&EPA
United States
Environmental Protection
Agency
Robert S. Kerr Environmental Research
Laboratory
Ada OK 74820
EPA-6 00/2-78-131 b
June 1978
Research and Development
Sewage Disposal on
Agricultural Soils:
Chemical and
Microbiologica
Implications
(Volume II
Microbiological
Implications)
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development, U.S. Environmental
Protection Agency, have been grouped into nine series. These nine broad cate-
gories were established to facilitate further development and application of en-
vironmental technology. Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in related fields.
The nine series are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4. Environmental Monitoring
5. Socioeconomic Environmental Studies
6. Scientific and Technical Assessment Reports (STAR)
7. Interagency Energy-Environment Research and Development
8. "Special" Reports
9. Miscellaneous Reports
This report has been assigned to the ENVIRONMENTAL PROTECTION TECH-
NOLOGY series. This series describes research performed to develop and dem-
onstrate instrumentation, equipment, and methodology to repair or prevent en-
vironmental degradation from point and non-point sources of pollution. This work
provides the new or improved technology required for the control and treatment
of pollution sources to meet environmental quality standards.
This document is available to the public through the National Technical Informa-
tion Service, Springfield, Virginia 22161.
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EPA-600/2-78-13113
June 1978
SEWAGE DISPOSAL ON AGRICULTURAL SOILS:
CHEMICAL AND MICROBIOLOGICAL IMPLICATIONS
VOLUME .II
MICROBIOLOGICAL IMPLICATIONS
by
R. W. Weaver, N. 0. Dronen, B. G. Foster, F. C. Heck, and R. C. Fehrmann
Texas A&M University
College Station, Texas 778^3
Grant No. R803281
Project Officer
Lowell E. Leach
Wastewater Management Branch
Robert S. Kerr Environmental Research Laboratory
Ada, Oklahoma 7^820
ROBERT S. KERR ENVIRONMENTAL RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
ADA, OKLAHOMA 7^820
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DISCLAIMER
This report has been reviewed by the Robert S. Kerr Environmental
Research Laboratory, U.S. Environmental Protection Agency4 and approved
for publication. Approval does not signify that the contents necessarily
reflect the views and policies of the U.S. Environmental Protection
Agency, nor does mention of trade names or commercial products constitute
endorsement or recommendation for use.
11
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FOREWORD
The Environmental Protection Agency was established to coordinate
administration of the major Federal programs designed to protect the
quality of our environment.
An important part of the agency's effort involves the search for
information about environmental problems, management techniques and new
technologies through which optimum use of the nation's land and water
resources can be assured and the threat pollution poses to the welfare
of the American people can be minimized.
EPA's Office -of Research and Development conducts this search
through a nationwide network of research facilities.
As one of these facilities, the Robert S. Kerr Environmental
Research Laboratory is responsible for the management of programs to:
(a) investigate the nature, transport, fate and management of pollutants
in groundwater; (b) develop and demonstrate methods for treating waste-
waters with soil and other natural systems; (c) develop and demonstrate
pollution control technologies for irrigation return flows; (d) develop
and demonstrate pollution control technologies for animal production
wastes; (e) develop and demonstrate technologies to prevent, control or
abate pollution from the petroleum refining and petrochemical industries;
and (f) develop and demonstrate technologies to manage pollution resulting
from combinations of industrial wastewaters or industrial/municipal
wastewaters.
This report contributes to the knowledge essential if the EPA is to
meet the requirements of environmental laws that it establish and enforce
pollution control standards which are reasonable, cost effective and
provide adequate protection for the American public.
c.
William C. Galegar
Director
Robert S. Kerr Environmental Research Laboratory
111
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ABSTRACT
The city of San Angelo, Texas, has been using agricultural land for
decades as a means of disposing of all its municipal sewage. The sewage
has only received primary treatment. Application rates have been such
that both row and hay crops have been grown. Additionally, the farm
has routinely supported approximately 500 cattle on its pastures.
Even though the farm consists of 259 ha, enough sewage £ffluent is applied
to it to satisfy the water requirements of crops grown on more than 600 ha.
Land application of sewage has public health implications. This study
was conducted to determine the public health implications of operation
of the sewage farm. This was accomplished by monitoring the soils and
waters on the farm to determine the incidence of salmonella and parasites.
Salmonella was isolated from various locations on the farm but the
frequency of isolation was not unusually high. Cattle grazing the pastures
on the sewage farm, generally, were not excreting salmonella in their
manure. Possible human parasites were not found in any effluent but
were present in sludge. The parasite population in cattle on the sewage
farm did not increase during the months the cattle were monitored. There
was an unusually high population of animal parasites in the sewage farm
soils as compared to similar soils off the sewage farm. This was thought
to be due to the higher animal density on the farm, the vegetative cover
on the farm and the relatively moist soil" conditions on the farm. Column
studies using soil from the farm indicated viruses could be leached
through the sewage farm soils. Methods used for detection of viruses on
the sewage farm were not sensitive enough to evaluate their potential
health hazard. The only public health hazard that was readily apparent
from this study was that seepage creeks originating from effluent applied
to the farm contained unusually high populations of coliforms. The seepage
water drains into the Concho River, which is used for recreational
activities and as a source of drinking water for cities downstream from'
San Angelo. This problem could be alleviated by expanding the size of
the sewage farm so that only enough water is applied to meet crop needs.
Presently, most of the applied water leaves the farm as seepage.
This report was submitted in fulfillment of Grant No. R803281 by
Texas A&M University under the sponsorship of the U.S. Environmental
Protection Agency. This report covers the period April 28, 1975, to
June 27, 1977, and work was completed as of June 27, 1977.
IV
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CONTENTS
Foreword 11:L
Abstract iv
List of Figures viii
List of Tables *
Acknowledgment s xiii
Sections
1. Introduction 1
2. Summary and Conclusions 3
3. Recommendations 5
k. Present Status of Information 6
Microbiological Studies 6
Leaching of Bacteria through Soils 6
Control of Bacteria Leaching through Soils 7
Survival of Fecal Indicator Organisms in Soil 8
Survival and Movement of Viral Particles through Soil 9
Parasitological Studies 10
Parasitic Protozoans 10
Parasitic Helminths 11
Parasite Life Cycles 13
5 . Materials and Methods l6
Site Description l6
Microbiological Studies 16
Field Study on Bacteria 16
Soil and Water Sampling 16
Sample Processing IT
Salmonella Isolation from Soil and Water 18
Salmonella Isolation from Bovine Manure - 20
Bacterial Analysis of Well Water Samples 20
Bacterial Analysis of Soil Core Samples 20
Laboratory Studies .with Bacteria 21
Soils 21
Inoculum Preparation ; 22
Column Preparation 2k
Adsorption to Soil 2k
Effect of Salts on Leaching = 25
Distribution in Columns 25
Saturation of Soils 25
Breakthrough Characteristics 27
Enumeration 27
Size 27
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Statistical Analyses 27
Field Study on Viruses 29
Soil and Water Sampling 29
Tissue Culture 29
Enumeration 29
Laboratory Studies with Viruses 29
Soils 29
Column Preparation 30
Inoculation and Enumeration 30
Distribution in Columns 30
Parasitological Studies 30
Detection of Possible Human Parasites in Sewage 30
Sewage and Water Sampling 30
Sample Processing and Examination 31
Detection of Possible Human Parasites in Sludge 31
Sampling 31
Sample Processing and Examination 31
Detection of Nematode Larvae in Soil 32
Sampling 32
Sample Processing and Examination 32
Detection of Parasites in Livestock Feces 33
Sampling of Livestock on Sewage Farm 33
Sample Processing and Examination 33
Monitoring of Parasite Buildup in Cattle Feces 33
Cattle 33
Sampling 33
Examination 33
6. Results and Discussion 3^
Microbiological Studies 3^
Field Study on Bacteria 3^
Populations in Sewage, Seepage Creeks, Lagoons and
the Concho River 31*
Populations in Well Water 37
Populations in Soil 37
Salmonella in Sewage, Seepage Creeks, Lagoons Soil
and the Concho River h 3
Salmonella in Bovine Feces ....' 46
Laboratory Studies with Bacteria it7
Introduction it7
Filter Selection ^7
Adsorption ^7
Bacterial Size ^9
Pore Size Distribution it9
Leaching Through Soils 51
Distribution of Bacteria in Columns 59
Effect of Salts on Leaching 60
Rate of Appearance in Leachate 6l
Saturation of Soil with Bacteria 65
Field Study on Viruses 65
Laboratory Studies with Viruses 67
Parasitological Studies 67
Detection of Possible Human Parasites in Sewage 67
vi
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Detection of Possible Human Parasites in Sludge 75
Detection of Nematode Larvae in Soil ^ 75
Detection of Parasites in Livestock Feces 75
Monitoring of Parasite Buildup in Cattle Feces 78
7. References , 87
Vll
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FIGURES
Number
1 The life cycle of Entamoeba histolytica 13
2 The life cycle of Strongyloides sp lU
3 The life cycle of Eimeria sp 15
k Water holding capacity of the four soils used in
laboratory studies on leaching of bacteria 23
5 Schematic of column assembly used in leaching
experiments 26
6 Total aerobic bacteria in water samples collected
each month from various locations on the San
Angelo sewage farm 35
7 Total coliforms in water samples collected each
month from various locations on the San Angelo
sewage farm. 36
8 Fecal coliform in water samples collected each
month from various locations on the San Angelo
sewage farm. 38
9 Pseudomonas aeruginosa in water samples collected
each month from various locations on the San Angelo
sewage farm 39
10 Enterococci in water samples collected each month
from various locations on the San Angelo sewage
farm ho
11 Location of fields, lagoons and seepage creeks on
the San Angelo sewage farm «. ill
12 Light micrographs of the bacteria used for size
determinations 50
13 The effect of soil column height on the number of
bacteria per ml of leachate 53
viii
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ih The effect of soil column height on the number of
bacteria per ml of leachate 56
15 The effect of soil column height on the number of
bacteria per ml of leachate 58
16 Number of bacteria present in consecutive 2 ml
increments of leachate , 63
17 Cumulative numbers of bacteria present in
consecutive 2 ml increments of leachate 6k
18 Bacteria present in the leachate after four
consecutive inoculations of Salmonella
typhimurium , 66
19 Approximate locations where soil and feces
samples were taken from the sewage farm for
detection of nematodes parasitic on animals 77
IX
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TABLES
Number
1 Volumes of Liquid Filtered and Media Used for
Determination of Salmonella sp. in Water from
the Concho River and in Water from Seepage
Creeks [[[ 12
2 Physical and Chemical Characteristics of Four Soils
Used in Laboratory Studies on Leaching of
Bacteria [[[ 21
3 Pore Size Distribution of the Soils Used in
Laboratory Studies on Leaching of Bacteria ..................... 22
k Initial Inoculum of Bacterial Suspension, per ml,
Added to Each Column ........................................... 25
5 Concentration of Inoculum Added to Each Soil ..................... 28
6 Populations of Bacteria in Surface Soil Samples
Collected from Several Sites on the Sewage
Farm and a Site off the Sewage Farm .............. . .......... '. . .' ^2
7 Total Aerobic Bacteria Present in Core Samples
of the San Angelo Series Soil on the San Angelo
Sewage Farm and a Farm Not Receiving Sewage
8 Total Aerobic Bacteria Present in. Core Samples
of the Rio Concho Series Soil on the San Angelo
Sewage Farm and a Farm Wot Receiving Sewage
9 Coliform Bacteria Present in Core Samples of the
Rio Concho Series Soil on the San Angelo Sewage
Farm and a Farm Not Receiving Sewage
10 Coliform Bacteria Present in Core Samples of the
San Angelo Series Soil on the San Angelo Sewage
Farm and a Farm Not Receiving Sewage
11 Number of Sewage, Water and Soil Samples That
Salmonellae Was Isolated From
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13 Decreases in Numbers of E_. coli When an Initial 10
ml Aliquot of the Bacteria Were Passed Through
Different Types of Filters .................................... 1|8
lU Percentage of Bacteria Adsorbed onto Soil Particles
Greater Than 1 urn In Diameter ................................. 1|8
15 The Proportion of Added Bacteria That Leached
Through Columns of an Arenosa Loamy Sand ...................... 52
16 The Proportion of Added Bacteria That Leached
Through Columns of a San Angelo Sandy Clay Loam ............... 55
17 The Proportion of Added Bacteria That Leached
Through Columns of a Houston Black Clay ........ j .............. 57
18 Total Numbers of Salmonella typhimurium in Sections
of 15 cm Soil Columns After Leaching .......................... 6l
19 Effects of Different Salt Solutions on the Leaching
of Salmonella typhimurium Through 15 cm Columns
of Soils [[[ 62
20 Presence of Virus in Collections of Leachate from
Glass Columns Filled With a San Angelo Sandy
Clay Loam and Repetitively Leached With
Physiological Saline .......................................... 68
21 Presence of Virus in Collections of Leachate from
Glass Columns Filled With a San Angelo Sandy
Clay Loam and Repetitively Leached With Water ................. 69
22 Presence of Virus in Collecitons of Leachate from
Glass Columns Filled With a Houston Black Clay
and Repetitively Leached with Physiological Saline ............ 70
23 Presence of Virus in Collections from Glass Columns
Filled With a Houston Black Clay and Repetitively
Leached With Water ............................................ 71
2k Presence of Virus in Soil Collected from Columns
of a San Angelo Sandy Clay Loam That Were Inocu-
lated With Virus and Leached With Physiological Saline
or Water [[[ 72
25 Presence of Virus in Soil Collected from Columns of
a Houston Black Clay That Were Inoculated With
Virus and Leached With Physiological Saline or Water .......... 72
26 Number of Four Possible Human Parasites in Raw
Sewage Entering the Sewage Treatment Plant During
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27 Relative Estimates of Four Possible Human
Parasites in Sludge 7U
28 Number of Strongyloidldae in Surface Soil
From the Sewage Farm and a Farm Not Receiv-
ing Sewage During November, December and
January l6
29 The Number of Gongylonema in Manure Collected
During Four Months from Cattle Being Grazed
on the San Angelo Sewage Farm and on an
Adjacent Control Farm Not Receiving Sewage 79
30 The Number of Eimeria in Manure Collected
During Four Months from Cattle Being Grazed
on the San Angelo Sewage Farm and on an
Adjacent Control Farm Not Receiving Sewage 80
31 The Number of Haemonchus in Manure Collected
During Four months from Cattle Being Grazed
on the San Angelo Sewage Farm and on an
Adjacent Control Not Receiving Sewage 8l
32 The Number of Parasites, in Two Genera,
Detected in Cattle Manure Collected in
January on the San Angelo Sewage Farm and
on an Adjacent Farm Not Receiving Sewage 82
33 The Number of Parasites, in Three Genera,
Detected in Sheep Manure Collected During
Three Months on the San Angelo Sewage Farm
and on an Adjacent Farm -Not Receiving Sewage 83
3^ The Number of Parasites, in Two Genera, Detected
in Sheep Manure Collected in January on the
San Angelo Sewage Farm and on an Adjacent
Farm Not Receiving Sewage 83
35 Average Number of Eimera sp. in Feces from
Ten Test Cattle for Seven Months After Arriv-
ing on the Sewage Farm 85
36 Average Number of Haemonchus sp. in Feces from
Ten Test Cattle from Seven Months After
Arriving on the Sewage Farm 86
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ACKNOWLEDGEMENTS
The numerous aspects of this project required input from many individu-
als. The officials with the city of San Angelo that assisted in the project
were Mr. Harry Behrend, farm manager, and Mr. Bob Pryor, municipal sewage
division manager. The assistance and cooperation of these city officials
was appreciated very much and was essential to this project. Mr. Dean
Gilliland, research assistant, was responsible for the bacteriological survey
of the farm and was assisted by Mr. Pete Kelleher, research assistant. Mr.
Harold Underwood, research assistant, and Mr. Russell Ingham, research assis-
tant, were reponsible for processing and analyzing various samples for para-
sites. Mrs. Maria Schroeder, Technician I, was responsible for conducting
the virus assays. Mr. Tom Regmund, research assistant, conducted the virus
leaching experiments with soil columns. Mr. Alan Waggoner, research associ-
ate, lived at San Angelo and was responsible for assisting in all samplings.
Mrs. Nancy Clinton and Mr. Marcin Varanka, both research associates, assisted
in development of the finil report. The contribution of all these personnel,
to the project, is very much appreciated and was essential to its success.
The overall responsibility for the bacteriology research was shared by Dr.
Bill Foster, Associate Professor in the Biology Department, Dr. R.W. Weaver
and Mr. Robert Fehrman, respectively Associate Professor and Reasearcih Associ-
ate in the Soil and Crop Sciences Department. The person having overall
responsibility for the virology research was Dr. Fred Heck, Associate Pro-
fessor in the Veterinary Microbiology Department. The assistance of Mr.
Lowell Leach, EPA project officer, in planning, implementing and reporting
the research of this project is much appreciated. This project was supported
jointly by the Texas Agricultural Experiment Station and The U.S. Environ-
mental Protection Agency.
Xlll
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SECTION .1
INTRODUCTION
The interest in land disposal of wastes from animals, industries,
and municipalities has grown rapidly within the past few years. Rigorous
standards discouraging discharge of sewage effluents into waterways have
contributed to the current interest in land disposal. The benefits of
increasing ground water recharge and increasing soil fertility have also
been factors in the widespread appeal of land disposal (Sanitary Engineering
Research Lab. 1955). The application of waste water to land has been viewed
as an economical treatment process in which the soil acts as a "living
filter" (Kardos 1967). But, the application of wastes onto land does not
offer a solution to all problems of waste disposal. There are inherent
health hazards.
Disease caused by pathogenic microorganisms known to exist in municipal
wastes is of primary importance to public health (Krone 1968). The poten-
tial for disease transmission by application of sewage to crops has long
been recognized (Benarde 1973 and Decker and Steele 1966) and depends on
available vectors of infection. Important vectors of infection associated
with land disposal of sewage are runoff and deep percolation of water.
Salmonellosis is a disease of major public concern. More than 200,000
cases of salmonellosis are reported annually, but between 1 and 2 million
people are actually infected with salmonella annually (Aserkoff, et al.
1970). More than half the cases have been sporadic, but the remaining
cases have been associated with epidemics that can usually be traced to
contaminated foods of animal origin or to contaminated waters (Steele 1968).
Animal wastes are an important factor in perpetuating and extending
the prevalence of salmonella. However, the transport and survival of
salmonella in sewage waters is also important (Caldwell 1938, Claudon et
al. 1972, Dunlop 1968, Hibbs and Foltz 1969, Kampelmacher and Van JToorle
Jansen 1970, Krone 1968, Moore 1971 and World Health Org.,1975). Indivi-
duals infected with salmonella excrete an enormous number of cells daily; as
many as 10 cells. Therefore, a tremendous inoculum is provided from one
infected individual for contamination of water. For example, an inoculum
of 10 cells in an 18.9 million liter, or 5 million gallon a day sewage
plant, such as the one in San Angelo, Texas, would contain 5 organisms per
ml. Only one organism need be ingested under optimal conditions to cause
disease. The normal minimal infective dose" for salmonella is approximately
10,000 (Moore 1971).
