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Full text of "Parasites and the land application of sewage sludge /"

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 

198 



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P37 
1981 



CANADA -ONTARIO AGREEMENT 
RESEARCH REPORTS 

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 
organizations . 

Enquiries pertaining to the Canada-Ontario Agreement RESEARCH 
PROGRAM should be directed to - 

Wastewater Technology Centre 

Canada Centre for Inland Waters 

Environment Canada 

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. 



78895 



£>65S 



PARASITES AND THE LAND 
APPLICATION OF SEWAGE SLUDGE 



H.J. Graham 
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 

Division (Water) 
Environmental Protection Service 
Environment Canada 
Ottawa, Ontario 
K1A 1C8 



Ontario Ministry of the Environment 
Pollution Control Branch 
135 St. Clair Avenue West 
Toronto, Ontario 
M4V 1P5 



e Minister of Supply and Services Canada 1981 

Cat. No. En 43-11 IIOE 

ISBN 0-662-11461-2 

BEAUREGARD PRESS LIMITED 



ABSTRACT 

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. 



i 



r£sum£ 



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. 



ii 



TABLE OF CONTENTS 



ABSTRACT 

TABLE OF CONTENTS 



List of Figures 
List of Tables 



3.1 Introduction 



Pa£e 

i 
iii 



IV 
V 

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 

2.1 Introduction 

2.2 Methods 

2.3 Results and Discussion 

2.4 Summary and Conclusions 



12 
13 
14 
26 



3 SURVIVAL OF PARASITES ON SLUDGED FIELDS 27 



27 



31 

32 



34 
41 
50 



3.2 Ova Recovered from Sludged Fields 30 

3.2.1 Methods 

3.2.2 Results and discussion 

3.3 Orangeville Experimental Plot 33 

3.3.1 Methods 

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 

REFERENCES 56 

APPENDIX I - PARASITE LIFE CYCLES 65 

APPENDIX II - PARASITE OVA RECOVERY FROM SLUDGE USING THE 

ZONAL ROTOR ei 



iii 



LIST OF FIGURES 

Figure Page 

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 
Application 48 

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 



IV 



LIST OF TABLES 

Table Page 

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 

Plot 39 

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 

Plot 49 



v 



1 INTRODUCTION 

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 
farmland, and 

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 
Ontario. 

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 
considered further. 

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 
sewage sludge. 

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 



3 



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 
cycles . 

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. 
histolytica . 



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 
at 30°C. 

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 



10 



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. 



11 



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 



12 



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. 

2.2 Methods 

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 
material floated. 

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 



i: 



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 
alternative . 

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 



14 



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 
considered unsuitable. 

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. 



15 




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. 



L6 



TABLE 1. PARASITE OVA FOUND AT LAKEVIEW SEWAGE TREATMENT PLANT 





Method 
Used 


Nurrber 

of 
Samp les 


Sample 

Vo 1 ume 
(mL) 








Results 






Samp le 
Type 


Type of 
ova 


Number of 

Samples 

Pos i t i ve 


Number 

per Pos i 

Samp I 


Ova 
it ive 
e 


Ova/L 


Ova/fc 


Raw 

5 1 udge 


D i rect 


9 


_ 


None found 
















Formal i n- 
ether 


31* 


5-7 


Ascaris 


1 




1 




150 






Zinc 
sulphate 


29 


5-7 


Ascar is 


2 




1 




150-200 






Acid- 
ether 


3 


2-5 


Trichur is 


1 




1 




500 






Zonal 
rotor 


6 


50-200 


Ascaris 
Trichur is 
Toxocara 
Hookworm 

Taenia 


4 
1 
3 

2 

1 




1-5 

2 
1-2 

1 

1 




10-100 

40 
5-20 
5-10 

10 


0.1-2 

1 
0.2-0.5 
0.2-0.3 

0.2 


D igested 
si udge 


Direct 


7 


- 


None found 


- 




m 




_ 


- 




Formal in- 
ether 


21* 


5-7 


Trichur is 


2 




1-2 




140-400 






Z inc 

su Iphate 


26 


5-15 


Ascaris 
Trichur is 
Toxocara 


4 
1 
1 




1-3 

1 
1 




140-400 

70 

200 


- 




Zonal 
rotor 


4 


50-400 


Ascaris 
Toxocara 
Trichur is 
Taenia 


4 

3 
1 
1 




4-9 
1-2 

1 
2 




10-180 

10-40 

20 

40 


0.2-6 
0.5-1.3 

0.7 

1.3 



Primary 
effluent Direct 

Zinc 

su Iphate 

Centrif u- 
gat ion 

Plant 

effluent Centrifu- 
gat ion 



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. 



