Parasites and the Land Application of Sewage Sludge
Research Report No. 110
I td 5 Program for the Abatement of Municipal Pollution
' G '^\ 3r Provisic Ontario Agreement
P37 on at Lakes Water Quality
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CANADA -ONTARIO AGREEMENT
These RESEARCH REPORTS describe the results of investigations
funded under the Research Program for the Abatement of Municipal
Pollution within the provisions of the Canada-Ontario Agreement on Great
Lakes Water Quality. They provide a central source of information on the
studies being carried out in this program through in-house projects by
both Environment Canada and the Ontario Ministry of Environment, and
contracts with municipalities, research institutions and industrial
Enquiries pertaining to the Canada-Ontario Agreement RESEARCH
PROGRAM should be directed to -
Wastewater Technology Centre
Canada Centre for Inland Waters
P.O. Box 5050
Burlington, Ontario L7R 4A6
Ontario Ministry of Environment
Pollution Control Branch
135 St. Clair Avenue West
Toronto, Ontario M4V 1P5
TD Parasites and the land
774 application of sewage sludge /
.G73 Graham, H. J.
PARASITES AND THE LAND
APPLICATION OF SEWAGE SLUDGE
Ontario Ministry of the Environment
RESEARCH PROGRAM FOR THE ABATEMENT
OF MUNICIPAL POLLUTION WITHIN THE
PROVISIONS OF THE CANADA-ONTARIO
AGREEMENT ON GREAT LAKES WATER QUALITY
Project No. 74-1-41
This document may be obtained from -
Training and Technology Transfer
Environmental Protection Service
Ontario Ministry of the Environment
Pollution Control Branch
135 St. Clair Avenue West
e Minister of Supply and Services Canada 1981
Cat. No. En 43-11 IIOE
BEAUREGARD PRESS LIMITED
Parasite ova, mainly Ascaris and Toxocara , were recovered from
many Ontario sewage sludges, and from sludged fields. These ova can
survive on pasture for several years, are probably destroyed on bare soil
within one year and possibly survive only a few weeks when the sludge is
mixed with the soil. This information, together with the type of
parasites present in Ontario and the numbers found in the digested sludge,
make noticeable parasite transmission, via sludge spread on farmland, a
remote possibility. Recommendations are made for the use and handling of
sludge that will reduce the possibility of transmission even further.
On a retrouve des oeufs de parasites, Ascaris et Toxocara
surtout, dans beaucoup de boues residuaires de l'Ontario et dans des
champs d'epandage. Dans les patu rages, ces oeufs peuvent survivre pendant
plusieurs annees; ils sont probablement detruits en nx>ins d'un an quand
ils se trouvent sur le sol nu et ne peuvent survivre que quelques semaines
quand les boues sont melangees au sol. Ces renseignements , compte tenu du
type de parasites presents en Ontario et de leur nombre et dans les boues
digerees, portent a croire qu'une transmission marquee de parasites par
l'epandage des boues sur les terres agricoles demeure une possibilite
lointaine. On emet des recommandations qui permettront de reduire encore
davantage les possibilites de transmission liees a 1 'utilisation et a
l'epandage des boues.
TABLE OF CONTENTS
TABLE OF CONTENTS
List of Figures
List of Tables
1 INTRODUCTION !
1*1 Possible Parasites in Ontario Sewage 1
1.2 Effect of Sewage Treatment 7
2 PARASITES FOUND IN ONTARIO SEWAGE SLUDGES 12
2.3 Results and Discussion
2.4 Summary and Conclusions
3 SURVIVAL OF PARASITES ON SLUDGED FIELDS 27
3.2 Ova Recovered from Sludged Fields 30
3.2.2 Results and discussion
3.3 Orangeville Experimental Plot 33
3.3.2 Results and discussion
3.4 Summary and Conclusions
4 PARASITES AND FARMLAND APPLICATION OF SLUDGE IN ONTARIO 52
4.1 General 52
4.2 Recommendations 54
4.2.1 Application of sludge 54
4.2.2 Sludge applied to pasture 54
4.2.3 Sludge applied to cultivated fields 55
APPENDIX I - PARASITE LIFE CYCLES 65
APPENDIX II - PARASITE OVA RECOVERY FROM SLUDGE USING THE
ZONAL ROTOR ei
LIST OF FIGURES
1 Unembryonated Ascaris Ovum Recovered from Raw Sludge 16
2 Trichuris Ovum Recovered from Raw Sludge 16
3 Hymenolepis diminuta Ovum, a Tapeworm of Rodents and
Occasionally of Man, Recovered from a Recently Sludged
Field Near Owen Sound 22
4 Decorticated Ascaris Ovum Containing Numerous Globules 32
5 Taenia Ova from a Cat 36
6 Decorticated Ascaris Ovum with fully Formed Larva Recovered
from the "Pasture" of the Orangeville Experimental Plot
539 Days after Application 43
7 Toxocara Ovum Containing an Active Larva, Recovered from
the "Pasture" of the Orangeville Test Plot 539 Days after
11. 1 Side View of Zonal Rotor and Associated Equipment Needed
to Withdraw the Gradient 83
11. 2 Graph Showing Typical Gradient Density as it was Removed
from the Column 84
LIST OF TABLES
1 Parasite Ova Found at Lakeview Sewage Treatment Plant 17
2 Parasite Ova Found in Ontario Sewage Sludge 18
3 Number of Enterobius vermicularis (Pinworm) Ova Recovered
when Incubated at 25°C and 37°C in Water and Sludge 24
4 Characteristics of Parasite Ova Recovered from Digested
Sewage Sludge 25
5 Parasite Ova in Orangeville Sludge Added to Test Plot 35
6 Summary of Temperatures Recorded at Orangeville Test Plot 38
7 Mean Monthly Temperatures Recorded on Visits to Test
8 Percent Weight of Water in Bare Soil from Experimental
Plot, with and without Sludge 41
9 Ascaris Ova found in Sludge Residue on Top of Bare Soil
at Experimental Plot 42
10 Ascaris Ova Recovered from "Pasture" Area of Experimental
Plot "" 44
11 Toxocara and Taenia Ova Recovered from "Pasture" Area of
Experimental Plot 47
12 Ascaris Ova Recovered in Core Samples from Experimental
In Ontario about 40 percent of municipal sewage sludge is
disposed of on farmland (Black and Schmidtke, 1974). This occurs mainly
in rural areas where this type of disposal suits the small scale of the
operation and the low haulage costs. It has been practised for many years
with mutual benefit to the farmer and the municipality. The farmer
receives a soil conditioner and fertilizer, and the municipality gets rid
of a problem waste product.
Many possibly hazardous contaminants in sewage sludge have been
suggested, from heavy metals to viruses. Frequently parasites are
mentioned as possible health hazards.
No documented evidence has been found linking human infection
directly or indirectly with digested sewage sludge, but parasites have
been reported from sludge and may cause some infections.
The purpose of this study was to:
1) summarize the relevant literature,
2) determine the incidence of parasites in Ontario sewage sludges,
3) determine the length of time the ova and/or cysts of these
parasites remain viable once the sludge has been applied to
4) to make recommendations to minimize any threat of parasitic
infection from sewage sludge spread on farmland.
1 • 1 Possible Parasites in Ontario Sewage
Parasites are a very diverse assortment of organisms including
members of the protozoa (some of which produce malaria), worms and even
arthropods such as fleas. However, the nature of their life cycles and
the intermediate hosts required mean that relatively few can be
transmitted in sewage sludge and fewer still can be transmitted in
Only those parasites present in the human and animal population
of Ontario, which may be transmitted via sewage sludge, will be
considered. Details on their life cycles are presented in Appendix I.
It is difficult to obtain exact figures on the level of parasitic
infection in the population of Ontario and harder still to find the number
of infections acquired, but some data are available from the average of
over 23 000 samples per year submitted to the Ministry of Health for
parasitic examination in 1970-1974 (Scholten and Yang, 1975).
The first parasites to be considered are those with "direct" life
cycles, that is to say, ones that require no intermediate host. This
category covers the important protozoan parasites.
These parasites normally live in the human intestine. Infection
is acquired when cysts, shed in the faeces, are ingested. Entamoeba
histolytica , Giardia lamblia and Balantidium coli were recovered by the
Ministry of Health an average of 435, 970 and five times per year,
respectively. Due to the low numbers of B. coli recovered, this parasite
is apparently scarce in the population of Ontario and will not be
Cysts of £_;_ histolytica , the human parasite causing amoebic
dysentery, have been reported from sewage by several authors (see Rudolfs
et al, 1950). Positive identification is difficult when the host is not
known since many animals have their own species of Entamoeba , such as E.
muris in rats and mice, and E. invadens in reptiles (Cheng, 1973). There
is also a free-living amoeba E^ moshkovskii that is morphologically
similar to E^ histolytica (Page, 1976). E*_ histolytica has a worldwide
distribution with infection rates varying from eight to 85 percent in some
populations (Cheng, 1973).
The other important protozoan parasite, Giardia lamblia , does not
invade the tissues of the body and, in adult hosts, is frequently
asymptomatic, but the infection can be serious in children. The human
infection rate in North America is thought to be about two to four percent
although rates up to 22 percent have been found (Healy, 1978).
At a September, 1978, conference on Giardia it was reported that
Giardia from many mammals will apparently infect man. Two recent
waterborne outbreaks at Berlin, N.H., and Camas, Washington, were probably
caused by cysts from beaver (Lippy, 1978, and Kirner et_ al_, 1978).
Documented outbreaks have been associated with improper or insufficient
water treatment. There have been no recorded cases of foodborne
transmission but person-to-person transmission has been reported in
day-care nurseries (Keystone et al, 1978).
Many Giardia cysts from man and other mammals would be expected
in sewage. Fox and Fitzgerald (1978) detected from 90 to over 500 cysts
per litre in some raw sewage samples from the Chicago area.
The only human tapeworm with a direct life cycle, Hymenolepis
nana , is found throughout the world, but there are no reports of this
parasite being found in sewage sludge. The ova are not very resistant to
heat or desiccation and cannot survive for long outside a host (Brown,
1975). Transmission would, therefore, most likely depend upon immediate
contact with the ova, rather than through contaminated food or water.
Children have a higher prevalence of infection than adults because they
are more likely to be exposed to direct faecal contamination. Indirect
infection of man could occur with this worm since the ova are also
infective to mice, rats and grain beetles, and cross-infection can occur
between these hosts (Noble and Noble, 1964). In faecal samples submitted
to the Ministry of Health from 1970-1974, H. nana was found in about 20
samples per year (Scholten and Yang, 1975). This low prevalence of
infection would result in very few ova in sewage. Therefore, at least in
Ontario, there is probably a minimum risk of aquiring the infection from
The largest group of parasites with direct life cycles, possibly
capable of being transmitted to man in sewage sludge, are the nematodes.
These include: the human roundworm Ascaris lumbricoides , the pig
roundworm A^ suum, the common whipworm Trichuris trichiura , the hookworms
Necator americanus and Ancylostoma duodenale , the threadworm Strongyloides
stercoralis , the pinworm Enterobius vermicularis , and the roundworms of
dogs and cats, Toxocara canis and J_^ cati .
A. lumbricoides , T. trichiura and the hookworms are the most
common helminths in Ontario and were found by the Ministry of Health in
human faecal samples an average of 350, 1730 and 630 times per year,
respectively, between 1970-74 (Scholten and Yang, 1975). These worms
become adults in the human intestine and shed ova which can be found in
the faeces. It has been estimated that each mature female A. lumbricoides
can produce more than 200 000 ova per day (Chandler and Read, 1961).
Under favorable conditions the ova develop in about three weeks to
infective larvae (WHO, 1967). In the case of A. lumbricoides and T.
trichiura the infective larvae remain in the ova and must be ingested by a
suitable host to complete their life cycles. The larvae of hookworms, on
the other hand, hatch into free-living larvae that eventually molt to
become non-feeding, infective larvae that must wait on the soil surface to
penetrate human skin, then migrate to the intestine to complete their life
The pig ascarid (A^ suum ) may be able to complete or partially
complete its life cycle in humans (Takata, 1951). Some larvae were
apparently able to reach the lungs and a few even reached the intestines
and grew to adults. However, the period ova were produced was relatively
short, indicating an abnormal cycle.