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A bacteriological survey was performed on the sewage treatment facility
at San Angelo, Texas for 12 months. This facility utilized lagoons for
sewage treatment and storage. Water from the lagoons was used to irrigate
row crops and forages. Some forages were used for hay but others were
grazed by cattle.
Because land disposal is a popular method of sewage treatment in the
western areas of Texas, the potential health hazard from this method of
sewage treatment was determined. This was accomplished by making
parasitological and microbiological measurements on the sewage farm soil,
on the river bordering the farm, on seepage creeks entering the river from
the farm, and on the cattle grazing on the farm. Groups of bacteria assayed
for included coliforms, fecal coliforms, Pseudomonas aeruginosa, and
fecal streptococci. Salmonella sp. were qualitatively studied by using an
enrichment technique. Also, laboratory experiments were used to gain
information on the leaching of bacteria and viruses through soil.
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SECTION 2
v
SUMMARY AND CONCLUSIONS
MICROBIOLOGY
The population of bacteria in the sewage lagoons on the San Angelo
sewage farm was only 10$ as large as the population in the raw sewage.
Total coliforms and fecal coliforms in the seepage creeks fluctuated from
month to month between 10 and 1,000 per ml. These populations of coliforms
were very high for normal seepage creeks. The ratio of fecal coliforms to
enterococci indicated the seepage waters were mainly polluted with sewage
and not cattle manure. Generally, the Concho River contained fewer than
50 total coliforms per ml and there was no indication that the population
of coliforms in the river was significantly increased by the sewage farm.
This was probably due to the large dilution by the river water. Salmonella
was isolated from raw sewage, from soil on the sewage farm, and from the
seepage creeks. The proportion of cattle grazing on the sewage farm
that were shedding salmonella in their manure was not unusually high.
Apparently, grazing cattle on the sewage farm did not result in their
becoming infected with salmonella. The sewage farm had no affect on the
microbiological content of deep wells used for drinking water in the area
surrounding the sewage farm. Coliform populations in the surface horizon
of sewage farm soils ranged from fewer than 100 to 70,000 per g of soil.
Similar soils off the sewage farm contained fewer than 100 per g of soil.
The highest population of coliforms was in the surface horizon of the
sewage farm soils. The number was reduced by more than 90% at 5 cm below
the surface of the Rio Concho soil. In laboratory investigations, the
San Angelo soil had a greater adsorbtive capacity for a strain of S_.
typhimurium than for a strain of E_. coli. The concentration of S_.
typhimurium and S_. enteriditis in the leachate from a 3 cm soil column was
reduced by more than 99%- However, the population of E_. coli was reduced
by 99-9$. The leachability of all the bacteria was similar for longer
columns.
f
Viruses were not isolated from samples of sewage, soil or water
collected from the San Angelo sewage farm. The method used in this study
required at least 10 viruses per g of material for detection. Laboratory
studies revealed that viruses could be leached through 15 cm columns of
the San Angelo clay loam. When either distilled water or physiological
saline was used as the eluent, viruses were leached through some columns
of this soil. Viruses were not leached through every soil column. The
San Angelo clay loam was more effective in adsorbing or inactivating viruses
than a Houston Black clay.
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PARASITOLOGY
Human parasites were present in sewage entering the San Angelo
sewage treatment facility. They were equally prevalent during the
winter and summer months. Yet, parasites were rarely detected in irrigation
effluent from sewage lagoons. Apparently the parasites settled with
the sludge in the lagoons. Sludge in abandoned sludge lagoons contained
organisms that appeared to be human parasites. From these observations,
we concluded that sewage effluent undergoing a simple pretreatment like
settling would be relatively free of parasites. But the sludge would
likely contain human parasites and would require special considerations for
land application. Inspection of cattle and sheep from the sewage farm
showed that these animals were not picking up human parasites and therefore
did not serve as reservoirs for them. A Trichuris sp. (nematode) and an
Entamoeba sp. (protozoan), which were similar to those found in humans,
were found in very low frequencies, but they were presumed to be non-
human parasites. There were greater densities of soil-borne larval nema-
todes of livestock on the farm as compared to a nearby control area. It
is probable that continued irrigation of this land and the ability of the
treated land to support more vegetation and livestock was beneficial to
the survival of these larvae. There was a higher density of nematode eggs
in feces from cattle and sheep on the sewage farm compared to livestock
off the farm, but parasites in cattle released on the farm did not increase.
The parasite population in the cattle when placeu on the farm was already
comparable to cattle being grazed on the farm. Attempts to reduce the
parasite population by using an antihelminthic drug was unsuccessful.
The main influence on animal parasites from irrigation of land with sewage
water appears to be the indirect effect of extending the survival and
density of livestock parasites by keeping the soil moist and providing more
vegetation to support a greater animal density which directly increases
problems associated with parasites.
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SECTION 3
RECOMMENDATIONS
Land application as the method of disposing of the sewage from San
Angelo should be continued. The semiarid and warm climate and relatively
permeable soils of the San Angelo area are ideal for utilizing land
disposal of sewage. Also, farmland is abundant in the area around San
Angelo and surface supplies of irrigation water are not readily available.
There was no evidence that human or animal health was endangered on the
sewage farm due to application of sewage materials. Animal parasites
were more abundant on the sewage farm than in surrounding land but this
may have been due to the higher animal density on the farm. The sludge
deserves special considerations before land disposal because it likely
contains most of the human parasites. The main problem with the past
procedure of sewage effluent disposal on the sewage farm was lack of
sufficient land. This resulted in large quantities of water leaving the
sewage farm as seepage. This seepage water frequently contained
thousands of coliform bacteria per ml. The sewage farm should be doubled
or tripled in size to allow for sufficient land area to adequately
utilize the effluent and to prevent sewage bacteria from leaving the
farm in seepage water.
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SECTION h
PRESENT STATUS OF INFORMATION
MICROBIOLOGICAL STUDIES
Leaching of Bacteria through Soils
The movement of bacteria through soils has important agricultural and
ecological implications. !j The realization that bacteria travel through soils
has been known for many years. In 1909, Dittlorn and Luerssen studied the
passage of bacteria through soils. During the 1930's a number of investi-
gations dealt with contamination and bacterial movement from bored hole
latrines. Caldwell (1938) and Caldwell and Parr (1937) observed that bacteri-
al travel was a function of the characteristics of the medium, and that
accumulations of solids and slimes in the medium slowed bacterial movement.
There has been considerable research conducted on the movement of
coliform bacteria through soils from sewage and related wastes. Krone,
Orlob, and Hodgkinson, in 1958, concluded that removal of bacteria from
water passing through soils was due in part to sedimentation in the pores.
They also determined that the sedimentation removal mechanism controlled
the subsequent passage of bacteria and that the soil surface played a
dominant role in the travel of bacteria, operating in a manner to restrict
continued infiltration of bacteria.
Jones (1968) studied the movement of coliform bacteria and organic
matter in an aquifer that was artificially recharged with playa lake water.
He concluded that the coliform bacteria were not likely to travel more than
31 meters in the fine sand of the aquifer, but bacterial travel might
increase in more- permeable soils.
In a study by Randall (1970) a municipal well was found to be contam-
inated with enteric bacteria after 19 years of trouble free operation. It
was found that bacteria from the sewage-polluted Susquehana River leached
55 meters to the municipal well after the river bed was excavated. This
excavation of the river bed favored induced infiltration by the contaminated
river water.
Reneau, et al. (1975) studied the movement of bacteria from septic tank
sources in Virginia. Their conclusions were: Coliform bacteria would
probably not move into the groundwater system because of restrictive layers
of soil, and the drainage water from the watershed would improve with
distance from the pollution source as a result of dilution, sedimentation,
and bacterial die-off.
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Evans and Owens (1972 and 1973) studied factors which affected the con-
centration of fecal "bacteria in land drainage water. Additions of pig
manure increased the concentrations of Escherichia coli and enterococci in
the drainage water. The flow rate of the discharge also affected the
concentration of bacteria in the drainage water.
Korkman (1971) studied the survival and leaching of enterococci from
applied liquid manure under field conditions. A field was topdressed with
50 metric tons/ha of liquid pig manure and irrigated with 100 mm of water.
Discharge pipes were 1 m below the surface. He reported that 3 percent
of the applied bacteria leached into the drainage pipes, and that the
bacteria did pass through the pores of clay soils.
There has been very little documentation of the movement of salmonella
in soils (Decker and Steele 1966). This has been because the number of
salmonella in sewage is much less than the number of coliform bacteria
(Boring 1971, Krone 1968, Moore 1971, Dunlop 1968, Water Quality Criteria
1972), and the detection of salmonella in the environment is much more dif-
ficult than the detection of coliforms. Yet salmonella has been found in
many water supplies throughout the United States (Claudon et al. 1972,
Hibbs and Foltz 1964, Thompson et al. 1975). A large waterborne outbreak
occurred in Riverside, California that affected over 16,000 individuals.
Boring, et al. (1971) investigated the presence of salmonella in municipal
wells during the epidemic. They foumi five or six municipal wells contami-
nated with Salmonella typhimurium. Water samples from around the city
demonstrated 10 times as many salmonella as E. coli. The source of contami-
nation was not known, however there was speculation that the water table
may have been contaminated by seepage hundreds of kilometers away from the
city.
Control of Bacterial Leaching through Soils
There are a number of factors which control the leaching of bacteria
through soils. Among these are filtration, the adsorptive capacity of
the bacteria to soil, soil water content, and soil water flux (Bitton, et
al. 197^, Burges 1950, Griffin and Quail 1968, Hattori and Hattori 1976,
Marshall 1971, Reneau et al. 1975, Wong and Griffin 1976). Krone (1968)
summarized the filtration process. Case I was that the bacteria were larger
than the pores and were strained at the soil surface. When the b'acterial
cells accumulated on the soil surface, they became the filter. Case II,
called bridging, occurred when the cells were slightly smaller than the
pores. In this case the cells traveled into the soil until a pore opening
was too small. Then the cell "bridged" between two soil particles, and cells
behind it were filtered out. Case III, called-straining and sedimentation,
included cases I and II, as well as removal of small bacteria by fluid
passing through the very fine pores and crevices. The adsorbing nature of
a .soil depended on the texture and composition of the soil, the nature of
the cations around the soil, the number of bacterial cells present, pH,
electrolytic concentration, and other factors.
Active bacterial movement is greatly affected by the water content of
a soil. Bacteria are restricted to movement in water filled pores or water
-------
films, and as the water content decreases more bacteria are retained in the
soil. Griffin and Quail (1968) presented suggestions for defining the
limitations of the physical regime permitting bacterial movement in soils.
They were: Bacteria depend on-a continuous, water-pathway where the water
filled pores have a greater pore neck diameter than 2 to 3 urn, and the lense
of water in very large pores must "be sufficiently large and be in contact
with a number of soil particles. They used soil and aluminum grit columns,
1 cm deep, and varied the moisture content. Their results demonstrated that
a critical volume of water was necessary for appreciable movement of bacter-
ia to occur. Griffin and Quail concluded that movement of bacteria in most
soils was very restricted if the soil was much drier than field capacity.
Hamdi (1971 and 197*0 studied the active movement of Rhizobium trifolii
in soils. His results generally agreed with those reported by Griffin and
Quail. Wong and Griffin (1976) studied the active movement of different
species of bacteria through soils. Bacillus subtilis and Azotobacter
did not move appreciably through natural soils at matrix potentials greater
than -150 cm of water.
There have been a few investigators that have used soil columns to
determine the movement and retention of certain bacteria. Bitton et al.
(197*0 was interested in the differences between bacterial movement in
saturated and unsaturated soils. Four soils were used, but a sandy soil was
used for most of the experiments. Tl^elr results indicated" that non-
encapsulated bacteria were least affected by soil water content. They
postulated that the encapsulated bacteria were larger and therefore more
subject to filtration. The bacteria did not move with the water when the
water content dropped below 15 percent in the sandy soil.
Survival of Fecal Indicator Organisms in Soil
It has been reported that the detection of fecal indicator organisms
suggests the occurrence of pathogenic organisms from sewage (Wilson et al.
1968). The potential health hazard is dependent on the retention of
sufficient numbers of pathogenic bacteria in water to transmit disease
(McPeters et al. 197^, Geldreich and Kenner 1969). In a laboratory study
this group noted the decline of fecal streptococci and fecal coliforms
isolated from domestic sewage. The reduction in numbers of pure cultures
of these indicator organisms followed a two-log reduction per,day. This was
well correlated with reduction kinetics for Salmonella typhimurium and
Shigella dysenteriae (McFeters et al. 197*0. By comparison the decline
was much more rapid in pure cultures than under natural conditions (McFeters
et al. 197*0- Similar results were observed by Hyde (1976) on the survival
of Streptococcus faecalis in sewage sludge. This pathogenic organism
survived at least 7 months after only one application (Hyde 1976). Addition-
al studies have been performed suggesting that disease producing bacteria
decrease exponentially outside the normal host but that this decrease is
related to the adversity of the environment (Mack et al. 1958). Adaption
to the outside environment, however, is possible, and potential hazard is
dependent on the level of contamination, the specific type of bacteria, and
on conditions in the soil and water (Mack'et al. 1958).
-------
The significance of Salmonella-contaminated water is realized in the
reports of Craun (197*0 and Reasoner (197*0- These independent researchers
concluded that surface waters could serve to transmit disease to man. Their
studies were performed on salmonellae shed in the feces of infected animals
which contaminated water supplies.
Some question has been raised as to the validity of the presence of
fecal organisms as an indication of the presence of pathogenic bacteria
(Fair et al. 1967 and 1971). Benarde (1973) reported the recovery and per-
sistence of pathogenic bacteria in the absence of detectable coliforms. He
suggested that coliforms do not indicate anything but the presence of fecal
matter. Another group has suggested that salmonella can become widely
distributed in surface waters in the apparent absence of coliforms and
could therefore pose a potential health hazard (Cherry et al. 1972). The
use of fecal indicators for determining potential presence or absence of
intestinal pathogens then remains questionable although at present no other
rapid method exists.
Survival and Movement of Viral Particles through Soils
Most wastes are treated, or stored, before application to land, and the
populations of microorganisms in the wastes are altered (Elliott and Ellis
1977). Pathogenic microorganisms are poor competitors outside the host
and either die or just survive rather, than proliferate in treated wastes.
Viruses are more resistant than bacteria to chlorination and significant
numbers remain active after chlorination (Foster and Engelbrecht 1973).
Wellings at al. (197*0 reported that there was a threefold reduction in
viruses due to chlorination. These results are not directly applicable to
the sewage treatment system used at San Angelo because chlorination has not
been used to treat the wastes before land application.
More than 100 different viruses are known to be excreted in the feces
of man (Benarde 1973, Rivers and Horsefall 1959)- Viruses of primary
concern are those regularly excreted in large quantities in the feces of
infected individuals; infectious hepatitis and entero virus (Berg 196*0.
The only documented cases of waterborne viral outbreaks have been due to
viral hepatitis (Clarke and Kabler 196*0.
A factor of importance in considering waste application to land is the
movement of viral particles through soils. There have been several investi-
gators that have studied the effects of soil on viral percolation. Most
agree that soil can very effectively filter viruses but some viruses do
pass through most soils (Drewry and Eliassen 1968, Duboise et al. 197**»
Dugan et al. 1975, and Young and Burbank 1973). McMichael and McKee
(1965) failed to isolate virus from primary effluent and secondary chlori-
nated sewage that was percolated through 6l cm of soil. Gilbert et al.
(1976) also experimented with removal of viruses from wastewaters by land
application. They determined that the number of viral particles were
reduced by four logs from percolating a viral solution through 9 mm columns
of sandy loam soil.
-------
Some investigations have determined factors affecting viral percolation.
Drewry and Eliassen (1968) reported that the cation concentration of the
liquid affected adsorption of viral particles and that soils with high clay
or silt content readily adsorbed viruses. They concluded that the removal of
viral particles from percolating waters was primarily due to adsorption and
that viruses were not likely to be leached into ground waters. Cooper et al.
(1975) and Wellings et al. (197*0 published data showing that viral movement
through soils was increased when distilled water was used for leaching.
Both Duboise et al. (1976) and Cooper et al. (1975) have demonstrated that
viruses were released from soils with addition of distilled water. The
leachability of different types of viruses may vary considerably. Young
and Burbank (1973) stated that polio viruses were less susceptible to ad-
sorption onto soil surfaces than other viruses.
Little information exists on factors influencing virus persistence in
soils. Gerba et al. (1975) has reported that kaolinite clay and cations
may prolong virus survival in sand. Virus adsorption onto clays plays an
important role in viral removal, but cannot be equated to their inactivation
(Bitton 1975). Bagdasar'yan (196*0 reported that enteroviruses adsorbed
on loamy and sandy loam soils remained infective and Schaub et al. (197*1-)
has shown that viruses adsorbed to clay were just as infectious as free
viruses.
PARASITOLOGICAL STUDIES
Parasitic Protozoans
One of the major protozoans in sewage is Entamoeba histolytica, the
causative agent of human amoebiasis which varies in its effect on humans
from mild abdominal discomfort involving diarrhea alternating with consti-
pation to chronic dysentery with mucous and blood. This parasite can be
extremely dangerous if it becomes extra-intestinal. Approximately 12
percent of the people in North America are infected with this parasite (Beld-
ing 1952). High levels of occurrence of this parasite have been reported
to be related to certain occupations and income levels. It is interesting
to note that only 9 percent of agricultural workers cultivating irrigation
fields where normal irrigation procedures were used have been shown to
harbor E_. histolytica, while over lU percent of municipal sewage plant
operators have been shown to be infected with this parasite (Mitchell 1972).
Tsuchiya (19^0) has demonstrated that 2 to 5 percent of humans are infected
with E_. histolytica but do not have clinical symptoms from it and act as
carriers that are not generally detected.
Entamoeba histolytica has been demonstrated from sewage in many parts
of the world. Several authors have noted the presence of this parasite in
sewage and have examined its transmission to man from sewage (Dixon and
McCabe 196*0. Faust and Russell (1957), Tobie (l8*K)), Scott and Littig
(1962), and other authors found that numerous animals served as vectors to
carry this parasite from sewage treatment plants to humans. Primarily flies
have been implicated but cockroaches, dogs, and rodents have also served
10
-------
as vectors. Pipkin (19^9) concluded that in areas where human sewage is
not protected and flies have easy access to it, there is a much greater
chance for spread of E_. histolytica. Brooke (196*0 suggested that primitive
sewage disposal systems such as surface or pit privies were responsible
for contamination of the rural environment with this parasite.
Faust and Russell (1957) showed that human fecal contamination such as
that incurred when sewage wast%water and sludge were applied to crops, was
responsible for the spread of amoebic dysentery. The threat was especially
large where large gatherings of people were established in close proximity
to the disposal site. In sewage, cyst viability reduced by 30 percent for
each 10" C rise in temperature (Cheng 1973).
