oEPA
United States
Environmental Protection
Agency
Municipal Environmental Research
Laboratory
Cincinnati OH 45268
Research and Development
EPA-600/S2-81-166 Oct. 1981
Project Summary
Parasites in Southern
Sludges and Disinfection by
Standard Sludge Treatment
R. S. Reimers, M. D. Little, A. J. Englande, D. B. Leftwich, D. D. Bowman, and
R. F. Wilkinson
The objectives of this study were to
(1) assess the presence and densities
of resistant stages of parasites in
municipal wastewater sludges (sew-
age) in the southern United States, (2)
investigate parasite inactivation by
lime treatment of sludges seeded with
intestinal parasites, (3) measure the
mass balance of helminth eggs through
various processes in a municipal
wastewater treatment plant, and (4)
assess, on the basis of laboratory and
field data, standard sewage sludge
treatment processes for their effective-
ness in inactivating parasites.
Sludge samples collected during
each of the four seasons from 27
municipal wastewater plants located
in Alabama, Florida, Mississippi,
Louisiana, and Texas were examined
for the presence and densities of
resistant stages of human and animal
parasites using parasitologic tech-
niques developed for this study. Viable
eggs of Ascaris and Toxocara were
recovered at least once from every
plant and viable eggs of Trichuris
vulpis and Trichuris trichiura were
recovered at least once from 26 and
15 plants, respectively. Viable eggs of
at least 10 other helminths and cysts
of a few protozoa were also found in
fewer numbers and less frequently.
Depending upon the parasite, the
inactivation of parasites during sewage
treatment fluctuated from season to
season, but, in general, most were
inactivated in the summer. Laboratory
studies verified the results of previous
investigations indicating that destruc-
tion of resistant parasite eggs is
primarily due to temperature (heat)
and not to a specific digestion process.
Very large lime doses were required
for the inactivation of viable Ascaris in
sludges and results were not always
consistent. Lime treatment thus ap-
pears to be an expensive and unreliable
treatment for Ascaris inactivation.
Laboratory experiments also showed
that at certain combinations of ultra-
sonic frequency intensity and exposure
time, Toxocara eggs could be destroyed,
but that the same ultrasonic conditions
did not affect Ascaris eggs.
An important finding of this study is
the poor suitability of Ascaris eggs
taken from the uteri of gravid female
worms as indicators of the character-
istics of Ascaris eggs discharged in the
feces of the host. The eggs removed
from the gravid females are not as
resistant to adverse factors as eggs
that have undergone a hardening
process in the intestines of the host
and been recovered from the feces.
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).
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Introduction
In the United States, land disposal of
sewage sludge has been practiced with
care because of controversies over
possible health and nuisance problems.
Pathogens are known to survive con-
ventional sludge stabilization and de-
watering processes including the use of
chemicals in these conventional
processes. Of the three general types of
pathogenic organisms—bacteria,
viruses, and parasites—found in sew-
age sludges, certain parasites are
known to be the most resistant to
conventional sludge treatment processes.
Also, parasites are the least studied of
the pathogenic organisms found in
sewage sludges. It was the general
purpose of this study to investigate the
types and densities of parasites in
sewage sludges in the southern United
States where both humidity and tem-
perature favor parasite survival. It was
also the purpose of this study to investi-
gate, through field data and laboratory
studies, the effectiveness of conven-
tional sludge stabilization and select
new processes for inactivating parasites
in sewage sludges.
Research Program
This research included both field and
laboratory studies. The field studies
consisted of a year-long investigation of
parasites in domestic waste sludges in
the southern United States. This in-
vestigation has resulted in new in-
formation concerning: 1) the types and
concentrations of resistant stages of
parasites in southern domestic sludge;
2) the seasonal fluctuation of these
parasites in sludge; 3) the effect of
abattoir wastes on the density of
parasites in sludge; and 4) other factors
affecting the prevalence and persistence
of parasites in sludges. Laboratory
studies investigated the effect of
selected sludge treatment processes on
parasite eggs and cysts found in sewage
sludge. The treatment processes in-
vestigated were aerobic and anaerobic
digestion, lime stabilization, ammonifi-
cation, sonication, and various combi-
nations of these processes.