The ova vary in their ability to withstand environmental factors.
Ascaris lumbricoides ova are extremely resistant to both heat and
desiccation and are often used as the bench mark for parasite destruction.
When no Ascaris ova survive, other parasite ova are assumed to be dead
also (Krige, 1964). Under experimental conditions, ascarid ova can
survive for many years but their normal survival in soil may be much less
(WHO, 1967). Ova of T. Trichiura and hookworms can only survive a matter
of days under freezing conditions.
These nematodes are found worldwide, although their highest
numbers are found in areas where conditions are warm and wet and there is
a tendency toward random defecation, producing highly polluted soils. The
ova have frequently been reported in sewage (Gunthor, 1971; Krige, 1964;
Rudolfs etal, 1950, 1951a, 1951b; Wang and Dunlop, 1954). It is not
possible to identify the ova types in sewage that are infective to humans.
The ova of A^_ lumbricoides from humans are identical to those from the pig
ascarid. Trichuris trichiura (or T^ ovis or 1\_ suis ) has also been
reported from a pig and the ova are similar to other trichurids, from
other hosts. The human hookworms also cannot be definitely identified by
the ova alone, due to the similarity with species from other mammals.
Indigenous transmission of these nematodes in Ontario is
conceivable, especially among children in day nurseries or playgrounds
where faecal contamination can occur (Freeman, 1977). Scholten et al
(1977) found transmission of T. trichiura and S. stercoralis and several
protozoa common in an Ontario mental institution.
The threadworm Sj_ stercoralis has also been found in faecal
samples from Ontario. An average of 26 positive samples per year were
found during 1970-1974 (Scholten and Yang, 1975). The threadworm is not
common in temperate zones but is prevalent in tropical and subtropical
areas (Brown, 1975). The life cycle is similar to that of hookworms;
however, the larvae usually hatch before being shed in the faeces and
there may be one or more free-living cycles before infective larvae are
produced. Identification is difficult in sewage or soil, due to the large
number of free-living nematodes common in these habitats.
Prolonged survival in our climate is not likely since larvae are
easily destroyed by cold, desiccation or direct sunlight and are short-
lived even under the most favorable conditions (Chandler and Read, 1961).
The ubiquitous pinworm Enterobius vermicularis is concentrated in
the temperate zones, specifically North America and Europe. The prevalence
in Canadian school children is as high as 30 to 60 percent (Chandler and
Read, 1961). A "conservative overall estimate" in the U.S.A. has been set
at 30 percent in children and 16 percent in adults (Warren, 1974). Ova
seem likely to be in sewage and may be transmitted via the sludge.
However, due to its peculiar habits in humans, the ova are not usually
very abundant in the faeces. The life cycle is also direct. The adult
worms reside in the human gut and the gravid female worms migrate to the
anus and deposit their eggs in the perianal region. This causes intense
itching which leads to finger faecal contamination, especially in
children. The ova can also be spread on clothing or sheets and even
airborne with dust. The ova survive longest (two to six days) under cool
humid conditions. They are not resistant to higher temperatures with few
surviving more than 12 hours at 25°C (Chandler and Read, 1961).
Parasites that have recently become known as human public health
problems are the dog and cat ascarids Toxocara canis and T\_ cati . In
these animals, the life cycle is similar to A^ lumbricoides (ova must be
ingested by host) but in humans the larvae are unable to complete their
life cycle and only wander through the body. Usually they cause no
noticeable damage; it has been found, using fluorescent antibody tests,
that about two percent of a healthy population have experienced toxocaral
infections (Churcher, 1976; Ghadirian et_ al_, 1976). However, problems can
arise when these parasites locate in the delicate tissues of the eye or
central nervous system. Apparent links have been found with epilepsy,
enlargement of the liver and asthmatic conditions (Woodruff, 1976).
Toxocara canis is a common parasite in dogs and can be passed
from the mother to fetal puppies. This accounts for the higher incidence
in puppies (50 percent) compared with adult dogs (10 percent) as found by
Ghadirian et_ al_ (1976) in the Montreal area. They also found over 25
percent of soil and sand samples from parks contained the distinctive
Toxocara ova. The ova are very resistant to adverse environmental
conditions and can survive for years.
Important parasites that can indirectly infect man and possibly
be transmitted by sewage sludge belong to the genus Taenia and include the
beef tapeworm T^ saginata and the pork tapeworm T^ solium . Man is the
only definitive host for the adult worms. Ova are passed in the faeces,
then are eaten by the appropriate intermediate host (mainly cattle). The
ova hatch in the gut liberating larval cestodes that migrate to the
musculature; there they encyst and develop into cysticerci, which infect
man when raw or undercooked meat is eaten. Man can occasionally serve as
the intermediate host of T. solium (Brown, 1975) and rarely of T. saginata
(Pawlowski and Schultz, 1972) if the ova are ingested. This is much more
serious than having the adult worm in the gut since the larval tapeworm
can migrate to any part of the body.
Human Taenia infections in Ontario, mainly T\_ saginata since T.
solium have been diagnosed very rarely, have been found an average of
about 35 times per year (Scholten and Yang, 1975). More than half of
these were discovered by finding ova in the faeces but this method does
not reveal all infected persons. The ova are frequently not distributed
through the faeces but are often enclosed in the proglottid or tapeworm
segment when it is shed in the faeces. These proglottids are very active
and can creep out of the anus of their own volition (Chandler and Read,
1961). Each one contains about 80 000 eggs and about six are shed per day
(Pawlowski, and Schultz, 1972). Under favorable conditions the ova will
remain viable for six months (Chandler and Read, 1961).
In a United States survey of 1.8 million stool samples, 0.023
percent were positive for Taenia and at least one-third of the cases were
indigenously acquired (Schultz, 1974). "Significant transmission" does
occur in the United States according to Schultz et al (1970). Where it is
possible to conduct epidemiological studies, cattle are usually infected
by coming in contact with untreated human faeces especially in such
confined areas as feed lots (Schultz et_ al , 1970). A similar outbreak was
reported in Ontario feed lot cattle (McAninch, 1974). According to Seddon
(1950) cattle on the Werribee sewage farm have become infected with T.
saginata cysticerci when they were exposed to the raw sewage used to
irrigate the land. It has been postulated that the drinking of sewage
effluent by cattle was most important in the spread of T^ saginata
(Silverman and Griffiths, 1955), and that partially digested sludge when
used in agriculture could spread T. saginata ova (Liebmann, 1964).
1»2 Effect of Sewage Treatment
The effects of sewage treatment processes on parasitic ova and
cysts have been reviewed by many authors (Greenburg and Dean, 1958; Hays,
1977; Kabler, 1959; Liebmann, 1964; Rao, 1973; Shephard, 1971).
Municipal systems collect dilute sewage and wastes, both domestic
and industrial, and deliver it to the treatment plant. The objectives of
sewage treatment, to separate clear effluent suitable for discharge to a
lake or river and to concentrate the other material in the sludge, are
mainly achieved by settling to produce the raw sludge. In a primary
treatment plant the effluent from this initial settling is chlorinated and
discharged; in a secondary or activated sludge treatment plant this
effluent is further refined by biological oxidation and more settling,
followed by chlorination and discharge. Excess activated sludge is
returned to the primary clarifier. All raw sludge is usually digested
anaerobically by bacteria in a large heated tank. Mesophilic digestion
occurs at about 35°C and thermophilic digestion (seldom used in Ontario)
at about 55°C. Digestion is carried out to reduce the number of
pathogens, to render the material fit for further treatment (i.e.,
dewatering, lagooning and land disposal) and to reduce the volume of
sludge. In such heavily populated areas as Toronto, because of the vast
amounts of sludge and the distance from agricultural land, the sludge is
often incinerated. At smaller treatment plants the digested sludge is
frequently hauled by tanker a few miles to a suitable agricultural field.
At these latter locations parasites may be a problem requiring special
handling techniques to avoid infections.
The raw sludge is the material that settles in either the primary
or secondary settling tanks. These tanks are usually not more than 3.7 m
(12 ft) deep with a hydraulic retention time of about two hours. Cram
(1943), in her experiments with E. histolytica cysts, and hookworm and
Ascaris ova, found that in raw sewage allowed to settle for over two
hours, large numbers of amoebic cysts and some hookworm ova were still
present in the upper level, while the Ascaris ova settled readily. Using
T. saginata ova, Newton et_ al_ (1949), found that two hours of settling
removed most ova from a 46~cm (18-inch) column of raw sewage. They
concluded that the majority of the tapeworm ova would be found in the
sludge. A settling rate in sewage of 0.6 to 0.9 m/rain (2 to 3 ft/min) has
been reported for these ova (Liebmam, 1964), which would be the slowest -of
the worm ova. It was concluded that if the settling time were less than
two hours or if the tank were subjected to disturbance from winds, large
numbers of worm eggs would pass into the plant effluent or the biological
oxidation portion of the plant.
Biological oxidation takes place in an aeration tank where the
retention time is five to ten hours. Newton et^ al_ (1949) found that five
months exposure to activated sludge had no noticeable effect on T«_ saginata
ova. Cram (1943) found it had no noticeable effect on the viability of E.
histolytica cysts, or hookworm or ascarid ova. Development of the ova
proceeded normally if left in the aerated activated sludge. In extended
aeration and contact stabilization plants, which usually have no primary
settling, no detrimental effects would be expected to helminth ova.
Kabler (1959) refers to work by Bhaskaran et_ al_, where high
reductions of Ascaris , Trichuris and hookworm ova were reported for
activated sludge. Shephard (1971), referring to the same paper, was of
the opinion that the removals were for the whole treatment process, not
just the aeration tank.
Biological oxidation is followed by a secondary settling tank
where most remaining helminth ova and some protozoan cysts would be
removed from the plant effluent. This removal would be enhanced if
chemical coagulants were added prior to the settling (Rao, 1973). Cram
(1943) found that an alum floe successfully removed cysts of E.
As of 1973 and 1975, many Ontario sewage treatment plants have
been required to reduce effluent phosphorus levels below 1 mg/L (Van
Fleet, 1973). This is usually accomplished by adding alum, lime or iron
salts just before one of the settling tanks. Where phosphorus removal is
part of the treatment process most parasitic ova and cysts will be removed
from the effluent and end up in the sludge.
Trickling filters, deep beds of gravel or other substrata that
the raw sewage or primary effluent flows through, are seldom used for
municipal sewage in Ontario. Newton et^ al_ (1949) found them ineffective
in the removal of T\_ saginata ova. Cram (1943) found that while 88 to 99
percent of E. histolytica cysts were removed, those in the effluent were
still viable. Worm eggs were removed much less effectively and the
aerobic environment allowed development of hookworm and Ascaris ova.
Bhaskaran et_ al_, on the other hand, found that 98 to 100 percent of the
parasitic ova were removed (Kabler, 1959).
Sand filtration of raw settled sewage, also seldom used in
Ontario, was found effective in removing ascarid and hookworm ova and
amoebic cysts if the sand were deep enough (Cram, 1943).
Most sludge in Ontario is subjected to anaerobic digestion,
although some undergoes aerobic digestion and some from extended aeration
plants is not digested. The average solids retention time in Ontario
anaerobic digesters is 15 to 20 days, although in new plants it would be
about twice as long. Cram (1943) found that ascarid ova were little
affected by three months of anaerobic, digestion and 10 percent were still
viable after six months. Hookworm ova were not as resistant but could
survive at least 41 days at 30°C. Development of both hookworms and
ascarids was suspended under anaerobic conditions. Entamoeba histolytica
cysts were much less resistant but could still remain viable for 10 days
Fox and Fitzgerald (1978) found cysts of Giardia in some raw
sewage samples but none were detectable in aerobically digested sludge,
indicating that they may not survive this treatment.