17 



TABLE 2. PARASITE OVA FOUND IN ONTARIO SEWAGE SLUDGE 









Sewage 

Treatment 

Plant 



Sample 
Type 



Activated Sludge 
Plants 



Method 
Used 



Number 

of 
Samples 



Sample 

Volume 

(mL) 



Type of 
Ova 



Results 



Number of 

Samples 
Positive 



Ova per 
Positive 
Sample 



Ova/L Ova/g 



Phosphorus 
Removal 
Chemical 



Barrie 



Orangeville 



a 



Peterborough 



Lane ) 



Newmarket 



Brantf ord 



Digested 


Formalin- 




sludge 


ether 


4 


•• 


Zinc 






sulphate 


1 


•■ 


Zonal 






rotor 


4 


Digested 


Formalin- 




sludge 


ether 


I 


•■ 


Zonal 






rotor 


1 


Digested 


Formalin- 




sludge 


ether 
Zonal 


1 




rotor 


J 


Digested 


Formalin- 




sludge 


ether 
Zonal 


1 




rotor 


1 


Digested 


Formalin— 




sludge 
■■ 


ether 
Zonal 


1 




rotor 


1 


Digested 


Formalin- 




sludge 


ether 
Zonal 


1 




rotor 


1 



2-3 Ascaris 



None found 



150-500 Ascaris 



Toxocara 



None found 



150 Ascaris 



None found 



150 Ascaris 



2-3 None found 



200 Ascaris 



2-3 None found 



300 Ascaris 



Toxocara 



2-3 None found 



200 Ascaris 



500 



1-2 2-13 0.1-0.7 
1 7 0.2 



20 



3 
10 



0.3 



0.02 



0.2 



0.03 

0.1 



Alum 



Alum 



Iron 



Iron 



Iron 



Iron 



TABLE 2. (CONT'D) 



Sewage 

Treatment 

Plant 



Unionville 



Results 



Sample 
Type 



Method 
Used 



Number 

of 
Samples 



Sample 

Volume 

(mL) 



Type of 
Ova 



Aerobically Formalin- 
digested ether 
sludge 



2-3 None found 







Zonal 
rotor 


I 


500 


Hookworm 


London 
(Vauxhall) 


Primary 
settled 
sludge 


Formalin- 
ether 


1 


2-3 


None 


found 






Zonal 
Rotor 


1 


400 


Ascaris 
Toxocara 


Waterloo 


Raw 
sludge 


Direct 


1 


- 


None 


found 




M 


Formalin- 
ether 


1 


7 


None 


found 




" 


Zinc 
sulphate 


1 


7 


None 


found 




Digested 
sludge 


Direct 


1 


— 


None 


found 






Formalin- 
ether 


1 


5 


None 


found 




11 


Zinc 
sulphate 


1 


5 


None 


found 


Elmira 


Raw sludge 


Direct 


3 


- 


None 


found 




•* 


Formalin- 
ether 


2 


5-7 


None 


found 




ii 


Zinc 
sulphate 


3 


1-7 


Hookworm 



Number of 

Samples 
Positive 



Ova per 
Positive 
Sample 



Ova/L Ova/g 



Phosphorus 
Removal 
Chemical 



Iron 



Lime 



Iron 



Iron 



TABLE 2. (CONT'D) 



Results 



Sewage 






Number 


Sample 




Treatment 


Sample 


Method 


of 


Volume 


Type of 


Plant 


Type 


Used 


Samples 


(mL) 


Ova 


Elmira 


Primary 


Direct 


2 


_ 


None found 


(cont 'd) 


thermophi- 
lic diges- 


Formalin- 










ted sludge 


ether 
Zinc 


2 


5 


None found 






sulphate 


2 


5 


None found 




Secondary 












digested 


Direct 


1 


- 


None found 




sludge 












» 


Zinc 












sulphate 


1 


5 


None found 



Number of Ova per 

Samples Positive 
Positive Sample 



Ova/L Ova/g 



Phosphorus 
Removal 
Chemical 



o 



Primary Plants 
Owen Sound 



Port Dover 



Raw 


Formalin- 




sludge 


ether 
Zinc 


1 




sulphate 


1 


Digested 


Zonal 




sludge 


rotor 


2 


(after 


Zonal 




land ap- 


rotor 


1 


plication) 






