v°/EPA
United States
Environmental Protection
Agency
Municipal Environmental Research \ 4
Laboratory '""•
Cincinnati OH 45268
Research and Development
EPA-600/S2-81-024 Mar. 1981
Project Summary
Helminth and Heavy Metals
Transmission From
Anaerobically Digested
Sewage Sludge
Paul R. Fitzgerald
This summary discusses the findings
of a study designed to determine the
transmission to an animal host of the
ova of the nematode worm Ascaris sp.
that have survived through a modern
sewage treatment process and are pre-
sent in the sludge. Four large experi-
ments and three smaller ones involv-
ing 178 specific pathogen-free (SPF)
pigs were used. Natural transmission
of Acaris sp. from soil treated with
liquid anaerobically digested sewage
sludge that had been stored for several
years occurred in a few pigs in each of
four experiments. Also, natural trans-
mission from Nu-Earth, a dried, stored
sewage sludge, also occurred in pigs
that were exposed to this material by
contact in the pens. In general, ova in
anaerobically digested sludge or in Nu-
Earth remained unembryonated until
after they were exposed to the air. With-
in 6 weeks after exposure to air, the
ova began to embryonate, and there-
after, a small percentage of the ova
that embryonated became infective
for pigs.
The occurrence of heavy metals in
the tissues of swine held in pens treat-
ed with anaerobically digested sludge
or Nu-Earth, which originated from a
large municipality, was also studied.
Chemical analyses of kidneys, livers,
hearts, diaphragm muscles, and bones
were conducted to determine the quan-
tities of the heavy metals cadmium.
zinc, copper, iron, lead, chromium, and
nickel that were present in the tissues
following exposure of the pigs to differ-
ent amounts of the sewage products
in or on the soil. Only cadmium accu-
mulated to a significant degree in some
tissues of swine exposed to sludge con-
taining heavy metals. No physiological
or pathological changes associated with
exposure to the sludge material were
detected. Examination of visceral fat
from control and experimental pigs in-
dicated that there was no unusual accu-
mulation of organic compounds includ-
ing polychlorinated biphenyls (PCB's)
and the insecticides Heptachlor and
Dieldrin.
This Project Summary was develop-
ed by EPA's Municipal Environmental
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).
Introduction
Improvements in sewage disposal
systems within the last 50 years have
contributed to improved use of biological
wastes to supplement the texture and
fertility of soils. In using aerobically or
anaerobically digested sewage sludges
for soil conditioning or fertilizer, one of
the important unanswered questions,
however, concerns possible adverse
-------
\
effects of dispensing of possible disease-
causing materials to the environment.
Farmers throughout the world have
used human and animal fecal material
as fertilizer for hundreds of years. Only
since the early 1900's has the use of
such materials been recognized as the
source of many diseases. More recently, it
has been determined that a few bacterial,
viral, protozoan, and helminth parasites
are able to persist in waste products
originating from humans or animals.
Although the majority of pathogenic
organisms of viral, bacterial, protozoan,
or helminth origin are destroyed by
exposure to an external environment,
many validated instances of serious
outbreak of disease have occurred
involving these organisms. In under-
developed countries, many parasitic
diseases are literally a "way-of-hfe."
During recent years, researchers
have found that ova of ascarid round-
worms are the most resistant organ-
isms in wastes from human and animal
sources. These ova are known to survive
in soil for many months, even years, and
thus serve as a potential source of
disease.
In addition to the bacteria, viruses,
protozoan, helminths, etc., other mate-
rials found in sludges may cause disease
in man and animals. Among these are
toxic chemicals such as heavy metals
(e.g., Cd, Pb, Hg, Cr, etc.) and organic
compounds (e.g, PCB's) of various kinds
and combinations. The route of these
chemicals to man or animals is through
direct ingestion of sludge, or through
the food chain that starts with crops
grown on sludge-amended soil. The
relationship between exposure or dose
and illness is complex and is not the
subject of this investigation However,
the chance of illness is clearly related to
the flow of the toxic substance up the
food chain. By establishing relationships
between the initial dose or exposure
and the absolute concentrations in the
intermediate and final hosts in the food
chain, the ultimate threat of illness can
be put in perspective.