Liebmann (1964) reported that if digestion was complete worm eggs
could be destroyed in three months in unheated digesters or two months in
heated ones. These heated digesters were probably the same as used by
Menschel (1965), where they were maintained at 26 to 28°C. In this
digester he found that 60 percent of the T^_ saginata eggs appeared to be
dead after 56 days. Silverman and Guiver (1960), also working with T.
saginata , found that these ova were non-viable when tested by an in vitro
technique after as little as five days in an experimental anaerobic
digester and failed to infect a calf, while the undigested control ova did.
Newton et^ al_ (1949), using only the morphological appearance of the ova,
found that some ova of Tj_ saginata could survive for months in digested
sludge but only half appeared normal after two months. There was also a
significant decrease in the number of ova with time.
Fitzgerald and Ashley (1977) found that ascarid ova, after 21 to
25 days in a simulated anaerobic digester at 38°C, were able to embryonate
and were subsequently infective in feeding experiments. However, one
sludge tested appeared to be lethal to these ova. They also found that a
parasitic protozoan, Eimeria , would not develop after four to five days in
sludge at 38°C.
Rudolfs et^ al_ (1951b), using Ascaris suum , found that: two hours
at 45°C had no effect; 50°C for 30 minutes retarded development; two
hours exposure at 50°C apparently killed the ova; and 55°C or 60°C killed
all the ova in 10 minutes. A U.S. Environmental Protection Agency manual
(1974) dealing with sludge treatment and disposal quoted a paper by
Roediger who found that at 50°C, cysts of Ej_ histolytica were destroyed in
five minutes and eggs of A. lumbricoides were destroyed in 60 minutes.
Keller (1951) concluded that 24 hours in a thermophilic digester
at 53 to 54°C resulted in the complete inactivation of Ascaris ova and that
this was due to the temperature, not the digestion process.
Gotaas (1956) reported that temperatures in excess of 60°C can
easily be achieved with aerobic composting. When sludge is composted and
all material is exposed to this temperature, destruction of all parasitic
organisms will occur (Almasi et _al_, 1971; Gotaas, 1956; Kawata ejt al ,
1977). In an extensive review of the effect of composting on pathogen
survival, Wiley (1962) concluded that even a few hours at these
temperatures would kill all parasites. A Water Pollution Control
Federation manual (1972) for the utilization of municipal wastewater sludge
recommends a minimum of 60°C for not less than 40 hours. Goleuke (1972)
felt that composted sewage sludge that would be exposed even indirectly to
humans should be heat sterilized since perfect control at all times would
be difficult to ensure.
Many authors have concluded that the only 100 percent effective
way of destroying pathogens in sludge is with heat (Sopper and Kardos,
1973; Hanks, 1967; Gunthor, 1971; Kabler, 1959; Rudolfs et_ al, 1951c;
Schatzle, 1969; Van Kleeck, 195«).
Sewage treatment plant effluents are frequently chlorinated,
especially when human exposure is expected. Rudolfs et^ al_ (1950), in an
extensive review of the data up to 1950, found that if proper experimental
procedures were used, chlorine residuals of 2 to 15 mg/L would kill E.
histolytica cysts given enough contact time. Kott and Kott (1967) found a
dose of b mg/L chlorine, with six hours contact time, would kill these
cysts. These data indicate an even shorter contact time may suffice.
Kabler (1959) found little data on the effect of disinfection practices on
parasitic ova, but what there was indicated a strong resistance to chlorine.
Krishnaswarai and Post (1968), found Ascaris ova very resistant to chlorine
residuals over 100 mg/L. Hays (1977) concluded that chlorine residuals
normally found in treated water and sewage were not harmful to ova or cysts.
Although the specific effects of sewage treatment processes have
not been examined in much detail, sufficient work has been done to say
that, of the parasite ova and cysts that gravitate to sludge, Ascaris ,
Toxocara and probably Trichuris ova can survive normal sewage treatment
including digestion; Taenia , Hymenolepis and hookworm ova would normally be
found after digestion but in reduced numbers; and E_. histolytica and
probably Giardia cysts would normally be destroyed. But due to plant upsets
short circuiting or by-passing, any parasite in the raw sewage could be
found in the digested sludge. Tha only effective way of ensuring parasite-
free sludge is with several hours of heat treatment at more than 60°C.
The situation is similar with treatment plant effluent. If a
primary plant was working properly, most nematode ova would settle into
the sludge, while some Taenia ova and a larger portion of protozoan cysts
may pass into the effluent. In a secondary plant most parasite ova and
cysts would be removed from the effluent, especially if a chemical
coagulant was used. But inadequate treatment could result in any parasite
ova or cysts in the raw sewage appearing in the plant effluent.
Disinfection with chlorine apparently has little effect.
2 PARASITES FOUND IN ONTARIO SEWAGE SLUDGES
2. 1 Introduction
The numbers and kinds of ova found in any sewage depend on the
level of infection of the population using the system, so one would not
expect the same results in the tropics and the far north. No reports were
found on the incidence of parasites in sewage in Ontario but data are
available from other parts of the world. In Haifa, Israel, the amoeba
E . histolytica has been found at an average of four cysts per litre in raw
sewage and less than one cyst per litre in final effluent (Kott and Kott,
1967). Cram (1943) found cysts similar to E. histolytica in the sludge
from several municipalities and army camps in California. Cysts of E.
histolytica were found in the sewage of Moscow, Russia, and Johannesburg,
South Africa, (Rudolfs e^ aJ_ 1950). Fox and Fitzgerald (1978) reported 90
to 530 Giardia cysts per litre in some samples of Chicago raw sewage.
Helminth ova have also been frequently reported in sewage. One
survey found 1933 ova per litre of Moscow sewage, including those of A.
lumbricoides , T. trichiura , E. vermicularis and Diphyllobothrium latum
(Shephard, 1971). In Tokyo, Japan, Aiba and Sudo (1964) found mainly A.
lumbricoides , fewer T. trichiura and rarely hookworm ova. The numbers of
Ascaris were 80/L in raw sewage, 700/L in digested sludge and none in the
effluent. Cram (1943) found no helminth ova in the municipal sludges of
California but Ascaris and other parasite ova appeared frequently in the
sludge from army camps. Grabow and Nupen (1972) found 119, 87 and 13 ova
per litre of Ascaris , Taenia and Trichuris , respectively, in the sewage
from a subtropical town in South Africa.
These values, while interesting, are of limited value in
assessing Ontario sewage. There is the additional problem of parasite ova
from other animals, indistinguishable from those from humans, also being
present. Liebmann (1964) estimated that only 10 percent of the ova found
in central European sewage and 30 percent of the ova found in the sewage
of southern Europe and subtropical regions were of human origin. On the
other hand, it was reckoned that most Ascaris ova found in a Pretoria,
South Africa, survey were of human origin (Shephard, 1971).
Several sludges from southern Ontario were studied to determine
the number and type of parasite ova and cysts present. Parastic protozoan
cysts are difficult to identify accurately, especially if scarce, when few
good specimens are present. Thsrefore, more effort was spent examining
samples for parasitic ova, which are relatively easy to identify.
Helminth eggs and protozoan cysts are frequently present in
faeces in relatively large numbers. Egg counts up to 100 000 per gram
may be found in sheep heavily infected with Haemonchus contortus (British
Veterinary Assoc, 1964). An estimated 15 000 000 cysts of £. histolytica
can be excreted daily by a single carrier (Kott and Kott, 1967). Detection
methods range from the direct smear to concentrating techniques, which
usually rely on tne differences in density of the ova and cysts compared
with the other faecal material.
Faust et_ a_l (1938, 1939) advocated a zinc sulphate centrifugal
flotation technique that would float the ova and cysts to the surface
while allowing the other faecal material to sink. Others using this
technique preferred such chemicals as sugar or sodium chloride (Chandler
and Read, 1961), and sodium nitrate or sodium dichromate (Alcaino and
Baker, 1974). However, this flotation technique is not universally
effective for recovering all parasite ova. The method will not float the
operculated ova of trematodes and probably of Diphyllobothrium (McDonald,
1920) nor the porous eggs of the taeniids (Chandler and Read, 1961). To
overcome the faults, Allen and Ridley (1970) modified the formalin-ether
technique so that all parasite ova and cysts sank while most faecal
Formalin-ether and flotation techniques (U.S. Department of the
Army, 1961) were used routinely in this study. They were not very
successful for recovering parasite ova from sewage sludge because only a
small sample volume (1 to 7 mL) could be examined in each test. Thus
estimates on the number of ova present were not accurate.
Rowan and Gram (1959) were able to analyze large volumes of
sewage for helminth eggs by having the samples flow over a saline solution
in a specially designed tray. The technique was applied to raw sewage
effluent and river water; it collected the helminth ova together with all
the dense material. Marquardt (1961) used a density gradient of sucrose
in centrifuge tubes to separate nematode ova from faecal debris. This
technique successfully separated the ova from both the lighter and heavier
faecal material but was limited to a relatively small volume.
Density-gradient centrifugation has been used repeatedly to
separate small organic and inorganic particles from other substances
(Lammers, 1%3, 1967, Lyttleton, 1970; Pertoft, 1970). Bowen et_ al_ ( 19 72 )
used density-gradient centrifugation to separate various planktonic
organisms. All these methods were limited to a relatively small sample
volume unless the particles were concentrated beforehand in a continuous-
flow centrifuge as done by Lammers (1962). Zonal centrifugation combines
these two steps. Since it is a continuous-flow centrifuge, large sample
volumes can be examined, and the sample flows over the top of a density-
gradient so the pertinent particles can be concentrated in a narrow band
by a suitable gradient. Price et al (1973) used this technique to separate
spinach chloroplasts from other plant material. This is essentially the
technique that was finally adopted to examine sewage sludge quantitatively
for parasite ova (Appendix II).
The actual numbers of ova in the sludge samples would be higher
than indicated since the technique was only 80 to 90 percent effective
when tested with known numbers of Ascaris and Toxocara ova. An efficiency
factor has not been included in any of the estimates recorded in this
report. The method allowed recovery of ova from most sludge samples so
that at least qualitative estimates could be made. The permeability of
Taenia ova makes their density less predictable than that of nematode ova,
rendering the zonal rotor technique quite inefficient for their recovery.
No suitable method was found to examine large quantities of sludge for
Taenia ova so the regular formalin-ether technique was used as a poor
2. 3 Results and Discussion
Sludge samples were initially taken from only the Lakeview sewage
treatment plant (Mississauga , Ontario) to develop suitable techniques to
detect parasite ova. The Lakeview plant is a large activated sludge,
secondary treatment plant having two-stage anaerobic sludge digestion.
Phosphorus removal at the plant using iron salts was initiated only before
the last samples were taken. Techniques to detect parasite ova in faecal
samples (direct smear, formalin-ether, zinc sulphate and acid-ether) were
used on the sludges exclusively over almost a year, with only a few
samples being positive for Ascaris (Figure 1), Trichuris (Figure 2) and
Toxocara ova (Table 1). When ova were found in the small-sized samples
( 1 to 7 mL) using these techniques, the estimate of ova per litre of
sludge was erroneously high, up to 500/L. These techniques were therefore
This led to the use of the zonal rotor (Appendix 11) with
density gradients of sucrose and sodium silicate and much larger sludge
samples of 50 to 400 mL (Table 1). Parasite ova were recovered from every
sample of Lakeview sludge and the numbers of ova, at least of Ascaris ,
were sufficient (four to nine per sample of digested sludge) that the
estimated number per litre be considered reasonably accurate.
Most raw sludge samples examined using the zonal rotor were from
Lakeview treatment plant. Ascaris was most abundant, being recovered from
over half of the samples and in those the estimated number was from 10 to
100/L (average 35/L) (Table 1).
Ascaris ova were recovered from all samples of digested sludge
from the Lakeview plant analyzed with the zonal rotor. The numbers ranged
from 10 to 180/L (Table 1) and the average (90/L) was over twice that
found in the raw sludge. This increase in concentration was not readily
apparent for other ova, possibly due to the low numbers recovered.