Raw 


Formalin- 




sludge 


ether 
Zinc 


1 




sulphate 


1 



None found 



1 None found 



50 Toxocara 



Hymenolepis 
150 diminuta 



150 



150 



None found 



None found 



40 



Alum 



Iron 



TABLE 2. (CONT'D) 



Sewage 

Treatment 

Plant 



Results 



Number 



Sample 


Method 


o 


Type 


Used £ 


Jam 


Digested 






sludge 


Direct 
Zinc 


1 




sulphate 


1 


Plant 


Centrifuga- 




effluent 


tion 


1 


Raw 






sludge 


Direct 
Formalin- 


1 




ether 


1 


•t 


Zinc 






sulphate 


1 


Plant 


Centrifu- 




effluent 


gation 


1 



Sample 

Volume 

(mL) 



Type of 
Ova 



Number of 

Samples 
Positive 



Ova per 
Positive 
Sample 



Ova/L Ova/g 



Phosphorus 
Removal 
Chemical 



Port Dover 
(cont'd) 



Parry Sound 



— 


None found 




Toxascaris 


3 


leonina 


150 


None found 


- 


None found 


5 


None found 


5 


None found 



330 



Alum 



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, 



22 



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 
recovered. 

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 
killed. 

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 
slaughterhouse. 

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 



23 



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 
this study. 

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 



WHEN 


INCUBATED AT 


25 c 


'C AND AT 


37 


X 


IN WATER AND 


SLUDGE 


Days of 




25 


°C 










37 


°C 




Incubation 


Water 






Sludge 


Water 






Sludge 


1 


159 






155 






173 






91 (10)* 


4 


55 (29) 






49 






62 (62) 






- 


6 


62 ( 2) 






3 






26 (24) 






- 


g 


81 ( 1) 






21 (1) 






3 






- 


13 


11 






22 






5 






— 



*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 



24 



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 
SEWAGE SLUDGE 

Average Median Range of Median 
Average Size Size Range Density where Densities where 
Parasite (ym) (ym) Ova Recovered Ova Recovered 

Ascaris 

with outer 66.4 x 53.7 (7)* 63 x 50 - 1.14 (8) 1.104 - 1.20 

coat 69 x 57 

without 

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 



25 



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 
recovered. 

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. 



26 



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 



27 



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. 



28 



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 



29 



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 
temperature ; 

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 



30 



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. 

3.2.1 Methods 

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 
sample. 



31 



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 
of globules. 




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. 



32 



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 
appeared viable. 

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 
normal. 

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, 



33 



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. 

3.3.1 Methods 

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 



34 



TABLE 5. PARASITE OVA IN ORANGEVILLE SLUDGE ADDED TO TEST PLOT 







Parasite 


Number 

Ova/L 

Sludge 


Number 

Ova/cm^ 

Soil Surface 


Area Sludge Applied 
(m 2 ) 


Date 


"Pasture 


Bare 
' Soil 


Bare 
Soil 

Mixed 


June 


15/77 


Ascaris 
Taenia 


3.3 x 
1.1 X 


10 3 
10 3 


4 
1 


2.8 


3.7 


0.5 


July 


5/77 


Ascaris 


2.2 x 


io 4 


29 


2.8 


3.7 


0.5 






Taenia 


1.4 x 


io b 


137 


0.1 






Aug. 


10/77 


Ascaris 


4.4 x 


10* 


57 


2.8 


3.7 


0.5 


Sept 


6/77 


Ascaris 


6.6 x 


io 4 


86 


2.8 


3.7 


0.5 






Toxocara 


5.9 x 


10 4 


215 


0.1 










Taenia 


1.3 x 


io b 


129 


0.02 







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 
days . 

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 



35 




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 



36 



taken of the sludge residue on the surface of the bare soil after each 
application. 

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 
hours . 