Another protozoan which has been detected in sewage is Giardia lamblia
the causative agent of human giardiasis. Depending on the geographic
location within the United States, infection ranged from 1 to 20 percent
(Healy 1969)- When pathogenic, this parasite causes diarrhea, abdominal
pain and loss of weight (Noble and Noble 1971) Its presence apparently
interferes with fat absorption in the small intestine. Generally, this
disease is more serious in children than in adults. This parasite has been
implicated with sewage contamination in a few cases (Moore 1969 and Scott
and Littig 1962). But Scott and Littig also indicated that animals, such
as the fly, could transport this parasite as well.
There are a number of other parasitic protozoans which potentially
could be found in sewage and sludge. Two of these are Entamoeba coli, an
amoeba which in most cases is not pathogenic in man, and Naegleria gruberi,
a pathogenic amoeba which causes fatal amoebic meningocephalitis in man
(Duma et al. 1969)
Parasitic Helminths
There are numerous parasitic worms which are of concern to public
health. Fortunately, several of the more pathogenic species (eg. Clonorchis
sinensis, human schistosmoes, and Paragonimus westermanii) have not been
introduced into North America apparently because of the lack of susceptible
intermediate hosts.
/
In the United States, Taenia saginata, the beef tapeworm of man,
causes clinical symptoms such as abdominal pain, digestive disorder, loss
of weight, and a variety of other symptoms (Belding 1952). Adult tapeworms
in the intestine of man discharge a million or more eggs per day in the
feces of infected humans. Cattle may become infected by grazing on pastures
where sewage wastewater has been used for irrigation or by drinking grossly
polluted water (Mitchell 1972). The intermediate stage of this parasite
normally develops in the muscle of cattle but man has been shown to be an
occasional accidental host for this stage as well.
Because of this parasite large numbers of cattle are condemned each
year by USDA inspectors. Man becomes infected with the adult tapeworm by
eating poorly cooked beef that contains the intermediate stage of this
parasite.
11
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The eggs of T_. saginata have frequently been observed in sewage (Hamlin
19^6). The eggs were able to survive for 335 days under cool moist condi-
tions (Silverman and Griffith 1955). Cheng (1973) states that the eggs of
this species were commonly transported by birds.
Ascaris lumbricoides is an intestinal nematode in man as an adult but
will also make a larval migration through the blood system and lungs before
reaching the intestine (Belding 1952). Adu#t females may discharge 200,000
or more eggs per day which can remain viable in water or soil for months.
This parasite has been found in about 20 percent of the sewage operators and
16 percent of the farmworkers where sewage irrigation is used (Mitchell
19T2). Monitoring of sewage in Poland is used as an index of the incidence
of ascariasis in the human population (iwanczuk and Stobnicka 1968). Raw
sewage from Darmstadt, Germany, has been shown to contain 5^0 eggs of this
parasite/100 ml of fluid (Mitchell 1972). The high density of this parasite
in Germany was attributed to application of raw sewage to gardens. Similar-
ly, viable eggs of A. lumbricoides have been detected in the Colorado River
downstream from where a chlorinated sewage effluent was discharged (Wang and
Dunlop 195*0. Some livestock, especially pigs, could become infected by
grazing on sewage irrigated fields and act as a reservoir for this parasite.
Other ascarids, such as Toxacara sp. which normally are parasites of
dogs, were shown on the national news last year to cause a serious disease
in children called larval migraines. This can be very serious especially when
the larvae invade the brain and central nervous system of the host. This
genus may be in municipal sewage if pet feces are disposed of in household
toilets, but pet feces can also reach the sewage treatment facility in gut-
ter washings.
Hookworms, Hecator americanus and Ancylostoma duodenale, are debilita-
ting parasites of humans. Symptons are loss of energy and anemia and resem-
ble malnutrition. This disease is endemic in the southeastern United States.
Humans are infected by a filariform larvae which penetrates through the skin.
In addition, hookworm infections are known to occur in higher than normal
levels where sewage is used for irrigation. In his book, "The Plague Kil-
lers", Greer Williams sums up the efforts of years of attempting to elimi-
nate human hookworms by pointing out the eradication is not possible and
that proper disposal of waste products from human and reservoir hosts and
that wearing of shoes are the only means by which this group of parasites
can be kept in check.
Other parasites (e.g. Diphyllobothrium latum, Dipylidium caninum,
Echinoecus sp.) are human parasites which could possibly be associated with
sewage wastewater and sludge.
Currently Fox and Fitzgerald (1976) from the University of Illinois
have been monitoring the kinds of parasites in sludge and have found A.
lumbricoides, Toxacara sp., Trichuris sp., Teania sp. and strongyloid type
nematode eggs, as well as some coccidia. Fitzgerald and Ashley (1976) have
been investigating the survival of the egg of Ascaris sp. in sludge.
12
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Parasite Life Cycles
The following are life cycle diagrams of the more prevalent parasites
found in this study. Figures 1, 2, and 3 are life cycle of Entamoeba
histolytica, Strongyloides sp., and Eimeria, respectively.
HUMAN HOST -
INTESTINE TROPHOZOITE
EXTRA -
INTESTINAL
TROPHOZOITE
STATE OF
INCYSTATION
PRE-CYST
START OF
EXCYSTATION
METACYST
COMPLETION OF
INCYSTATION
EXTERNAL
ENVIRONMENT
CYST
Figure 1. The life cycle of Entamoeba histolytica.
Human infected by ingestion of cyst stage from polluted water, infected
food handlers, flies contaminating food, night soil cultivation and direct
introduction of cyst. The same life cycle has been demonstrated for E_. coli.
Giardia sp. has a very similar life cycle to that described above for
_E. histolytica. Extra-intestinal implications are known only for E.
histolytica.
13
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DEFINITIVE HOST - INTESTINE
FEMALE ADULT
EGGS IN TISSUES
if-
FS LARVAE'
PENETRATES
HOST
RHI LARVAE FROM
EGG MIGRATES TO
LUMEN OF
INTESTINE -
PASSED IN FECES
. OF HOST
EXTERNAL ENVIRONMENT
LARVAL FORMS IN SOIL
RHI AND RH2
RH3 LARVAE START
OF.FREELIVING CYCLE
FREE LIVING LARVAL
FORMS
Figure 2. The life cycle of Strongyloides sp.
Infection of definitive host by F3 larvae either from environment or
by internal or external autoinfection.
Haemoncus contortus has a similar life cycle to that found in the
parasitic portion of Strongyloides sp. except that eggs rather than larvae
are passed in the feces and hatch in the environment.
-------
DEFINITIVE HOST
MACROGAMETOCYTES
MICROGAMETOCYTES
REINFECTION OF CELLS
\
ZYGOTE
OOCYST
IN CELL SCHIZC30NY PRODUCES MEROZOITES
OOCYSTS INGESTED RELEASING
SPOROZOITES WHICH PENETRATE
INTESTINAL CELLS
EXTERNAL ENVRIONMENT
SPOROGONY FORMING THE
SPORULATED OOCYST IN
SOIL
Figure 3. The life cycle of Eimeria sp.
Infection' of the definitive host is by ingestion of the sporulated
oocyst.
15
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SECTION 5
MATERIALS AND METHODS
SITE DESCRIPTION
The main study area was the sewage farm operated by the City of San
Angelo, Texas. This city has a population of approximately 67,000 inhabi-
tants. The sewage disposed of on the sewage farm was predominantly from
households with less than seven percent of it contributed from industries.
The average daily flow of sewage to the farm was approximately 0.2U m /sec.
Treatment of the sewage was minimal before application to the land.
During the time of the study, a new sewage treatment facility was under
construction and much of the old facility was inoperable. The incoming
sewage was put through a primary filter and distributed to the sewage la-
goons . Water for irrigation was pumped from the lagoons. Retention time
in the lagoons was not known because there was no set schedule for using
the lagoon water for irrigation.
The farm consisted of 259 ha of land used for pasture and hay produc-
tion and 77 ha of cultivated land. Approximately 600 cattle were normally
being grazed on this land. Generally, small calves were not kept on the
farm. All the land on the farm was highly productive because neither
water nor fertilizer nutrients were limiting.
The fields of the sewage farm were levied to prevent surface runoff
of water. Irrigation was accomplished by flooding. Internal drainage of
the soils was very good, and much more water was applied than was needed to
meet the requirements of the crops. The excess water drained through the
soils and surfaced in seepage creeks on lower areas of the farm. The
seepage creeks drained into the Concho River.
MICROBIOLOGICAL STUDIES
Field Study on Bacteria
*
Soil and Water Sampling
Water, sewage, and soil samples were collected for bacteriological
analysis at monthly intervals. Samples were evaluated as to'total aerobic
bacterial counts, bacterial indicators of human pollution, and potential
human pathogens. These included total and fecal coliforms, Salmonella sp.,
Shigella sp., enterococci, and Pseudomanas aeruginosa.
16
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A total of eleven water and sewage samples were collected per month.
These included one sample of raw sewage taken at the entrance to the
sewage plant; the second, third, fourth, and fifth samples were taken from
each of the four sewage lagoons; the sixth sample was taken from a deep
well used for watering livestock; the seventh, eighth, and ninth samples
were taken from each of three seepage creeks entering the Concho River; the
tenth sample was taken from the Concho River approximately 6 miles upstream
from the sewage facility; and the eleventh sample was taken from the Concho
River approximately 6 miles downstream from the sewage facility. In
addition, irrigation water was collected when irrigation of the fenced
plots occurred.
Samples were aseptically collected in sterile milk dilution bottles.
Raw sewage, sewage from each lagoon and seepage creek, as well as river
water samples, were collected at a depth of 15-30 cm. The well sample
was collected from the delivery pipe after flushing with approximately 37
liters of water.
A total of 17 soil samples were collected on each sampling. These
included a sample from the fenced control plot and samples from each
fenced irrigation area. Cattle were grazed on all areas except the fenced
control plot. In addition, an off-farm control sample was taken.
Soil cores (2.5 cm diameter), 10 cm deep, were collected and trans-
ferred to sterile, airtight plastic sample bags. All samples were refrig-
erated on ice until processing.
Sample Processing
Samples were returned to the laboratory at Texas A&M University and
processed within 36-^8 hours after collection. Ten ml of each water or
sewage sample and 10 g of each soil sample were transferred to 90 ml sterile
Nad (0.85$) blanks and diluted serially to effect a log dilution range
from 10-1 through 10-8. Ten grams of soil from each sample were dried and
weighed to determine water content so that microbial counts could be
reported as organisms per gram dry weight soil.
Total aerobic bacterial counts were determined by the pour plate
method. One ml from each sample dilution was added to ~L% nutrient glucose
agar (Difco) and incubated for US hours at 37°C. After incubation the colonies
were counted.
Total coliform counts were made by making serial dilutions and spread
plating onto Eosin Methylene Blue (EMB) or Endo Agar (Difco). Incubation
was for 2k hours at 37"C. All colonies exhibiting a typical green sheen were
counted as presumptive coliforms. Ten percent of the presumptive coliform
colonies were transferred to Difco Triple Sugar Iron Agar (TSIA). Cultures
demonstrating acid/acid and no hydrogen sulfide reactions were transferred
into lactose broth for final confirmation. Fecal coliform counts were
obtained by making serial dilutions on EMB or Endo Agar. Plates were
17
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incubated at 44.5"C for 2k hours. All colonies demonstrating a green
metallic sheen were counted as presumptive fecal coliforms. Confirmation
consisted of picking colonies to TSIA and lactose broth as per the total
coliform confirmation. This was followed by determining the IMViC reactions
of those typical coliforms. Only those organisms yielding an IMViC of
++, + , and -+ and grown at 44.5"C were reported as fecal coliforms.
The presence of fecal streptococci was determined by making serial
dilutions and spread plating onto m-Enterococcus Agar. Plates were incu-
bated overnight at 37"C. Presence of the organism was confirmed by
hydrolysis of esculin on Bile Esculin Azide Agar (Difco) and by making
catalase tests.
The population of Pseudomonas aeruginosa was determined by spread
plating of serial dilutions onto Pseudosel Agar (Biquest Laboratories).
Colonies producing diffusible blue-green pigments were counted, and
confirmation was by the cytochrome-oxidase test.
Salmonella Isolation from Soil and Water
Samples were taken of both raw sewage and irrigation effluent for
isolation of Salmonella sp. Samples were collected and maintained at
ambient temperature for approximately 48 hours before processing, but after
processing the remainder of the samples were stored at approximately 10" C.
Ten, twenty, and thirty ml samples were enriched in tetrathionate broth (TT),
tetrathionate broth + 1% lactose (TTL), tetrathionate broth + 1% lactose and
1.% glucose (TTLG), tetrathionate broth at pH 4.55 and tetrathionate broth
at pH 5-0. Enrichments were incubated at, 37" C for 18 hours after which
time log dilutions were made (10 to 10 ) and plated onto Xylose Lysine
Desoxycholate (XLD) agar. Plates'were incubated 2k to 48 hours at 37"C
and suspect salmonella colonies picked to Triple Sugar Iron Agar (TSIA).
Organisms yielding typical salmonella reactions on TSIA (AK++ or AK-+)
were subjected to further biochemical and serological analysis.
Twenty-one and 1*5 days after sampling 10 to 30 ml aliquots of both
samples were re-examined for the presence of salmonella by tetrathionate
broth enrichment followed by plating onto XLD agar. Biochemical and
serological analyses were performed as above on presumptive salmonella
isolates.
Two sample locations, sites 13 and 6, were chosen for collection of
soils for the isolation of Salmonella sp. Two enrichment media, tetra-
thionate (TT) and tetrathionate broth + \.% lactose (TTL), and two selective
plating media, Bismuth sulfite (BS) agar and Xylose Lysine Desoxycholate
(XLD) agar were used for isolations. Thirty and 50 g of each of the soils
were placed into each enrichment, 1 part soil to 10 parts enrichment.
After ingubationgat 31°C for approximately 18 hours log dilutions ranging
from 10~ to 10 were made and plated onto each of the media. After
incubation at 37°C for 2k to 48 hours, suspect colonies from each dilution
were picked to Triple Sugar Iron Agar (TSIA). Those TSIA which gave
typical salmonella reactions were treated to biochemical analysis.
18
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Concentration and enrichment techniques were used to isolate
Salmonella sp. from the Concho River and seepage creeks. The enrichment
media were tetrathionate broth (TT)» tetrathionate broth + 1% lactose
(TTL) and nutrient broth with 1$ lactose (NL).
River and seepage creek waters were filtered through .1*5 um filters
by negative pressure and placed into the various enrichments according to
Table 1. The volume filtered was limited by the quantities of suspended
solids.
TABLE 1. VOLUMES OF LIQUID FILTERED AND MEDIA USED FOR DETERMINATION
OF SALMONELLA SP. IN WATER FROM THE CONCHO RIVER AND IN
WATER FROM SEEPAGE CREEKS
Sample
No. 7
No. 8
No. 19
No. 20
No. 11
No. and Site
(Seepage Creek No. 3)
(Seepage Creek No. 2)
(Upstream Concho)
(Downstream Concho)
(Seepage Creek No. l)
Volume Filtered
UL
2L
2L
3L
2L
2L
500 ml
250 ml
250 ml
500 ml
300 ml
250 ml
kOO ml
200 ml
200 ml
Medium
TT
NL
TTL
TT
NL
TTL
TT
NL
' TTL
TT
NL
TTL
TT
NL
TTL
All preparation of media, filtration, and enrichment were performed
at San Angelo to prevent loss of potential salmonella due to prolonged
storage. Enrichment media was kept refrigerated during transportation
back to the laboratory at College Station where they were incubated at?
37"C for approximately 18 hours. After incubation, log dilutions (10~ to
10~^) were plated onto BS agar and XLD agar and again incubated at 37"C
for 2k to 1*8 hours. At the time of plating of the enrichment, a serial
transfer of 10 ml of primary enrichment was inoculated into freshly
prepared secondary enrichments of the same medium. Again after 18 hours
of incubation at 37*C log dilutions were plated onto BS and XLD agar.
Suspect colonies from each dilution of all plates were picked to TSIA and
19
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biochemical analysis performed where indicated. No Salmonella sp. were
confirmed from any of the enrichments.
Due to the lack of success with prior enrichment techniques, samples
for Salmonella sp. were collected and enriched according to the method
of Moore (1971) and Moore et al. (1969). -The procedures consisted of
two highly absorbent commercial tampons (Kotex) tied to a cotton cord.
These were suspended in the raw sewage inlet, primary settling tank,
sewage lagoons, seepage creeks, and up and downstream Concho River. The
cord was anchored with weights and equipped with a conventional plastic
float to maintain the tampons in the flowing water or sewage. These were
allowed to remain in place for 3 to 5 days. At the end of this period, the
tampons were enriched in 200 ml of freshly prepared TT broth and incubated
at ^5°C for U8 hours. Dilutions of the enrichment were prepared and plated
onto XLD and BS agar. Colonies typical for Salmonella sp. were picked and
characterized biochemically.
Salmonella Isolation from Bovine Manure
To determine whether or not cattle could become potential reservoirs of
salmonellae after being pastured on the sewage farm, a study was initiated.
Ten cows, not previously exposed to the farm environment, were chosen for
the study. The cattle were transferred to farm pastures after a manure sam-
ple was collected. Six samplings were made over a 7 month period. When a
sufficient quantity of manure was available, 50 g was enriched in 200 ml of
TT broth for 18 hours:, dilutions werevmade^ and plated onto BS and XLD
agars. At the time of initial plating, serial transfers of 10 ml were made
to freshly prepared TT broth and processed as previously described. Suspect
colonies were examined biochemically for typical salmonella reactions.
Bacterial Analysis of Well Water Samples
Wells were sampled toth on and off the sewage farm. -The water samples
were examined for totaj. lerobic bacteria, and fecal and total coliforms.
The procedure for enumeration and confirmation was as previously described
for water samples.
Bacterial Analysis of Soil Core Samples
Core samples from two. soils present on the San Angelo sewage farm
were enumerated for coliform bacteria. The soil series used were Rio Concho
and San Angelo. Six cores from each soil were taken; three from inside
the sewage farm and three from outside the sewage farm. The cores were
sectioned and the bacteria in each section were enumerated. The sections
were from the soil depths: 0-5, 5-10, 10-15, 15-20, 20-30, 30-il-O. UO-50
50-100, and 100-150 cm. Soil from each depth was mixed thoroughly, and
10 g of soil from each section was added to 95 ml of saline. This was
ground in a Waring blender for 1 minute. A dilution sequence from the soil
slurry was conducted and was plated on m-endo agar (Difco). Pour plates
were used on the soil slurry from the blender. Spread plates were used
on higher dilutions. Plates were incubated at 37" C for 2k hours. Colonies
exhibiting a characteristic green sheen were considered coliform bacteria.
20
-------
Laboratory Studies with Bacteria
Soils
Soils chosen for this investigation represent important agronomic and
economic soils of Texas. Soil types were: Arenosa loamy sand, San Angelo
sandy clay loam, Houston Black clay, and Beaumont clay. The San Angelo
soil was collected from the San Angelo sewage farm, where sewage had been
applied for the last 15 years. The soil samples were collected from the
A horizon, air-dried, and ground to pass through a 2 mm mesh sieve.
