United States
Environmental Protection
Agency
Health Effects Research
Laboratory
Cincinnati OH 45268
EPA-600 1 80-004
June 1980
Research and Development
Human Enteric Virus
Survival in Soil
Following Irrigation
with Sewage Plant
Effluents
EP 600/1
80-004
AGSNCY
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RESEARCH REPORTING SERIES
Research reports of "he Off ce of Research and Deveiopmeni U 5
Protection Agency, have been gicuped into nine series These nine b'oac cate
gones were established to facilitate f'jrtner development anc application o! en-
vironmental tecnnolocy Elimination o1 traditional grouping was consc oasK
planned to foster technology trance- ard a ria^irn jrn m*erfai e -n related ;on Teen no ogv
3 Ecological Ftesearch
4 Environment! Momtoi ng
b Socioeco lomic Environmental Studies
6 Scientific and Technical Assessment Reports (STAF3)
7 interagen :y Fnergy-Env-ironment P^searc'i and Development
3 Gpec'al Ri.-oorts
9 Misct-iianec'.s F!epcrt"_-
Trvs repo't ''as bee- assigre-n tc the E NVIRONMtMTAL HFALlf- F.FFf'^5 PE
SEARCH scries Tn,; series j-soribes pro|ects ana studies reiafng to f •_- Mi-r
This document is available to the public through the National Technical Informa-
tion Service, Springfield, Virginia 22161
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EPA-600/1-80-004
June 1980
HUMAN ENTERIC VIRUS SURVIVAL IN SOIL FOLLOWING
IRRIGATION WITH SEWAGE PLANT EFFLUENTS
by
Bernard P. Sagik
Barbara E. Moore
and
Charles A. Sorber
Center for Applied Research and Technology
The University of Texas at San Antonio
San Antonio, Texas 78285
Grant No. R-803844-03
Project Officer
Elmer W. Akin
Field Studies Division
Health Effects Research Laboratory
Cincinnati, Ohio 45268
HEALTH EFFECTS RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
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DISCLAIMER
This report has been reviewed by the Health Effects Research Laboratory,
U. S. Environmental Protection Agency, Cincinnati, Ohio 45268, 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 endorse-
ment or recommendation for use.
11
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FOREWORD
The U. S. Environmental Protection Agency was created because of
increasing public and government concern about the dangers of pollution
to the health and welfare of the American people. Noxious air, foul
water, and spoiled land are tragic testimony to the deterioration of our
natural environment. The complexity of that environment and the inter-
play between its components require a concentrated and integrated attack
on the problem.
Research and development is that necessary first step in problem
solution and it involves defining the problem, measuring its impact, and
searching for solutions. The primary mission of the Health Effects
Research Laboratory in Cincinnati (HERL) is to provide a sound health
effects data base in support of the regulatory activities of the EPA.
To this end, HERL conducts a research program to identify, characterize,
and quantitate harmful effects of pollutants that may result from ex-
posure to chemical, physical, or biological agents found in the environ-
ment. In addition to valuable health information generated by these
activities, new research techniques and methods are being developed that
contribute to a better understanding of human biochemical and physio-
logical functions, and how these functions are altered by low level
insults.
This report provides an evaluation 'of the fate of naturally-occur-
ring viruses in undisinfected treated sewage effluents applied to the
soil through irrigation. Sewage treatment removes many but not all
viruses from the effluent. Therefore, a knowledge of the survival and
fate of these viruses is important in assessing the health risk to farm
workers and consumer of food crops and ground water in areas where
domestic wastes are applied to the land. This study has provided a
significant contribution to the data base required to fully assess the
health considerations of this waste disposal practice.
