagoon condi-
tions).
5) A study of the inactivation of parasite
eggs within 3 months of storage in
the absence of retrieving them from
either sludge or soil (the object here
is to conclusively establish whether
they were inactivated or whether
they became more difficult to iso-
late).
6) Studies of die-off rates in sludge-
amended soils.
7) Biochemical and physiological anal-
yses of parasite eggs to determine
what is altered in eggs exposed to
sludge as compared with eggs stored
in controls (soil or water).
8) Research to improve the analytical
methods for measuring the recovery
and viability of parasite eggs in
sludge (e.g., improve the standard
deviation of the method).
The full report was submitted in
fulfillment of Cooperative Agreement
CR806954-02 by the University of
Cincinnati under the sponsorship of the
U.S. Environmental Protection Agency.
Edna S. Kaneshiro is with the University of Cincinnati, Cincinnati, OH 45221; and
the EPA author Gerald Stern (also the EPA Project Officer, see below) is with
the Water Engineering Research Laboratory, Cincinnati, OH 45268.
The complete report, entitled "Survival of Parasite Eggs in Stored Sludge," (Order
No. PB86-137 148/AS; Cost: $16.95, 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:
Water Engineering Research Laboratory
U.S. Environmental Protection Agency
Cincinnati, OH 45268
U. S. GOVERNMENT PRINTING OFFICE:1986/646-l 16/50787
-------
United States
Environmental Protection
Agency
Center for Environmental Research
Information
Cincinnati OH 45268
Official Business
Penalty for Private Use $300
EPA/600/S2-85/142
0000329 PS
------- |