The soils had a wide variety of physical characteristics (Tables 2 and
3). The pH was determined according to the procedure of Davis (19^3). The
soil texture was determined by the Bouyoucos hydrometer method '(Day 1965).
Organic matter was measured by using a colorimetric variation of the
Walk.ley-Black method (193lj.). Hydraulic conductivity (cm/hr) and permeability
(cm ) of the soils were determined by the procedure of Klute (1965).
Electrical conductivity was determined on a 2:1 liquid-soil suspension
using a conductivity bridge. Soil moisture characteristics were measured
by applying pressure to soils on porous plates (Neilson 1958 and Richards
19^9). Total porosity was determined by measurement of the bulk density
using the paraffin clod method (Russell 19^9). By using soil moisture
characteristic curves (Figure 4), pore-size distributions were derived by
the method of Vomocil (1965).
TABLE 2. PHYSICAL AKD CHEMICAL .CHARACTERISTICS OF FOUR SOILS
USED IN LABORATORY STUDIES OK LEACHING OF BACTERIA
Property
Sand
Silt
Clay
Arenosa
loamy sand
81*$
6%
10%
San Angelo
sandy clay
loam
^7$
18$
35$
Houston
Black
clay
11*
35$
5U*
Beaumont
clay
20$
30$
50$
pH
Organic matter
Hydraulic conductivity
Electrical conductivity
Pore space
U.9 7-3
1.3$ 3.6$
5.81 cm/hr 0.6l
230 umhos/cm 680
53$ 58$
7.5
3.1$
0.51
U80
52$
5-5
2.U*
0.16
220
21
-------
TABLE 3. PORE SIZE DISTRIBUTION OF THE SOILS USED IN LABORATORY
STUDIES ON LEACHING OP BACTERIA
Pore-size
(ym)
30,000 - 150
150 - 75
75 - 50
50 - 22
22 - 12
12-9
9 - 6
6-3
3 - .6
.6 - .3
Arenosa
loamy sand
11*
7$
18*
31*
Itf
1*
1*
0*
i #
l/o
0$
San Angelo
sandy clay
loam
15*
10*
10*
5*
15*
1*
1*
1*
3*
U*
Houston
Black
clay
12*
13*
1%
8*
15*
1*
1*
1*
3*
U*
Beaumont
clay
U*
10*
7*
lltf
27*
1*
!*
1*
7*
6*
Total
65*
65*
Inoculum Preparation
Four strains of fecal related bacteria were used in this experiment.
Laboratory strains of Escherichia coli 11303 arid Salmonella typhimurium
were obtained from Dr. B. G. Foster, Biology Dept., Texas A&M University.
Strains of Salmonella enterjditis and Streptococcus fecalis were isolated
from the San Angelo, Texas sewage farm.
Biochemical tests were conducted for confirmation of the bacterial
species.. The salmonella strains were streaked on brilliant green agar
(Difco) for growth. Tests were conducted using triple Sugar Iron agar
(Difco), Lysine Iron agar (Difco), urea agar (Difco), Simmon's citrate
agar (Difco), tryptophane for indole production, MR-VP medium (Difco),
dulcitol, arabinose, mannitol and malonate. The isolates were then tested
for agglutination by Salmonella 0 Antiserum (Difco), and typed. The strain
of E_. coli was streaked on MacConkey's agar (Difco) for growth. The same
biochemical tests were then conducted as with the salmonella. Streptococcus
fecalis was streaked on m-enterococcus agar (Difco), and for confirmation as
a group D streptococcus bile esculin azide agar (Difco) was used. Addi-
tional biochemical tests to determine the species were grown on mannitol,
arabinose, saccharose, and glycerol hemolysis, and gelatin liquification.
All biochemical reactions were incubated at 37"C for 2h hours.
The E. coli and the strains of salmonella were grown in a 50 ml
Erlenmeyer flask containing 20 ml of nutrient broth. An initial inoculum
was added, and the organisms were incubated at 37*C for 10 to Ik hours
on a rotary shaker, until a cell concentration of 10 organisms per ml was
22
-------
u>
# Beaumont clay
* Houston Black clay
San Angelo sandy clay loam
A Arenosa loamy sand
100
200
300 400 500
Water tension (cm)
600
700
10000
Figure H. Water holding capacity of the four soils used in
laboratory studies on leaching of bacteria.
-------
reached. Streptococcus feealls was grown in tryptic soy broth
These bacteria only obtained a cell concentration of about 3 x 10° organisms
per ml.
Dilutions of bacteria were performed using physiological saline (,0.85
percent WaCl in distilled water) to establish a total bacterial concen^tra-
tion of lO^ organisms per ml. Five ml of the bacterial suspension was \then
added for each cm of soil in the columns. The amount of bacterial suspen-
sion added to each column is shown in Table k.
Column Preparation
Columns were developed that would retain soil, pass water and bacteria,
and that could obtain a -1/3 atmospheric tension on the soil with applied
suction. The columns consisted of a modified millipore swinnex (25 mm)
filtering assembly, with a 22 mm diameter pyrex tube inserted into the
top portion of the assembly. The bottom section was attached to a small
glass tube which passed through a rubber stopper. This entire column
assembly could then be attached to a 500 ml filtering flask. Figure 5
shows a representative column assembly.
A 3 um nucleopore filter was chosen as the best supporting grid to
pass the liquid and bacteria. Filters were tested for optimal removal of
gravitational water, but that would also pass bacteria. Filters tested
were: 3, 5» and. 8 um nucleopore, 8 um cellulose filters (Selectron), and
5 and 10 um teflon filters (Millipore).
Soil column heights of 1, 2, 3, k, 5» 10 and 15 cm were used. The
representative soils were added to the columns in 5 g increments and
compacted to a reproducible bulk density. Compaction was by means of a
#1 rubber stopper attached to a glass rod dropped 10 times from a height
of 15 cm. This procedure was repeated until the desired column height was
obtained.
The inoculum was carefully poured onto the soil surface so that the
soil surface would not be disturbed. The water was allowed to percolate
through the soil until the wetting front visually reached the bottom of
the column, then a vacuum of 0.8 to 0.9 atmospheres was applied until
visible drainage from the column had stopped. Bacteria in the leachate
and in the soil were then enumerated.
Adsorption to Soil
The capacity of bacteria to adsorb to particles greater that 1 um in
diameter was estimated by the following procedure. Twenty-five g of soil
and 25 ml of a liquid suspension containing 10° bacteria per ml were mixed
in a 250 ml centrifuge tube. This mixture stood for 5 minutes before an
additional 220 ml of saline was added. The soil was suspended by shaking
on a mechanical shaker for 5 minutes, and centrifuged (IEC #2 head no. 226,
1500 rpm, 3:30 minutes) to remove any particles greater than 1 um (Day
1965). The supernatant was decanted, mixed, and the bacteria were enumer-
ated. An additional 220 ml of saline was added to the sediment in the
2k
-------
TABLE U. INITIAL INOCULUM OF BACTERIAL SUSPENSION, PER ML, ADDED TO EACH
COLUMN3"
Soil column
height
1 cm
2 cm
3 cm
h cm
5 cm
10 cm
15 cm
Milliliters of
inoculum added
5
10
15
20
25
50
75
a 6 ;
-10 org/ml
tube and the procedure was repeated. Salmonella typhimurium and Escherichia
coli were the bacteria used in this experiment.
Effect of Salts on Leaching
All previous experiments in this study were conducted using physiologi-
cal saline as the. transport medium. Therefore, additional tests were con-
ducted using other salts in solution at varying concentrations. These tests
were conducted to determine the effects of different salts on the leaching
characteristics of bacteria through the soils. One salt mixture contained
an equal number of sodium and calcium ions consisting of 2/3 CaCl and 1/3
NaCl. Three concentrations were used: 0.31 N, 0.155 N, and 0.077 N.
These concentrations represented 1, 1/2, and lAv respectively, the normality
of physiological saline. The other salt solution was 0.01 M phosphate
buffer (K/pHPOl}.) i*1 distilled water. These salt solutions were inoculated
with S_. typhimurium. and passed through 5 cm soil columns. The bacteria in
the leachate were then enumerated.
Distribution in Columns
A study was conducted to determine the numbers of bacteria present
in different sections of a, soil column after an inoculum of bacteria had
passed through the soil. Fifteen cm columns, inoculated with S_. typhimurium,
were sectioned and the bacteria present in each increment of soil were
enumerated. Soil sections examined were 0-3, 3-5» 5-10, and 10-15 cm.
Saturation of Soils
Salmonella present in the leachate were determined after h additions
of Salmonella typhimurium were made to soil columns. Four 25 ml additions
of salmonella were added to 5 cm soil columns of Arenosa sand and San Angelo
soil. Four 5 ml additions of salmonella were added to 1 cm columns of
Houston Black clay. Columns were allowed to drain before the next
addition of bacteria was made.
25
-------
Pyrex tube - 22 mm
inside diameter
Top View
Top - Swinnex 25 mm
filtering assembly
Teflon 0-ring
3 ym nucleopore
Bottom Swinnex
filtering assembly
#7 Stopper
Surface of
filtering
assembly
Glass tube
Figure ? Schematic of column assembly used in
leaching experiments. (Actual scale)
26
-------
Breakthrough Characteristics
Breakthrough characteristics vere conducted with 5 cm soil columns to
determine the movement of the bacteria in relation to the movement of the
water. Twenty-five mis of bacterial suspension in saline were added to
the soil surface. Bacteria in each 2 ml increment of the leachate were
enumerated.
Enumeration
Bacteria in the leachate that passed through the soil columns were
enumerated by making serial, ten-fold dilutions (Table U). A 1 ml Eppendorf
pipette and 9 ml sterile saline blanks were used for the dilution sequences.
A 0.1 ml Eppendorf pipette was used to deliver the suspensions onto the
desired plating medium. The spread plate method was used for spreading the
inoculum on the plates.
Bacteria from the initial dilution that was used to inoculate the soils
were enumerated. From this, the total number of bacteria applied to the
soils could be determined so that all tests could be standardized by
comparing a fixed constant to the initial concentration of bacteria. Ini-
tial concentrations of bacteria added to the soil are presented in Table 5.
The plating media used for this investigation consisted of brilliant
green agar (Difco) for enumeration of salmonella, m-endo agar (Difco) for
E_. coli, and m-enterococcus agar (Difco) for enumeration of S_. fecalis.
The media were prepared 1 day in advance and allowed to dry at 37*C over-
night. Colonies exhibiting characteristic reactions with the respective
medium were counted.
Size
Preparations of the bacteria were photographed using phase/contrast
light microscopy to determine the respective size distribution of the
bacteria. The bacteria were grown in their respective medium, and a drop
was placed on a microscope slide. Two percent agar (liquid) was added to
the suspension and covered by a coverglass.
Statistical Analyses
To determine significant differences between bacterial numbers present
in the leachate from the column experiments, statistical analyses were
performed on the differences between the proportions of bacteria leaching
through the soil. The proportions of bacteria were determined, by comparing
the amount of bacteria present in the leachate to the total amount of
bacteria that leached through the soil at a particular depth. The standard
deviation of bacteria at, each d«pth was used to determine significance be-
tween the amount of bacteria that leached through the soil at a particular
depth. Differences were determined by Duncan's multiple range test using
the 95 percent confidence level. Differences in numbers of bacteria in
the salt experiments were determined by the Student's t-test.
27
-------
TABLE 5. CONCENTRATION OF INOCULUM ADDED TO EACH SOIL
Depth
Soils
Arenosa
loamy sand
San Angelo
sandy clay loam
Houston1
Black clay
1 cm
E_. coli
S. fecalis
S_. typhimurium
S. enteriditis
5.8
1.1
0.9
1.6
million/ml
1.0
1.6
0.9
l.U
1.0
0.5
1.8
3.0
2 cm
E_. coli
S. fecalis
S_. typhimurium
S. enteriditis
2.0
1.1
1.7
1.6
1.5
1.6
0.9
3.6
1.2
0.5
1.8
3.5
3 cm
E_. coli
S. fecalis
S_. typhimurium
S_. enteriditis
k cm
E_. coli
S_. fecalis
S_. typhimurium
S_. enteriditis
5 cm
E_. coli
S. fecalis
S_. typhimurium
S. enteriditis
8.0
2.7
1.7
1.6
8.0
2.7
0.6
1.6
i.l
1.7
0.6
1.1*
2.0
2.0
0.9
3.6
1.2
2.0
0.9
3.6
9-1
1.7
9-0
1.7
1.0
0.5
1.8
3.5
1.0
0.5
1.8
2.9
1.0
0.5
1.0
1.6
10 cm
E_. coli
S_. fecalis
S_. typhimurium
S_. enteriditis
15 cm
E. coli
S_. fecalis
S_. typhimurium
S. enteriditis
1.1
2.0
2.2
3.0
1.1
2.6
2.2
3.0
8.8
2.0
0.9
1.6
1.1
2.6
U.3
1.6
1.0
0.5
1.0
2.1
1.0
0.5
2.6
2.1
28
-------
Field Study on Viruses
Soil and Water Sampling
Water and soil samples were collected at the San Angelo site at monthly
intervals. These samples were collected at the same sites and times as the
bacteriological samples, primarily from the seepage creeks and the sewage
lagoons. Sample sizes normally consisted of 10 to 15 ml. Each water sample
was frozen (-70*0) and thawed prior to filtration and inoculation.
Each soil sample was ground separately in a mortar and then mixed in 5
ml of Hank's balanced salt solution. After thorough mixing the soil
particles were allowed to settle. The supernatant fluids were decanted into
a centrifuge tube and centrifuged (1000 RPM) for 10 min. This fluid was
decanted and stored at -70"C.
Tissue Culture
The protocol for the tissue culture system was developed using Buffalo
monkey kidney cells (BGMK) or African Green monkey kidney cells in culture.
The growth medium selected consisted of Eagle minimum essential medium
with Earle's salts supplemented with fetal bovine serum, L-glutamine, and
tryptose phosphate broth. The maintenance medium was identical to the
growth medium except the serum level was reduced from 10 percent final
concentration to 1 percent.
Enumeration
A preliminary filtration study was conducted to determine the effect
of serial filtration of viral samples to eliminate bacterial contaminants.
Both O.it5 urn and 0.22 urn millipore filters were used in this study. Cul-
tures of Group 3 Reovirus exhibiting h+ CPE on BGMK were first frozen and
then thawed before filtration. The outgrowth of the cultured cells to
confluency was accomplished in k8-72 hours. Separate aliquots of each virus
dilution were inoculated onto the BGMK cells. The test was read 6 days
after inoculation.
The reduction of viruses, due to serial filtration, prompted the
filtration technique for samples to be altered to a single passage of the
liquid through a 0.22 um membrane. The filtrate was inoculated directly
onto substrate tissue in replicates of 3-5 tubes per sample. An inoculum
of 0.25 ml of filtrate was used throughout. All cultures were read after
6 days. Those samples exhibiting CPE in culture were either passaged or
re-inoculated onto other BGMK cultures.
Laboratory Studies with Viruses
Soils
The two soil types used in these experiments were a San Angelo fine
sandy loam and Houston Black clay. Their characteristics are given
in Tables 2 and 3.
29
-------
Column Preparation
Column assemblies were identical to those used in the bacteriological
column studies with the same supporting grid (3 um nucleopore). Soil column
heights of 1, 2, 3, U, 6, 10, and 15 cm were used. The representative soils
were added to the columns in 5 g increments and compacted to a reproducible
bulk density.
Inoculation and Enumeration
Columns were separately charged with h ml sample (l ml of Reovirus,
TCID 50 1 x 10 U.09/ml in 3 ml Hank's ESS). Five ml of water or saline
solution (0.85$ NaCl) were added to the column surface to provide eluant
for each 5 ml fraction collected. Eight to twelve fractions were collected
per column. The eluate fractions were stored at -60'C until assayed in
tissue culture.
Group 3 Reovirus was used because of its stability between pH 2.2 and
8.0 and its survival abilities. Assays were conducted on BGMK tissue in
culture. Mono layers were grown in Leighton tubes in Eagle minimum
essential medium containing 10 percent v/v fetal bovine serum, 200 units
of penicillin G and units of streptomycin per ml. Each column leachate
fraction was filtered through a 0.22 pm millipore filter. Tissue cultures,
inoculated with 0.25 ml filtrate, were incubated at 37'c for 6 days and
monitored for virus associated cytopathic effects (CPE).
Distribution in Columns
Soils from each column were divided into thirds to represent the top,
center and bottom of each soil column. These were retained in bulk and
frozen (-60'C) until assayed for residual virus in tissue culture.
For assay the soil was thawed and mixed with 5 ml of Hanks BSS and
shaken by hand. After the soil settled, the supernatant fluid was decanted,
filtered through a 0.^5 pm HA millipore membrane, and U replicates of
Leighton tubes were inoculated with 0.25 ml of filtrate. These were
monitored for any viral associated cytopathic effects (CPE).
PARASITOLOGICAL STUDIES
Detection of Possible Human Parasites in Sewage
Sewage and Water Sampling
Fluid samples were taken from the incoming raw sewage, the primary
settling tank, storage lagoons and the irrigation effluent and examined
for possible human parasites. A 500 ml sample was taken monthly from each
location in an attempt to quantitate the number of parasites in the sewage.
Samples were placed in sterilized 500 ml glass bottles and transported to
the laboratory on ice for examination. All samples were refrigerated at 5*C
until examined. Most samples were examined within k8 hours after collection.
30
-------
Sample Processing and Examination
Samples were thoroughly mixed and 25, 50, 75 and sometimes 100 ml
subsamples were concentrated using hi mm diameter millipore filters having
5 urn pores. The concentrate was removed from the filter with the edge of
a 22 x 22 cm glass coverslip and resuspended in 1 ml of Lugol's iodine
solution. Subsamples of over 100 ml were normally too cluttered with
sediments to be adequately examined microscopically. Suspensions were
uniformly mixed with a pipet to prevent clumping and 6 to 10 wet mounts
were made to determine if protozoan cysts, helminth eggs or larvae of
possible human parasites were present. If none were.observed the subsample
was discarded and the next larger volume subsample was concentrated and
examined. If parasites were observed in wet mounts, portions of the con-
centrated sample were examined using the white blood cell counting grids
of a hemocytometer. Usually, 3 to 5 hemocytometer observations were made
'on each suspension using the 8 white blood cell grids. In some preparations,
some of the 8 grids could hot be examined because of excess debris. The 3
volume per white blood cell counting grid was Immxlmmx0.1mm= 0.1 mm .
The references used in identification of protozoan cysts and helminth eggs
and larva'e were primarily "Basic Clinical Parasitology" (Brown 1969);
"Textbook of Clinical Parasitology" (fielding 1952) and "Laboratory Guide to
Medical Protozoology and Helminthology" (National Eaval Medical Center,
Bethesda, Maryland). Unfortunately, nuclei.could not be distinguished
in 50 to 70 percent of the suspected protozoan cysts; thus, size and general
appearance were used in classification. Size and general appearance may not
be a good indicator for the diagnosis of human protozoan parasites.