R. J. Garner
Director
Health Effects Research Laboratory
iii
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ABSTRACT
The wastewater treatment processes at Kerrville and Uvalde, Texas,
have been evaluated in terms of their efficacy in reducing TOC, BOD , sus-
pended solids, orthophosphate, nitrogenous compounds, total coliform, fecal
coliform, bacteriophage and human enteric viruses. The Kerrville facility,
an underloaded parallel oxidation ditch-trickling filter system, is highly
effective; achieving a 90-95% reduction in BOD and an 80% reduction in TOC.
By comparison, the Uvalde plant, utilizing an overloaded trickling filter
followed by a series of six ponds, reduces incoming levels of BOD and TOC
by 85% and 75%, respectively. Both plants reduce phage levels by a factor
of 300 to 500. Enteric viruses are reduced by greater than 99% at Kerrville
and at least 99% at Uvalde. These waters are used for irrigation without
disinfection.
Soil samples at the Kerrville and Uvalde application sites have yielded
both fecal coliforms and bacteriophages. In addition, two confirmed entero-
virus isolations were made at the Kerrville site.
The Kerrville lysimeters (at 1.5 ft, 3.0 ft, and 4.5 ft depths) have
yielded large numbers of bacteriophage isolates. In addition, ten lysimeter
samples yielded a total of 29 confirmed viral isolates. This is a strikingly
high number of isolations of indigenous enteric viruses, relative to the
irrigation pond which was demonstrably low in viruses (when assayed on the
same cell lines).
Monitoring wells at the Kerrville site show water quality to be im-
proved over that in the irrigation pond. Nitrate values are somewhat elevated,
however. Further, indicator organisms (fecal coliform and fecal streptococci)
were isolated from all wells at some time and with relatively high frequency
(not less than 40% of sampling times from any well). No confirmed virus
isolations were made from any of the monitoring wells.
Geological descriptions of the site are included as are well log and
hydrologic analyses.
These studies of wastewater treatment plants processing dilute to mo-
derate strength sewage in efficient treatment schemes represent a "best
possible case" for the use of undisinfected, domestic wastewater effluents
for irrigation. The isolation of enteroviruses in water from lysimeters but
not from the monitoring wells suggests that depth to groundwater should be
a critical factor in the selection of irrigation sites. From data developed
in this study, it appears that a depth of 4.5 ft is not sufficient for effec-
tive viral attenuation in soils such as those described in this report.
IV
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CONTENTS
Foreword ..... ill
Abstract iv
Figures vi
Tables viii
Abbreviations and Symbols xii
Acknowledgment xiii
1. Introduction 1
2. Conclusions 2
3. Recommendations 4
4. Background 5
Introduction 5
Land Application of Wastewater 12
Potential Use of Land Application Sites 20
5. Objectives 21
6. Description of Study Sites 22
Kerrville, Texas 22
Uvalde, Texas 33
7. Methods and Materials 35
Lysimeters 35
Monitoring Wells 35
Surface Water Measurements 39
Sampling 42
Wastewater Analyses 44
Soil/Sediment Analyses 54
Special Irrigation Study 59
8. Results and Discussions 60
Wastewater Analyses 60
Irrigation Pond Studies 70
Irrigation and Precipitation 78
Soil Analyses 78
Lysimeters 88
Monitoring Wells 109
Viral Recoveries, Identification and
Characterization 124
References 129
Appendices
A. Viral Identification and Characterization 135
B. Growth of Bacterial Indicators in Lysimeter Waters. . . . 137
v
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FIGURES
Number Page
1 Wastewater treatment facilities, Kerrville Site 23
2 Aerial view of Kerrville site 24
3 Location of wells, lysimeters, soil series and soil
sampling points, Kerrville site 26
4 Wastewater treatment facilities, Uvalde site 34