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 



37 



OC 



TABLE 6. SUMMARY OF TEMPERATURES RECORDED AT ORANGEVILLE TEST PLOT 



Date 



"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 



Hours at 
>40°C >50°C 



Bare Soil Temperature 
2 cm under surface 
Mean Mean Hours at 

Max Min <-5°C >40°C 



June/77 30 

July 31 

August 22 

September 23 

October 16 

November 30 

December 6 

December*** 25 

January/ 78*** 31 

February*** 28 



March*** 

April 

May 

June 

July 

August 



31 

11 



16 



22 


8 


25 


14 


22 


11 


18 


11 


13 


3 


6 


-1 


-1 


-6 


-3 


-10 


-6 


-14 


-7 


-17 





-11 


4 


-4 


21 


8 


22 


10 


26 


11 


22 


12 



12 
11 










-1 


-3 


-11 


-1 


-2 


-7 


-14 


-1 


-1 


-3 


-16 


-1 


-1 


2 


-10 


-1 


-1 









-1 






22 


11 






25 


13 



10 



25 



16 



11 


33 


5 


12 


33 


16 


14 


9 





II 10 

.4 2 

5 -1 

•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 



Date 



Mean 
Number of Air 
Observa- Temp 
tions (°C) 



"Pasture" Soil Temperature (°C) 



In Shade 
of Long 
Vegetat- 
ion 



Under Short 
Vegetation 
on Surface 



Just 
Under 
Soil 
Surface 



Down 
1 cm 



Down 
2 cm 



Down 
3 cm 



Down 
8 cm 



Bare Soil Temperature (°C) 
Just 
Under 

Soil Down Down Down Down 
Surface 1 cm 2 cm 3 cm 8 cm 



CO 



May/ 77 

June 

July 

August 

September 

October 

November 

December* 

January/78* 

May 

June 



33 
29 
29 

23 
13 
12 
4 
-8 
-7 
20 
20 



34 
43 
30 

24 

13 

13 

4 



57 
41 
24 
14 
13 
3 



39 
47 



40 
30 
25 

14 
11 

3 
-1 





34 
27 
22 

14 

11 

3 





31 
25 
21 
14 

11 
3 





23 

27 
24 
20 
14 
10 
4 




21 
22 
19 

14 
9 
4 

0.5 
0.5 



47 
47 
31 
25 
13 
11 
3 



39 
38 







29 




39 


34 


32 


26 


28 


26 


24 


22 


24 


23 


23 


19 


13 


13 


13 


13 


11 


10 


10 


9 


3 


3 


3 


4 



*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 
(WHO, 1967). 



40 



TABLE 8. PERCENT WEIGHT OF WATER IN BARE SOIL FROM EXPERIMENTAL 
PLOT, WITH AND WITHOUT SLUDGE 



Date 



June/77 

July 

August 

September 

October 

November 



Number 


of 


Without 


With 


Observations 


Sludge 


Sludge 


2 




0.8 


0.6 


5 




5.4 


5.4 


4 




14.1 


11.3 


4 




11.1 


22.2 


3 




11.2 


10.4 


2 




14.8 


17.1 



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" 
area. 

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. 



41 



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 

8 0.8 

July 5, 1977 9 1 

16 none found 

August 10, 1977 1 8.1 

7 3.1 
15 1.4 

21 0.6 

September 6, 1977 1 61.9 

8 86.9 
15 117.4 

22 97.1 
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. 



42 



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. 



43 



TABLE iO. 


ASCAR1S OVA 


RECOVERED FROM "PASTURE" AREA 


OF EXPERIMENTAL 






PLOT 










Vegetation Or 


ily 




Vegetation and 


Soil 


Date 




Days 
Until 
















Sludge 




% with 


% 


Larvae 




% with 


Larvae 


Applied 




Sampled 


Ova/g 


Larvae 


Active 


Ova/g 


Larvae 


Ac t i ve 


June 15/77 




1 
8 










2.1 
none found 








July 5/77 




2 

9 
16 
22 
71 
85 










4.0 
2.2 
0.1 
0.03 
none found 
1.0 





i) 



96 




o 




0.04 






92 


0.1 










0.1 


100 


25 






100 


none found 








0.3 


11 









107 


0.5 


20 

















113 


none found 








0.4 


100 


20 






126 


0.1 










0.1 


33 









141 


none found 








0.2 


100 









161 










none found 










167 










none found 










184 










none found 










190 










none found 










196 










0.1 












202 










0.1 


Q 





August 10/77 


1 










23.0 












7 










1.3 












L5 










0.1 












21 










none found 










49 










0.1 


7 5 









56 


0.1 







i) 