Results
Field Studies
Parasitological Findings
Sludge samples collected during each
of the four seasons from 27 municipal
wastewater treatment plants located in
Alabama, Florida, Mississippi, Louisiana,
and Texas were examined for the
presence and density of the resistant
stages of human and animal parasites
using parasitologic techniques devel-
oped for this study. The selection of the
wastewater treatment plants for sludges
was made on the basis of the method of
wastewater treatment used, size of the
treatment plant, type of population
served, and geographic and climatologic
features of the region.
The results of the parasitologic
examination are shown in Tables 1
through 3. Many of the eggs or cysts of
parasites found in sludges were identi-
fied only as to genus or type because the
resistant stages of closely related
parasites are often so similar that it is
not possible to tell them apart. For
example, the eggs of Ascaris lumbricoides
(human roundworm)andA lumbricoides
var suum (pig roundworm) are virtually
indistinguishable and, consequently,
when Ascaris eggs are found, it could
require examination with an electron
microscope to distinguish between the
two species. The probable identity of
each type of helminth egg and protozoan
cyst found in the sludges is shown in
Table 1.
Ascaris, Toxocara, Trichuristrichiura,
and Trichuris vulpis were the parasites
most commonly found in the sludges
(see Table 2). The eggs of Ascaris,
Toxocara, and Trichuris vulpis, either
viable or non-viable, were recovered
one or more times from each plant
studied. Eggs of T. trichiura were found
in all but one of the plants. Viable eggs
of Ascaris and Toxocara were recovered
at least once from every plant, and
viable eggs of T. vulpis and T. trichiura
were recovered at least once from 26
and 15 plants, respectively.
In Table 3 are listed other parasites,
either viable or non-viable, found in the
sludges. Of these parasites, Hymenolepis
diminuta was most frequently found; its
eggs were observed in 23 of the 27
plants studied. Viable eggs of H.
diminuta were found in primary sludges
in 15 plants and in treated sludges in 4
plants. H. diminuta is a tapeworm of
rats, and its presence in sludge in 23
plants is an indication of the frequent
occurrence of rats in or near sewage
systems and treatment plants. Other
parasite eggs that are more likely to be
from a rodent source include the
Trichosomoides-\\V.e eggs and some of
the Capillaria eggs. Hymenolepis nana
eggs could have been from either
humans or rodents.
Densities of Parasites in Sludges
The total number of parasite eggs
recovered from the digested sludge
samples ranged from 0 to more than
230,000 eggs/kg dry weight of sludge,
depending on the source of sludge and
season of the year. The average number
of total parasite eggs was approximately
14,000/kg dry weight of sludge. The
percentage of the total parasite eggs in
the sludge samples that were viable
ranged from 0% to 100%, but was
generally greater than 45% for primary
sludge and 69% for treated sludge (see
Table 4). Primary and secondary un-
digested sludge samples were found to
contain in order of decreasing average
densities: 9,700 Ascaris spp. eggs,
1,200 Toxocara spp. eggs, 800 7".
trichiura eggs, and 600 T. vulpis eggs/
kg dry weight of sample. The average
numbers of these parasites in stabilized
sludge samples were: 9,600 Ascaris
spp. eggs, 2,600 T. trichiura eggs, 700
Toxocara spp. eggs, and 700 T. vulpis
eggs/kg dry weight of sludge sample.
Densities of these four most prevalent
parasites fluctuated greatly. The stan-
dard deviation of their densities is
greater than the observed averages and|
the ranges are from zero to 10 times the!
average. Other parasite eggs and cysts
were observed in the sludge samples,
but in low concentration.