Detection of Possible Human Parasites in Sludge
Sampling
During the last half of this project it became apparent that examina-
tion of sludge for possible human parasites would be important. Six to
ten core samples were taken each month from a sludge lagoon that had been
allowed to go dry in September of 1975. Sampling of the sludge material
started in January of 1976 and was continued through August of 1976. The
depth of sludge in this lagoon was between 15 to 25 cm. Core samples were
placed in 500 ml widemouth glass jars and transported on ice to the labora-
tory for examination;' samples were refrigerated in the laboratory at yc
until used.
Sample Processing and Examination
One-gram subsamples were taken from the surface of core samples and
approximately 15 cm below the surface for comparison. Examination of these
subsamples were by direct iodine wet mounts and zinc-sulfate flotation
(Cable 1958). In the zinc-sulfate technique, each of two 1-gram subsamples
were suspended in 10 ml of lukewarm water in a centrifuge tube. These
suspensions were centrifuged for approximately 1 minute at 2600 rpm and the
supernatant fluid discarded. This process was repeated until the super-
natant was, clear. After the final rinse the tube was filled to capacity
with a zinc-sulfate solution (specific gravity = 1.180) and the sediments
31
-------
resuspended. Centrifuge tubes were covered with a glass coverslip to pick
up floating materials. The coverslip just touched the surface of the
liquid. The remaining material was centrifuged at 1800 rpm for 1 minute.
After centrifugation coverslips were again floated on the liquid in the
tubes. This time they were allowed to float for 10 to 15 minutes and then
placed on a glass microscope slide with a little iodine stain for
microscopic inspection.
Detection of Nematode Larvae in Soil
Sampling
Soils were sampled to determine if possible human and/or livestock
parasites could be detected. Sampling was initiated in October 1975 and
was terminated in January 1976 in favor of a controlled study monitoring
parasite levels in cattle. On-farm parasite levels were compared t%> the
levels observed on an adjacent control site where sewage had not been used
for irrigation. Detection of nematode larvae was primarily by the Baerman
method (Cable 1958).
Samples were taken from a 30 cm square area from which all vegetation
but that within 2 cm of the soil surface was removed. Samples included
remaining grass, forbes plant debris, and the top 2m of soil. We attempted
to take at least 10 on-farm samples and 4 off-farm samples each month.
Sampling was occasionally disrupted by excessive irrigation or rain on
fields of the sewage farm* Field samples were transported on ice in plastic
zip-lock bags to the laboratory where they were stored at 5"C until exam-
ined.
Sample Processing and Examination
Samples were thoroughly mixed (vegetation and soil) and 50 ml packed
subsamples were placed on two layers of gauze over a wire screen mounted
in a large glass funnel. Funnels were freshly filled each time with UOrC
water so that the water level in the funnel just touched the bottom of the
wire screens. The stems of funnels were fitted with surgical tubing which
was clamped off to hold the water in the funnel. The funnels with sub-
samples were allowed to stand undisturbed for at least 1 hour. Nematode
larvae were attracted towards the warm water and accumulated in the stem of
the funnel. The larvae were harvested in a centrifuge tube after releasing
25 ml portions of water from rubber tube into centrifuge tubes. Most of
the larvae were in the first 25 ml but additional 25 ml portions were
drained into centrifuge tubes until no more larvae were present. Further
concentration was possible by centrifugation of these 25 ml samples at 1800
rpm. Nematode larvae harvested were counted in a gridded watch glass with
a 5X wide dissecting microscope fitted with 20X ocular lenses. When
necessary, for identification, larvae were examined with a compound
microscope.
32
-------
Detection of Parasites in Livestock Feces
Sampling of Livestock on the Sewage Farm
A study was established to compare the parasite levels of sheep and
cattle on the sewage farm to those grazing on a nearby farm. Sampling was
initiated in September 1975 and terminated in January 1976. Samples were
taken from freshly deposited feces and placed in 500 ml glass containers
and transported on ice to the laboratory for examination. Samples were
stored at 5" C in the laboratory until examined.
Sample Processing and Examination
On thawing, samples were thoroughly mixed and subsamples used for
analyses. Examination of these samples was by direct iodine wet mounts
by the Baermann funnel technique and by zink-sulfate flotation as previously
described in this report under the sections, on sludge and soil processing
and examination.
Monitoring of Parasite Buildup in Cattle Feces
Cattle
Ten cattle were assayed for parasite levels before being released on
the sewage farm. The average weight of the cattle was approximately kOQ
pounds. Each animal was treated with 8 mg/kg of Levasole, marked with ear
tags and released on the farm for observation. Animals 1 through 5 were
retreated with the drug after 2 months to see if an antihelminthic drug
would hold parasite levels down. Three cattle already on the farm were
randomly selected to serve as controls to determine how quickly parasite
levels would increase to those already present.
Sampling
Monthly fecal samples were removed directly from all ten cattle,
placed in 500 ml sterilized glass containers and transported to the
laboratory for examination. Samples were stored at 5*C until used.
Examination
The feces were examined for parasites by direct iodine wet mount
examination, zinc-sulfate flotation and Stoll's egg counting technique
(Cable 1958). Counting eggs by the Stoll technique was accomplished as
follows: A Stoll flask was filled to the % ml mark with N/10 sodium
hydroxide, feces was added until the level in the flask reached the 60 ml
mark, ten glass beads were added and the flask was stoppered and mixed by
hand'shaking. From this suspension 0.15 ml was transferred to a microscope
slide using a Stoll's pipette and covered with a 22 x i+0 mm glass coverslip.
Eggs were counted using a compound microscope. The number of eggs was
multiplied by 100 to obtain the number of eggs per gram of feces. This gave
only an estimate of the number of eggs present. We assumed that more eggs
would indicate more parasites.
33
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SECTION 6
RESULTS AND DISCUSSION
MICROBIOLOGICAL STUDIES
Field Study on Bacteria
Populations in Sewage, Seepage Creeks, Lagoons and the Concho River
Application of municipal sevage effluent to agricultural lands may have
an affect on the microbiological quality of ground water. The population of
microorganisms in the effluent has a bearing on the impact of the effluent
upon ground waters. In these investigations sewage effluent was monitored
monthly from the point raw sewage entered the treatment facility until some
of the water reappeared as seepage water draining into the Concho River.
Populations of total aerobic bacteria, fecal coliforms, enterococci, and
Pseudomonas aeruginosa were determined to evaluate the bacteriological
impact of the effluent on the ground water quality.
The sewage lagoons effectively reduced the populations of all groups
of bacteria by approximately 90 percent. Unfortunately, the retention time
in the lagoons could not be determined because there was no set schedule for
piping sewage into the lagoons or taking effluent from them for irrigation.
The total aerobic counts from the different sample areas are presented
in Figure 6. The raw sewage usually contained thenhighest numbers of total
aerobic bacteria, ranging from 5 x 10 to over 10 bacteria per ml. The
four sewage lagoons were very similar to one another in regards to total
bacteria ranging between 1 x 10 to 10 _organisms per ml. The bacterial load
present in the seepage creeks demonstrated considerable fluctuation, from
about 1 x 10 to 10 organisms per ml. The total aerobic load of the Concho
River was relatively constant, with few differences between the upstream
and downstream counts. The two noticeable exceptions were a sharp increase
in numbers in the downstream samples during the month of June, and the
decrease in numbers in the downstream samples during October. The down-
stream population fluctuations in June were similar to that'in the seepage
creeks.
The total coliform counts from the different sample areas are presented
in Pig. 7. The raw,_sewage contained the greatest numbers of organisms,
ranging from 2 x 10 to 5 x 10 organisms per ml. The numbers of coliform
organisms present in lagoons 1 through 3 were about the same, ranging from
10 to 10 . However, the coliform numbers were considerably reduced in
-------
U)
J F M A M J J ASOND
Figure 6, Total aerobic bacteria in water samples collected each month from various locations
on the San Angelo sewage farm. The lines represent raw sewage . n ... , lagoons 1
2 , 3___, U , seepage creeks 1 , 2 , 3>.>>».....»..., and upstream Concho
River »» , and downstream Concho River w» .
-------
(JO
ON
0)
a
.2
^
o
4-1
u
flj
m
i
o
oT
6
5
4
3
^^
J FMA/WJJASOND
Figure 7. Total coliforms in water samples collected each month from various locations on the
San Angelo sewage farm. The lines represent raw sewage , lagoons 1 , 2.
3 , and k , seepage creeks 1 ~~~v, 2- - , 3-^^^, and upstream Concho River
, and downstream Concho River x%v%x»«..
-------
k compared to the other lagoons. The effluent had to pass through lagoons
2 and 3 to^reach ^U The coliform counts in the seepage creeks fluctuated
between 10 and 10 organisms per ml, but no trends could be established
between these counts and the coliform count in the raw sewage. There was
a slight increase in bacteria for the month of September and October for
the downstream Concho samples.
The fecal coliform counts from the selected areas revealed a consider-
able amount of fluctuation (Fig. 8). The fecal coliform population present
in the raw sewage varied between 10 to 10 organisms per ml. Fecal
coliform numbers in lagoons 1 through 3 coincided very well with the
fluctuations in the raw sewage, but lagoon k was very low in fecal coliform
bacteria. The fecal coliform population in lagoon k was usually an order
lower than the other lagoons. The population of fecal coliforms in the
seepage creeks were generally much lower than in the lagoons.
The populations of Ps. aeruginosa in monthly samples varied consider-
ably (Fig. 9). There was a sharp increase in Ps. aeruginosa for the month
of April, and again in June for most samples. An increase in bacterial
numbers in the lagoons was also observed in December. A greater number of
Ps_. aeruginosa were present in the downstream samples for April, as compared
to the upstream samples. However, in some cases the upstream samples con-
tained greater numbers of these bacteria than did the downstream samples.
The enterococci populations from the different samples are presented
in Fig. 10. Again the raw sewage contained the greatest number of
recoverable bacteria. A sharp decrease was observed for numbers of enter-
ococci in the raw sewage for the month of September. However, the popula-
tions of these bacteria in the sewage lagoons did not decrease noticeably.
Seepage Creek 2 contained a substantial number of enterococci compared to
Seepage Creek 1. Even so, Seepage Creek 2 only contained 6 x 10 enterococ-
ci per ml. Enterococci were not isolated' from the Concho River or Seepage
Creek 3.
Populations in Well Water
Because the microorganisms from sewage were present in the seepage
waters we examined water samples collected from 15 wells in the surrounding
area to determine if sewage organisms were reaching the wells. The total
aerobic counts in the. wells were extremely low, ranging from less than 1 x
10 to 3 x 10 organisms per ml. No total or fecal coliforms were detected
in any of the wells. Thus the sewage farm did not affect the deep ground
water used for drinking.
Populations in Soil
Soil samples representing IT collection sites were collected monthly
to determine impact of sewage effluent on the population of sewage microor-
ganisms (Fig. 11). Because there was no significant difference (5% level
of significance) in populations between months, the data were averaged for
brevity (Table 6). A control sample was collected from both on the sewage
farm (site 20). and off'it '(site 66). The control area on the farm was fenced
37
-------
U)
Co
0)
a
(0
^
0)
*->
o
(0
00
o>
o
M
Figure 8. Fecal coliforms in water samples collected each month from various locations on the
San Angelo sewage farm. The lines represent raw sewage , lagoons 1, 2
3 } and U , seepage creeks 1^., 2 - -, 3 >">»>», and upstream Concho River
HI, , and downstream Concho River
-------
U)
0)
a
ro
'i_
o
*->
o
(0
m
O)
o
J FMAMJJ AS OND
Figure 9. Pseudomonas aeruginosa in water samples collected each month from various locations
on the San Angelo sewage farm. The lines represent raw sewage , lagoons 1 ,
2 , 3 , and k , seepage creeks 1
River "«» , and downstream Concho River
and upstream Concho
-------
-p-
o
0)
a
.2
>_
a>
4->
o
(0
CD
O)
O
7
6
*M»^«*y^Mf!s>^M^
J FMAMJ J ASOND
Figure 10. Enterococci in water samples collected each month from various locations on the
San Angelo sewage farm. The lines represent raw sewage , lagoons 1 ,
2 , 3 , and U , seepage creeks 1 ~~~~, 2 - -, 3^>-^>-^ a^^- upstream
Concho River , and downstream Concho River «%.««»..
-------
Figure 11. Location of fields, lagoons and seepage creeks on the San Angelo
sewage farm.
Ill
-------
ro
TABLE-6. POPULATIONS OF BACTERIA IN SURFACE SOIL SAMPLES COLLECTED FROM
SEVERAL SITES ON THE SEWAGE FARM AND A SITE OFF THE SEWAGE FARM
Site
20
17
13
9
6
10
11
12
15
16
19
18
8
7
5
Off J'arm
Total
Aerobic
O
3x10^
6 X 10n
3x10^
5 x 101
U x 10'
U x 10ft
2 xlO°
2 x IDA
k x log
2 x 10
3 x 10A
1x10?
5 x 10 '
6 x lei
1 x 10g
LL "V" TO
ri
1 x 10°
Total
Coliform
A
1 x 10,
3 x 10; a
7 x lo3(l)a
U x 10,(1)
u x ioe
3 x 10,(1)
3 x 10;
2 x 10^(1)
5 x 10^(1)
1 x 10|
9 x 10,(1)
1 x 10, (1) '
2 x I0j(l)
1 x 10,(1)
3 x 107(1)
1 x 10
(M
Fecal
Coliform
\
1 x 10,(3)
1 x 10^(3)
3 x 10^(3)
2 x 10P(2)
7 x 107(3)
3 x 10,(1)
2 x 10,(1)
1 x lOHl)
1 x 10Z(3)
8 x 10^(1)
8 x 10,(2)
3 x 10^(3)
1 x 10J(2)
( *4 )
( Ij. \
1 x 10 (1)
(M
Pseudomonas
aeruginosa
JNO / g SO1J. Z
1 x -lor
8 x io;(l)
9 x 10^(1)
8 x 102(2)
(k)
9 x 102
3 x 10^(2)
5 x 10p(l)
5 x 10,(1)
U x 10,
1 x 10^(2)
1 x 10,
9 x 10|
5 x 10,(2)
7 x 10,(1)
U x 10-3
(M
Enterococci
^_
1 x 10^(2)
1 x 10,(3)
3 x 10^(1)
^ x lof (1)
7 x 10,(3)
1 x 10p
1 x 10p(2)
1 x 10p(3)
8 x lo;(2)
U x lo;(2)
7 x 10p(2)
2 x HOI)
1 x 10p(3)
1 x 10p(l)
2 x 10,(1)
1 x 10J(1)
(U)
aThe number in parenthesis is the number of samples, out of the four collected, that contained
fewer than 100 organisms per g. The number of organisms indicated is the average of the
samples that contained more than 100 per g.
-------
to keep cattle out, but was irrigated with effluent. The population of
the total aerot>ic bacteria was not different between on the sewage farm
and off i| (5% level of significance) and was in the range between 3 x 10
to k x 10 per g of soil. However, the distribution of selected types of
bacteria recovered on the sewage farm was very different as compared to off
the farm. Large populations of fecal associated bacteria occurred on the
sewage farm and in the fenced plot (site 20)on the sewage farm. Populations
of enterococci amounted to only 10 percent of the population of the fecal
and total coliforms. The populations of bacteria present in soil collected
from different sites on the sewage farm were not significantly different
(5$ level of significance). No coliforms or enterococci were isolated
from the site off the sewage farm. Thus, irrigation of the sewage farm
soils with sewage lagoon effluent contributed significantly to the numbers
of fecal associated bacteria in the soils.
Core samples from two soil series on the sewage farm were examined to
determine the distribution of total aerobic and total coliform bacteria in
soil profiles. Off farm controls were also included. Differences between
total aerobic bacteria on and off the farm were not significantly different
(5% significance level) (Tables 7 and 8). However, there were substantial
decreases in bacterial numbers with increasing soil depth. This probably was
due to the nutrients available in the soil for the growth of the indigenous
bacteria, and not due to leaching of these bacteria through the soil.
Coliform bacteria were substantially greater in numbers in the soil of
the sewage farm than in the soil not treated with sewage (Tables 9 and 10).
There were differences in numbers of bacteria between the two soil series
obtained from the sewage farm. This difference may have been due to irri-
gation. The Rio Concho soil had been recently irrigated with sewage
effluent; however, the San Antonio soil had not been irrigated for at least
3 weeks* The bacteria present in the San Angelo soil remained relatively
constant for the first 30 to ^0 cm of soil, but were not detected below
this. Numbers of coliform bacteria in the Rio Concho soil decreased by a
magnitude of 1.5 logs in the uppermost 10 cm of the soil. The population of
coliform bacteria decreased slightly throughout the remainder of the soil
cores.
Salmonella in Sewage, Seepage Creeks, Lagoons, Soil and the Concho River
A pathogenic bacterium commonly present in sewage and likely to be of
public health significance is salmonella. Therefore, attempts were made to
isolate salmonella from various locations on the. sewage farm. Salmonella
sp. were isolated from raw sewage, primary settling tank, all lagoons,
in Seepage Creeks 1 and 2, and in the soil (Table 11). Best recovery of
salmonella was accomplished by using tetrathionate broth without supplements
using a 30 ml enrichment volume. Organisms were isolated from both the
raw sewage and irrigation effluent. Organisms demonstrating the typical
salmonella biochemical pattern were sent to the Houston City Health Lab-
oratory for Somatic "0" and Flagellar "H" antigenic analysis. Serotype
Salmonella saint paul 1, ^, 5, 12: e, h: 1, 2 was isolated from the raw
sewage. Serotype Salmonella panama 1, 9, 12: 1, V: 1, 5 was isolated
from the irrigation effluent. Biochemically presumptive Salmonella sp. were
-------
TABLE 7- TOTAL AEROBIC BACTERIA PRESENT IN CORE SAMPLES OF THE SAN ANGELO
SERIES SOIL ON THE SAN ANGELO SEWAGE FARM AND A FARM NOT RECEIVING
SEWAGE
Sampling depth
cm
0 -
5 -
10 -
15 -
20 -
30 -
1*0 -
50 -
100 -
5
10
15
20
30
40
50
100
150
sewage farm
1
3,300
1,1*00
980
850
WO
270
230
97
33
2
3,200
1,200
720
740
990
430
48
15
-
3
h
1__ -i f\ i _
x 1U /g
3,100
1,700
1400
1*50
140
800
340
100
4o
1
.n
soil
1,900
1,000
675
4oo
163
990
166
2,700
990
farm
2
3,500
980
1,000
700
190
126
960
16
9
3
200
615.
360
850
110
670
770
350
1*9
TABLE 8. TOTAL AEROBIC BACTERIA PRESENT IN CORE SAMPLES OF THE RIO CONCHO
SERIES SOIL ON THE SAN ANGELO SEWAGE FARM AND A FARM NOT RECEIVING
SEWAGE
sewage farm
cm
0
5
10
15
20
30
40
50
100
_ c
- 10
- 15
- 20
- 30
- ho
- 50
- 100
- 150
1
3,800
630
4oo
330
9
10
6
3
11
2
280
60
4o
13
8
11
7
10
2
3
X .LU /g
>2,000
>2,000
2,000
150
7
3
9
2
2
1
.