In the work summarized here, one
objective was to determine the possibility
of transmission of one pathogen (Ascaris
lumbricoides suum), a resistant para-
site, from soil treated with sludge to a
susceptible host (a pig). This parasite is
almost identical in nature and in its
mode of infection to the Ascaris lumb-
ricoides, which infects man. Conse-
quently, information developed on trans-
mission bears directly on the dose-
infection relationship for pigs and is
most likely very similar to the dose-
infection relationship in humans. A
second objective was to determine the
uptake of heavy metals and organic
compounds in the pigs exposed to
sludge. From this information, the
possible exposure of humans who ulti-
mately consume the pigs can be esti-
mated.
Procedure
A total of 178 pigs free from parasitic
infections (specific-pathogen-free:SPF)
were used to study whether anaerobic-
ally digested sludge, added to soil at
different concentration levels, could be
a potential source of infestation with the
nematode Ascaris lumbricoides suum.
The soil was treated with various levels
of liquid anaerobically digested sludges
(from 22 to 65 dry metric tons per
hectare) or with dried sludge (Nu-Earth)
added to the soil to a depth of 15.3 cm.
Pigs were confined in pens (9.8 x 14.6
m) to which the sludge had been added.
Pigs living in each of these environ-
ments, for approximately 4 months,
were compared with pigs living in
similar control pens, under the same
environmental conditions, but without
sludge added to the pens.
Results
• In each of two of the experiments,
eight young pigs were necropsied 2
to 3 weeks after introduction into
the pens and lung and liver tissues
were Baermannized to recover
migrating stages of the worms.
Larvae were recovered from the
lungs of 2 of 16 necropsied. Of 134
pigs that survived the full experi-
ment to necropsy, 43 contained
ascarid worms in the intestines,
including four from the control
pens. These four represent a con-
tamination of the control area that
apparently occurred accidentally
during the course of the study.
These results represent an infection
rate of 28% in the experimental
groups and 3% in the control groups.
Of the 134 pigs, 11 were housed in
pens treated with previously stored
Nu-Earth; of these, 6 were infected.
• An attempt was made todetermine
the rate of transmission of the
human ascarid worm, obtained
from a source in Columbia, South
America, to pigs. Fifteen SPF pigs
were confined in two sludge-free
pens. Embryonated or unembryo-
nated ova in water were sprayed
onto the soil or vegetation at the
rate of 572,000 ova in each of the
two pens. The ova were extracted
from the uteri of adult female
human Ascaris lumbricoides; the
portion of embryonated were em-
bryonated in 1% formalin with
continuous aeration for 30 days.
The pigs were allowed to remain in
the environment for 4 months,
During that time, all of the vegeta-
tion was consumed and the pigs
did what pigs do: ate soil, breathed
dust, rolled in water and mudholes
after rainstorms, etc. At necropsy,
4 months after being placed in the
pens, no worms were found in
either group of pigs.
1 Three pigs were fed anaerobically
digested sludge in which there
were naturally occurring ascarid
ova as part of their normal food
diet. The source of the ascarid ova,
of course, could have been humans
or pigs. Before the feeding step, the
liquid anaerobically digested sludge
was aerated by bubbling air through
it for 104 days. It was then fed to
each of the three pigs during a 30
day period. Forty-seven days after
the end of the feeding period, the
pigs were necropsied and the gastro-
intestinal tracts were examined for
ascarid worms. Worms found in
the intestine of one pig indicated
that ova naturally present in the
sludge could be infective for pigs.
The rate of embryonation of ascarid
ova (removed from the uteri of
gravid female worms) was deter-
mined in the presence of untreated
soil, soil treated with liquid anaero-
bically digested sludge, or Nu-
Earth. At the end of 6 weeks'
exposure to the soil to which liquid
sludge or Nu-Earth had been added,
an average 71% of the ova had
embryonated and were presumed
to be infective. The worms were
determined to be viable by observa-
tion of movement of larval stages
within the ova. None of the ova
were fed to susceptible pigs to
ascertain infectivity.