The Influence of Abattoirs on
Parasite Concentration
Unusually high levels of Ascaris eggs
were found in the sludges of one
wastewater treatment plant. A detailed
study revealed that an abattoir in the
community processed large numbers of
swine and that the wastes from this
abattoir entered the municipal sewer
system. In Table 5, the levels of parasite
eggs in the wastewater treatment
sludges receiving the abattoir wastes
are compared with 6 other wastewater
treatment plants of similar size in the
same geographic area. In the plant
receiving abattoir wastes, an average of
81,800 Ascaris eggs/kg dry weight of
sludge was recovered, while in the 6
plants receiving little or no abattoir
wastes an average of 7,900 Ascaris
eggs/kg was recovered. The Ascaris
eggs in the treatment plant receiving
the abattoir wastes were undoubtedly
mostly A. suum eggs that came from
infected swine. It is interesting that the
level of the T. tr/chiura-ltke eggs was
nearly the same in each plant. This|
would indicate that very few, if any, of
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Table 1. Parasites Found in Sludge Samples from 27 Municipal Plants in Southern United States
Parasite Found
Probable Identity
Definitive Host
Ascaris eggs
Toxocara eggs
Trichuris trichiura
Trichuris vulpis eggs
Toxascaris-//*e eggs
Ascaridia-//'*e eggs
Trichosomoides-///te eggs
Cruzia-//*e eggs
Capillaria spp. eggs
(3 or more types)
Hymenolepis diminuta eggs
Hymenolepis nana eggs
Hymenolepis sp. eggs
Taenia sp. eggs
Acanthocephalan eggs
Entamoeba coli-//Are eggs
Giardia cysts
Coccidia oocysts
Ascaris lumbricoides'1
Ascaris suum1
Toxocara canis2
Toxocara cati2
Trichuris trichiura
Trichuris suis3
Trichuris vulpis
Toxascaris leonina
Ascandia galli
Heterakis gallinae
Trichosomoides crassicauda
Anatrichosoma buccalis
Cruzia americana
Capillaria hepatica
Capillaria gastrica
Capillaria spp.
Capillaria spp.
Capillaria spp.
Hymenolepis diminuta
Hymenolepis nana
Hymenolepis spp.
(poss. more than one species)
Taenia saginata4
Taenia pisiformis4
Hydratigera taeniaeformis"
Macracanthorhynchus hirudmaceus
Entamoeba coli5
Entamoeba spp.
Giardia lamblia
Giardia spp.
Isospora spp.
Eimeria spp.
Humans
Pigs
Dogs
Cats
Humans
Pigs
Dogs
Dogs and Cats
Domestic poultry
Domestic poultry
Rats
Opossums
Opossums
Rats
Rats
Domestic poultry
Wild birds
Wild mammals
(opossums, racoons, etc.)
Rats
Humans and rodents
Domestic and/or wild
birds
Humans
Cats
Dogs
Pigs
Humans
Rodents, etc.
Humans
Dogs, cats, mammals
Dogs, cats
Domestic and wild birds, mammals
'Eggs of A. lumbricoides and A. suum are indistinguishable.
2Toxocara eggs were probably mostly T. canis.
3~f. suis eggs were probably only rarely seen.
'Eggs of these worms are indistinguishable.
sAn intestinal amoeba that is a commensal, not a parasite.
these eggs were those of T. suis, the
swine whipworm.
destroying parasite eggs (see Figure 1 ).
The number of viable Ascaris and
be confirmed. The use of drying beds,
however, is consistently very effective
Effects of Sludge Treatment
Processes on Parasites
The results of this investigation on
parasites in southern domestic sludges
indicate that, in general, conventional
sludge stabilization treatment processes
(e.g., mesophilic anaerobic or aerobic
digestion) were not very effective in
Toxocara per unit dry weight of sludge
actually increased during these
processes due to the loss of dry mass of
sludge solids that occurs in the diges-
tion processes. The concentration
processes of vacuum filtration and
centrifugation appear to have removed
or destroyed eggs, but due to the low
number of samples analyzed and
variable nature of the effect, this cannot
for destroying parasites in sludges.