186
280
94
99
280
66
20
23
14
farm
2
430
150
65
48
35
15
8
2
35
3
89
59
31
74
32
11
8
6
6
44
-------
TABLE 9- COLIFORM BACTERIA PRESENT IN CORE SAMPLES OF THE RIO CONCHO SERIES
SOIL ON THE SAN ANGELO SEWAGE FARM AND A FARM NOT RECEIVING SEWAGE
Sampling depth
cm
sewage farm
1
2
3
farm
123
x ±u /g soil
0 -
5 -
10 -
15 -
20 -
30 -
ho -
50 -
100 -
5
10
15
20
30
ko
50
100
150
5,000
290
150
90
38
33
23
20
2
1,500
130
30
20
10
25
5
10
-------
found in a 50 g soil sample from site 6 when enriched in TT broth.
Although no salmonellae were isolated from the Concho River, their presence
in the seepage creeks suggest that they were added to the river.
The isolation of Salmonella enteriditis serotype Saint Paul from both
the raw sewage and lagoon effluents suggested that salmonella survived and
passed through the system. Therefore, there was the potential for salmonella
to be introduced into the Concho River. The load of fecal indicators in the
seepage creeks and Coneho River were essentially the same, and in spite of
the small population of these indicators, salmonella was isolated. This
suggests that in low numbers, fecal coliform-enterococci indicators are
not reliable in determining the possibility of salmonella contamination.
Salmonella in Bovine Feces
Cattle are capable of being infected by Salmonella sp. and may shed the
organism in their manure. The presence of salmonellae in sewage, water, and
soil provides a source of the organisms for the cattle on the sewage farm to
become infected. To determine if cattle, placed on the sewage farm, became
infected with salmonella, the manure from 10 cattle was monitored from the
day the cattle arrived on the farm. Fresh manure samples were taken at
bi-weekly intervals for a total of 10 samplings. Salmonella was isolated
from one animal before it was released on the farm.
Only one animal shed salmonella in its manure. It was not, however,
the same animal that was infected before release on the farm. Manure from
this animal only contained salmonella at one sampling. Therefore, the
sewage farm did not increase shedding of Salmonella sp. in cattle feces.
TABLE 11. NUMBER OF SEWAGE, WATER AND SOIL SAMPLES THAT SALMONELLAE WAS
ISOLATED FROM
Sample
No. Positive
Sample
No. Positive
Raw Sewage
Primary Settling Tank
Sewage Lagoon #1
Sewage Lagoon #2
Sewage Lagoon #3
Sewage Lagoon #k
Irrigation Effluent
1
5
5
5
5
5
1
Deep Well 0
Seepage Creek 1 1
Seepage Creek 2 1
Seepage Creek 3 1
Concho River Upstream 0
Concho River Downstream 0
Soil (Site 13) 1
-------
Laboratory Studies with Bacteria
Introduction
The field investigations revealed that microorganisms from sewage ef-
fluent leached through soils of the sewage farm, but field data were not
obtained to relate the leaching characteristics of a disease organism, sal-
monella, to that of common fecal bacteria. Therefore, laboratory experi-
ments were initiated to measure the relative leachability of different
bacteria added to soil. More than one soil was used to determine if there
was an interaction between soils and bacteria on the leaching of bacteria.
Filter Selection
Before leaching experiments could be initiated, columns of soil had
to be constructed in a manner that they could be leached without loss of
soil. To accomplish this, filters had to be used on the bottom of the
columns. Many filters were compared for their ability to pass bacteria,
and to allow for sufficient suction on the soil for it to drain to field
capacity. Three types of filters were tested: Nucleopore, teflon, and
cellulose (Table 12). The teflon filters greatly impeded water movement.
Because of this, the teflon filters were not suited for this experiment.
The cellulose filters v&re fibrous filters like the teflon filters, but
liquids could pass through them easily. The nucleopore filters consisted
of a thin sheet of polycarbonate with the holes "punched" through the sheets.
The 3 ym nucleopore filter was chosen for the soil column experiments be-
cause it allowed the greatest amount of liquid from the soil column to
pass through when suction was applied. Also, only a small decrease in
bacterial numbers occurred when the bacteria were passed through the 3 vtm
nucleopore filter (Table 13).
Adsorption
Bacteria are prevented from leaching through soils by the filtering
action of the soil and also by adsorption of bacteria onto soil particles.
Therefore, the adsorption of bacteria to soil particles greater than 1 ym
in diameter was measured by adding bacteria to soil and separating the non-
adsorbed bacteria by differential centrifugation. Adsorbed bacteria varied
between 9 percent of the total applied bacteria for the Arenosa sand, to
over 99-9 percent for the Beaumont clay (Table lU). There were differences
in adsorption between bacterial species. The greatest differences occurred
with the San Angelo soil; approximately 60 percent and 80 percent,
respectively, of the E. eoli and 5. typhimurium were adsorbed. Increased
adsorption for S_. typhimurium also occurred for the Houston Black clay;
approximately 90 percent and 98 percent, respectively, of the E_. coli and
S_. typhimurium were adsorbed. Relatively small differences in bacterial
adsorption occurred in the Arenosa sand and the Beaumont clay.
Bacterial cells normally range in size between 1.6 to 0.9 ym. There-
fore, 1 Urn soil particles were chosen to represent the minimum clay fraction
that could easily be separated from the non-adsorbed bacteria. The sus-
pension of soil and bacteria was centrifuged until all soil particles
-------
TABLE 12. PERCENTAGE WATER REMAINING IN A SAN ANGELO SANDY CLAY LOAM
COLUMN 1 cm IN DEPTH, AFTER DRAINAGE
a
Filter Water content
3 vim Nucleopore (polycarbonate) 26.8$
5 ym Nucleopore (polycarbonate) 32.6$
8 ym Selectron (cellulose) 29.5$
5 ym Millipore (teflon) 35-5$
Percent water retained in soil against 1/3 atm. pressure was 27.
TABLE 13. DECREASES IN NUMBERS OF E.. COLI WHEN AN INITIAL 10 ml ALIQUOT
OF THE BACTERIA WERE PASSED THROUGH DIFFERENT TYPES OF FILTERS
Q
Filter Water content
3 ym Nucleopore 1.4 x ~LQs (9.0 x 10 )
5 ym Nucleopore 1.4 x 10,. (2.9 x 10^)
8 ym fibrous 5-5 x HT (l.l x lO^)
aThe initial number of bacteria was 1.5 x 10 /ml (3.0 x 10 ) and the
number in parenthesis is the standard deviation
TABLE Ik. PERCENTAGE OF BACTERIA ADSORBED ONTO SOIL PARTICLES GREATER
THAN 1 ym IN DIAMETER
Soil Type E_. coli S_. typhimurium
Arenosa 7(?.5)a 11 (6.3)
San Angelo 63 (1.4) 82 (1.9)
Houston Black 90 (0.7) 98 (1.4)
Beaumont 99.9 (O.Ol) 99.9 (0.08)
Q
Standard deviations are in parenthesis
-------
greater than 1 ym were pelleted. This procedure could be considered a
differential centrifu.gation process, since the particle density for clay is
approximately 2 g/cm , and the particle density for bacterial cells is
approximately 1.08 g/cm .
The true adsorption values for these soils may have been somewhat
lower than the measured values, especially the clay soils, due to possible
adsorption of many of the finer clay particles to the bacteria. The
bacteria may have become massive enough after adsorbing many small clay
particles to centrifuge out with the bacteria adsorbed to the 1 ym
particles. An increase in surface area and a larger net negative charge
for the clay soils was probably responsible for the increased bacterial
adsorption over that occurring for the sandy soils.
Evidence for finer clay materials adsorbing onto bacterial cells has
been shown in transmission electron micrographs (Marshall 1971). These
smaller clay fractions adsorbing onto the bacterial surfaces may simply
move with the organism through the soil, not impeding the movement of the
bacteria. This phenomenon might actually have promoted the movement of
the bacteria, if a thin coat of the negatively charged particles prevented
adsorption of the bacterium to the large particles by repulsive forces.
Bacterial adsorption increased with increasing clay content. This
was not unexpected. In Marshall's review of sorptive interactions many
investigators have reported increased bacterial adsorption with increasing
clay content. The clay content appears to be much more important than the
organic matter content, since the San Angelo and Houston Black soils
contained the higher organic matter content, but did not adsorb the most
bacteria.
A plausible explanation for the differences in bacterial adsorption
in the same soil may be that the surface charges of the two bacterial
species were different. Marshall (19T1) has reported a distinct relation-
ship between the amount of clay sorbed per unit area of cell surface and
the nature of the surface ionogenic groups of the bacteria.
Bacterial Size
«
The size of bacteria probably has an effect on the movement of bacteria
through a soil. Therefore, the size of the bacteria used in these investi-
gations was measured with the light microscope and is illustrated in
Figure 12. Escherichia coli was the largest bacterium, averaging 1.8 by
0.9 ym. Salmonella typhimurium and Salmonella enteriditis were similar in
size, measuring 1.6 by 0.9 ym. Streptococcus fecalis was coccoidal in
shape and measured 1 ym in diameter. A number of bacterial pairs were
observed in the streptococcus suspension, but this may have been due to the
procedure in preparing the organisms for observation.
Pore Size Distribution
The pore size distribution of soils would be expected to influence the
quantity of bacteria that could leach through. The pore size distribution
-------
tf'JS,
* * *
A
Figure 12. Light micrographs of the bacteria used for size determina-
tions. A. Escherichia coli B. Streptococcus fecalis C. Salmonella
enteriditis D. Salmonella typhimurium. Line represents 1 ym.
-------
of the soils used inthese investigations is shown in Table 3. The
majority of the total pore space was comprised of pores greater than 12 ym
in diameter. For most soils, less than 9 percent of the pore space was
comprised of pores less than 3 ym. For example there were virtually no
pore spaces less than 6 ym in diameter in the Arenosa sand. Therefore,
there was little possibility of this soil filtering out single bacterial
cells. Filtration of clusters of bacteria could have occurred if the
clusters were relatively large. Krone (1958) has shown that filtration of
bacterial clusters could be an important process in soils.
All of the soils contained a large fraction of pores having diameters
of 50 to 12 ym that accounted for between 20 to kl percent of the total
pore volume of the soils. These larger pores would be the pores most
conducive to leaching of bacteria. In all soils, except for the Arenosa
sand, there was a fraction of the pore space, between 5 and 9 percent,
that was comprised of pores smaller than the diameters of the bacteria.
These smaller pores would impede the movement of even a single bacterium
by filtration. This would represent case II filtration proposed by Krone.
Therefore, the finer textured soils or the one with a greater percentage of
pore space comprised of small pores would have the greatest potential to
filter out bacteria.
Leaching Through Soils
Small differences were observed for the leaching characteristics of
the four bacteria through the Arenosa loamy sand (Table 15 and Fig. 13).
Approximately 5 percent of the bacteria passed through 5 cm of this soil.
The numbers of bacteria, when leached through 15 cm columns of this soil,
were reduced over 2 logarithmic units or 99-5 percent.
A two-phase curve could be seen in the plot of bacterial numbers in
the leachate vs. soil depth (Figure 13). The first section of this curve
was the relatively rapid decrease in bacterial numbers that occurred for
each increment of soil in the first 5 cm. The second phase was a more
gradual decrease in bacterial numbers per increment of soil. This phase
began between 5 and 10 cm of soil.
Analysis between the proportions of bacteria that passed through this
soil indicated that there were significant differences between the bacteria
(Table 15). The leaching characteristics for 1C. eoli and S_. typhimurium
were similar thoughout this soil. There were a few statistically signifi-
cant differences between iS_. feealis and the bacteria E_. coli and S_.
typhimurium; however, at most soil depths there were no differences. More
S_. enteriditis than the other bacteria leached through the first 5 cm of
this soil. Similar quantities of S_. feealis as well as S_. enteriditis
leached through 10 and 15 cm of soil.
For the 10 and 15 cm depths the leachability of S_. feealis and S_.
enteriditis were similar, and the leachability of S_. typhimurium were
similar. However, the effect per cm of soil height was the same between
these two depths. This indicates that the differential rate of leaching
for the organisms would have occurred before the 10 cm soil depth to allow
51
-------
TABLE 15. THE PROPORTION OF ADDED BACTERIA THAT LEACHED
THROUGH COLUMNS OF AN ARENOSA LOAMY SAND
VJ1
ro
Soil Depth
Organism
E. coli1
S. fecalis
S_. typhimurium
S. enter iditip
'
1 cm
.551* b
.906 c
.21*9 a
.987 c
(-3UT)2
2 cm
.329 b
.566 a,b
.255 a
.698 b
(.209) (
3 cm
.208 a
.216 a
.123 a
.653 b
.273)
1* cm
.081* a
.127 a
.082 a
.313 b
( .111)
5 cm
.061* a
.060 a
.013 b
.161* b
(.060)
10 cm
.OOU a
.OlU b
.005 a
.007 a
(.005)
15 cm
.0013 a
.0031* b
.0006 a
.0038 b
(.0016)
Refer to table 3 for the quantity- of teacteria added to each column. Values, in columns,
having a letter in common were not significantly different at the 5$ level
2
The standard deviation is in parenthesis
-------
V/l
LO
E
i_
o
a 3
.5
a>
S 2
00
I
o
T-
O)
Arenosa sand
+ E.coli
* S. fecalis
<> S. typhimurium
^ S.enteriditis
10
15
cm of soil
Figure 13. The effect of soil column height on the number
of bacteria per ml of leachate.
-------
for the separation at the 10 cm depth. A trend for this to happen occurred
in the first 5 cm of soil.
There were greater differences in the leaching characteristics of the
bacteria through the San Angelo sandy clay loam than occurred with the
Arenosa sand (Table l6 and Figure 1*0. Only 0.8 percent of the bacteria
passed through 5 cm columns of this soil. The bacterial numbers were
reduced over 5-5 logarithmic units, or 99.999 percent, by passage through
15 cm columns.
A three-phase curve for E_. coli, S,. enteriditis, and S_. fecalis could
be seen in the plot of bacterial numbers in the leachate vs. soil depth
(Figure lU). The first phase could be considered the relatively large
reduction in numbers from that initially applied to that present in the
leachate from the 1 cm column depths. This reduction was most apparent in
the curve for !E. coli. The second phase was considered the relatively
slower linear decrease in bacterial numbers for the 1 and 5 cm depths.
In comparison of this soil to the previous one, a more rapid decrease in
numbers of bacteria was observed for the first 5 cm of the San Angelo soil.
Bacterial numbers decreased at about the same rate between the third phase
of the San Angelo soil and the first phase of the Arenosa sand.
The leaching characteristics for S_. typhimurium, S_. enteriditis, and
§_ fecalis were all similar for most of the soil depths. The decrease in
numbers of E_. coli for the first 5 cm of soil was substantially greater
than the other bacteria. The reason for this phenomenon was not
determined; however, it was not due to the size of the bacteria since all
the bacteria were about the same size. At the 15 cm depth, the numbers
of S_. enteriditis and E_. coli present in the leachate were not statistically
different. However, the numbers of S_. typhimurium and S_. fecalis in the
leachate from 15 cm columns were significantly greatly than the other two
bacteria.
Almost all of the bacteria were retained in the first few cm of.the
Houston Black clay. The rate of decrease of bacteria in the leachate was
very rapid for all of the bacteria (Table IT and Figure 15). Escherichia
coli, S_. fecalis, and S_. typhimurium were not detected in the leachate
from columns longer than 2 cm. However, S_. enteriditis was detected in
the leachate from all column lengths with three organisms per 10 ml of
leachate present from 15 cm columns. The population of S_. fecalis and
S_. enteriditis were also similar for the first 2 cm of soil. However, S_.
fecalis was not detected beyond this point.
A two-phase curve could only be seen in the plot of the numbers of
S_. enteriditis in the leachate vs. soil depth (Figure 15). All the other
bacteria were eliminated so quickly from the soil that a straight line was
observed for the bacterial decreases.
Beaumont clay is a dense, poorly structured soil with very low
permeability. Because of the tortuous path taken by the liquid, the large
fraction of small pores, and the high adsorption rate, no bacteria passed
through even 1 cm of this soil.
-------
TABLE 16. THE PROPORTION OF ADDED BACTERIA THAT LEACHED
THROUGH COLUMNS OP A SAN ANGELO SANDY CLAY LOAM
vn
Soil Depth
Organism
E. coli
S. fecalis
S . typhimurium
S. enteriditis
1 cm
.OlUla
.2237b
.1229b
(.1711)2'
2 cm
.0017a
.0603c
.0576c
.0223b
(.0286)
3 cm
. 0007a
.0303c
. Ol62b
. 0109b
(.0107)
k cm
.0002a
,0038b
. 0066c
. 0032b
(.0026)
5 cm
.OOOOla
. 0032c
. 0009b
. 0008b
(.0012)
10 cm
. OOOOOla
.OOOOHa
. 00009a
.OOOOSa
(.ooooU)
15 cm
3.7 x 10 a
7.1 x 10~5c
1.0 x 10~5b
3.1 x 10 a
"T^efer to table 3 for the quantity of bacteria added to each column. Values, in columns,
having a letter in common were not significantly different at the 5$ level
p
The standard deviation is in parenthesis
-------
cr\
San Angelo sandy clay loam
+ E.coli
* S. fecalis
<> S. typhimurium
S. enteriditis
cm of soil
Figure lA. The effect of soil column height on the number
of bacteria per ml of leachate.
-------
TABLE IT. THE PROPORTION OF ADDED BACTERIA THAT
LEACHED THROUGH. COLUMNS OF A HOUSTON BLACK CLAY
vn
1
Soil Depth
Organism 1 cm
E. coli .0051 a
S. fecalis .0655 b
S. typhimurium .0033 a
S. enteriditis .1038 c
,.**>'
2 cm 3 cm h cm 5 cm 10 cm 15 cm
7.5 x 10~6a
.0053 b
2d
£ ,7
.0966 c .0027 .0006 .00003 1.0 x 10 l.U x 10~ '
(.0^72) (.0003) (.0003) (.00002) (3.8 x 10~7) (7.0 x 10~8)
Kefer to table 3 for the quantity of bacteria added to each column. Values in columns having
a letter in common were not significantly different at the 5$ level.
p
No bacteria were detected in the leachate
Standard deviation is in parenthesis
-------
CD
Houston Black clay
+ E.coli
* S.fecal is
<> S-typhimurium
A S-enteriditis
cm of soil
Figure 15 The effect of soil column height on the number
of "bacteria per ml of leachate.
-------
There were significant differences in bacterial movement through the
four soils used in this investigation. The sandy soil retained the fewest
bacteria; whereas, the Beaumont clay retained all of the bacteria. The
retention capacity of the soils increased with increasing clay content.
The order of increasing retention was: Arenosa loamy sand, San Angelo
sandy clay loam, Houston Black clay, and Beaumont clay.
Adsorption was probably an important factor in bacterial retention in
the clay soils. The hydraulic conductivity in these soils was relatively
low. Therefore, the bacteria moving through such soils would have a
greater opportunity to interact with the soil particles. As the adsorption
results indicated (Table lU), under conditions where the bacteria come in
contact with many soil particles, a very large percentage of the bacteria
could be adsorbed.