Tissues taken from the pigs at ne-
cropsy were analyzed for heavy met-
als and organic compounds. Dia-
phragm muscle, heart, liver, kidney,
and bone were selected for analysis
for heavy metals, and visceral fat
-------
was selected for analysis for the
organic compounds (Tables 1 and
2).
Cadmium in the kidney and liver was
the only heavy metal showing significant
accumulation. Some heavy metals pre-
sent in sludges may accumulate in the
tissues at levels related to the level of
exposure, i.e., animals exposed to great-
er concentrations of sludge had greater
tissue concentration of some heavy
metals. In these studies, pigs exposed to
dried sludge (Nu-Earth) accumulated
the greatest quantities of heavy metals.
For example, the average accumulation
of cadmium in the kidneys was 9.8 ppm
dry weight. On a wet weight basis this
would be less than 3 ppm. In humans,
clinical evidence of damage to the
kidney is said to begin when approxi-
mately 200 ppm, wet weight, have
accumulated.
The results of chemical analysis for
organic compounds (PCB's, Dieldrin,
Heptachlor) showed there was no differ-
ence between experimental and control
animals in terms of accumulation. These
compounds were not accumulated in
the animal tissues to levels greater than
is normally found in the environment.
Conclusions
Exposure of swine to different levels
of anaerobically digested sludge from a
large municipality has shown that a
potential for transmission of the nema-
tode Ascaris sp. exists. The results of
this study show that some ova from this
parasite worm are able to withstand the
rigorous treatment of a modern sewage
treatment plant and are subsequently
infective to pigs. The exposure of swine
to anaerobically digested sludge was
infinitely greater than a normal human
exposure would be, yet the levels of
infestation with Ascaris sp. were low.
With ova present at an approximate
level of 4000/m2 of surface, embryona-
tion of some ova occurred, some were
ingested by pigs and eventually caused
light infestations in the gastrointestinal
tracts. Although these worms did not
cause detectable pathology in the ani-
mals they infested, a potentially serious
parasite problem could develop in a
swme herd continuously exposed to
anaerobically digested sludge. This
could occur not because of the immedi-
ate damage caused by worms transmit-
ted from the sludge, but because of the
potential for buildup of numbers of ova
in the environment as sexually mature
female worms, originating from ova in
Table I. Summary of chemical analyses for heavy metals in tissues of pigs exposed
to different levels of anaerobically digested sewage sludge originating
from the Metropolitan Sanitary District of Greater Chicago. Numbers in
parenthesis represent deviations in numbers of animals.
ppm, dry weight
Metal & Group
Number of
Animals Kidney
Liver
Diaphragm
Muscle
Heart Bone
Cadmium
Control 14
22-25 DMT/Ha sludge 18
33 DMT/Ha sludge 4
65 DMT/Ha sludge 4
Nu-Earth 4
NBS control (bovine)
Chromium
Control 12
22-25 DMT/Ha sludge 12
33 DMT/Ha sludge 4
65 DMT/Ha sludge 4
Nu-Earth —
Copper
Control 14
22-25 DMT/Ha sludge 18
33 DMT/Ha sludge 4
65 DMT/Ha sludge 4
Nu-Earth 4
NBS control (bovine)
Iron
Control
22-25 DMT/Ha sludge
33 DMT/Ha sludge
65 DMT/Ha sludge
Nu-Earth
Lead
Control 14
22-25 DMT/Ha sludge 18
33 DMT/Ha sludge 4
65 DMT/Ha sludge 4
Nu-Earth 4
NBS control (bovine)
Nickel
Control 12
22-25 DMT/Ha sludge 12
33 DMT/Ha sludge 4
65 DMT/Ha sludge 4
Nu-Earth —
Zinc
Control 14
22-25 DMT/Ha sludge 18
33 DMT/Ha sludge 4
65 DMT/Ha sludge 4
Nu-Earth 4
NBS control (bovine)
0.47
2.30
2.10
3.83
9.80
0.23
0.34
