United States
Environmental Protection
Agency
Health Effects Research
Laboratory
Research Triangle Park NC 27711
Research and Development
EPA/600/S1 -85/022 Jan. 1986
Project Summary
Investigation of Parasites in
Sludges and Disinfection
Techniques
R. S. Reimers, M. D. Little, A. J. Englande, Jr., D. B. McDonell,
D. D. Bowman, and J. M. Hughes
The objectives of this research grant
were to: 1) assess the presence and
densities of resistant stages of para-
sites in municipal wastewater sludges
(sewage) in northern United States;
2) compare the results of this study
with the results of our previous study of
sludges in southern United States;
3) evaluate several decontamination
techniques for their effectiveness in in-
activating parasites in waste sludges;
and 4) develop a standard method for
the parasitologic examination of waste
sludges. Sludge samples from all
phases of treatment (i.e., primary, etc.)
were collected during the fall, winter
and summer from 48 municipal waste-
water treatment plants located in New
York (13 plants), Ohio (12 plants), Min-
nesota (11 plants), and Washington (12
plants). Resistant stages of twenty
types of parasites were found in these
samples which may represent 25 to 30
different human and animal parasites.
This Project Summary was devel-
oped by EPA's Health Effects Research
Laboratory, Research Triangle Park, NC,
to announce key findings of the re-
search project that is fully documented
in a separate report of the same title
(see Project Report ordering informa-
tion at back).
Introduction
The problems associated with the oc-
currence of human and animal para-
sites in domestic wastewater sludges
have received little attention in the
United States prior to 1970. Even
though studies had examined waste-
water effluents and sludges from mu-
nicipal treatment plants in different
parts of the country and had found the
eggs of several intestinal helminths,
these studies have little practical value
other than recording the presence of the
parasites found, since the techniques
used to recover the parasites and to
quantitate them varied from study to
study.
In a recent study for the U.S. Environ-
mental Protection Agency (EPA) by Tu-
lane University, "Parasites in Southern
Sludges and Disinfection by Standard
Sludge Treatment," the presence and
densities of parasites in sludge samples
from 27 municipal wastewater plants in
Florida, Alabama, Mississippi, Louisi-
ana, and Texas were investigated over
one year, and several standard proc-
esses were evaluated in regard to their
effectiveness in inactivating parasites.
The results indicated that further testing
was required to develop reliable ap-
proaches to the inactivation of parasites
in municipal sludges, and that addi-
tional information on the occurrence of
parasites in sludges in other areas of the
United States was needed before the
potential health risks associated with
the land application of wastewater
sludges could be judged.
The purpose of the present study was
to determine the types and densities of
parasites in sewage sludges in northern
United States, to evaluate factors which
may affect their densities in final
sludges, i.e., sludges destined for final
disposal, and, through laboratory ex-
periments to determine the effective-
ness of several sludge treatment proc-
esses in inactivating parasites.
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Field studies consisted of collecting
sludge samples representing all phases
of the treatment process (primary stabi-
lization, post-stabilization, etc.) during
the fall, winter and spring seasons, re-
spectively, from 48 municipal waste-
water treatment plants located in New
York (13), Ohio (12), Minnesota (11) and
Washington (12). Plants of four size cat-
egories, <1 MGD, 1-10 MGD, 10-50
MGD, and >50 MGD, were included in
the study. Each sample was analyzed
for parasites and several abiotic param-
eters, i.e., pH, temperature, total sus-
pended and volatile solids, etc. In labo-
ratory studies, aerobic digestion, lime
and caustic stabilization, ammonifica-
tion, irradiation with cobalt 60 and ultra-
sonification were investigated regard-
ing their effectiveness in inactivating
parasites in municipal sludges.
Results
Field Studies
Parasitological Findings
In sludge samples collected from
each of the 48 treatment plants in the
four northern states, 20 different types
of parasite eggs or cysts were detected
(Table 1), but many of them could be
identified to the genus level only. Ap-
proximately 90% of all sludge samples
examined contained stages of one or
more parasites. The four most com-
monly found parasites were Ascaris
spp., Trichuris trichiura, Trichuris vulpis
and Toxocara spp. The eggs of one or
more of these four parasites were found
in about 89% of the samples examined
from the four states. The geometric
means of the numbers of eggs per kg
dry weight of sludge of each of these
four parasites found in different types of
sludge samples from the four states and
the percentages of samples positive for
these parasites are presented in Table 2.
The percentages of treatment plants in
the four states with samples positive for
these eggs are shown in Table 3.
