PAR AS ITO LOGICAL EXAMINATION
OF COMPOST
U.S. ENVIRONMENTAL PROTECTION AGENCY
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PARASITOLOGICAL EXAMINATION OF COMPOST
A Solid Waste Research
Open-File Report
written by
MIRDZA L. PETERSON, Senior Research Microbiologist
U.S. ENVIRONMENTAL PROTECTION AGENCY
Office of Research and Monitoring
Solid Waste Research
1971
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Parasitological Examination of Compost
Mirdza L. Peterson
Solid Waste Research
Office of Research and Monitoring
Cincinnati, Ohio 45213
Marketed and stockpiled compost and a mixture of compost and raw
sewage sludge known to contain parasites were examined to compare the
effectiveness of two concentration methods and one direct method for
detecting helminthic forms and protozoan cysts. Nonviable helminthic
ova and larvae were found in the stockpiled compost, and viable forms
of helminthic parasites were found in the marketed compost. The
formalin-ether centrifugal sedimentation method was usually more
effective than the brine gravity flotation method for recovering both
ova and larvae; the direct film method was the least efficient.
Municipal solid waste, with or without addition of sewage sludge,
is being composted on a small scale in the United States today. Com-
posting is merely a microbiological process wherein certain bacteria,
fungi, and actinomycetes convert all biodegradable organic matter to
a stable humus. Compost is being utilized mainly in agricultural and
land reclamation projects.
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The health hazards that may exist in composting and in the use of
compost, especially when sewage sludge is added to municipal solid waste,
have been the concern of many investigators.
Gotaas (10) and Golueke and Gotaas (9) have shown that a temperature
of 140 F (60 C) for 1 hr should kill all nonspore-bearing pathogens.
These reports indicated that Asoavis Iwribriooides eggs were destroyed
when the compost pile was subjected to temperatures of about 113 F (45 C)
for 50 min, and Endamoeba histolytioa and Endamoeba ooli cysts were •
more easily destroyed than Asoavis eggs. Chang (3, 4) showed that 20
days at 45 C was required for unheated sewage sludge digestion to remove
all nonhatching Asaaris ova, and that a contact time of 115 min at 45 C
was needed to achieve a complete killing of Endamoeba histolytica cysts
in water. Strauch (19), Hanks (11), and Fair and Geyer (6), however,
have shown that municipal solid waste as well as sewage sludge can con-
tain pathogenic organisms that endanger the health of man and animals.
Krishnaswami and Post (15) stated that slaughterhouse waste waters often
contain many helminthic parasites (ova, larvae, and adults) and these
may enter receiving watercourses through disposal of waste.
Recently, Gaby (8) showed that Endolimax nana, Endamoeba histolytica,,
and Necator ameriaanus ova inserted in composting refuse and sludge were
killed within an 8-day exposure period at a minimum temperature of 120 F
(49 C) at the 2- to 4-inch depth, and within 7 days at a minimum tem-
perature of 131 F (55 C) at the middepth (2 to 3 ft); however, some
morphologically intact parasitic ova such as those of AsaariSy Tviohupis,
Neoator, Anaylostoma, and Hymenolepis persisted to the end of the 49th
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day of the composting process. (Facilities were not available to de-
termine the viability of these parasite forms.)
Our preliminary investigation was designed to find concentration
methods, through comparative studies, that could effectively be used
to recover helminthic parasites and their ova and larvae in composted
waste.
The effectiveness of several methods presently used to recover ani-
mal parasites and ova from clinical specimens, sewage, vegetables, and
compost samples were compared (1, 5, 7, 12, 16-18, 20, 21). Of these,
two concentration methods (the brine gravity flotation method by Willis
(21) and the formalin-ether centrifugal sedimentation method by Ritchie
(17)) and also the direct film method by Harris and Coleman (12) were
selected for comparison in experimental studies designed to recover and
morphologically identify helminth ova and protozoan cysts in a compost—
digested sewage sludge mixture. These methods were then applied.in field
studies to analyze stockpiled compost, with and without digested sewage
sludge, and marketed compost.
It should be noted that the viability of ova was determined by embryo
motility in direct observation with a light microscope. No testing was
made to determine the hatchability of the ova recovered.
MATERIALS AND METHODS
Collection and Preparation of the Sample
Compost—digested sewage sludge mixture (experimental study). A
50-g sample of digested sewage sludge known to contain Ascari,s and
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Hymenolep'is ova was mixed with 250 g of compost. .The mixture was divided
into three equal portions. After each of the three portions was thor-
oughly mixed, a final 10-g subsample was removed and placed in a 500-ml
wide-mouthed flask containing 100 ml physiological salt solution and
glass beads. The flask was placed on a rotary shaker at medium speed
for 5 min to emulsify the material. The flask was then removed from
the shaker and allowed to stand undisturbed for 45 min. Each of these
portions was examined by three methods: brine gravity flotation, formalin-
ether centrifugal sedimentation, and direct film.