In the field investigation, data were
collected on both raw (undigested)
sludges and on sludges stabilized by
either aerobic digestion or anaerobic
digestion under ambient or mesophilic
temperatures followed by dewatering
on drying beds. These data are shown in
Table 6. During the winter and fall,
parasite inactivation tended to be most
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Table 2.
Parasite
Number of Municipal Plants in Which Eggs of Ascaris, Toxocara,
Trichuris trichiura ano'Trichuris vulpis were Found (27 Plants Studied)
fa//1
Winter Spring Summer Entire Year
Ascaris
Toxocara
Trichuris trichiura
Trichuris vulpis
/72/253/26" 22/25/2S 14/26/27 14/25/25 26/27/27
11/22/24 17/27/279/24/24 9/23/25 23/27/27
6/10/16 8/12/18 7/10/19 6/10/16 12/15/26
19/21/22 19/23/24 19/23/26 12/24/25 25/26/27
'Samples from only 26 plants examined in fall.
*Number of plants in which viable eggs were found in treated sludges.
3Number of plants in which viable eggs were found in any sludge sample.
^Number of plants in which viable or non-viable eggs were found in any sludge
sample.
Table 3.
Miscellaneous Parasites Found in Sludges from 27 Municipal
Treatment Plants Sampled
Parasite
No. of plants in which found
Toxascaris leonina eggs
Ascaridia-//'/ce eggs
Cruz\a-like eggs
Trichosomoides-//'/re eggs
CapiMaria eggs (shells with pits)
Capillaria eggs (shell with striations)
Hymenolepis diminuta eggs
Hymenolepis nana eggs
Taenia sp. eggs
Acanthocephalan eggs
Entamoeba co\\-like cysts
Giardia cysts
Coccidia oocysts
2
7
1
7
7
11
23
6
1
1
23
9
6
variant. Except for Toxocara, the densi-
ties of all viable parasite eggs were
reduced more in the summer and spring
than in the fall and winter. Table 6
indicates the influence of anaerobic or
aerobic digestion on drying bed treat-
ment for parasite eggs. The percent
reduction of viable eggs of four pre-
dominant parasites with respect to the
drying bed process was generally not
influenced by either aerobic or anaerobic
stabilization. However, some reduction
in effectiveness during the fall and
winter was noted with anaerobically
digested sludges, yet with aerobically
digested sludges, a seasonal fluctuation
was noted only with T. trichiura.
A correlation was found between the
density of inactivated parasites and
moisture contents in drying bed sludges.
Figure 2 shows the correlation of the
logarithm of the numbers of viable
Ascaris eggs in raw versus drying bed
sludge in the same plants (grouped by
the moisture content of the drying bed
sludges). The inactivation of viable
parasite eggs in the raw sludges
increases with decreasing moisture
content of the drying bed sludges. The
densities of viable Ascaris and Toxocara.
eggs in the drying bed sludges as related^
to the sludge moisture content was
analyzed for each season. Table 7
shows that lowest moisture levels at
which all Ascaris or Toxocara eggs were
inactivated was 5% in the fall, 7% in the
Table 4. Parasite Concentrations in Primary and Secondary Sludge as Compared to Treated Sludge
Number of Viable and Non-Viable
Eggs/kg Dry Weight of Sample
Parasite
Ascaris spp.
(human and pig
roundworm)
Trichuris
trichiura (human
whipworm)
Trichuris vulpis
(dog whipworm)
Toxocara spp.