5 Details of polypropylene, pan lysimeter 36
6 Generalized geology at the Kerrville irrigation site ... 37
7 Monitoring well completion diagram 40
8 Generalized well log for monitoring wells at Kerrville
site 40
9 Water and sediment sampling points in Kerrville and
Uvalde ponds 73
10 Fecal coliform and coliphage removals in Uvalde Ponds . . 75
11 Diagrammatic representation of the succession of layers
in some layer lattice silicates 34
12 Soil aggregate distribution curves, Kerrville soils ... g£
13 Soil particle size distribution curves for
lysimeter #1 95
14 Soil particle size distribution curves for
lysimeter -#2 96
15 Soil particle size distribution curves for
lysimeter #3 96
16 Fecal streptococci inactivation in lysimeter water .... 103
17 Groundwater contours, Kerrville site 112
vi
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Number Page
18 Summary of precipitation and irrigation, Kerrville
site 114
19 Hydrographs of wells 1, 2 and 3 115
20 Hydrographs of wells 4 and 5 116
vii
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TABLES
Number Page
1 Pesticide and PCB Content, Dry Sludge ............ 7
2 Concentration of Trace Elements in Plants .......... 8
3 Pathogen Removal by Conventional Wastewater Treatment (%) . . 9
4 Effect of Wastewater Treatment on Various Organisms ..... 10
5 Results of Wastewater Microbiological Screen ......... 11
6 Estimated Wastewater Pathogens Applied to Soil ........ 13
7 Effect of Temperature on the Survival of Poliovirus I
in Soils at 15% Moisture Content .............
8 Effect of Soil Moisture on the Survival of Poliovirus I
at 20C .......................... 16
9 Sampling Points and Sampling Method ............. 43
10 Comparison of Bentonite Concentration Technique
Recovery Efficiency .................... 44
11 Comparison of Membrane Filtration and Spread Plating
Techniques ........................ 5©
12 Comparison of Fecal Streptococci Isolations on
Selective Media ...................... 5Q
13 Holding Procedures for Routine Chemical/Physical Analysis . . 53
14 Poliovirus Recovery from Kerrville Soil ........... 55
15 Enumeration of Total Aerobic Bacteria from Soils ....... 56
16 Enumeration of Fecal Streptococci in Soil Samples ...... 57
17 Mean Results of Wastewater Chemical/Physical Analyses,
Kerrville Site ...................... 61
18 Mean Results of Wastewater Chemical/Physical Analyses,
Uvalde Site ........................ 62
viii
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Number
19 Mean Results of Wastewater Bacteria and Bacteriophage
Analyses, Kerrville Site 64
20 Mean Results of Wastewater Bacteria and Bacteriophage
Analyses, Uvalde Site 65
21 Bacteriological Screen, Kerrville and Uvalde Sites 67
22 Results of Wastewater Enterovirus Assay, Kerrville Site. ... 68
23 Effectiveness of Wastewater Treatment Processes in
Reducing Enterovirus Recovery (Selected Sampling Days),
Kerrville Site 69
24 Results of Wastewater Enterovirus .Assay, Uvalde Site 71
25 Effectiveness of Wastewater Treatment Processes in
Reducing Enterovirus Recovery (Selected Sampling Days),
Uvalde Site 72
26 Organism Distribution in the Kerrville Irrigation Pond. ... 74
27 Microbial Profiles in Uvalde Ponds (June, 1978) 77
28 Application of Wastewater and Precipitation
(October 1977-August 1978) - Kerrville Site 79
29 Selected Properties of Soil Samples SO
30 Soil Metallic Ion Concentrations (yg/5 grams) 81
31 Minerals Present in the Silt Size Range Dominant
Mineral Present Listed First 82
32 Minerals Present in the Clay Size Fraction;
Dominant Mineral Present Listed First 83
33 Ranges of Results of Soil Bacteria and Bacteriophage
Analysis, Kerrville Site 87
34 Ratio of Soil Total Coliform to Fecal Coliform,
Kerrville Site 89
35 Soil Data, Special Irrigation Study I, Kerrville Site .... 90
36 Soil Data, Special Irrigation Study II, Kerrville, Texas. . . 91
IX
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Number
Page