Digested sewage sludges from 13 other treatment plants in
southern Ontario were examined. Ten of these locations used an activated
sludge process for sewage treatment; the other three were primary plants.
Digestion was: anaerobic except at Unionville where it was aerobic;
mesophilic (35°C) except at Elmira which was thermophilic (55 °C); and
two-stage except at Unionville and Parry Sound which were single-stage.
The digested sludges were used almost exclusively on agricultural land.
The zonal rotor was used to analyze sludge samples from nine
locations (Table 2). Ascaris were again recovered roost frequently and
were found in samples from seven of the plants. The average number per
litre was eight and the average number per gram of dry sludge was 0.29.
Toxocara ova were found at four plants at an average of 15/L of wet sludge
and 0.2/g of dry sludge. Hookworm and Hymenolepis ova (Figure 3) were
each recovered only once.
FIGURE 1. UNEMBRYONATED ASCARIS OVUM RECOVERED FROM
RAW SLUDGE. Size 67 x 54 pm. This ovum
developed an active larva after incubating
one month at room temperature.
FIGURE 2. TRICHURIS OVUM RECOVERED FROM RAW SLUDGE
Size 56 x 27 um.
TABLE 1. PARASITE OVA FOUND AT LAKEVIEW SEWAGE TREATMENT PLANT
Vo 1 ume
Pos i t i ve
per Pos i
5 1 udge
D i rect
Formal i n-
0.05 None found
50 None found
50 None found
50 None found
*0n most initial samples both the formalin-ether and the zinc sulphate tests were done.
TABLE 2. PARASITE OVA FOUND IN ONTARIO SEWAGE SLUDGE
2-3 None found
2-3 None found
2-3 None found
1-2 2-13 0.1-0.7
1 7 0.2
TABLE 2. (CONT'D)
2-3 None found
TABLE 2. (CONT'D)
Number of Ova per
1 None found
TABLE 2. (CONT'D)
50 None found
FIGURE 3. HYMENOLEPIS DIMINUTA OVUM, A TAPEWORM OF
RODENTS AND OCCASIONALLY MAN, RECOVERED
FROM A RECENTLY SLUDGED FIELD NEAR OWEN
SOUND. Size about 70 um in diameter.
The other methods of examining sewage sludge revealed Ascaris ,
hookworm and Toxascaris ova (Table 2).
Most nematode ova require aerobic conditions for the ova to
embryonate. Hookworms will develop and hatch in 24 hours if the
conditions are suitable (Chandler and Read, 1961). The ova from the
sludge appeared normal. The nematode ova, Ascaris, Toxocara and
Trichuris , were all undeveloped when found and several developed normally
when incubated at room temperature. These ova were all from anaerobically
digested sludge (Tables 1 and 2). An apparently normal undeveloped
hookworm ova was recovered from aerobically digested sludge. Thermophilic
digestion (55°C) was used at Elmira (Table 2) on an experimental basis
(Smart and Boyko, 1977). Although the zonal rotor was not used to analyse
sludge from this plant, a hookworm ovum was recovered from the raw sludge,
while no parasite ova were found in the heat-treated sludge.
All the plants shown in Table 2 used a chemical coagulant to
reduce phosphorus in plant effluent. Eight used iron salts, four alum,
and one lime. Lime has been used to kill hookworm ova in night soil
(Chandler and Read, 1961). Rudolfs et al (1950) reported that lime
prevented the development of hookworm ova, while Seddon (1950) found that
small quantities of lime were ineffective against Taenia ova but
sedimentation plus lime treatment "might entirely remove T_. saginata eggs
from sewage". Lime was used at the London, Ontario, sewage treatment
plant (Table 2) where normal appearing Ascaris and Toxocara ova were
To test the effect of lime on ova in sludge, two batches of
Orangeville sludge were used. The same number of Ascaris suum ova was
added to each and sufficient lime was added to one to raise the pH to
nine. They were incubated at 31 °C. When examined after three, eight, 11
and 17 days, there was essentially no difference in the numbers of ova
found. The experiment was repeated with the pH increased to 11.2. After
three days, fewer ova were found, but after eight and 14 days the
differences were minimal. The results indicate that to have any effect
the pH must be increased to above 11, and even then not all ova are
The numbers of Ascaris ova found in the Lakeview digested sludge
samples analyzed using the zonal rotor were much higher than those found
in sludge from other plants. However, the number of Toxocara ova was
similar, with 10 to 40 per litre being found at Lakeview (Table 1) and tw<
to 40 per litre of digested sludge (Table 2) from the other plants. The
higher number of Ascaris ova at Lakeview probably is due to waste from a
Ascaris ova were commonly found in digested sludge but Trichuris
were only rarely found. The pre valance of T. trichiura in the human
population of Ontario appears to be almost five times higher than A.
lumbricoides , judging by the samples analyzed by the Ministry of Health
(Scholten and Yang, 1975), leading to the speculation that many of the
Ascaris ova lound were pig paras ites.
Because of the high incidence of pinworm ( E. vermicularis )
infection in North America, some ova were expected in the sewage sludge.
These ova have been found in the sewage of Moscow (Shephard, 1971).
Although Liebmann (1964) listed Oxyuris ( Enterobius ) vermicularis as
common in sewage, and Hays (1977) listed it as a parasite egg often found
in sewage, none was found in either raw or digested sludges analyzed in
A short experiment was devised to determine why this should be.
Pinworm ova collected from worms picked off the perianal region of a
heavily infected child were evenly distributed in 20 test tubes; half
contained distilled water, the other half contained digested sewage sludge
from Orangeville. Samples from each group were incubated at 25°C and 37 °C
and analyzed at various times (Table 3). The ova in sludge at 25°C
survived longer than those at 37 °C but few completed development to
larvae. No ova were found after four days in sludge incubated at 37 °C.
Many ova survived six to eight days in water at both 25° and 37 °C. At
37 °C development of larvae was almost complete after four days.
TABLE 3. NUMBER OF ENTER0B1US VERMICULAR1S (Pinworm) OVA RECOVERED
'C AND AT
IN WATER AND
62 ( 2)
81 ( 1)
*Number of ova with fully embryonated larvae are shown in parentheses.
Since most sludge is digested at 35°C for 15 to 20 days it is
not surprising that no pinworm ova were found in Ontario sludges.
In most cases, the exact species of parasites found in the sewage
sludges, and hence the host they could infect, could not be determined.
The Ascaris ova found had the typical morphology of the human parasite
( A. lumbricoides ) and had an average size of 6b. 4 x 53.7 ym (Table
4). However, they were also identical to the ascarid from pigs, A. suum ,
which is physiologically different from the human strain but may complete
the first portion of development in man (Morgan and Hawkins, 1949), and
may even produce some ova for a short time (Takata, 1951). The Trichuris
ova recovered had an average size of 56 x 27.7 pm, which would not be T.
vulpis from the dog or fox but could be T_*_ trichiura from man, T\_ suis
from pigs or Capillaria contorta from chickens. Most of the Toxocara ova
recovered had an average size of 84.3 x 70.3 urn which is identical to T.
canis of dogs and foxes, while one Toxocara ova was smaller with a size
of 76 x 70 um which is similar to T. cati of cats. The one hookworm ovum
that was measured (60 x 38 um) was probably from a dog or cat. There are
many species of Taenia , all with identical ova, so that neither definitive
nor intermediate hosts could be determined. The two ova that can be
identified with some certainty, Hymenolepis ditninuta from rats and mice,
and Toxascaris leonina of dogs and cats, were recovered only once.
TABLE 4. CHARACTERISTICS OF PARASITE OVA RECOVERED FROM DIGESTED
Average Median Range of Median
Average Size Size Range Density where Densities where
Parasite (ym) (ym) Ova Recovered Ova Recovered
with outer 66.4 x 53.7 (7)* 63 x 50 - 1.14 (8) 1.104 - 1.20
coat 69 x 57
outer coat 57 x 44 (1) - 1.12 (5) 1.037 - 1.182
Trichuris 56 x 27.7 (3) 56 x 27 -
59 x 28
Toxocara I 84.3 x 70.3 (4) 81 x 60 - 1.069 (5) 1.026 - 1.104
88 x 76
Toxocara II 76 x 70 (1) - 1.117 (1)
Hookworm 60 x 38 (1) - 1.085 (2) 1.040 - 1.131
*Number of ova are shown in parentheses.
Table 4 gives the median densities of the tubes containing the
density gradient from the zonal rotor where the ova were found. These
were determined with a ref ractometer (Appendix II). The Ascaris ova with
the outer coat intact were the heaviest ova found in this study, being
recovered in solutions with an average density of 1.14. The range was
quite wide, from 1.1 to 1.2, although 87 percent were recovered between
1.10 and 1.15. The Ascaris ova with the outer coat removed were slightly
less dense. Tne Toxocara ova were lighter still, being recovered from
solutions with an average density of 1.07.
There were insufficient data for any difference in parasite
survival to be apparent between primary and secondary sewage treatment or
between single or two-stage digestion.
2.4 Summary and Conclusions
A method using a zonal rotor was developed to recover parasite
ova from sewage sludge. This technique was reasonably efficient at
recovering nematode ova but not as successful on the porous taeniid ova.
The numbers of ova recovered allowed reasonably accurate estimates of the
number of ova present.
Parasite ova are present in digested sewage sludge from Ontario
sewage treatment plants. Ascaris ova were found in most sludges analyzed
using the zonal rotor at an average of 33/L of liquid sludge or 1.3/g of
dry sludge. Toxocara ova were found in sludges from half the plants.
The estimated concentration was 20/L of wet digested sludge and 0.6/g of
dried sludge. Trichuris , Taenia , Hymenolepis and hookworm ova were
recovered only once from the digested sludges analyzed using the zonal
rotor. Using other techniques, Ascaris , Trichuris , Toxocara and
Toxascaris ova were found.
The Ascaris , Trichuris and Taenia ova could have come from
either man or animals, while the Toxocara , Toxascaris , Hymenolepis and
probably tne hookworm ova were not of human origin. The ova appeared
normal, and Ascaris and Toxocara ova embryonated when incubated.
The digestion process appeared to have concentrated the ova at
one treatment plant from which samples of both raw and digested sludge
were analysed. More than twice as many Ascaris ova per unit volume were
Anaerobic digestion of sludge had no apparent affect on any
parasite ova except those of pinworms. Probably aerobic digestion would
have no effect either, while thermophilic digestion probably would
destroy all parasites.
3 SURVIVAL OF PARASITES ON SLUDGED FIELDS
3 . 1 Introduction
This chapter describes investigations to determine if parasite
ova could be recovered from farm fields in southern Ontario normally
fertilized with digested sludge, and field experiments into the effects of
the natural conditions in Ontario on the viability of parasite ova.
The survival of parasite ova and cysts, once the digested sludge
has been applied to the soil, depends on many factors, including the
resistance of the parasites, the type of soil, the depth of the parasite
in the soil and the moisture and temperature of the soil. Temperature at
the soil surface depends largely on the effective solar radiation and the
soil moisture. The effective solar radiation is affected by the colour,
slope and vegetation cover, while the soil moisture is dependent on the
surface and soil drainage, the organic content of the soil and the season
(Buckman and Brady, I960). These conditions would differ for each
location where sludge is applied, making parasite survival variable.
Rudolfs et_ al_ (1950) summarized most of the work prior to 1950
concerning parasite survival in soil. Little work has been done on E.
histolytica , but in one study the cysts only survived six to eight days in
warm, moist soil. Hookworm larvae only lasted six to 12 weeks under
favorable conditions, only three weeks at 35°C and only one week at 0°C.
Ascarid ova, including Ascaris and Toxocara , could resist freezing
conditions and remained viable over the winter, especially under snow.
Clay, loam or humus soils that retain their moisture, together with a
shaded soil surface, increased the survival of Ascaris ova. Trichuris ova
required more moisture for development and died under moisture conditions
favourable for Ascaris .