(dog and cat
roundworm)
Nature of Sludge*
Primary and Secondary
Treated
Primary and Secondary
Treated
Primary and Secondary
Treated
Primary and Secondary
Treated
A verage2
9,700
9,600
800
2,600
600
700
1,200
700
Standard
Deviation
26,300
27.400
2,900
9,800
1,000
1,300
2,300
1.500
Range
200,000 -
230,000 -
26,000 -
84,000 -
5.7OO -
10,500 -
5,400 -
8,500 -
0
0
0
0
0
0
0
0
Percent Viable
Eggs
45
69
50
48
90
64
88
52
1 Primary and Secondary sludges include sludges from primary clarification, Imhoff digestion, activated sludge, contact stabilization.
and extended aeration. Treated sludges include sludges from mesophilic aerobic and anaerobic digestion, vacuum filtration.
centrifugation, lagoons and drying beds. t
ZNumbers rounded off to nearest 100.
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Influence of Abattoir Wastes on Parasite Concentrations in Primary and Secondary Sludges
Parasite
Ascaris spp.
(human and pig
roundworms)
Trichuris trichiura
or
Trichuris suis
(human or pig
whipworms)
Toxocara spp.
(dog and cat
roundworms)
Significant Source
Contribution
Domestic2
Abattoir3
Domestic
Abattoir
Domestic
Abattoir
Average No. of Viable
and Non-Viable
Eggs/ kg Dry Weight of Sludge^
7,900
81.800
1.500
1,600
1,800
500
Number of
Plants
6
1
6
1
6
1
^Numbers rounded off to nearest 100.
^Domestic plants found in the geographic area.
^Treatment plant in the geographic area receiving waste from large swine slaughter and packing houses.
80-
60-
40-
20-
-20-
-40-
-60-
-80-
-100-
-120-
-140-
-160-
-180-
-200
Aerobic
Digestion
MM
-t-u
mm*
(0
u.
2 •- <»
lilt
J2 o . .
< i- h- i-
DryingBed
After
Aerobic
Digestion
PPM
^Pl
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Table 6. Percent Reduction of Viable Parasite Eggs by Total Sludge Treatment
Processes1
Process
(Period)
Total
Fall
Winter
Spring
Summer
Aerobic
Fall
Winter
Spring
Summer
Anaerobic
Fall
Winter
Spring
Summer
Ascaris
67/76/69*
58/101/16
36/108/17
87/16/18
84/26/18
70/45/13
97/5/3
34/60/4
82/37/4
80/29/2
66/83/55
49/111/13
37/121/13
89/24/14
84/28/15
Toxocara
91/28/74
82/39/16
89/37/22
95/14/19
98/6/17
90/21/16
69/34/3
98/31/4
88/25/5
100/0/4
91/31/55
83/42/12
86/42/17
98/6/14
97/7/12
T. trichiura
39/118/26
31/87/7
(261/196/7
80/28/6
93/15/5
(25)/105/4
(66)/-/1
(155)/-/1
54/-/1
67 /-/I
52/119/21
48/82/6
(5)/206/6
85/27/5
100/0/4
T. vulpis
44/87/58
(1)3/ 135/8
7/108/16
70/41/18
74/49/16
63/46/11
81/-/1
48/74/2
63/44/4
68/54/4
34/96/43
(31J/145/6
(7)/113/13
71/42/13
74/51/11
1Aerobic or anaerobic digestion followed by drying beds.
'Average/standard deviation/number of samples.
^Numbers in parenthesis indicate percent increase.
Moisture
Content
20-40%
Moisture
Content
40-60%
34 56789
Log of Number of Viable Ascaris Eggs in Raw Sludge
10
Figure 2.
Plot of log of number of viable Ascaris eggs in raw sludge grouped by
moisture content of drying bed sludges.
effectively concentrating the eggs in the
contact stabilized and reaerated sludge.
Therefore, it would appear that the
processes of contact stabilization,
activated sludge, or extended aeration
tend to maintain a uniform level of
parasite eggs in the sludges of the
treatment plant.