The removal mechanisms for bacteria passing through soils may selectively
remove a portion of the bacterial population. This was indicated by the
nonlinear plots of column height and population of bacteria in the leachates
(Figures 13, lU, and 15). A theory proposed by Gerba and Lance (1976), for
viruses, was that the surface soil layers remove the majority of the highly
charged viral particles and the remaining more neutrally charged viruses,
could pass further into the soil. This theory may also apply to the
adsorption of bacteria, since bacterial cells have charged surfaces
(Hattori and Hattori 1976, Hattori 1973, and'Marshall 1971).
Soil structure has a large influence on the leaching of bacteria.
The Houston Black clay is a Vertisol, and, in field condition, large cracks
can occur during periods of dryness. The columns from these experiments
were packed with dry soil and did not contain large cracks. Therefore, the
results reported here have demonstrated the maximum retention of the
bacteria, because the bacteria had to travel through the soil pores rather
than escape through large cracks or cleavage plains.
The overall results indicate that the bacteria basically behaved
similarly in their leaching characteristics through soils. However, slight
differences in leaching characteristics did occur for the different bacteria
and were not related to the size of the bacteria. For any soil type, a
particular organism could not be predicted to leach through the soil more
readily than another. In these experiments S_. fecalis rather than E_. coli
behaved most similarly to salmonella and may be the better indicator for
the leaching rate of salmonella through soils.
Distribution of Bacteria in Columns
Because salmonella is of particular public health significance, it was
used as the test organism to determine the distribution of bacteria within
leached soil columns, to determine the effect of salts in the leaching
solution on the movement of bacteria through soil columns, to determine the
rate at which bacteria move through soil columns relative to the movement
of the leaching solution, and to determine the effect of leaching a soil
more than once with a bacterial suspension.
59
-------
To determine the distribution of viable salmonella in soil columns
after an inoculum was passed through, columns were sectioned and the
bacteria in each section were enumerated. To accomplish this, 15 cm columns
were constructed as in the leaching experiments, and 75 nil of a salmonella
suspension was leached through the soils. The bacteria in the 0-3, 3-5» 5-
10 and 10-15 cm sections were enumerated.
The distribution of bacteria in sections of Arenosa sand ranged from
2.6 x 105 to 8.2 x 103 bacteria per g of soil (Table 18). The percentage
of the applied bacteria in each section was 26, 6.5, 1.9» &n(i 0.8 percent,
respectively for the 0-3, 3-5, 5-10 and 10-15 cm sections. The numbers of
bacteria in the San Angelo soil ranged from 1.1 x H)5 bacteria per g of
soil in the 0-3 cm section, to 3.0 x 10^ bacteria per g of soil in the 10-15
cm section. Percentages of applied bacteria retained in the 0-3, 3-5, 5-10,
and 10-15 cm sections of the soil, were, respectively, 11, 0.12, 0.02 and
0.03- Bacteria were only present in the uppermost section of the Houston
Black clay. Eleven percent of the total applied bacteria were recovered in
this segment.
The results from enumerating the bacteria present in the soil sections
indicated similar trends to the results obtained by determining the total
numbers of bacteria present in the leachate passed through soil columns o'f
various heights. That is, the first increments of soil removed proportion-
ately more bacteria than soil located further down the columns.
The detectable salmonella present in the columns were considerably less
than expected, since the recovery of salmonella from columns varied between
26 to 11 percent of the cells added. This phenomenon is probably best
explained by the die-off of the bacteria, since the bacteria were usually in
contact with the soil for a period of at least 1 hour.
Effect of Salts on Leaching
Variations in the retention and the leachability of bacteria may be
affected by the ionic strength and the nature of the ions in the transport
liquid (Cooper et al. 1975) Therefore, fluctuations in the leachability
of salmonellae using different salts as the transport medium were compared
to saline solution. Zohar et al. (1971) also reported that differences in
the salt concentrations of the transport medium affected the leachability of
bacteria.
There were no statistically significant differences (Ov05 level) between
the salts on leachability of salmonella through the two soils (Table 19).
Our results indicate that the ionic strength of the transport liquid does
not greatly affect bacterial numbers leached through the Arenosa loamy sand
or the San Angelo clay loam.
60
-------
TABLE 18. TOTAL NUMBERS OF SALMONELLA TYPHIMURIUM IN SECTIONS OF 15 cm
SOIL COLUMNS AFTER LEACHING3- "
Soils
Soil
depth
0 - 3 cm
3 - 5 cm
5 -10 cm
10 -15 cm
Arenosa
loamy
sand
2.6
(1.5
6.2
(1.7
1.9
(7.0
8.2
(1.1
xioj
x 104)
x lojj
x 104)
x lot
x 1CT)
x 10_
x 10J)
San Angelo Houston
sandy clay Black
loam clay
i.i x 10!? 1.1 x 10?
(3.0 x 10-3) (1.6 x 10 )
1.2 x lo| N.D.b
(2.1+ x 10 )
2.3 x 10g N.D.
(1.0 x 10 )
3.0 x 10p N.D.
(1.0 x 10 )
Standard deviations are present in parenthesis
N.D. not detected
Rate of Appearance in Leachate
cm
Bacteria present in each 2 ml increment of leachate passed through 5
soil columns were enumerated to determine the rate bacteria pass through
a soil relative to the rate -of the leaching solution. The initial 2 ml
increment from the Arenosa sand contained approximately 200 bacteria per
ml, but the third increment contained 2350 bacteria per ml (Figure 16).
As the soil dried, bacterial numbers decreased very rapidly. Of the initial
25 ml of liquid applied to the soil, about 21 ml leached through the soil.
These results provide evidence that the soil solution passes through many
pores that the bacteria are restricted from passing through.
Bacterial leaching through the San Angelo soil behaved similarly. The
first increment of leachate contained about 73 bacteria per ml, and the
third increment contained 166 bacteria per ml (Figure 16).
A cumulative curve of the numbers of bacteria in the leachate demon-
strates that after the third increment for the Arenosa sand, and after the
fourth increment for the San Angelo soil, the leaching rates for the bacteria
were relatively constant (Figure 17). The slopes of the log of bacteria vs
soil depth show that more bacteria passed through the Arenosa sand per
increment of leachate than through the San Angelo clay loam.
6l
-------
TABLE 19. EFFECTS OF DIFFERENT SALT SOLUTIONS ON THE LEACHING OF
SALMONELLA TYPHIMURIUM THROUGH 15 cm COLUMNS OF SOILS
Salt solutions
Saline
(0.31 N NaCl)
Salt solution
(0.31 N CaCl2 and
NaCl - equimolar)
Salt solution
(0.15 N CaCl2 and
NaCl - equimolar)
Salt solution
(0.07 N CaCl2 and
NaCl - equimolar)
Phosphate buffer
(0.01 M K2HPO^)
Bacteria per
Arenosa sand
1.3 x 10
2.k x 10
1.7 x 10
1.3 x 10
1.3 x 10
mlb
San Angelo clay
loam
9.3 x 102
2.6 x 103
7.1 x 102
2.3 x 103
8.2 x 102
The F test (0.05 level) indicated there were no statistically significant
differences, between leaching solutions, on the leaching of salmonella
Initial bacterial concentration of 10 organisms per ml was used
62
-------
ON
-------
£ 4
a
CO
>_
0)
s
(0
CQ
O)
O
San Angelo
6 8 10 12 14 16
ml leachate
18 20
Figure 17. Cumulative numbers of bacteria present in
consecutive 2 ml increments of leachate.
-------
Saturation of Soil with Bacteria
Four increments of bacteria were passed through columns of three soils
to determine the effects of consecutive additions of bacteria on the reten-
tion of the bacteria. The quantity of salmonella passing through Arenosa
or San Angelo soils with each new inoculation remained relatively constant
(Figure 18). A decrease in numbers of bacteria from 1 x 105 to 5 x 10^ per
ml of leachate was observed from the third to the fourth inoculation of
the Arenosa soil. The numbers of bacteria that passed through 5 cm, Sanp
Angelo columns increased steadily for all four increments, from 7.5 x 10
bacteria per ml for the first increment, to 2.h x 103 bacteria per ml in
the fourth increment. Numbers of bacteria that passed through 1 cm of
Houston Black clay increased on addition of the first three increments of
bacteria, from 6.1 x l(n bacteria per ml, to 1.5 x 105 bacteria per ml.
The fourth increment, probably due to clogging of pores by previously added
bacteria, resulted in a rapid decrease in numbers of bacteria to 7-9 x 102
bacteria per ml.
These three soils reacted differently to repeated additions of bacteria.
A possible explanation for the differences between soils is that the pores
in the Arenosa sand were large enough to allow continued bacterial leaching
without retaining most of the bacteria. In the fourth increment, a
noticeable decrease of bacteria occurred, probably due to the bacteria
clogging some of the smaller pores. The increases observed in the San
Angelo soil were probably due to much of the water passing through pores
that were too small for the bacteria. Repeated inoculations provided more
water to complete leaching of nonadsorbed bacteria. The reason for the
increase of bacteria passing through the Houston Black, clay with addition
of the second and third incrementsof bacteria, was probably the same as
for the San Angelo soil. The low numbers of bacteria leaching through the
soil in the fourth increment was probably due to clogging of soil pores by
bacteria or to the dispersion of aggregates by the sodium present in the
transport leaching solution.
Field Study on Viruses
Viruses were not detected in water or soil samples collected at various
locations on the sewage farm. However, this does not mean that viruses
were not present, only that the technique used on the water samples for
these investigations was not sensitive enough to detect fewer than 10 viral
particles per ml. Passage of the viruses through the millipore filters to
remove bacteria probably further reduced the likelihood of detecting
viruses. For example, we found that passage of Reovirus through a -U5y
millipore filter reduced the population from 1 x 105 to 1 x 103 per ml.
To quantitate the population of virus in the sewage waters and soils would
have required concentration methods not available in our laboratory.
Therefore, we directed our efforts to determining the leachability of
viruses through columns of soil in the laboratory.
-------
4
a
(0
v_
a>
**
o
(0
OQ
O)
O
5cm Arenosa
5 cm San Angeto
1 cm Houston
Black
2 3
inoculations
Figure 18. Bacteria present in the leachate after four
consecutive inoculations of Salmonella
typhimurium.
66
-------
Laboratory Studies with Viruses
Analyses on the leachates collected from columns of a San Angelo sandy
clay loam, ranging between 1 cm and 15 cm in length, revealed that Reovirus
3 was leached through some of the columns (Tables 20 and 21). There does
not appear to be any difference in leaching of the virus whether saline
or water was used as the eluate. However, there was a large amount of
variability between columns as to the presence or absence of the virus in
the leachate. Oftentimes one replication contained viral particles in the
leachate and the other replication did not. Even some fractions of leachate
from a column contained the virus but other fractions did not.
Leaching of Reovirus 3 through columns of a Houston Black clay was
similar to its leaching through the San Angelo soil (Tables 22 and 23).
These column studies prove that viruses can be leached through at least
15 cm of these soils when the soils have been prepared in a manner that
would minimize leaching; the columns were filled with screened soil and were
uniformly packed. This eliminated large pores and cracks normally present
in soil in the field. The San Angelo soil is one of the main soil series
on the San Angelo sewage farm.
The results of the 'assays used to determine if viruses were retained
in the soil columns after passage of the eluate were inconsistent (Tables
2k and 25). In only a single instance was virus detected in the San Angelo
soil. However, the frequency of detecting virus in the Houston Black soil
taken from the leached columns was much greater than for the San Angelo
soil. Viruses were not detected in the Houston Black soil taken from the
3 and U cm columns.
It seems reasonable that if the viral particles were not present in the
leachate they should have been isolated from the soil. Yet in most instan-
ces this did not occur for the San Angelo soil (Tables 20, 21 and 2U).
Therefore, in some way the San Angelo soil must have inactivated or
adsorbed many of the virus particles. This phenomenon only occurred for
the 2 cm column of Houston Black soil that was leached with water.
Apparently the San Angelo soil was much more effective in adsorbing or
inactivating the virus. Both soils were calcareous, but the Houston Black
soil contained substantially more clay and organic matter than the San
Angelo soil. The time required for the eluate to leach through the San
Angelo soil was less than for the Houston Black soil. The reason for the
inactivation of virus in the San Angelo soil needs to be determined using
quantitative methods.
PARASITOLOGICAL STUDIES
Detection of Possible Human Parasites in Sewage
The number of possible human parasites detected in raw sewage was un-
usually high (Table 2.6). The numbers of Entamoeba histolytica, E. coli
and G_. lamblia cysts were extremely variable from one month sample to the
nextT but E. histolytica was detected in almost every sample. Giardia
w
67
-------
TABLE 20. PRESENCE OF VIRUS IN COLLECTIONS OF LEACHATE FROM GLASS COLUMNS
FILLED WITH A SAN ANGELO SANDY CLAY LOAM AND REPETITIVELY LEACHED
WITH PHYSIOLOGICAL SALINE
Column Number of
height collection
cm
1 1
2
2 1
2
3 1
2
U l
2
3
6 1
2
3
10 1
2
3
15 1
2
3
Quantity of Presence of
saline added virus Replicate
ml 1
5
5
5
5 +
5
5 +
5
5
5
10
c _
5
15 +
5
5 +
20
5
5
2
+
+
+
_
-
_
-
-
^
-
-
+
+
-
+
-
+
increment of liquid added was the quantity required to obtain a
leachate. An additional 5 ml of liquid was added to the column for each
additional collection
68
-------
TABLE 21. PRESENCE OF VIRUS IN COLLECTIONS OF LEACHATE FROM GLASS COLUMNS
FILLED WITH A SAN ANGELO SANDY CLAY LOAM AND REPETITIVELY LEACHED
WITH WATER
Column
height
cm
1
2
3
k
6
10
15
Number of
collection
1
2
1
2
1
2
1
2
3
1
2
3
1
2
3
2
3
Quantity of
water added
5
5
5
5
5
5
5
5
5
10
5
5
15
5
5
20
5
5
Presence of
virus Replicate
1 2
+
+
+ +
: t
+ +
+
+
+ +
^_ MM
+ +
increment of liquid added was the quantity required to obtain a
leachate. An additional 5 ml of liquid was added to the column for each
additional collection
Sample not collected
69
-------
TABLE 22. PRESENCE OF VIRUS IN COLLECTIONS OF LEACHATE FROM GLASS COLUMNS
FILLED WITH A HOUSTON BLACK CLAY AKD REPETITIVELY LEACHED WITH
PHYSIOLOGICAL SALINE
Column
height
cm
1
2
3
h
6
10
Number of
collection
leaching
1
2
1
2
1
2
1
2
3
1
2
3
1
2
3
Quantity of
saline added
ml
5
5
5
5
5
5
5
5
5
5
5
5
15
5
5
Presence of virus
Replicate
1 2
+
- -
+ +
+
b
+
_ _
+ b
+ +
+ +
+ +
- -
w. _
- -
+
ji'irst increment of liquid added was the quantity required to obtain a
leachate. Aa additional 5 Ml of liquid was added to the column for each
additional collection
Sample not collected
70
-------
TABLE 23. PRESENCE OF VIRUS IN COLLECTIONS FROM GLASS COLUMNS FILLED WITH
A HOUSTON BLACK CLAY AND REPETITIVELY LEACHED WITH WATER
Column
height
cm
1
2
3
k
6
10
Number of
leaching
1
2
1
2
1
2
1
2
3
1
2
3
1
2
3
Quantity of
water added8"
5
5
5
5
5
5
5
5
5
10
5
5
15
5
5
Presence of virus
Replicate
1 2
_ .*.
-
_ ^
-
+ +
+
+ +
b b
+ +
b
+
b
+
+
+ +
aFirst increment of liquid added was the quantity required to obtain a
leachate. An additional 5 ml of liquid was added to the column for
each additional collection
"K
Sample not collected
71
-------
TABLE 2k. PRESENCE OF VIRUS IN SOIL COLLECTED FROM COLUMNS OF A SAN ANGELO
SANDY CLAY LOAM THAT WERE INOCULATED WITH VIRUS AND LEACHED WITH
PHYSIOLOGICAL SALINE OR WATER
Column
height
cm
1
2
3
k
6
10
Position in
column
entire
entire
entire
entire
entire
top
middle
bottom
Saline
1
a
-
-
-
a
a
a
a
Replicate
2
a
-
-
-
a
a
a
a
. Water
1
a
-
-
-
a
a
a
a
Replicate
2
a
-
-
-
-
-
-
a,
'Sample not analyzed or data missing
TABLE 25- PRESENCE OF VIRUS IN SOIL COLLECTED FROM COLUMNS OF A HOUSTON
BLACK CLAY THAT WERE INOCULATED WITH VIRUS AND LEACHED WITH
PHYSIOLOGICAL SALINE OR WATER
Column
height
cm
1
2
3
k
6
10
Position in
column
entire
entire
entire
entire
top
middle
bottom
top
middle
bottom
Saline Replicate Water Replicate
1 21 2
; : I :
72
-------
lamblia and E. coll were detected in over half of the samples. Generally,
|. histolytica and G. lamblia were the most prevalent parasites detected.
Fewer parasites were detected during June and September, but this may have
been due to chance because of the small samples collected, rather than a
seasonal trend.
The population estimates given in Table 26 are above what would be
normally^expected in sewage and are partially due to the sampling method.
The sensitivity of the hemocytometer method has definite limitations as
a quantitative tool. Using a 25 to 1 concentration, the detection of only
one organism indicated a density of 50 parasites per ml of sample. The
concentration of larger volumes of sewage would have provided a more repre-
sentative sample but other methods of filtering and concentration would
have to "be used.
Although materials identified as possible human parasites fitted
existing descriptions, the nuclear elements of many protozoan cysts could
not be clearly observed. Also, suspected protozoan cysts (eg. 1C. histolyti-
ca) did not always appear exactly as those seen when examining fresh feces
from infected humans. It is possible that at least some of the materials
identified as protozoan cysts could have been spores or other structures
which resembled cysts.
Table 2.6 includes only those parasites which were consistently observed
in raw sewage. Strongyloides sp. larvae, unidentified nematode eggs,
Taenia sp. eggs, and unidentified trematode eggs were also detected by
direct examination of concentrated raw sewage, but these parasites did not
consistently show up when using hemocytometer counting grids. The eggs of
other relatively common helminths, such as pinworm and hookworm, were not
detected. Perhaps the membranes enclosing the eggs of some helminths, like
hookworms and pinworms, were more delicate than those of the helminths
detected in this study and may have ruptured under osmotic stress when
placed in sewage wastewater. It is likely that pinworm infections were
present in the local population at San Angelo.
Possible human parasites in the primary settling tank were also detec-
ted. Entamoeba histolytica, E_. coli, and Giardia sp. were detected in
wet mounts from samples taken any month from August of 1975 to January of
1976 except for September and November of 1975 and June of 1976. No
samples were taken for February, March and May of 1976 because of shutdown
of the primary settling tank to accommodate ongoing new construction at the
sewage farm. Attempts to use the hemocytometer method on samples was
generally unsatisfactory because of the amount of debris present. There-
fore, only a qualitative examination was possible. Entamoeba histolytica
and Giardia sp. were observed in about 1 out of ho wet mounts from these
samples and E_. coli in approximately 1 out of 30 wet mounts. As car is sp.
and Strongyloides sp. were not detected in these samples, but a Taenia sp.
egg was found in the October 1975 sample.