Two World Health Organization (WHO) expert committees also
summarized some of the work on survival of helminth ova in soil (WHO,
1964, 1967). The development of hookworms was inhibited at 10°C and at 45
to 50°C the larvae were killed. They also could not survive freezing
temperatures. With Ascaris ova the greatest mortality occurred in warm
seasons and all were killed in soil temperatures of 45°C or higher. Only
undeveloped ova were able to survive 90 days at -12° to -15°C. Under
laboratory conditions Ascaris ova remained infective for years but in soil
they survived for relatively short periods. Some eggs were reported to
withstand more than two years exposure on fields in a temperate region.
Spindler (1929), working with Ascaris ova in soil, found that
even with continuous faecal contamination there was no appreciable buildup
Of infective ova, leading him to believe that the ova do not survive for
many years and may not even live through a winter.
beaver (1975) showed that on bare soil, rain can concentrate
nematode ova under a thin protective- layer of silt which may, under
suitable conditions, enhance human infection.
The concern about survival of parasites on food crops is probably
most significant where night soil with very high numbers of ova is used to
fertilize vegetables, although even this is considered relatively unimport-
ant in the transmission of Ascaris eggs (Chandler and Read, 1961). Rudolfs
et_ al_ (1950) reported that few contaminated vegetables were found from a
farm that used sewage for irrigation. Only one-third of the Ascaris ova
were viable. The same authors (1951a) applied Ascaris ova to tomatoes and
lettuce growing under field conditions during a hot dry summer. No
infective ova were found on the vegetables after 19 days, only 10 percent
of the ova could be recovered, and only a few were able to embryonate when
incubated. Viable ova were present on the vegetable surface three to four
weeks. Another study (WHO, 1964) reported that in a modern European city
market, most radishes and half the lettuces were contaminated with Ascaris
ova. Choi (1972), examining vegetables in Korea, also found helminth eggs
and larvae on 23 to 92 percent of samples.
Ova near the soil surface can be moved considerable distances by
heavy rains and could contaminate above-ground vegetables. Beaver (1975)
quotes several authors who indicate that soil particles can be transported
several metres over a flat surface, and while most reached heights of less
than 30 cm a few could go higher than 60 cm.
A study by Rudolfs et^ al_ (1951b), on the removal of Ascaris ova
from vegetables, found that most anionic and non-ionic detergents and
germicidal rinses were not noticeably better than water for removing ova.
Cationic detergents did remove most ova but a heat treatment of 55°C to
60°C for 10 minutes was the only method that could ensure complete
decontamination. Choi (1972) found that washing vegetables significantly
reduced but did not eliminate the parasites.
Transmission of T aenia saginata , the human tapeworm, is a major
concern in putting sewage sludge on pasture used for grazing beef cattle.
Seddon (ly50) showed that transmission occurred when pasture was irrigated
with raw sewage. Transmission from digested sewage sludge, however, is
more controversial. Many feel it is possible (Burd, 1968; Fair et al,
1971; Greenburg and Dean, 195b; Hanks, 1967; LePage , 1956, Newton et al,
1949; Silverman, 1955, Thornton, 1952) but no known cases of infections
could be linked with digested sewage sludge. Various reports (Silverman,
1954; 1955, Silverman and Griffiths, 1955) have stressed that, in Britain,
direct association of tapeworm carriers and cattle is uncommon. It was
postulated that since the ova survived many sewage treatment processes
cattle became infected when they had direct access to the sewage effluent
or sludge, or when a transport host (e.g., a gull) ingested the proglottid
with thousands of ova, at the sewage works and excreted the ova on
pasture. Crewe and Owen (1978) also studied infected cattle in Britain.
They postulated that the ova in treated sewage would be evenly and thinly
spread in the sludge and the infection in cattle would be similar. They
found an uneven distribution of the infection in cattle, some being
heavily infected but most being uninfected, leading them to believe
another source of ova was probable, possibly via gulls. In Ontario, due
to differences in sewage treatment processes, there is only a remote
possibility of birds having access to tapeworm proglottids.
In North America several epidemiological studies have sought the
source of infective ova to cattle. Schultz et^ al_ (19b9) reported on
several cases of bovine cysticercosis in the United States, including one
involving 743 infected cattle. Of those studied in detail, all could be
traced to faecal contamination of feed lots or cattle feed by infected
employees. The size and distribution of the infection depended on the
source of ova. In one case, the infections were caused by contaminated
sewage effluent. McAninch (1974) found the faecal contamination of feed
lots by one hired hand was responsible for several outbreaks of
cysticercosis in Ontario.
An extensive review by Pawlowski and Schultz (1972) on taeniasis
and cysticercosis included a section on the ability of the ova to with-
stand environmental conditions. Due to widely differing conditions of
temperature, moisture, age of ova, method of evaluating viability and
other relevant ecological conditions, the reported results vary widely and
often conflict. However, a few general statements can be made along with
some specific data.
The ova are reasonably resistant to many environmental factors.
Moisture level is reported to be an important factor in ova survival, but
little work has been done on this aspect. Much more work has been done
with temperature which probably is equally important. In different
studies the ova have apparently survived:
1) 168 days at 4 to 5°C;
2) 60 days at room temperature;
3) 62 to 64 days at -4°C;
4) 16 to 19 days at -30°C;
5) 12 days at -4.5°C, but a few survived for 76 days at this
6) 58 days in grass during the summer and 159 days during the late
winter to early summer in Denmark.
In Russia, where summer and winter temperatures differ greatly, it has
been found that the ova survive much longer during winter.
Most of these data were conjectural since the viability of the
ova was usually based on the morphological appearance or the ability of
the oncosphere to hatch, and rarely on feeding experiments. Once certain
morphological changes occur, the ova are definitely not viable. Their
viability may be impaired long before, and this can be measured only by
numerous feeding experiments. In the case of Taenia saginata , this is a
large and costly undertaking since the only intermediate hosts are cattle.
3. 2 Ova Recovered from Sludged Fields
No data were found concerning the survival of the natural levels
of parasite ova in digested sewage sludge once it has been applied to
farmland. Rudolfs et^ al_ (1950) reported that Vassilkova found 57 ova per
6600 g of soil irrigated with sewage. Spindler (1929) recovered Ascaris
ova from soil around certain dwellings when looking for the source of
infection to the occupants. Ghadirian et^ al_ (1976), Yang et_ al_ (1979) and
others have found Toxocara ova in soil from parks and sandboxes. A WHO
committee (1967) reported that, due to the natural sorting action on soil
particles by rain and wind, Ascaris ova are not randomly distributed, and
are difficult to recover even though they may be present in large numbers
The aim of this investigation was to determine how long ova
remained viable by taking soil samples from farmland where sewage sludge
known to contain parasite ova had been spread.
Because of difficulties in the recovery of Taenia ova and the
problem of identification of protozoan cysts, soil samples were only
analyzed for parasite ova that could be recovered by flotation. Of prime
interest were Ascaris , Toxocara and Trichuris , which had all been
recovered from digested sludge.
Samples were taken from two farms that had received digested
sludge from the Barrie sewage treatment plant. At Farm A the last sludge
application was about l\ years before the samples were taken, while at
Farm B the samples were taken from the same day to many months after.
For the samples from Farm A the method used was similar to that
of Spindler (1929). Antiformin (a 30 percent sodium hypochlorite
solution) was mixed with the sample to separate the ova from the soil
particles and sodium dichromate solution (specific gravity 1.35) was then
used to remove the ova. On shaking, the mixture tended to form a foam
which trapped the ova. The foam was removed, diluted with water,
centrifuged and the ova recovered by zinc sulphate flotation.
For samples from Farm B and subsequent soil samples (Section 3.3)
the method used was similar to that in the WHO report (1967). The soil
sample (10 to 20 g) was continuously stirred in a 30 percent sodium
hypochlorite solution for 15 to 30 minutes to separate the ova. The soil
particles larger than 150 um and smaller than 20 um were removed with the
appropriate sized sieves and the residue concentrated by centrifugation at
2000 rpm for two minutes. This was well shaken in a sugar and 30 percent
hypochlorite solution (specific gravity 1.2A) and centrifuged at 2000 rpm
for three minutes. Material on the surface was removed by a coverslip and
examined for ova. At least three slides were examined for each soil
3.2.2 Results and discussion
The Barrie digested sludge, analyzed in 1977, contained both
Ascaris and Toxocara ova (Table 2).
At Farm A three areas were investigated: a pit where large
quantities of sludge had been dumped (probably when weather conditions
prevented land application), and two fields were sludge had been applied.
One crop of corn had been harvested from the fields and they were again
ploughed before the samples were taken in May, 1977.
Material in the pit was essentially dry sludge; when a 52-g
sample was examined, one Ascaris ovum was found (Figure 4). The egg
contents appeared abnormal and no development occurred when the egg was
incubated for three to four weeks at room temperature.
From the ploughed fields, surface scrapings of 54 g and 26 g were
negative for parasitic ova. A core sample 2 cm in diameter and weighing
55 g from one of the fields contained two Ascaris ova. Neither ova were
viable, one contained a disintegrating larva, and the other a compact ball
FIGURE 4. DECORTICATED ASCARIS OVUM CONTAINING NUMEROUS
GLOBULES. Recovered from pit at Farm A, It years
after sludge last applied. Size 55 x 45 pm.
Ascaris ova were recovered from sludged fields li years after
the last sludge application, even when the fields had been ploughed twice.
None was viable and only one had probably been infective.
At Farm B, two areas were examined during July and August 1977.
The first was a field with a corn crop that had received sludge eight to
10 months previously. The other was a pasture where sludge was still
being applied. Three surface soil samples (10 to 12 g) taken from the
corn field were analyzed but no parasite ova were found.
In the pasture, samples were taken of the dry or liquid sludge,
together with the vegetation and soil. One sample of soil, vegetation and
sludge (bb g) was taken the same day as the sludge application. Two ova
were found, one Ascaris and one Toxocara ; both were undeveloped and
Some samples were taken about two days after the sludge had been
applied. Two samples of vegetation and soil (12 and 31 g) were negative,
but one of two sludge samples was positive. This 30 g sample contained
one Ascaris and three Toxocara ova; all were undeveloped and appeared
Three of four sludge and soil samples (6 to 63 g) taken several
weeks after the sludge was applied were negative for any parasite ova.
The positive sludge and soil sample (26 g) contained two Ascaris ova, both
with inactive but fully-formed larvae. These ova probably had been on the
field for at least three weeks since all Ascaris found in fresh digested
sludge were undeveloped and it takes about three weeks under optimum
environmental conditions for development to be completed (WHO, 1967).
Viable but undeveloped Ascaris and Toxocara ova were recovered
from farmlands up to two days after sludge was applied. Fully developed
Ascaris ova were found where sludge had been applied weeks before, indicat
ing that at least some parasite ova would be infective where digested
sludge was applied. After a year and a half only nonviable Ascaris ova
were found. Since these findings were based on limited data, a more
detailed approach was required as outlined in the following section.
3.3 Orangeville Experimental Plot
Parasite ova survival studies have frequently used the ascarid
from pigs, Ascaris suum (Almasi et^ al_, 1971; Fitzgerald and Ashley,
1977; Krishnaswami and Post, 1968; Rudolfs et. al, 1951a), since they are
almost identical to the commoa human parasite (A. lumbricoides ), are
easily acquired at any slaughter house, are one parasite likely to be
found viable on polluted soils, and are relatively safe to work with.
Several have referred to it as an indicator organism, reasoning that if it
is destroyed no other parasite ova would survive (Almasi et_ al_, 1971;
Krige, 1964; Kawata et al, 1977). Caldwell and Caldwell (1928) suggested
that there is a biological difference between the pig and human ascarid
with the latter possibly being more susceptible to environmental factors.
Ascarid ova, both A^ suum and Toxocara , were the main parasite
ova used in the present study, but ova of Taenia were also tested because
of the possibility of cattle becoming infected by sludge on pasture.