Stockpiled compost (field study). Random 10- to 20-g samples from
each of eight different piles of composted municipal solid waste, with
and without the addition of digested sewage sludge, were collected with
sterile tongs at a depth of 15 to 30 cm and placed in sterile, 32-oz,
capped, glass bottles with wide mouth or in sterile, scalable, 18-oz
polyethylene bags. The random samples from each of the eight compost
piles were composited into eight 100- to 200-g samples. After thorough
mixing, they were prepared as above.
Marketed compost (field study). Random samples were collected
from five different marketed compost materials in a manner similar to"
the stockpiled compost. After thorough mixing, they were prepared in
a manner similar to that of the compost—digested sewage sludge mixture.
Examination of Sample
Direct film method (12). Approximately 0.05 ml of the sediment
from the suspended material was added to a drop of physiological saline
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solution on the center of a slide and stirred well. Coarse fibers,
paper, sand, etc., in the test material were removed, and a cover glass
was applied. The specimen was then examined under low power of the
microscope. If protozoa, ova, or larvae were found, a drop of Lugol's
iodine solution was added to the film, and the organisms were tentatively
identified.
Brine gravity flotation method (21). The original suspension was
well mixed and strained through an 18- by 16-mesh nylon sieve into a.
250—ml beaker. The sieve was washed with approximately 10 ml of physio-
logical salt solution and divided into two 50-ml centrifuge tubes. One
tube was centrifuged at 2,000 rpm for 1 min; the other was saved for
the formalin-ether centrifugal sedimentation. After centrifugation,
the supernatant was decanted and salt brine was added to fill the tube.
The contents of the tube were stirred to produce an even suspension
and allowed to stand entirely undisturbed for 30 to 60 min. With the
use of a freshly flamed wire loop (4-mm i.d.), the material in surface
film from several locations was placed onto a clean slide, covered with
cover glass, and then examined under low power of the microscope.
Formalin-ether centrifugal sedimentation method (17). The original
suspension, saved from the previous test, was centrifuged at 2,000 rpm
for 1 min. The supernatant was decanted, and the sediment resuspended
in approximately 10 ml of physiological salt solution. The suspension
was transferred to a 15-ml conical centrifuge tube and centrifuged for
1 min at about 2,000 rpm. After centrifugation, the supernatant fluid
was discarded, and 10 ml of 10% formalin solution was added. It was
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allowed to stand 10 min or longer for fixation. After this, 3 ml of
ether was added to the tube; the tube was stoppered, shaken vigorously
for 20 to 30 sec, and centrifuged for 2 min at about 1,500 rpm. After
centrifugation, the supernatant was decanted, and 0.05 ml of sediment
was transferred to a slide. The preparation was covered with a cover
glass and examined under low power of the microscope.
RESULTS
Compost-sewage sludge mixture. In experimental studies of compost—
digested sewage sludge mixture, Ascarts-ova were found in each of the
three portions tested by the brine gravity flotation and the formalin-
ether centrifugal sedimentation methods (Table 1). The efficiency for
the recovery of helminth ova from the compost—sewage sludge mixture
was compared on the basis of the number of Asoaris ova. The results
showed that formalin-ether centrifugal sedimentation was the best for .
the recovery of Asaaris ova from the first subsample and for Hymenolep-is
ova from the second subsample. The two concentration methods were found
to be equally efficient for the third subsample. As for the protozoan
Endomoeba ooT/L cysts, the formalin-ether centrifugal sedimentation method
also appeared to be the most efficient.
Stockpiled compost. The occurrence of helminth ova and larvae
in an 8-sample series of stockpiled compost of various ages is summarized
(Table 2). These samples were shipped to our laboratory from a composting
plant designed to decompose municipal solid waste, with or without addi-
tion of digested sewage sludge, by a batch-type, high-rate mechanical
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TABLE 1. Recovery of helminth ova and protozoan cysts from
compost—digested sewage sludge mixture (per 0.05 ml)
Subsample
ritJLlloo.
Direct film
Brine gravity flotation
1
0
2
2
Ascaris
0
2
3
0
2
Formalin-ether centrifugal
sedimentation
Direct film
Brine gravity flotation
Formalin-ether centrifugal
sedimentation
Direct film
Brine gravity flotation
Formalin-ether centrifugal
sedimentation
0
0
0
0
Eymenolep-is
0
0
Endamoeba aoli
1
0
0
2
0
0
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TABLE 2. Helminth ova and larvae in various-aged, stockpiled compost
Sample
no.
1
2
3
4
5
6
7
8
Sewage
sludge
added
Yes
Yes
Yes
Yes
No
No
No
No
Curing
(months )
1
1
2
3
6
6
6
7
_ Organisms recovered/0.05 ml
temperature • • •
(C) Ova Larvae
38 Hymenolepis
diminuta (26)
76 -
64 - Strongyloides (l)c
66 -
49 -
63 -
63 -
64
Recovered by the formalin-ether centrifugal sedimentation method.
None found.
Recovered by the brine gravity flotation method.
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aerobic bacterial digestion system that employed digestor tanks. The
waste decomposed to compost in 6 to 10 days at temperatures of 66 C
and above in the winter and at 71 C in the summer. Four to five days
were required before temperatures reached their maximum values at all
depths within the composting mass. The samples were collected from
stockpiled composted material that had completed the digested cycle,
had undergone a final grind, and had aged for various periods of time.