No possible human parasites were detected in fluid samples from storage
lagoons or in irrigation water from storage lagoons. It is possible that
because of their large size, many of the human parasites settled out in the
73
-------
TABLE 26. NUMBER OF FOUR POSSIBLE HUMAN PARASITES IN RAW SEWAGE ENTERING
THE SEWAGE TREATMENT PLANT DURING 1975 AND 1976
Sampling
Time
Ent amoeba
histolytica
Entamoeba
coli
HT- __ .
Ascaris
sp.
Giardia
sp.
1YU JJ C± JUU-
June '75
July '75
Aug. '75
Sept. '75
Oct. '75
Nov. '75
Dec. '75
Jan. '76
Feb. '76
Mar. '76
Apr. '76
May ' 76
June '76
TABLE 27.
Sampling
Time
Jan. '76
f
Feb. '76
Mar. '76
Apr. '76
May ' 76
June '76
60
13
30
20
25
50
15
30
18
30
50
0
0
RELATIVE ESTIMATES
Ent amoeba
histolytica
Surface 15 cm
iAoa 1/10
1/UO 1/20
-
o iAo
-
0 1/27
30
20
0
0
0
50
25
30
20
2Q
20
0
0
OF FOUR POSSIBLE
Entamoeba
coli
Surface 15 cm
1/20 1/20
0 0
-
1/20 1/20
-
0 0
0
80
0
0
25
0
0
0
0
0
0
0
0
HUMAN PARASITES
Ascaris
SP.
Surface 15 cm
0 1/80
0 0
-
0 1/36
-
0 0
0
.80
80
80
0
25
50
25
uo
ho
30
0
30
IN SLUDGE
Giardia
sp.
Surface 15 cm
1/27 1/10
0 1/40
-
1/UO 1/18
-
0 0
number of positive wet mounts per number of wet mounts examined
-------
primary settling tank with the sludge or sediments in the storage lagoons.
If substantiated, this finding could be important in developing methods
for the treatment of human sewage to remove human parasites.
Detection of Possible Human Parasites in Sludge
Although we were not able to quantify the number of possible human
parasites in the dried up abandoned sludge lagoon, some were detected (Table
27). Both E_. histolytica and Giardia sp. appeared to be most prevalent in
sludge 15 cm below the surface. With the drying of the sludge lagoon,
parasites in the exposed sludge were apparently most susceptible. Viability
of possible parasites was not tested but they appeared normal. Ascaris sp.
was found only at the 15 cm depth and in low frequency. E_. coli was
observed relatively frequently at both sludge depths. ~~
Substantial difficulty was encountered in looking for human parasitic
nematodes in sludge because of the large numbers of freeliving and plant
parasitic nematodes present. Eimeria sp. was the most prevalent parasite,
but it may have come from cattle that had access to the sludge lagoon.
Detection of Rematode Larvae in Soil
Approximately one fourth of the larvae detected on the farm appeared to
be in the genus Strongyloides, but we could not determine if they were
human parasites (Table 28). The majority of the Strongyloides larvae were
Strongyloid-like and were probably Haemonchus contortis. Generally, there
appeared to be a higher density of these nematode larvae on the sewage farm
than off. Figure 19 shows the sewage farm field locations that were
sampled for nematodes.
*
The eggs and larval stages of these nematodes are susceptible to temper-
ature and more importantly to drying. The soil on the sewage farm has been
treated with wastewater over a long period of time which has increased the
soil's organic matter and ability to retain moisture. This, along with
keeping the soil relatively moist from frequent application of sewage
wastewater, provides a favorable environment for larval nematode to survive.
Thus, the nematodes escaped the natural ranges of humidity and precipitation
that occur in the San Angelo area. It is also probable that the lusher
vegetation on the sewage farm was beneficial to larval parasites by pro-
viding protection from direct sunlight.
Some difficulty in identifying parasitic larvae was encountered because
of the large number of free living soil nematodes. Attempts': to separate
soil nematodes from parasitic forms by treating with different concentra-
tions of HC1 were not successful.
Detection of Parasites in Livestock Feces
Comparisons of parasite levels in fresh cattle (Tables 29, 30, 31, 32)
and sheep feces (Table 33 and 31*) collection on the sewage farm and off
the sewage farm indicated a higher density of parasites on the sewage farm.
Only the major genera of parasites observed were included in the tables,
75
-------
TABLE 28. NUMBER OF STRONGYLOIDIDAE IN SURFACE SOIL FROM THE SEWAGE FARM
AND A FARM
JANUARY
Location
Control Farm
Field 1
Sewage Farm
Field 1
Field 2
Field 3
Field h
Field 5
Field 6
Field 8
NOT RECEIVING
Sample
Number
1
2
3
u
1
2
3
k
1
2
3
k
1
2
3
^
1
2
3
1*
1
2
3
li
1
2
3
^
1
2
SEWAGE DURING
November
3
23
9
ill
__
5
2
__
3
52
.__
1*07
0
NOVEMBER,
December
-No/50cc so
0
0
0
0
36
27
0
0
0
30
0
0
68
0
12
12
27
0
0
15
35
0
29
0
0
26
0
0
DECEMBER AND
a
January
3(18)
13(9)
32(9)
m m m
_ _
__
_
__
«.«.
^^
__
__
__
__
__
The data is an average and the number in parenthesis is the number of sam-
ples taken. Only 1 sample collected from the control farm was positive but
h and 7 of the samples, respectively, from fields 1 and 2 on the sewage
farm were positive.
76
-------
CONCHO
RIVER
Figure 19. Diagram of sewage farm.
Approximate locations where soil and feces samples were taken,
from the sewage farm, for detection of nematodes parasitic on animals.
77
-------
but Hematodirus (nematode), Skrjabinema sp. (nematode), unidentified nema-
tode eggs, unidentified oocysts (protozoan), and Entamoeba sp. (protozoan)
were also observed. Figure 19 shows the locations of sewage farm fields
listed in these tables.
Gonglyonema was found only on the sewage farm and detected in about
one out of every five fecal samples (Table 29). This parasite was not
detected off the sewage farm. Because the cattle on the sewage farm and
those on the control area had different histories, we cannot be sure that
the sewage application was the cause of the apparent higher incidence of
Gongylonema on the sewage farm. However, Gongylonema nematode is
reasonably widespread and both populations of cattle had likely been
previously exposed to it. An adequate number of monthly samples was not
taken off the sewage farm to make statistically valid comparisons.
In contrast to Gongylonema, the protozoan Eimeria was found in nearly
all feces samples (Table 30). Eiremia is generally non-pathogenic unless
present in very large numbers and is a common parasite of livestock.
The nematode Haemonchus is a detrimental parasite of cattle. This
parasite seemed to be more prevalent in cattle on the sewage farm as com-
pared to cattle off the sewage farm (Table 31), but an adequate number of
monthly observations was not made on the control farm to allow for
statistical comparisons. Egg counts were used as an indication of parasite
burden and may not be valid for this parasite
To more adequately examine the difference between on-farm and off-
farm levels of Eimeria and Haemonchus in cattle feces, nine samples were
taken from each location on one occasion (Table 32). The results showed
a significantly higher density of both parasites in cattle on the sewage
farm.
The population of parasites in fecal samples from sheep appeared
larger on the sewage farm (Table 33). However, too few monthly samples
were taken for valid statistical comparison. Strongyloides was detected
in two samples collected on the farm. This parasite was not detected in
cattle feces.
As with the cattle, a one-time sampling of nine fecal samples from
sheep was taken from the control farm arid one field .on the sewage farm.
Eimeria and Haemonchus were much more numerous in the samples collected on
the sewage farm (Table 3k). Haemonchus was not detected in any fecal
sample from sheep being grazed on the control farm not receiving sewage.
The data collected demonstrate larger populations of parasites on the sewage
farm, but does not prove that this was due to sewage application. The two
groups of sheep may have had different histories of contact with parasites
before arriving at the two farms.
The previous experiment summarized in this report suggested that appli-
cation of sewage effluent to pasture being grazed by cattle may have
78
-------
TABLE 29- THE NUMBER OF GONGYLONSMA IN MANURE COLLECTED DURING 1* MONTHS
FROM CATTLE BEING GRAZED ON THE SAN
ADJACENT CONTROL FARM NOT RECEIVING
Location
Control Farm
Field 1
Sewage' Farm
Field 1
Field 2
Field 3
Field h
Field 5
Field 6
Field 7
Field 8
Sample
Number
1
2
3
.
1
2
1
2
3
-
2
3
k
1
2
1
2
1
2
1
2
1
2
September
0
0
0
0
0
_M
v
|
0
0
...
__
__
ANGELO
SEWAGE
October
No . per
__
3
3
__
__
3
3
0
0
SEWAGE FARM
November
g manure
0
0
0
__
0
0
__
0
0
0
0
AND ON AN
December
0
0
0
0
0
0
0
0
0
10
0
0
0
0
0
1
_«.
79
-------
TABLE 30. THE NUMBER OF EIMERIA IN MANURE COLLECTED DURING k MONTHS FROM
CATTLE BEING GRAZED ON THE SAN ANGELO SEWAGE FARM
ADJACENT CONTROL FARM NOT RECEIVING SEWAGE ,
Location
Control Farm
Field 1
Sewage Farm
Field 1
Field 2
Field 3
Field It
Field 5
Field 6
Field 1
Field 8
Sample
Number
1
2
3
1
2
1
2
3
1
2
3
It
1
2
1
2
1
2
1
2
1
2
September October November
AT /
rio . pep/ g mELirmre
2-6
lit
3
9,
13 TNTC
, 2 7
2
2
6
3
TNTC
TNTC
6 lit
12 21
__ _._ __
It6
29
2
0
AND ON AN
December
k
16
3
u
8
6
21
90
IT
2lt
25
2lt
61
33
88
6k
__
___
TNTC means too numerous to count
80
-------
TABLE 31. THE NUMBER OF HAEMONCHUS IK MANURE COLLECTED DURING k MONTHS
FROM CATTLE BEING GRAZED ON THE SAN ANGELO SEWAGE FARM AND ON AN
ADJACENT CONTROL FARM NOT RECEIVING SEWAGE
Location
Control Farm
Field 1
Sewage Farm
Field 1
Field 2
Field 3
Field U
Field 5
Field 6
Field 7
Field 8
Sample
Number
1
2
3
1
2
1
2
3
1
2
3
U
1
2
1
2
1
2
1
2
1
2
September
0
2
0
It
0
5
4
0
2
0
0
_ _
__
__
October November
nT_ /
6
5 ^5
5 6
__
1
1
56
11
28
3k
5
0
1
0
December
3
0
It
5
3
266
50
159
ItO
3
h
10
h9
0
139
"*"""
81
-------
TABLE 32. THE NUMBER OF PARASITES, IN TWO GENERA, DETECTED IN CATTLE MANURE
COLLECTED IN JANUARY ON THE SAN ANGELO SEWAGE FARM AND ON AN
ADJACENT FARM NOT RECEIVING SEWAGE
Location
Control Farm
Field 1
Sewage Farm
Field 1
Number
1
2
3
4
5
6
7
8
9
1
2
3
4
5
6
7
8
9
Eimeria
3
15
20
0
0
1
0
0
7
11
14
19
30
11
20
17
.29
35
Family
Hamonchus
TtT / -0
~ iMo - per/g leces
1
0
0
2
11
0
0
7
0
47
32
21
0
273
154
20
0
15
82
-------
TABLE 33. THE NUMBER OF PARASITES, IN THREE GENERA, DETECTED IN SHEEP
MANURE COLLECTED DURING 3 MONTHS ON THE SAN ANGELO SEWAGE PAEM
AND ON AN ADJACENT FARM NOT RECEIVING SEWAGE
Control Farm Sample
Genus
Eimeria
Haemonchus
Strongyloides
Month
Oct.
Nov.
Dec.
Oct.
Nov.
Dec.
Oct.
Nov.
Dec.
1
__
6
l
__ .
6
2
_
0
0
2
1\T
1 * ^J IJ ^? JL
__
0
8
imu[ii_
0
0
__
0
0
Sewage Farm Sample
1
I ^£»
TNTCa
0
19
1
131
311
5
0
0
2
TNTC
19
0
0
137
2
0
0
aTNTC
means too numerous to count
TABLE 3U. THE NUMBER OF PARASITES, IN TWO GENERA, DETECTED IN SHEEP MANURE
COLLECTED IN JANUARY ON THE SAN ANGELO SEWAGE FARM AND ON AN
ADJACENT FARM NOT RECEIVING SEWAGE
Location
Control Farm
Field 1
Sewage Farm
Field 1
Sample
Number
1
2
3
4
5
6
7
8
9
1
2
3
U
5
6
7
i
8
9
Eimeria
11T_
1MO .
0
0
0
0
0
5
3
1
1
21
18
3
3^
166
118
TNTCa
TNTC
32
Family
Haemonchus
pGr/g leces
0
0
0
0
0
0
0
0
0
105
9U
1
k
0
37
1
61
52
aTNTC means too numerous to count
83
-------
increased the population of parasites in the cattle. To better evaluate
this hypothesis, a controlled study was designed in which cattle were
monitored monthly for parasites, beginning the day the cattle were brought
to the sewage farm. The number of Eimeria in the cattle did not increase
with time on the sewage farm (Table 35), nOr did the population of Haemonchus
(Table 36).
Each animal was wormed with an antihelminthis drug when brought to the
sewage farm. Yet no reduction in the population Of parasites was observed
after worming. Five of the ten cattle were wormed a second time with a
noticeable reduction in parasites. Either the worming agent was riot
effective or the month interval between worming and ttaking the measurement
was enough time for the parasite population to lower and increase to the
previous levels.
The cattle in this experiment contained more parasites when brought
onto the farm than did the cattle in the off-farm control areas (Table 32).
Perhaps the method of collecting fecal samples was one reason for this
difference. Pecal samples were taken directly from the cattle in this
experiment, but were taken from droppings in the. earlier, experiments.
The moisture and pH of manure changes rapidly after being voided by the
animal.
-------
TABLE 35- AVERAGE BOMBER OF EIMERIA SP. TIM FECES
oa
FROM 10 TEST CATTLE FOR 7 MONTHS AFTER
ARRIVING ON THE SEMASE FARM
Test Animals
On Arrival
March
April
May
June
July
August
#1
6
95
95
TNTC
TNTC
12
32
#2
265
8
b
7
3U
8
10
#3
35
TNTC
TNTC
30
TNTC
126
159
#u
606
9
9
31
25
1
7
#5
I"
36
120
120
98
TNTC
185
1
#6
#7
fo. per/g fece
TNTC 121
11
11
TNTC
98
88
19
22
22
1U
68
13
U
#8
IB ' '
126
TNTC
TNTC
TNTC
99
TNTC
266
#9
202
55
55
TNTC
52
^7
16
#10
70
9
9
--
81
65
TNTC
-Controls
#1 #2
-
-
172
39
29
TNTC
-
-
10
3
61
W
aThese two cattle were already on the farm when the ten cattle arrived
TNTC means too numerous to count
-------
TABLE 36. AVERAGE NUMBER OF HAEMONCHUS SP. IN PECES
00
ON
FROM 10 TEST CATTLE FOR 7 MONTHS AFTER
ARRIVING ON THE SEWAGE FARM
On Arrival
March
April
May
June
July
August
#1
W
56
67
165
loU
78
201
#2
6
h
73
23
39
1*
8
#3
69
lU6
50
120
232
5U
hQ
ffh
22
51
-
78
178
5
18
Test
#5
U2
33
-
63
107
17
12
Animals
#6
No . per/g
78
^ 78
U9
207
ia
61
ia
#7
feces
118
152
35
78
100
30
16
#8
1*5
63
1+0
77
99
95
ia
#9
26
33
-
U7
52
70
36
#10
5k
1+8
U8
-
81
53
30
Q
Controls
#1 #2
.
-
-
U8 108
39 3
30 127
28 1
These two cattle were already on the farm when the ten cattle arrived
-------
SECTION 7
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93
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO.
EPA-600/2-78-131b
3. RECIPIENT'S ACCESS)OI*NO.
4. TITLE AND SUBTITLE
SEWAGE DISPOSAL ON AGRICULTURAL SOILS: CHEMICAL
AND MICROBIOLOGICAL IMPLICATIONS (VOLUME II MICRO-
BIOLOGICAL IMPLICATIONS)
5. REPORT DATE
June 1978 issuing date
6. PERFORMING ORGANIZATION CODE
R> w> Weaver
N. 0. Dronen
B. G. Foster
F. C. Heck
R. C. Fehrmann
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Texas A&M University
Department of Soil & Crop Sciences
College Station, Texas 77843
10. PROGRAM ELEMENT NO.
1BC611
11. CONTRACT/GRANT NO.
R803281
12. SPONSORING AGENCY NAME AND ADDRESS
Robert S. Kerr Environmental Research Lab. - ADA, OK
Office of Research and Development
U.S. Environmental Protection Agency
Ada, Oklahoma 74820
13. TYPE OF REPORT AND PERIOD COVERED
Final - 1975-1977
14. SPONSORING AGENCY CODE
EPA/600/15
15. SUPPLEMENTARY NOTES
16. ABSTRACT
The city of San Angelo, Texas, has been using agricultural land for decades as
a means of disposing of all of its municipal sewage after primary treatment. Water
applications have been high enough to satisfy crop requirements for a 600 ha farm
even though the farm consists of only 259 ha. The farm routinely supports about
500 cattle on its pastures and produces both row and hay crops. Land application
of ""sewage has public health implications, and this study was conducted to evaluate
these concerns. This was accomplished by monitoring the soils and waters on the
farm to determine the incidence of Salmonella and parasites. Salmonella was isolated
from various locations on the farm but the frequency of isolation was not unusually
high. Possible human parasites were not found in any effluent but were present in
the sludge in holding lagoons. The parasite population in cattle on the farm did
not increase during the months the cattle were monitored. There was an unusually
high population of animal parasites in the soils as compared to off-farm control
soils. This is thought to be due to the higher animal density, the vegetative
cover, and relatively moist soil conditions on the farm. Column studies using soil
from the farm indicated viruses could be leached through the soils. Their potential
health hazard could not be determined due to insensitive detection techniques.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.lDENTIFIERS/OPEN ENDED TERMS C. COSATI Field/Group
Land use/sewage effluents
Sewage treatment/microorganism control
Bacteriology/soil microbiology
Land pollution abatement
San Angelo, Texas
Land application
Municipal wastewater
Rural land use
Environmental health
57H
57K
57N
57U
44G
68D
68G
8. DISTRIBUTION STATEMENT
RELEASE TO PUBLIC
19. SECURITY CLASS (ThisReport)'
UNCLASSIFIED
21. NO. OF PAGES
108
20. SECURITY CLASS (Thispage)
UNCLASSIFIED
22. PRICE
EPA Form 2220-1 (9-73)
94
-140/1357
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