The experimental plot was approximately 8 km (five miles)
northeast of Orangeville in Dufferin County on a farm established about
190U. The 7-ha (17-acre) field containing the plot had not been
cultivated for about 15 years. The soil is well drained and is classified
as a fine sandy loam; a soil sample analyzed by the Applied Sciences
Section, MOE was mainly fine sand and silt with some organic material.
The test plot was 4.5 x 10.5 m (15 x 35 ft) and was surrounded by
a wire fence. During the spring and summer of 1977 parasite-seeded sewage
sludge was applied (Table 5). Three different areas were studied with
sludge applications to: "pasture", bare soil, and bare soil mixed to a
depth of 5 to 8 cm (two to three inches) after the sludge application.
The grass in the "pasture" was cut in the spring and at the time of sludge
application; thereafter it was allowed to grow. The sod was removed from
half the plot and vegetation was kept off the bare soil by applications of
atrazine (a common herbicide used in commercial corn production) in the
spring of 1977 and 1978 at a dosage of 2.8 kg/ha (2.5 lb/acre).
Digested sludge from the Orangeville sewage treatment plant was
used, acquired the same day or the day before application.
The parasite ova used in the test were from several sources.
Live ascarid worms from pigs were collected from Canada Packers on April
5, 1977. The uterus was removed from each adult female worm and, to
TABLE 5. PARASITE OVA IN ORANGEVILLE SLUDGE ADDED TO TEST PLOT
Area Sludge Applied
(m 2 )
ensure ova similar to those in the faeces, only the ova from the distal
third of the uteri were retained for experimental use. To confirm this
decision, ova from the distal, middle and proximal portions of the uteri
were incubated at 29 and 23°C. Some ova from the distal portions had
larvae after 11 and 15 days at 29° and 23°C, respectively. Development
was slower in ova from the middle and proximal portions. Larvae were
first apparent in the ova from the middle portions of the uteri after 26
days for those incubated at 29°C and after 40 days for those incubated at
23°C . Some development did occur in most ova from the proximal portions
but there were few larvae by the end of the observation period of 40
The Toxocara ova were concentrated from dog faeces collected from
the Oak Ridges Animal Hospital on August 8, 1977. Taenia ova for the
first two applications on June 15 and July 5, 1977, were concentrated from
dog faeces on February 13, 1976, and most still appeared normal. Those
for the last application, October 20, 1977, were from a worm expelled by a
cat just the day before (Figure 5).
Nematode ova can be effectively concentrated (80 percent
recovery) by using dilute sodium hypochlorite to separate the ova from
the soil, then separating off the finer and coarser materials with the
FIGURE 5. TAENIA OVA FROM A CAT. Ova from this source were
added to sludge applied to the Orangeville
experimental plot. Size about 35 x 30 jjm.
appropriate sieves and finally using a sugar and hypochlorite solution to
separate the ova from the remaining debris. Despite extensive testing,
no efficient method to recover Taenia ova from soil was discovered. The
method finally used was essentially the same as for the nematodes except
that a quick settling period removed the heaviest soil particles, and the
residue from the supernatant was subjected to a formalin-ether procedure.
At the experimental plot most samples for parasite ova were of
four types. In the "pasture" area, samples were taken initially of the
grass and the soil associated with the roots, and later of the grass
alone cut 2 cm above the soil. The first sample would include the soil
surface where most ova were expected; the second sample would be the
portion cropped by animals or collected with hay. A corer, sampling a
3.3-cm^ area, was used to sample the "pasture", bare soil and the
mixed areas. The core was divided into 2-cm lengths, each analyzed
separately to determine both the number of ova near the soil surface and
if the ova had been washed down into the soil. Some samples were also
taken of the sludge residue on the surface of the bare soil after each
Various temperatures were continuously recorded for one year
after the parasite-seeded sludge was applied. The results are tabulated
in Table 6 and, although the data are not complete, they do indicate the
range of temperatures that can be expected.
The air temperatures were recorded at the test plot during the
winter of 1977-1976 and they were sufficiently similar to those recorded
at the Orangeville sewage treatment plant (8 km away) that the latter
could be used as the air temperature at the experimental site.
Soil temperatures in the test plot were recorded in several
areas (Table b). A continuous record was kept of the temperature under
"pasture" where the grass was left to grow. A record of several months
is also presented of the temperature just under grass ("pasture") that
was kept snort ( 1 to 2 cm) and under the surface of the bare soil.
The soil temperature under the long grass was close to or
slightly higher than the air temperature except in winter when it
remained just below freezing, regardless of air temperature, because of
the thick insulating snow cover. Maximum temperatures were never above
40°C, but frequently were in the mid-30's.
Maximum surface soil temperatures under short grass were
considerably higher than the maximum air temperatures especially during
June and July, the period of longest daylight. Then, the temperature was
above 40°C for 33 hours; in July it was above 50°C for a total of 16
During the summer the soil temperatures under the bare soil were
also higher than the air temperature and probably approached those under
short "pasture". During the fall the soil temperatures were similar to
the air temperatures.
High soil temperatures were reported by Caldwell and Caldwell
(1928) during parasitological studies in south Alabama, where
temperatures up to b3°C were noted when the air temperature was 3b°C.
During weekly visits to the test plot, temperatures were taken
at various depths on the "pasture" and bare soil. The monthly averages
are presented in Table 7. Temperatures were taken in areas with and
TABLE 6. SUMMARY OF TEMPERATURES RECORDED AT ORANGEVILLE TEST PLOT
"Pasture" Soil Temperature (°C)
Just under surface**
Mean Air Temperature (°C) 2 cm under surface
Number Orangeville* Test Plot Mean Mean Hours at Mean Mean
of days Max Min Max Min Max Min <-5°C Max Min
Bare Soil Temperature
2 cm under surface
Mean Mean Hours at
Max Min <-5°C >40°C
January/ 78*** 31
•1 -4 15
* Temperature data from Orangeville sewage treatment plant provided by Canadian Climate Centre, Environment Canada.
** Grass kept short over this temperature probe.
*** Snow cover.
TABLE 7. MEAN MONTHLY TEMPERATURES RECORDED ON VISITS TO TEST PLOT
Number of Air
"Pasture" Soil Temperature (°C)
Bare Soil Temperature (°C)
Soil Down Down Down Down
Surface 1 cm 2 cm 3 cm 8 cm
*Snow cover of 35 to 75 cm.
without sludge to determine any differences. There was essentially no
effect in wet weather as occurred in August and September, 1977. In hot
dry weather (June 1977) adding sludge immediately reduced the surface
temperature by 20-30°C and it was still 5-6 G C below that of the untreated
areas after 24 hours.
The highest temperatures were recorded under dry vegetation
where the grass was kept short; a maximum temperature of 68°C was
recorded in the middle of June, 1977, when the air temperature was only
27°C. Temperatures much higher than the air temperature were also
obtained just under the soil surface, especially under the bare soil.
Temperatures in the high 40's were not uncommon in hot dry weather under
bare soil receiving direct sunlight.
Soil surface temperatures in the range of 50 °C were only noted
during May, June and July, and only during hot dry weather under full
sunlight. In the shade of long grass the temperatures at the soil
surface were always much lower (Table 7). Soil temperatures during these
months were lower the further down they were taken.
From August to mid-November the soil temperatures were between
30° and 2°C. When there was considerable snow cover, temperatures at the
soil surface were just below freezing. Although data are not available
for early spring, soil surface temperatures would remain at about the air
temperature until the soil surface dried out.
Soil moisture was determined by drying the soil samples in an
oven at 104°C and the results are shown in Table 8. These were grab
samples taken approximately every week so the results only give a rough
indication of the soil moisture, but they do correlate with the soil
temperatures in Table 7. As expected, the highest surface temperatures
on the bare soil (47°C in June, 1977) correspond with the lowest soil
moisture (0.8 percent). From August to November when the soil moisture
was relatively high, the soil surface temperature was approximately the
same as the air temperature. It has been reported that Ascaris ova 1 cm
deep in soil were killed when the soil moisture fell below four percent
TABLE 8. PERCENT WEIGHT OF WATER IN BARE SOIL FROM EXPERIMENTAL
PLOT, WITH AND WITHOUT SLUDGE
3.3.2 Results and discussion
The sludge was applied over the surface as evenly as possible
but due to slight irregularities it could be thicker in some areas. This
probably accounts for some of the variation in results.
If the bare soil surface was fairly dry (June 15, 1977), moisture
from the sludge quickly wet the top soil layer and the sludge showed
little lateral movement on relatively level ground. However, if the soil
surface was saturated with moisture at the time of application the liquid
sludge quickly flowed into any hollows in the surface. Heavy rain before
the sludge had dried (e.g., June 6, 1977) caused substantial spreading of
the sludge even on the relatively level test plot. This did not occur in
the "pasture" where the grass held the sludge where it was applied.
As the sludge dried it shrank, cracked into small pieces and
became patchy. Once this occurred the patches of dried sludge were quite
stable and were still visible on the soil surface after more than 300
days. The same type of sludge residue could be found in the "pasture"
The sludge residue on the bare soil was examined for ova after
each application and the results are tabulated in Table 9. Few ova were
found after the first application. The numbers were higher following the
second application and after nine days one Ascaris ova per gram of sludge
residue was found. Five of the seven ova found appeared non-viable.
This was not apparent in any subsequent samples since most ova appeared
normal. After 16 days no ova were found in this section. The mean
maximum air temperatures (Table 6) were highest in June of 1977 and 1978.
TABLE 9. ASCARIS OVA FOUND IN SLUDGE RESIDUE ON TOP OF BARE SOIL
AT EXPERIMENTAL PLOT
Date of Days After Ascaris
Application Application Ova/g
June 15, 1977 1 0.5
July 5, 1977 9 1
16 none found
August 10, 1977 1 8.1
September 6, 1977 1 61.9
284 none found
442 none found
In 1978, when temperature recorders were operational, this coincided with
the maximum soil temperatures. This probably occurs every year. The
destruction of ova was possibly due to these high temperatures. It has
been reported that ascarid eggs are killed at soil temperatures of 40°C
or higher (WHO, 1967).
The third application showed a gradual reduction in the number
of ova per gram of sludge residue in samples taken over 21 days. The ova
per gram of sludge residue of the fourth application showed no reduction
over 2 JL days. This is possibly due to the lower temperatures in September
Many of the ova found on the bare soil showed some development and
although no fully developed larvae were found, this could be due to the
limited sampling. After 284 and 442 days (June and October, 1978) no ova
were found in the soil surface and any remaining sludge residue.
Apparently Ascaris ova, and probably all parasite ova, cannot
survive a full year on well drained bare soil exposed to full sunlight.
This is probably due to high soil temperatures in spring and early summer
when the upper soil layers dry out.
Four Ascaris seeded sludge applications were made to the
"pasture" area (Table 10). In the samples that contained vegetation and
surface soil, the numbers of ova were usually highest immediately after
the application, followed by a decline in numbers with time. This decline
was rapid at first and then appeared to level off at a relatively low
number of ova per gram of sample. After almost a year and a half Ascaris
ova were still being found (Figure 6). When the sludge was applied to
pasture the grass wm^mw lush and green which would protect the soil
surface from high temperatures and moisture loss.
FIGURE 6. DECORTICATED ASCARIS OVUM WITH FULLY FORMED
LARVA RECOVERED FROM THE "PASTURE" OF THE
ORANCEVILLE EXPERIMENTAL PLOT 539 DAYS AFTER
APPLICATION. Size 62 x 44 m .
When the vegetation alone was examined, half of the samples were
negative for ova and in the other half the numbers of ova found were
considerably lower than in the samples that included the soil surface.
This ..hows that only a small percentage of the Ascaris ova (and possibly
other parasite ova), when applied to pasture, would be found on the
vegetation. The ova are probably thrown onto the vegetation along with
soil particles by rain.