Ascaris eggs were found in a higher
percentage of samples in Washington
than in the other three states and the
mean number of eggs was also higher
in Washington sludges. 7. trichiura
eggs were found more commonly in
New York than in the other three states.
7. vulpis eggs were found in more
sludge samples in Ohio and New York,
but the egg densities did not differ sig-
nificantly in the sludges in the four
states. Toxocara eggs were the most
frequently found parasite with viable
Table 1. Parasites Found in Sludge Samples from 48 Municipal Plants in Northern
United States
Parasite Found
Probable Identity
Definitive Host
Ascaris eggs
Toxocara eggs
Trichuris trichiura eggs
Trichuris vulpis eggs
Toxascaris-//te eggs
Parascaris equorum eggs
Ascaridia-Wce eggs
Gongylonema-//fce eggs
Physaloptera-//7re eggs
Trichosomoides-//fce eggs
Capillaria spp. eggs (3 or
more types)
Hymenolepis diminuta eggs
Hymenolepis nana eggs
Hymenolepis sp. eggs
Diphyllobothrium-/;7re eggs
Spirometra-Mce eggs
Schistosoma mansoni
egg (1)
Entamoeba coli-like cysts
Giardia cysfs
Coccidia oocysts
Ascaris lumbricoides'
Ascaris suum
Toxocara canis2
Toxocara cat!2
Trichuris trichiura
Trichuris suis3
Trichuris vulpis
Toxascaris leonina
Parascaris equorum
Ascaridia galli
Heterakis gallinae
Gongylonema neoplasticum
Gongylonema pulchrum
Physaloptera spp.
Turgida turgida
Trichosomoides crassicauda
Anatrichosoma buccalis
Capillaria hepatica
Capillaria gastrica
Capillaria spp.
Capillaria spp.
Hymenolepis diminuta
Hymenolepis nana
Hymenolepis spp. (poss.
more than one species)
Diphyllobothrium latum
Diphyllobothrium spp.
Spirometra mansonoides
Schistosoma mansoni
Entamoeba coli4
Entamoeba spp.
Giardia lamblia
Giardia spp.
Isospora spp.
Eimeria spp.
'Eggs of A. lumbricoides and A. suum are indistinguishable.
2Toxocara eggs were probably mostly T. canis.
3T. suis eggs were probably only rarely seen.
4An intestinal amoeba that is a commensal, not a parasite.
Humans
Pigs
Dogs
Cats
Humans
Pigs
Dogs
Dogs and cats
Horses
Domestic poultry
Domestic poultry
Rat
Cattle, pigs, etc.
Dogs, cats, etc.
Opossums
Rats
Opossums
Rats
Rats
Birds
Wild mammals
(opossums, racoons,
etc.)
Rats
Humans and rodents
Domestic and/or wild
birds
Humans, dogs, bears
Dogs, bears, birds
Dogs and cats
Humans
Humans
Rodents, etc.
Humans
Dogs, cats, mammals
Dogs, cats
Domestic and wild
birds, mammals
eggs being found in one or more sam-
ples from each plant studied (Table 3).
The densities of Toxocara eggs were
considerably lower in samples in
Minnesota than in samples in the other
three states.
Parasite Densities vs. Popula-
tion Served by Plants
Utilization of multiple regression
analysis to examine the relationship of
the population served by the waste-
water treatment plants to the densities
of total and viable Ascaris and 7. trichi-
ura eggs in undigested sludges were
not related to the size of the population
served by the plant; however, the densi-
ties of total and viable 7. vulpis and
Toxocara eggs decreased significantly
in the undigested sludges from north-
ern (and/or southern) states as the pop-
ulation served by the treatment plants
increased. In digested sludges, the den-
sities of viable and total Ascaris and 7.
trichiura eggs increased significantly
with an increasing population size, but
the densities of viable 7. vulpis eggs
and viable total Toxocara eggs were not
significantly related to the population
served.
Parasites in Stabilized Sludges
In general, no differences were noted
in the densities of eggs in aerobically
and anaerobically digested sludges ex-
cept for viable Ascaris and Toxocara
eggs. Significantly lower densities of vi-
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Table 2, Ascaris spp., Trichuris trichiura, T. vulpis and Toxocara spp. Eggs in Samples of Undigested Sludge from Digesters
PARASITE
Source or
Type of Sludge
Undigested
N= 162
% positive
Geometric Mean2
Digester
N= 137
% positive
Geometric Mean2
Post-Digestion
A/= 773
% positive
Geometric Mean2
All Sludges
N= 142
% positive
Geometric Mean2
Ascaris spp.