2.2 


70 









64 


none found 








1.3 


14 









71 


0.1 


100 







1.4 


33 









11 


none found 








0.5 


50 









90 


none found 








0.7 












105 


none found 








1.1 


100 









125 










none found 










148 










0.1 


100 









154 










0.4 


67 









160 










positive* 


7 









166 










1.9 


10 


40 


September 




1 










16.3 








6/77 




8 










6.8 





p 






15 


1.0 










14.0 


u 


Q 






22 


0.2 










9.0 









44 



TABLE 10. (CONT'D) 



Vegetation Only Vegetation and Soil 

Date Days 

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 



37 


none 


found 


44 





8 


50 


none 


found 


63 





2 


78 


none 


found 


98 






121 






127 






133 






139 






232 






278 






539 







5.3 


3 





2.6 


17 





2.7 








2.8 


42 





0.5 


67 


25 


0.02 


100 





23.55 








positive* 


2 





4.6 


1 


50 


0.9 








positive* 








1.0 


89 






*0va found but sample weight not recorded. 



45 



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 



46 



TABLE 11. TOXOCARA AND TAENIA OVA RECOVERED FROM "PASTURE" AREA OF 



EXPERIMENTAL PLOT 










Date 


Days 


Vegetation and Soil 


Sludge Parasite 


Until 






% with 


Applied Ova 


Sampled 


Ova/g 




Larvae 


July 5/77 Taenia 


2 


0.2 


* 




9 


0.3 








16 


none fou 


md 






22 


none found 




Oct. 20/77 Taenia 


19 
26 
34 


none fou 
0.1 
0.1 


nd 




Sept. 6/77 Toxocara 


1 


21.9 









8 


17.7 









15 


65.6 









22 


43.8 









29 


16.3 









44 


9.9 




o 




50 


43.4 









63 


8.9 









78 


none fou 


nd 






442 


1.6 




100 




539 


5.9 




92 




558 


2.1 




100 


*Taenia ova are fully developed 


and infective 


when they 


leave 


the 



definitive host. 

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. 



48 



TABLE 12. ASCARIS OVA RECOVERED IN CORE SAMPLES FROM EXPERIMENTAL PLOT 



Date 

Sludge 

Applied 



Days 
Until 
Sampled 



Depth (cm) 



Ova/cm^ (ova/g) 



'Pasture" 



Bare 
Soil 



Mixed 



June 5/77 



July 5/77 



Aug. 10/77 



Sept. 6/77 



20 



16 



22 



15 

21 
1 

8 

15 

22 

98 



0-2 
2-4 

0-2 
2-4 

0-2 
2-4 

0-2 

2-4 

0-2 
2-4 
4-6 

0-2 

2-4 

0-2 
2-4 
4-6 

- 2 
2-4 

0-2 
2-4 

0-2 

0-2 
2-4 

0-2 
2-4 

0-2 
2-4 

0-2 

2-4 

0-2 
2-4 
4-6 



1.6 (1.0) 

NF 

0.3 (0.2) 
NF 

0.3 (0.2) 
NF 

0.3 (0.2) 

54.8 (30.2) 
NF 

3.0 (1.7) 

NF 

8.5 (2.7) 

NF 

1.2 (0.5) 

NF 



NF 



1.5 (0.8) 
0.3 (0.1) 



2.4 (1.0) 

NF 

1.2 (0.4) 
NF 

0.3 (0.1) 
NF 

0.9 (0.3) 

NF 



NF 



NF 

0.6 (0.4) 
2.1 (0.8) 

NF 
NF 

0.3 (0.1) 

NF 
NF 



0.6 (0.2) 
0.3 (0.1) 

NF 

0.9 (0.5) 
0.3 (0.1) 

0.9 (0.3) 

NF 



9.1 (2.8) 
2.4 (1.6) 

13.0 (4.7) 
NF 

12.4 (3.5) 

6.1 (1.7) 

8.2 (2.7) 

NF 

0.9 (0.2) 

NF 



NF - none found 



49 



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 
applied. 

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, 
wet weather. 



51 



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 
pathogen-free sludge. 

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 
sludge ; 

- 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. 



52 



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, 
1978). 

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 



53 



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. 

4.2 Recommendations 

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 



54 



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. 



55 



REFERENCES 

Aiba, S. , and R. Sudo (1964), Discussion following paper by H. Liebmann, 
"Parasites in sewage and the possibilities of their extinction". Adv. in 
Water Pollut. Res. , 2:282-284. 