RECOVERED FROM "PASTURE" AREA
Ac t i ve
TABLE 10. (CONT'D)
Vegetation Only Vegetation and Soil
Sludge Until /» with % Larvae % with % Larvae
Applied Sampled Ova/g Larvae Active Ova/g Larvae Active
September 29 0.8 17 100 3.4
6/77 (cont'd) 37 none found 7.2
*0va found but sample weight not recorded.
Development of the ova on the test plot was not uniform and was
spread over a much longer period than those incubated at constant
temperatures. After the second application (July 5) ova with larvae were
not definitely found until the 85th day, although some ova found on the
16th and 22nd days had almost fully-formed larvae. Ova with larvae were
found until late November (141 days). After the third and fourth
applications (August and September, 1977) ova with larvae were found
between the 40th and 50th days (early fall) and they persisted until
early January. For ova from the fourth application, there were none with
larvae in the spring of 197b but the percentage was again high in the one
sample taken during the winter of 1979. Most of the larvae found were
inactive. Rudolfs et_ al_ (1950) and a WHO committee (1967) reported that
larval movement indicates viability. Keller (1951) reported that,
according to Owen, the lack of larval mobility is not a sure test whether
the larvae are alive or not. In the present study, all ova that
contained larvae were considered viable, since motility appeared to be
quite random (Table 10).
It appears that Ascaris ova (and possibly other parasite ova)
can survive a considerable period on grass that is left to grow. Fully
developed larvae were present after about 50 days or less, but in one
application did not appear to survive the winter. Fully developed larvae
were again found the following year, presumably from ova that completed
development after the winter of 1978.
Toxocara ova in sewage sludge were applied on September 6, 1977,
only on the "pasture" and samples taken only of the vegetation together
with the top layer of soil (Table 11). During fall, 1977, the numbers of
ova were high, reflecting the high numbers applied, and most were
developing. When the site was next sampled after more than a year, the
number of ova was drastically reduced but almost all contained active
larvae (Figure 7). Therefore, some Toxocara ova applied in sludge to
grass that has been allowed to grow and provide a protected environment
could be expected to survive for several years.
The first Taenia application occurred with the first Ascaris
application on June 15, 1977. Formalin-ether tests were carried out on
soil samples, but no Taenia ova were found. Two other Taenia ova
TABLE 11. TOXOCARA AND TAENIA OVA RECOVERED FROM "PASTURE" AREA OF
Vegetation and Soil
July 5/77 Taenia
Oct. 20/77 Taenia
Sept. 6/77 Toxocara
*Taenia ova are fully developed
applications were made to the "pasture" (Table 11). As stated, the
methods for Taenia ova recovery from soil were very inefficient, and very
few ova were found. A total of eight ova were recovered from the July
application after two and nine days but none were found on the 16th and
22nd days. From the fall application a total of three ova were recovered
after 26 and 34 days. All ova appeared normal. From these limited data,
it would appear that Taenia ova cannot survive more than a few weeks when
applied to well-drained pasture exposed to full sunlight during the early
summer, but some can survive for at least a month when the sludge is
applied during cool damp conditions.
Since the length of time Taenia ova could survive was of prime
importance, a different method of finding the ova and determining their
viability was tested. It was decided to use T* taeniaeformis , which uses
FIGURE 7. TOXOCARA OVUM CONTAINING AN ACTIVE LARVA,
RECOVERED FROM THE "PASTURE" OF THE
ORANGEVILLE TEST PLOT 539 DAYS AFTER
APPLICATION. Size about 90 x 75 um.
a cat as a definitive host and a mouse as an intermediate host, as a
model. Fresh ova from worms recovered from cats were incubated at
various temperatures in water and sludge; added to sludge and then
applied to soil in pots and incubated at various temperatures; and added
to sludge and applied to the "pasture". Samples from all these sources
were manipulated to concentrate the ova and the material was fed to
laboratory mice. Each feeding usually contained about 350 ova, although
some contained up to 1500. No cysticerci were found in any mice from the
first series of experiments nor in several others using different strains
of laboratory mice. This work should be repeated when a suitable mouse
host is found or sufficient ova are used to result in infection.
After each application of sludge containing Ascaris ova, core
samples were taken from the "pasture", bare soil and mixed areas (Table
12). With one exception ascarid ova were only recovered from the top
2 cm of the cores in the "pasture" and bare soil areas. The one ovum
that was found below this level was probably carried there by the corer.
TABLE 12. ASCARIS OVA RECOVERED IN CORE SAMPLES FROM EXPERIMENTAL PLOT
NF - none found
This shows that there is no appreciable downward movement of Ascaris ova,
and probably other ova, even in well-drained soil. When sufficient ova
were applied in the sludge (August 10 and September 6) ova were recovered
from below the 2 cm level in the mixed area. However, none were
recovered below 2 cm after 15 days, suggesting a lower survival rate,
possibly due to such soil organisms as acarines and certain fungi (WHO,
1967). In April 1979 (day 595) two samples were taken in the mixed area
of the fourth Ascaris application to confirm that no ova were present.
The sample of the top centimetre of soil weighed 14 g while the sample
between 2 and 3 cm weighed 17 g. No Ascaris ova were found.
Under the test plot conditions, it appeared that some ascarid
ova survived a considerable time when applied in sludge to pasture, but
their survival was limited when the sludge was applied to bare soil and
possibly even more limited when the sludge was mixed with the soil.
3.4 Summary and Conclusions
The survival times of parasites once the sludge is applied to
farmland were determined by two methods. In the first method, samples
from fields where sludge had been applied at various times were examined.
In the second, a small experimental plot was used where various field
conditions were set up and monitored after parasite-seeded sludge was
Undeveloped Ascaris and Toxocara ova were recovered from a
recently sludged field. Fully developed Ascaris ova were recovered from
an area where the sludge had been applied several weeks before. On
another farm, where sludge had not been applied for a year and a half,
only a few nonviable Ascaris ova were found.
On farm fields where sludge has been applied, parasite ova can
be found that have developed normal-appearing larvae. They may not
survive It years.
At the experimental plot large numbers of Ascaris , Toxocara and
Taenia ova were mixed with sewage sludge, and applied to the surface of
grass and bare soil, and mixed with the top layer of soil. Conditions
were monitored and samples taken periodically to determine the state and
number of remaining ova.
Temperatures recorded at the test plot showed that the highest
temperatures, frequently above 50 C C (lethal to Ascar is ova), occurred
near the soil surface where there was short or no grass cover, in direct
sunlight during dry weather in the spring and early summer. The
temperatures were moderate in the shade of long grass, rarely being above
40°C. When the surface was wet, the soil temperatures were close to the
air temperatures, well below the temperatures lethal to Ascaris ova.
During the winter, under a thick insulating snow cover, surface soil
temperatures were just below freezing.
Core samples showed that there was no appreciable downward
movement of the parasite ova applied to the soil surface even in
well-drained soil. On pasture the number of ova found on grass alone was
much lower than when surface soil was included in the sample, indicating
that most ova in the sludge would remain at or near the soil surface.
On well-drained bare soil exposed to full sunlight, Ascaris ova
apparently will not survive a full year and possibly not even a few weeks
if the sludge is applied at times when the soil moisture is low allowing
high surface soil temperatures. When the sludge was mixed with the soil,
Ascaris were not recovered below the top layer after 15 days, suggesting
a shorter survival of parasite ova when this method is used.
Some Ascaris ova in sludge applied to pasture left uncut or
ungrazed could be expected to survive for several years, although the
numbers would decrease with time. When sludge was applied in the spring
and summer, fully formed larvae, presumably infective, could be found
after one to two months, although they may not persist through the
winter. More would be expected to develop the following year. Ova of
Toxocara would probably have survival times similar to Ascaris ova, while
other less resistant parasite ova would not survive as long. The limited
data suggest that Taenia ova may not survive more then a few weeks in'
sludge applied to well-drained pasture during hot dry weather, but some
appear to survive at least a month when sludge is applied during cool,
4 PARASITES AND FARMLAND APPLICATION OF SLUDGE IN ONTARIO
4 . 1 General
Digested sewage sludge that is spread on farmland in Ontario
contains parasite ova that appear viable. In the present study, ova of
Ascaris , Toxocara , Trichuris and hookworms were found frequently, while
ova of Taenia , Hymenolepis and Toxascaris were rarely recovered. Only
some of these may eventually be infective to humans. Parasitic protozoan
cysts may also be present. In fact any human pathogen present in the
population can end up in raw sewage and may survive sewage treatment.
Only heat-treatment at 60°C for several hours will guarantee a
In a recent survey, Antonic found that digested sludge was
applied to Ontario farms at an average application rate of 12 L/m 2 »annum
(10 650 gal/acre-yr). There are probably between 10 and 200 parasite
ova/L of digested sludge (mainly Ascaris ) (Tables 1 and 2); the sludges
examined in this study using the zonal rotor had an average of 21 ova/L.
Using this value together with the average sludge application rate,
approximately 252 ova/m^»annum would accumulate on the soil surface if all
the ova survived. If the soil were ploughed to a depth of 20 mm, with
complete mixing, there would only be 2.5 ova/m^ in the top 2 mm of
soil. Ova in this layer would probably stand a chance of completing their
life cycles. These ova, even most of the Ascaris and Toxocara , would not
survive for long and represent no appreciable hazard.
Larger numbers of ova could occur at the soil surface if any one
or combination of the following conditions were present:
- a focus of infection, causing a larger number of ova in sewage
- application of excess sludge;
- insufficient ploughing of sludged land, leaving more sludge at or
near the surface,
- weather conditions favourable for parasite survival.
On pastures where sludge is applied, the Ascaris and Toxocara ova
accumulation could be considerable. Some ova would probably survive for
more than one year but the likelihood of these reaching man is remote,
unless contaminated grass, food or sludge were consumed.
Ascaris , Trichuris and Toxocara ova would pose no threat in fresh
sludge since they need at least three to eight weeks of aerobic conditions
for the infective larvae to develop. The ova of Tj^ saginata , on the other
hand, would pose the most threat to cattle in fresh sludge. Infected
cattle would show no outward symptoms in a light infection (Lepage, 1956).
McAninch (1974) reported that only 12 carcasses and 307 portions were
condemned in Canada during 1970-71 due to T. saginata cysticerci, out of a
total of 3.5 million cattle inspected. Such inspection forms one line of
defence against infected meat reaching the consumer. The most reliable is
adequate cooking (60°C plus), which kills any cysticerci present.
Freezing at -10°C for 10 days also destroys the cysticerci.
The chances are very remote that any individual animal or human
will acquire more than a few ova from sludge. These helminths, with the
possible exception of Strongyloides , cannot increase in numbers within the
body, so a heavy infection can only be acquired by ingesting large numbers
of infective ova. Beaver (1975) points out that for nearly all helminths,
infections only cause disease symptoms when the numbers of parasites are
high enough. With Trichuris and hookworms there is a direct relationship
between worm burden and clinical disease (Martin, 1975). Children over
six years of age can harbor up to 500 Trichuris without evident disease
(Beaver, 1975), although light Ascaris infections infrequently produce
serious complications (Martin 1975).
The parasitic protozoans possible in sludge, E. histolytica and
G. lamblia , can increase in number in the gut of man, so a light infection
could eventually show disease symptoms. Rendtorff (1954) was not able to
infect human volunteers fed a single cyst of G^_ lamblia but was successful
if 10 or more cysts were fed. Helminth infections also appear to be
dose-dependent. Feedings of hundreds or even thousands of ova have been
reported necessary to cause infection with Taenia crassiceps (Freeman,
The mere presence of parasite ova and cysts in sludge does not
mean infection is probable. In fact, most parasite ova and cysts have
poor success in infecting a new host, so large quantities are produced.
One female hookworm will produce 20 000 ova a day for a least five years,
which would amount to over 36 000 000 offspring from one worm, if all
succeeded (Chandler and Read, 1961). A beef tapeworm can produce
three-quarters of a million eggs per day (Crewe and Owen, 1978), but it is
a rare parasite in North America. Obviously the more ova present in
sludge, the better the chance of infections, but the numbers present in
Ontario digested sludge may be insufficient. Chandler and Read (1961)
estimated the chances of an Ascaris ovum reaching maturity in the final
host at many millions to one. The odds against an ovum reaching maturity
in humans via sewage sludge would be even greater.