Total1 Viable
51%
940
56%
1700
54%
560
54%
WOO
49%
670
50%
1400
45%
310
48%
710
Trichuris trichiura
Total1 Viable
35%
500
42%
480
29%
260
36%
440
27%
650
28%
400
19%
130
25%
400
T. vulpis
Total1 Viable
33%
430
48%
460
47%
240
42%
400
31%
440
45%
440
37%
190
37%
370
Toxocara spp.
Total1 Viable
81%
880
85%
1200
69%
330
79%
880
78%
870
74%
740
50%
190
69%
670
1 Viable and nonviable eggs.
2Antilog of the mean of the log of positive samples, expressed as number of eggs/kg dry weight of sludge.
Table 3.
Parasite
Ascaris
T. trichiura
T. vulpis
Toxocara
Percentage of Plants in Four Northern States with
trichiura, T. vulpis and Toxocara.
Viable and Nonviable Eggs
85%
88%
85%
100%
Eggs of Ascaris, Trichuris
Viable Eggs
77%
73%
81%
100%
able Ascaris and Toxocara eggs were
found in anaerobically digested
sludges.
Parasites in Drying Beds
In all cases a significant relationship
between the density of viable eggs and
the moisture content of the sample was
observed. As the moisture content de-
creased, the densities of viable parasite
eggs decreased.
Step-up multiple regression analysis
was applied to the density of viable
eggs in drying beds vs. the density of
viable eggs in a simultaneously col-
lected sample of undigested sludge.
The purpose was to test the significance
of the effects of the percent moisture
content of the drying bed, the age of the
sludge in the bed, the season of the
sampling, and the type of digestion
process before the drying bed.
With additional controls for the age of
the sludge, season of collection and
type of digestion, the percent moisture
content of the drying bed sludge was
not found to be significant with respect
to densities viable Ascaris eggs, al-
though it was significant for densities
Tbxocara eggs (p < 0.001). Fewer viable
Toxocara eggs were found with de-
creasing sludge moisture content. The
age of the sludge in the drying bed was
not found to have a significant effect for
either Ascaris (0.25 > p > 0.10) or Toxo-
cara (p > 0.25) eggs. The type of diges-
tion process was not significant with re-
spect to Ascaris (p > 0.25) but was
significant with respect to Toxocara
eggs (0.05 > p > 0.01) with fewer viable
eggs in the drying bed associated with
anaerobic processes than with aerobic.
Significantly, fewer Ascaris eggs were
found in fall than in winter
(0.05 > p > 0.01), while no seasonal dif-
ferences were found for Toxocara eggs.
Well over half of all simultaneously col-
lected samples showed more than 75%
fewer viable Ascaris, T. trichiura, T.
vulpis and Toxocara eggs in drying bed
samples than in digester samples. Only
five samples from the northern drying
beds had a moisture content of less
than 20%, which was previously re-
ported to be necessary for effective par-
asite egg inactivation. Three of these
samples were from covered drying
beds; while the other two samples were
from plants in eastern Washington.
Higher densities of viable Ascaris
eggs in undigested sludges were signif-
icantly associated with higher densities
of viable Ascaris eggs in drying bed
sludges (p = 0.001). This was also true
for the eggs of T. trichiura (p < 0.03)
and T. vulpis (p < 0.03). The densities of
viable Toxocara eggs did not show a
significant relationship, however.
Step-up multiple regression analysis
was utilized to test the significance of
the effect of 1) percent moisture content
of the drying bed sludge, 2) age of the
sludge in the bed, and 3) digestion proc-
ess (aerobic vs. anaerobic) employed
on the undigested sludge on the inacti-
vation of parasites. No trend was noted
for sludge age and the digestion proc-
ess, but decreasing moisture content of
the drying bed sludge was significantly
associated with decreased densities of
viable Ascaris (0.01 >p> 0.001), 7.
trichiura(Q.Qb > p > 0.01), T. vulpis
(p < 0.001) and Toxocara (p < 0.001)
eggs.
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Sludges Destined for Ultimate
Disposal
The type of sludge destined for dis-
posal was dewatered sludge in 31
plants, liquid sludge (primary or di-
gested) in 13 plants, and both de-
watered and liquid sludge in 4 plants. In
the majority of cases, the sludge was
land-applied with only about one-third
being placed in landfills, lagoon stor-
age, or the ocean.