0.29
0.58
1.50
0.27
0.04
0.06
0.07
0.15
0.06
0.12
0.17
0.14
0.09
0.05
0.05
0.22
0.09
0.06
0.08
0.09
0.18
0.06
37.00 26.00
33.00 25.00
28.00 37.00
33.00 23.00
44.00 33.00
188.00
7.00
10.00
9.00
8.00
7.00
16.00
17.00
19.00
16.00
18.00
14
18
4
4
4
166.00 488.00
155.00 541.00
144.00549.00
172.00586.00
198.00 453.00
99.00
103.00
91.00
119.00
73.00
180.00
187.00
169.00
180.00
152.00
0.31
0.31
0.29
0.32
0.44
0.33
0.34
0.36
0.31
0.47
0.35
138.00 264.00
133.00 263.00
117.00248.00
128.00210.00
140.00 286.00
131.00
0.22
0.28
0.17
0.20
0.21
0.39
0.41
0.30
0.60
123.00(9}
/2S.001111
119.00
108.00
113.00
0.20
0.23
0.18
0.38
0.26
75.00
74.00
65.00
74.00
77.00
0.03('
0.02"
0.04
3.50''
5.30"
3.80
44.00('
44.00"
42.00
0.63''
0.69(t
1.77
98.00''
118.00"
122.00
the sludge, began to lay eggs which
would "seed" the environment. Sub-
sequent swine, exposed to the area,
could be exposed to much greater
numbers of ova, and a potential parasite
problem could develop into a serious
actual problem.
Under the conditions of this study, the
health of the animals did not appear to
be threatened by the parasites transmit-
I US GOVERNMENT PRINTING OFFICE 1981 -757-0 1Z/7I06
-------
Table 2. Summary of chemical analyses for organic compounds in visceral fat of
pigs exposed to different levels of anaerobically digested sludge originating
from the Metropolitan Sanitary District of Greater Chicago.
ppm, wet weight
Exposure
Control
22 DMT/Ha sludge
Nu-Earth
Number of
Pigs
17
9
10
Polychlorinated
Biphenyls
0.158
0.111
0.120
Heptachlor
Epoxide Dieldrin
0.063 0.051
0.005 0.006
0.002 0.003
ted through the sludge. If humans were
exposed to fields with the same sludge
application densities, they would ingest
far less sludge than did the pigs. Con-
sequently, it is not likely that man would
become seriously parasitized from expo-
sure to anaerobically digested sludge
under current sanitary conditions in the
United States.
The results of this study have shown
that cadmium was the only one of seven
heavy metals which accumulated to a
significant degree in tissues of swine
exposed to sludge containing heavy
metals. Of the five tissues examined for
heavy metals, only kidney and liver
accumulated cadmium at levels greater
than in controls. Although the accumula-
tion in these organs was statistically
greater than in controls, the accumula-
tion was far below a lethal level.
The presence of polychlorinated bi-
phenyls and the organic insecticides
Heptachlor and Dieldrin in tissues of
experimental animals, at levels com-
parable to controls, indicates that these
organic compounds did not appear to be
an immediate threat to animal health
under the conditions of these studies.
The facts revealed herein suggest that
serious exposure to anaerobically di-
gested sludge, containing organic com-
pounds at levels described in this study,
would cause no significant detrimental
effect on the animals.
The full report was submitted in
fulfillment of Grant No. R805315-01 by
the University of Illinois under the
sponsorship of the U.S. Environmental
Protection Agency.
Paul R. Fitzgerald is with the University of Illinois, Urbana, IL 61801.
Gerald Stern is the EPA Project Officer (see below).
The complete report, entitled "Helminth and Heavy Metals Transmission from
Anaerobically Digested Sewage Sludge," (Order No. PB 81-161 846;
Cost: $8.00, 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
Postage and
Fees Paid
Environmental
Protection
Agency
EPA 335
Official Business
Penalty for Private Use 8300
Return Postage Guaranteed
Third-Class Bulk
Rate
U f> iv'JVTK
KLbJtJiv 5
23u S UL
CHICAGO IL o06u4
------- |