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56 



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58 



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59 



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62 



APPENDIX I 
PARASITE LIFE CYCLES 



APPENDIX i 
PARASITE LIFE CYCLES 



Protozoa 



Entamoeba histolytica 
Giardia lamblia 

Tapeworms 

Hymenolepis diminuta 
Hymenolepis nana (dwarf tapeworm) 
Taenia saginata (beef tapeworm) 
Taenia solium (pork tapeworm) 

Nematodes 

Ascaris lumbricoides (human roundworm) 

Ascaris suum (pig roundworm) 

Enterobius vermicularis (pinworm) 

Necator americanus and Ancylostoma duodenale (hookworms) 

Strongyloides stercoralis (threadworm) 

Toxascaris leonina 

Toxocara Canis and T. cati (roundworms of dogs and cats) 

Trichuris trichiura (whipworm) 



65 



PROTOZOA 



ENTAMOEBA HISTOLYTICA 



MAN 

(amoebic trophozoites 

15-60/^ rn in diam.) 



ABLE TO REPRODUCE IN 
THE BODY AND INVADE TISSUES 



INFECTIVE CYSTS 
SHED IN FAECES 



CYSTS 
INGESTED 



Asymptomatic infections of the intestine are most 
common, although invasion of the liver and lungs can 
produce severe disease symptoms. 



66 



PROTOZOA 



GIARDIA LAMBLIA 

MAN 
(trophozoite 15 x 10// m) 



ABLE TO REPRODUCE 
IN INTESTINE 



INFECTIVE CYSTS 
SHED IN FAECES 



CYSTS 
INGESTED 



In adults asymptomatic infections predominate, but 
in children there can be numerous gastrointestinal 
complaints . 



67 



TAPEWORM 



HYMENOLEPIS DIMINUTA 



RODENTS AND 

OCCASIONALLY MAN 

(10-60 cm long 

tapeworm in gut) 



EMBRYONATED 

OVA PASSED 

IN FAECES 



INTERMEDIATE HOST 
EATEN 



OVA INGESTED BY 

ARTHROPOD 

INTERMEDIATE HOST 

(flea, beetle) 



Infection in man produces few or no definite symptoms 



btt 



TAPEWORM 



HYMENOLEPIS NANA (dwarf tapeworm) 



MAN 
(2 cm long tapeworm in gut) 

i 



EMBRYONATED 

INFECTIVE OVA 

PASSED IN FAECES 



♦ 



INTERNAL 

AUTOINFECTION 

POSSIBLE 




•-OVA INGESTED 
BY MAN 



/ 



INGESTED BY X 

GRAIN BEETLE 

(may act as intermediate host) 



Light infections produce either no symptoms or vague abdominal 
disturbances . 



TAPEWORM 



TAENIA SAG1NATA (beef tapeworm) 



MAN 

(adult tapeworm 

5-10 m in gut) 



EMBRYONATED 
INFECTIVE OVA 
PASSED IN FAECES 



EATING 
UNCOOKED BEEF 






OVA INGESTED 

BY COW 

INTERMEDIATE HOST 

(cysticerci encyst m flesh) 



Infection in man is usually asymptomatic but occasionally with 
vague alimentary upsets. 



70 



TAPEWORM 



TAENIA SOLIUM ( (pork tapeworm) 



MAN 
(3 m adult 
tapeworm in gut) 



EMBRYONATED 
INFECTIVE OVA 
PASSED IN FAECES 




OVA INGESTED 
BY PIG 

INTERMEDIATE 

HOST 

(cysticerci in flesh) 

\ OVA INGESTED 

\ BY MAN 

OCCASIONAL 

INTERMEDIATE 

HOST 

(cysticerci in flesh, 
human cysticercosis) 



UNCOOKED 
PORK EATEN 



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. 



71 



NEMATODE 



ASCARIS LUMBRICOIDES (roundworm) 



MAN 

(20-35 cm long 
adults in gut) 



UNDEVELOPED 
OVA PASSED 
IN FAECES 



OVA EMBRYONATE 
(3 weeks or more) 



LARVAE COU6HED-UP 
AND SWALLOWED 



LARVAE MIGRATE THROUGH 
BODY FROM INTESTINE 
TO LUNGS 



OVA INGESTED 
BY MAN 



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. 



72 



NEMATODE 



ASCARIS SUUM (roundworm of pigs) 



PI6 

(20-35 cm long 

adults in gut) 



UNDEVELOPED 
OVA PASSED 
IN FAECES 




OVA INGESTED 
BY PIG 



LARVAE COUGHED -UP 
AND SWALLOWED 

4 



LARVAE MIGRATE THROUGl 
BODY FROM INTESTINE 
TO LUNGS 

/ 
/ 
/ 

/ 
/ 



/ 



/ 



y OVA INGESTED / 
BY MAN 



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. 