The percentages of the samples of
these final sludges that contained viable
eggs of Ascaris, T. trichiura, T. vulpisor
Toxocara and the geometric mean of
these eggs are presented by state in
Table 4. In samples from all four states,
Toxocara eggs were found most fre-
quently (55%), with Ascaris eggs, 7.
vulpis eggs and 7. trichiura eggs being
found somewhat less frequently, 48%,
38% and 22%, respectively. Ascaris
eggs occurred in higher levels in final
sludges than the other three parasites,
and the densities of Ascaris eggs in final
sludges in Washington were signifi-
cantly higher than in these sludges in
the other three states.
The number of plants that distributed
sludge to the public for use at home in-
cluded five in Ohio, four in New York,
two in Washington, and one in Minne-
sota. Viable eggs of Ascaris, T. trichi-
ura, T. vulpis and Toxocara were found
in 39%, 6%, 56% and 50%, respectively,
of the samples of these sludges. The ge-
ometric means of viable eggs of each of
these parasites found in positive sam-
ples were 492, 92, 300 and 580 eggs/kg
dry weight, respectively.
Laboratory Studies
Continuous aerobic digestion at 25°C
with detention times of 10, 20 and
30 days did not noticeably affect the vi-
ability of Ascaris eggs in the sludge.
However, when digestion was carried
out at 35°C, a 30-50 percent inactivation
of the eggs was noted within 10 days.
The addition of caustic (NaOH) to
sludge previously digested at 25°C had
little effect on the viability of the Ascaris
eggs, even over a period of 10 days.
However, when caustic was added to
sludge from the 35°C digester, 97% of
the eggs were killed within 5 days and
100% were killed within 10 days. In lime
stabilization studies, no effect on the As-
caris eggs was noted until a lime
dosage of 1,000 mg lime per gram of
sludge solids was used. At that dosage,
97% of the eggs were killed within
5 days.
When ammonium sulfate at a dosage
of 50 mg ammonia per gram of sludge
solids was added to sludge previously
aerobically digested at 25°C for 10 days,
there was little effect on the Ascaris
eggs during the first five days. After
10 days, 62% of the eggs were inacti-
vated. When the ammonia dosage was
increased to 500 mg/gram solids, com-
plete or near complete inactivation was
observed after 10 days.
When ammonia gas was added to
sludges previously aerobically digested
at 25°C at detention times of 10,20 or 30
days, a dosage of one percent was nec-
essary to obtain effective inactivation of
the Ascaris eggs. When ammonia was
added to sludges digested at 35°C, a
higher degree of inactivation was found
to occur than with comparable dosages
in sludges digested at 25°C.
The exposure of Ascaris eggs to an
ultrasonic field at 33 KHz and 600 watts
for 11 minutes inactivated 77 percent of
the eggs.
In studies using gamma radiation
from a Cobalt 60 source, it was found
that a dosage of 200 Krads or greater
was completely effective in killing As-
caris eggs in sludge (3% solids). Eggs
from the feces of pigs and from the uteri
of worms were equally susceptible to
the radiation, as were the intact and de-
coated eggs.
Table 4. Percentage of Sludge Samples Destined for Disposal in Each of Four Northern
States that Contain Viable Eggs of Ascaris, Trichuris trichiura, T. vulpis and
Toxocara and Mean Number of Eggs of Each Parasite
STATE
Eggs
MN
OH
NY
WA
All
Samples
Ascaris
% positive 49%
Geometric Mean' 375
T. trichiura
% positive 14%
Geometric Mean 140
T. vulpis
% positive 12%
Geometric Mean 255
Toxocara
% positive 51%
Geometric Mean 315
41%
235
12%
150
63%
270
280
49%
560
31%
390
56%
230
49%
455
47%
1920
33%
280
28%
395
50%
515
48%
565
22%
265
38%
270
55%
370
1Antilog of the mean of the log of positive samples; expressed as number of eggs/kg dry
weight of sludge.
S. GOVERNMENT PRINTING OFFICE: 1986/646-116/20763
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R. S. Reimers, M. D. Little, A. J. Englande. Jr., D. B. McDonell, D. D. Bowman, and
J. M. Hughes are with School of Public Health and Tropical Medicine, Tulane
University, New Orleans, LA 70112.
Norman E. Kowal is the EPA Project Officer (see below).
The complete report, entitled "Investigation of Parasites in Sludges and
Disinfection Techniques,"(OrderNo. PB86-135407'/AS; Cost:$22.95, subject
to change) will be available only from:
National Technical Information Service
5285 Port Royal Road
Springfield, VA22161
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
United States
Environmental Protection
Agency
Center for Environmental Research
Information
Cincinnati OH 45268
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