Ova of Eymenolep-Ls diminuta (26 per 0.05 ml) were found in a sample
that had been cured for 1 month; rhabditiform larva of StTongy1o-Ld.es
(1 per 0.05 ml) appeared in a sample collected from a pile that had
been cured for 2 months. These developmental forms appeared to be micro-
scopically nonviable. Note that temperatures of 38 and 64 C were recorded
in the stockpiled compost at the respective time of sampling.
Marketed compost. The incidence of helminth ova and larvae in
marketed compost materials is shown in Table 3. Of the five samples
analyzed, one showed the ovum of Trichians trichuria (1 per 0.05 ml),
Taenia sp. (1 per 0.05 ml), Hymenolepis diminuta (2 per 0.05 ml), and
rhabditiform larvae of Stvongyloides (1 per 0.05 ml); another showed
the larvae of hookworm (2 per 0.05 ml). The ovum of T. -tviohmria and
the larvae of StTongyloi-des and hookworm were found to be viable. Compost
(H) was derived from waste to which activated sewage sludge had been
added before composting. Although the exact constituents of Compost
(L) were not learned, the bulk of this compost was known to be composted
animal excreta and peat moss.
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TABLE 3. Recovery of helminth ova and larvae in marketed compost materials
Type of sample Organisms recovered/0.05 ml
(source) . T
Ova Larvae
a b
Compost (H) - Hookworm, viable (2)
Compost (H) - -
Compost (L) Trichuris tridhwpla, Strongyloides, viable (1)°
viable (1)
b
Taenia (1)
Hymenolepis diminuta (2)
Compost (F)
Milorganite -
None found.
Recovered by the formalin-ether centrifugal sedimentation method.
£
Recovered by the brine gravity flotation method.
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DISCUSSION
Compost derived from municipal solid waste, with or without sewage
sludge, contains a great variety of materials, most of which are either
lighter or denser or smaller or larger than cysts, ova, and larvae of
parasites. In using a concentration method, it was of importance to
separate, as completely as possible, the cysts, ova, and larvae of para-
sites from all other elements of compost. This was accomplished by
screening, by sedimentation, and by flotation. The two concentration
methods, brine gravity flotation and formalin-ether centrifugal sedi-
mentation, incorporated all three of these principles. The screening
was achieved by filtering the sample through an 18- by 16-mesh nylon
sieve; sedimentation, by treatment with 10% formalin and ether, followed
by centrifugation; and flotation, by application of a saturated aqueous
solution of sodium chloride. Our experimental studies showed that the
formalin-ether centrifugal sedimentation method was usually more effec-
tive than the brine gravity flotation method for recovering both ova
and larvae; the direct film method was least efficient. The viability
of ova was based on direct observations of i/n vivo embryo motility with
a light microscope.
To compare the parasite recovery ability of the two concentration
procedures, a series of samples were examined: marketed and stockpiled
compost, and a mixture of compost and raw sewage sludge known to con-
tain parasites.
Nonviable helminth ova and larvae were demonstrated in various ages
of stockpiled compost. One sample contained ova of Hymenolepis dimlnuta
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and other larva of Strongylo-ides. Since no hatchability tests were made
during this preliminary study, the significance of these parasite forms
cannot be ascertained.
The incidence of helminth ova and larvae in marketed compost was
significant. Two out of five samples were positive. One sample contained
viable ova of Triehuris triehiurla and viable Strongytoides larvae; viable
hookworm larvae were found in another sample. As Chang (2) pointed out,
if hookworm or StTongylo-ides larvae are present in the marketed compost,
applying the product where humans walk barefooted or where the larvae
may be carried by food or water can spread the infection. Gotaas and
others (9, 10, 14), however, found that compost derived from municipal
solid waste without sewage sludge is generally accepted as being innocuous
from a health standpoint; but, the compost from solid waste and sewage
sludge or from animal excreta has the potential hazard of a residual
pathogen content.
Kabler (13) stated that the sludges derived from digestion processes
are apt to contain large numbers of parasites. To render this material
innocuous, it must be heat treated at 57.2 C for 1 hr or exposed to
extended composting or drying. Drying for 12 to 15 months is sufficient
to render Myeobaoteiriiffn tuberculosis nonviable; however, the moisture
content in sludge must be at a very low level to destroy completely the
viability of the helminthic ova.
These preliminary findings indicate the need of further studies
to determine the potential public health hazard of parasites in improperly
disposed of solid waste and in compost derived from municipal solid waste
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and sewage sludge. This is especially true in relation to monitoring
all solid waste components for disease potential. Such studies would
include: a standardized methodology for parasitological examination
of waste materials; studies on survivability and transmission of parasites
through disposed, untreated waste; studies on migration of parasites
through soil from leached solid wastes; and, finally, studies of treatment
methods and environmental factors affecting parasite destruction in
solid waste.
ACKNOWLEDGMENTS
I wish to thank Harry Stierli and John Ruf for providing the samples
for testing and Doctors Shih L. Chang and Paul W. Kabler for reviewing the
manuscript.
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