73 



NEMATODE 



ENTEROBIUS VERMICULARIS (pinworm) 



MAN 

(5-13 mm long 

adults in gut) 



EMBRYONATED 
INFECTIVE OVA 
DEPOSITED IN 
PERIANAL REGION 



OVA 
INGESTED 



A relatively harmless parasite but migrating female worms 
can cause intense itching in the perianal region. 



74 



NEMATODE 



NECATOR AMERICANUS AND 



ANCYLOSTOMA DUODENALE (hookworms) 



MAN 
(10 mm adult in gut) 



UNDEVELOPED 
OVA PASSED 
IN FAECES 



COUGHED-UP AND 
SWALLOWED 



OVA EMBRYONATE 
AND HATCH 
(eventually produce 
infective larvae 
0.5 mm long) 



LARVAE MIGRATE THROUGH 
BODY TO LUNGS 



LARVAE 
PENETRATE 
THE SKIN OF MAN 



Light infection are often asymptomatic but the worms can cause 
numerous allergic and gastrointestinal complaints. 



75 



NEMATODE 



STRONGYLOSES STERCORALIS (threadworm) 



MAN 
(2mm long 
adult in gut) 

\ 



LARVAE PASSED 

IN FAECES 
\ 



\ 



\ 



N 



\ 



\ 



\ 



FREE LIVING 
CYCLE 




\ 



\ 



LARVAE COU6HED-UP 
AND SWALLOWED 



\ 



LARVAE MIGRATE THROUGH 
BODY TO LUNGS 



INFECTIVE LARVAE 
PENETRATE SKIN 



Light infections are often asymptomatic but the worms can 
cause numerous allergic and gastrointestinal complaints. 



76 



NEMATODE 



TOXOCARA CANIS AND T. CATI 
(roundworms of dogs and cats) 



DOG OR CAT 

(10-20 cm long 

adults in gut) 



UNDEVELOPED OVA 
PASSED IN FAECES 



OVA EMBRYONATE 
(1 week or more) 

V 

V 

\ 
\ 

\ " OVA INGESTED 

\ BY DOG OR CAT 

\ 



LARVAE COUGHED-UP 
AND SWALLOWED 



LARVAE MIGRATE THROUGH 
BODY FROM INTESTINE 
TO LUNGS 



OVA INGESTED 
BY MAN 



LARVAE MIGRATE 
THROUGH BODY 



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. 



77 



NEMATODE 



TRICHURIS TRICHIURA (whipworm) 



MAN 
(3-5 cm long 
adults in gut) 



UNDEVELOPED 
OVA PASSED 
IN FAECES 



OVA INGESTED 



OVA 

EMBRYONATE 

(3-5 weeks) 



Light infections are usually asymptomatic 



78 



APPENDIX II 
PARASITE OVA RECOVERY FROM SLUDGE USING THE ZONAL ROTOR 



APPENDIX II 



PARASITE OVA RECOVERY FROM SLUDGE USING THE ZONAL ROTOR 

General 

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. 

Details 

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 

200 1.07 

200 1.15 

150 1.22 

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. 



82 



FLOW 
DEFLECTOR 



* VACUUM (APPROX. 15 cm) 



700 



500- 



300- 



GRADUATED CLEAR 
ACRYLIC COLUMN (mL) 



CLAMP 



SEPARATORY FUNNEL 
FILLED WITH WATER 
TO TRAP BUBBLES 



SAMPLE ADDITION 




GRADIENT AND 
PISTON FLUID 
ADDITION 



ZONAL 
ROTOR 



T 
3.5 cm 

k 



GRADIENT 



SAMPLE SUPERNATANT 



FIGURE II. 1. SIDE VIEW OF ZONAL ROTOR AND ASSOCIATED EQUIPMENT NEEDED TO 
WITHDRAW THE GJIADIENT. 





1.20-1 


> 




»- 


1.15 


> 




< 




cc 




(D 




O 


1.10 


LL 




o 




LJJ 




CL 


1.05 



1.00* 




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. 



84 



TD Parasites and the land 

774 application of sewage sludge / 

.G73 Graham, H. 3. 

P37 78895 
1981