There is no documented evidence that parasitic infections have
been transmitted by digested sewage sludge. There appears, however, to be
a remote possibility, although the risk of transmission may be very low
and would be reduced further by the following precautions.
The following recommendations are for use with properly digested
sewage sludge from a conventional sewage plant. They are based only on
the parasites in the sludge. If the sewage treatment is inadequate, more
precautions will be needed. Similarly, if the treatment process is more
harmful to parasites (i.e., thermophilic digestion or composting) less
stringent precautions will suffice.
4-2.1 Application of sludge
The following recommendations are directed to personnel who
actually apply the sludge to the farm fields.
a) Ingestion of, or food contamination with, liquid sludge should be
prevented, because of the possible presence of protozoan cysts.
They pose a greater threat in raw than digested sludge.
b) Food contamination with dry sludge, from a sludge-drying bed,
should be avoided due to the probable presence of infective ova
of Ascaris , Trichuris and Toxocara .
c) Skin contact with sludge from a drying bed should also be avoided
due to the possible presence of hookworm larvae.
4.2.2 Sludge applied to pasture
The following recommendations concern the surface application of
the sludge. If subsurface-injection is used, and no sludge remains on the
surface, there should be no restrictions on use of the pasture. The threat
of parasitic infection from digested sludge to domestic animals is far
less than that of acquiring infections in the normal farm environment.
The exception is Taenia saginata which can only be transmitted to cattle
by human faecal contamination.
a) Cattle should not be allowed on a freshly sludged field due to
the possible presence of T. saginata ova. The threat of
infection will decrease with time especially in hot, dry weather.
In conditions adverse to ova survival, the ova will probably only
survive a few weeks, however, in favorable conditions they could
last more than one month. The data for this recommendation are
incomplete and more work using feeding experiments is necessary
to determine if transmission is possible via sewage sludge.
b) Well-dried hay from a field where sludge was applied several
months before can be used for cattle feed since it is probably
free of viable Taenia ova.
c) Animals such as sheep and goats, even though they crop the grass
closer than cattle, probably stand little threat from parasitic
organisms in urban sewage sludge. Probably the same restrictions
as for cattle would be advisable.
d) Intimate human contact with sludged fields (e.g., picnicking,
children playing) should be avoided for several years after the
last application, due to the survival of parasitic nematode ova,
especially Ascaris and Toxocara .
4.2.3 Sludge applied to cultivated fields
Fields cultivated after the last sludge application would have a
relatively small number of parasite ova near the soil surface.
a) These fields may be used with no time restrictions to grow animal
feeds and grain, even grain destined for human consumption.
b) Recently sludged fields should not be used to grow vegetables for
human consumption. This would include root crops as well as
above-ground vegetables. The parasite ova are unlikely to last
longer than one year except in moist, shaded soil, where longer
survival of ova would necessitate a two-year waiting period.
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PARASITE LIFE CYCLES
PARASITE LIFE CYCLES
Hymenolepis nana (dwarf tapeworm)
Taenia saginata (beef tapeworm)
Taenia solium (pork tapeworm)
Ascaris lumbricoides (human roundworm)
Ascaris suum (pig roundworm)
Enterobius vermicularis (pinworm)
Necator americanus and Ancylostoma duodenale (hookworms)
Strongyloides stercoralis (threadworm)
Toxocara Canis and T. cati (roundworms of dogs and cats)
Trichuris trichiura (whipworm)
15-60/^ rn in diam.)
ABLE TO REPRODUCE IN
THE BODY AND INVADE TISSUES
SHED IN FAECES
Asymptomatic infections of the intestine are most
common, although invasion of the liver and lungs can
produce severe disease symptoms.
(trophozoite 15 x 10// m)
ABLE TO REPRODUCE
SHED IN FAECES
In adults asymptomatic infections predominate, but
in children there can be numerous gastrointestinal
(10-60 cm long
tapeworm in gut)
OVA INGESTED BY
Infection in man produces few or no definite symptoms
HYMENOLEPIS NANA (dwarf tapeworm)
(2 cm long tapeworm in gut)
PASSED IN FAECES
INGESTED BY X
(may act as intermediate host)
Light infections produce either no symptoms or vague abdominal
TAENIA SAG1NATA (beef tapeworm)
5-10 m in gut)
PASSED IN FAECES
(cysticerci encyst m flesh)
Infection in man is usually asymptomatic but occasionally with
vague alimentary upsets.
TAENIA SOLIUM ( (pork tapeworm)
(3 m adult
tapeworm in gut)
PASSED IN FAECES
(cysticerci in flesh)
\ OVA INGESTED
\ BY MAN
(cysticerci in flesh,
Infection with an adult tapeworm is either asymptomatic or
with a mild, but chronic, digestive disorder. It can cause
the potentially more serious cysticercosis, if reverse
peristalsis or faecal contamination of food allow the ova t<
hatch in the human intestine.
ASCARIS LUMBRICOIDES (roundworm)
(20-35 cm long
adults in gut)
(3 weeks or more)
LARVAE MIGRATE THROUGH
BODY FROM INTESTINE
Light infections often symptomless but allergic reactions are
possible during larval migration. Adult worms may block the bile
duct or migrate to other organs of the body.
ASCARIS SUUM (roundworm of pigs)
(20-35 cm long
adults in gut)
LARVAE COUGHED -UP
LARVAE MIGRATE THROUGl
BODY FROM INTESTINE
y OVA INGESTED /
In man the parasite usually cannot complete its life cycle but
respiratory symptoms may be caused if the larvae reach the lungs
and a few may even continue on to become adult worms in the
intestine. They only remain for a relatively short period, since
man is an abnormal host.
ENTEROBIUS VERMICULARIS (pinworm)
(5-13 mm long
adults in gut)
A relatively harmless parasite but migrating female worms
can cause intense itching in the perianal region.
NECATOR AMERICANUS AND
ANCYLOSTOMA DUODENALE (hookworms)
(10 mm adult in gut)
0.5 mm long)
LARVAE MIGRATE THROUGH
BODY TO LUNGS
THE SKIN OF MAN
Light infection are often asymptomatic but the worms can cause
numerous allergic and gastrointestinal complaints.
STRONGYLOSES STERCORALIS (threadworm)
adult in gut)
LARVAE MIGRATE THROUGH
BODY TO LUNGS
Light infections are often asymptomatic but the worms can
cause numerous allergic and gastrointestinal complaints.
TOXOCARA CANIS AND T. CATI
(roundworms of dogs and cats)
DOG OR CAT
(10-20 cm long
adults in gut)
PASSED IN FAECES
(1 week or more)
\ " OVA INGESTED
\ BY DOG OR CAT
LARVAE MIGRATE THROUGH
BODY FROM INTESTINE
In man a cause of visceral larval migrans, which may be
asymptomatic or cause numerous symptoms depending on the
number and location of the larvae.
Toxascaris leonina has a life cycle similar to Toxocara ,
although the larvae usually migrate back to the intestine
directly, without going through the lungs. It may infect
other hosts, possibly even man, where it becomes encysted
in the abdominal viscera.
TRICHURIS TRICHIURA (whipworm)
(3-5 cm long
adults in gut)
Light infections are usually asymptomatic
PARASITE OVA RECOVERY FROM SLUDGE USING THE ZONAL ROTOR
PARASITE OVA RECOVERY FROM SLUDGE USING THE ZONAL ROTOR
Equipment used was a Damon/IEC, PR-J refrigerated centrifuge,
together with aa IEC, CF-6 zonal rotor.
The rotor, spinning in the centrifuge, holds a density gradient
near its outer margin. The sample is pumped into the rotor continuously
and it flows over the top of the gradient. Some of the particles in the
sample are accelerated into the gradient and are trapped. The heaviest
particles actually pass through the gradient to the outside wall of the
rotor, while the very light particles pass out with the supernatant. The
particles retained in the gradient depend on the specific gravity of the
gradient, the speed of rotation and the rate of sample addition. Trapped
particles are then allowed to migrate to their equilibrium densities
(banding time). By pumping in a dense solution (piston fluid) and
applying an appropriate vacuum, the gradient can be withdrawn from the
rotor. It is then divided into fractions and analyzed.
The material used to make the gradient was initially sucrose,
later sodium silicate; they were equally effective. A discontinuous
gradient consisting of:
Amount (mL) Specific Gravity
100 (water) 1.00
was added to the rotor when the rotor speed was 1600 rpm.
The sludge sample (100 to 500 mL) was filtered through a 180-ym
pore sieve. The residue in the sieve was well rinsed. The filtrate was
then further diluted to 4-5 L and kept mixed with a magnetic stirrer.
The sample was then pumped into the rotor at a rate of 50 mL/min, with a
rotor speed of 500 rpm
To help prevent formation of bubbles when the gradient was being
withdrawn under vacuum, all solutions were first subjected to a vacuum of
75 cm of mercury to remove excess gases.
The banding time, to aliow the particles in the gradient to
stabilize their positions, was at least one hour at 1600 rpm.
The gradient was removed from the rotor into a graduated, 5-cm
diameter, plexiglass column, by adding a piston solution (specific
gravity 1.26) that was heavier than any part of the gradient (Figure
II.l). At the same time a vacuum of about 15 cm of mercury was applied
to the top of the plexiglass column. The amount of vacuum was critical,
since too much would cause unnecessary bubbling and too little would
allow the gradient to go out the waste tube at the bottom. The speed of
rotation was kept low (500 rpm) to keep the vacuum required at the
minimum. A flow deflector was added at the bottom of the column, to
reduce mixing as the gradient flowed into the column.
Bubbles, which would mix the gradient in the column, were an
initial problem. They were caused by any slight air leaks in the rotor
or by the gases coming out of solution. The problem was solved by adding
a separatory funnel filled with water to the hose from the rotor to the
column. Although the displaced water would dilute the gradient, there
was usually not enough to cause appreciable mixing.
The zonal rotor has a 655 mL capacity so it was easy to
determine from the graduated column when all the gradient had been
removed. The column could then be disconnected from the vacuum and the
rotor. The bands of particles in the gradient were clearly visible.
The gradient was then removed from the column into 50-mL
centrifuge tubes. Before each tube was filled a drop of gradient was
taken and its specific gravity determined using a hand-held refracto-
raeter, calibrated for sodium silicate or sucrose solutions, so that the
specific gravity range for each 50-mL portion was known. Although a
discontinuous gradient was added to the rotor, the final gradient was
more continuous due to mixing (Figure 11.2). The structure of the
gradient, when removed from the column, revealed if the sample analysis
was acceptable and allowed the prediction of which tubes were most likely
to contain parasite ova. Due to the experimental nature of the method,
all of the gradient was examined for ova. If the method were used on a
routine basis, examination of only some of the tubes would be necessary
to find specific parasite ova.
* VACUUM (APPROX. 15 cm)
ACRYLIC COLUMN (mL)
FILLED WITH WATER
TO TRAP BUBBLES
FIGURE II. 1. SIDE VIEW OF ZONAL ROTOR AND ASSOCIATED EQUIPMENT NEEDED TO
WITHDRAW THE GJIADIENT.
GRADIENT VOLUME (mL)
FIGURE II. 2. GRAPH SHOWING TYPICAL GRADIENT DENSITY AS IT WAS REMOVED
FROM THE COLUMN. SAMPLE ANALYZED WAS 300 ML OF DIGESTED
NEWMARKET SLUDGE. FOUR PARASITE OVA WERE FOUND.
Each 50-mL portion of the gradient was then diluted to 200 mL,
to reduce the specific gravity, and centrifuged at 3000 rpra for five
minutes. The sediment was washed with water and examined either by a
direct mounting technique or the zinc sulphate or sugar/sodium
hypochlorite flotation tests. Three or four microscope slides were
examined from each 50-mL portion of gradient.
TD Parasites and the land
774 application of sewage sludge /
.G73 Graham, H. 3.