Laboratory Studies
Laboratory studies on selected waste-
water sludges were conducted to
determine the factors involved in the
inactivation of parasite eggs and cysts in
sewage sludges. Bench scale results of
semi-continuous aerobic (ten day
hydraulic retention time) or anaerobic
sludge digestion (fifteen day hydraulic
retention time), lime stabilization,
ammonification, sonication, and combi-
nations of the above processes for the
destruction of Ascaris suum and
Toxocaris canis eggs in sludges are
briefly as follows:
1) Aerobic digestion inactivated
parasite eggs at temperatures of
55 °C or greater within two hours
and at 45 °C within two days.
2) Anaerobic digestion inactivated
Ascaris and Toxocara eggs at
temperatures greater than 45 °C,
but only retarded egg development
at temperatures less than 45 °C.
3) Lime treatment of sludges, pre-
treated by aerobic digestion at 28
°C and 35 °C to produce maximum
embryonation (and thus maximum
sensitivity to environmental fac-
tors), did not produce consistent
inactivation of Ascaris eggs. The
sludges were lime treated and
then held under aerobic or anaero-
bic conditions. With one exception,
inactivation increased with in-
creasing contact time (up to 20
days) and increasing lime dose,
although 3,000 mg Ca(OH)2/gram
sludge solids was required for
essentially complete inactivation.
The exception, 35 °C aerobically
digested sludge stored under
anaerobic conditions, showed
poor and erratic inactivations at
the various storage times and lime
doses. Lime treatment thus ap-
pears to be an expensive and
unreliable method for Ascaris
inactivation.
4) The results of the ammonification
studies were inconclusive. In
aerobically digested sludges, the
viable Ascaris eggs densities were
reduced 95% within 5 days even in
the control (no ammonia added).
The anomalously high reductions
with the control casts doubt on this
set of data. In the anaerobically
digested sludges, no reduction in
Ascaris viability was observed at
any dosage of ammonia up to
5,000 mg of ammonia sulfate per
gram of suspended solids.
5) Ultrasonication was effective in
destroying Toxocara eggs at49kHz
within a 6 minute exposure, but
Ultrasonication was not effective
in destroying Ascaris eggs under
these same conditions. The test
samples consisted of a suspension (
of approximately 10,000 eggs of
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Table 7.
The Relationship Between the Viability of Ascaris and Toxocara Eggs in Drying Bed Sludges and the Moisture Content
of the Sludge, in Different Seasons
Season
Number of Drying
Bed Samples
Analyzed
Number of Samples
With No Viable
Eggs
Lowest Moisture
Contents Below
Which No Viable
Eggs Observed
Number of Samples
With No Viable
Eggs At or Below
Lowest Moisture
Contents
Number of Samples
With Viable Eggs
Fall
Winter
Spring
Summer
Fall
Winter
Spring
Summer
Fall
Winter
Spring
Summer
the respective
of water.
The full report
For Both Ascaris and Toxocara Eggs
24 7 5% 1
22 4 7% 1
22 12 8% 8
21 14 15% 8
For Only Ascaris Eggs
21 7 5% 1
22 5 7% 1
22 12 8% 8
21 14 15% 8
For Only Toxocara Eggs
24 12 5% 1
22 12 21% 3
22 17 21% 11
21 18 20% 10
parasite in 100 ml
was submitted in
fulfillment of Grant No. 805107 by the
School of Public Health and Tropical
Medicine, Tulane University, under the
partial sponsorship of the U.S. Envi-
ronmental Protection Agency.
R. S. Reimers. M. D. Little, A. J. Englande, D. 8. Leftwich, D. D.
11
18
10
7
14
17
10
7
12
10
5
3
Bowman, and
R. F. Wilkinson are with the Tulane University School of Public Health and
Tropical Medicine, New Orleans, LA 70112.
Gerald Stern is the EPA Project Officer (see below).
The complete report, entitled "Parasites in Southern Sludges and Disinfection
by Standard Sludge Treatment." f Order No. PB 82-102 344; Cost: $17.00.
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:
Municipal Environmental Research Laboratory
U.S. Environmental Protection Agency
Cincinnati, OH 45268
-------
United States
Environmental Protection
Agency
Center for Environmental Research
Information
Cincinnati OH 45268
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