37 Ranges of Results of Soil Bacteria and Bacteriophage
Assays, Uvalde Site 92
38 Ratio of Soil Total Coliform to Fecal Coliform,
Uvalde Site 93
39 Chemistry - Kerrville Lysimeter Soils 97
40 Mean Results of Chemical/Physical Analyses, Lysimeter—
Kerrville Site 99
41 Chemical/Physical Data, Special Irrigation Study I,
Kerrville Site 100
42 Chemical/Physical Data, Special Irrigation Study II—
Kerrville Site 101
43 Ranges of Results of Bacteria and Bacteriophage
Analyses of Lysimeter Samples, Kerrville Site 104
44 Selected Results of Lysimeter Microbiological Analysis 105
45 Irrigation Schedule for Special Irrigation Study I,
Kerrville Site 10.7
46 Microbiological Data, Special Irrigation Study I,
Kerrville Site 107
47 Microbiological Data, Special Irrigation Study II,
Kerrville Site 108
48 Lithologic Logs of Monitoring Wells 110-111
49 Results of Chemical/Physical Analyses - Monitoring Wells . . . .117
50 Groundwater Metallic Ion Concentration (mg/1) 119
51 Identification of Fecal Coliform Isolates 120
52 Percentage of Positive Isolations of Indicator Organisms
from Kerrville Monitoring Wells 120
53 Results of Bacterial Analyses, Kerrville Monitoring Wells. . . .121
54 Selected Bacteriological Results, Kerrville Monitoring
Wells 123
x
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Number Page
55 Volumes Routinely Concentrated for Enteroviruses
from Wells 124
56 Comparison of Indigenous Virus Recoveries (pfu) on
HeLa and BGM Cells 125
57 Identification of Viral Isolates from the Kerrville
Irrigation Site 127
58 Temperature Characterization of Poliovirus I
Isolates, Kerrville Irrigation Site 128
xi
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ABBREVIATIONS
BGM —
BOD —
C
CEC —
cfu —
can ——
COD —
COLE -
CPE -
fm
g
gm -•
gpd -
gpm —
1
meq -
MF
mg
mgd -
Buffalo Green Monkey ml
biochemical oxygen demand-5 day MLSS -
centigrade mm
cation exchange capacity MPN -
colony forming units msl -
centimeter ND
chemical oxygen demand PCB
- coefficient of linear expansion PEG -
cytopathic effect
formation
gravity
grams
gallons per day
gallons per minute
liter
milliequivalents
membrane filtration
milligram
million gallons per day
pfu
PH
ppm -
psi
PVC -
rpm -
TKN -
TOC -
TP04 -
TSS -
VSS -
- milliliter
- mixed liquor suspended solids
- millimeters
- most probable number
- mean sea level
- none detected
- polychlorinated biphenols
- polyethylene glycol
- plaque forming units
- hydrogen ion concentration
— parts per million
- pounds per square inch
- polyvinyl chloride
- revolution per minute
- total Kjeldahl nitrogen
- total organic carbon
— tryptose phosphate broth
- total suspended solids
- volatile suspended solids
xii
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ACKNOWLEDGEMENTS
The grantees wish to acknowledge the efforts of the members of the
staff of the Center for Applied Research and Technology and thank them for
their dedication and professionalism. In particular, we would single out
Messrs. Dudley, Funderburg, Ibarra, Moring, Turk and Ms. Hulsey. We are
indebted, too, to W. W. Hammond for his contributions to our physical
understanding of the Kerrville site and for his study of well cores.
This research could not have been completed without the continuing
support of so many individuals in the cities of Kerrville and Uvalde,
Texas. Mr. T. H. Caffall and Mr. Marvin Angermiller have been extremely
helpful at Uvalde; at Kerrville, where this effort was concentrated, a
large number of city employees have contributed immeasurably. Of parti-
cular value, has been the willing assistance of Mr. Calvin Neely and
Mr. Jay Clanton of the Department of Public Works. At the Kerrville
Wastewater Treatment facility, the efforts and patience of Mr. Frank
Meeker and Mr. Arthur Karcher are greatly appreciated by the authors and
all of the members of the CART staff. They have all managed to be un-
failingly helpful and even cheerful despite our often excessive demands.
Above all, we are grateful to Dr. Elmer Akin for his close review of
this project, for his many astute observations and for his perspicacious—
even trenchant—criticisms. By these means, he has contributed greatly.
xiii
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SECTION 1
INTRODUCTION
Sewage farming has been used widely in the Far East for centuries and
in Europe for over one hundred years. While practiced in the past on a
limited scale in the United States, land application of wastewaters now
has become attractive from both an environmental and economic perspective.
Further, its popularity has been enhanced by recent amendments to the
Federal Water Pollution Control Act (PL 92-500).
Increased interest in the public health concerns associated with the
land application of wastewaters has paralleled the popularity of the pro-
cess. Of particular public health concern is the possible survival and
accumulation of pathogens in the soil and their movement to groundwater
supplies. An urban treatment plant receives significant quantities of
Clostridia, Enterobacter, Klebsiella^ Leptospira^ Mycobacteria. Proteus,
Ppovidenaia, Staphyloaocoij as well as human enteroviruses. The degree to
which these are passed on through the plant to land application sites is
a function of the efficiency of the wastewater treatment chain prior to
application to soils.
Pathogen movement through soils has been reported for distances up to
1500 feet. Release and subsequent movement of bacteria is not unexpected
as this is a reversible phenomenon and, at least in part, ion dependent.
The same theoretical considerations appear to govern viral movement through
soils. Fewer data are available in the literature for viruses than for
bacteria because of the problems inherent in their apparent lesser fre-
quency of occurrence and the nature of concentration and assay techniques
(which, in turn, may account for the reported lower frequency).
Because more municipalities are turning to land application, especially
where economically attractive, the potential for groundwater pollution is
increasing. The degree of that potential is not known. The studies
carried out under this grant were designed to evaluate 'the survival and
transport of human enteric viruses through the soil at an operating land
application site in South Central Texas. Although it focuses specifically
on human enteroviruses, it seeks to corroborate and place in perspective the
results by concomitant analyses for bacterial indicators, bacteriophages
and selected chemical parameters. Samples of raw wastewater, treated
effluent, holding pond water, soils, water from the zone of percolation and
from the underlying aquifer have been analyzed. Only under field conditions
can the potential pollution hazard be studied meaningfully.
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SECTION 2
CONCLUSIONS
As essentially all of the field effort undertaken during this study
was directed toward the Kerrville site, the conclusions drawn are based pri-
marily on observations made at that site. Specific information regarding the
Uvalde site can be found in the text of this report.
Geological descriptions of the wastewater application site indicate that
it is typical of the South Central Texas area with its limestone formations.
Hydrologic studies indicate that groundwater moves toward Third Creek in a
southerly direction acros§ the irrigation site. It is either discharged to the
creek as seeps or continues as subsurface flow in the alluvium down-valley.
The treatment processes are effective in treating moderate strength
domestic wastewater. The resulting effluent is moderately low in coliforms
and bacteriophages and low in enteric viruses. Despite this, there were
isolations of fecal coliforms, bacteriophage and enteric viruses from soil and
lysimeter samples in significant numbers. The levels of phage found in lysi-
meters, even to a depth of 4.5 ft, were remarkably high. No strong evidence
for attenuation by passage through soils up to 4.5 ft could be found either
during the routine monitoring or in the special lysimeter studies which utilized
an intensive irrigation regimen. In addition, positive isolations of enteric
viruses from lysimeter samples were made and the viruses were identified.
In view of the low levels of enteroviruses detected in the irrigation pond, a
conservative role of soil in the survival of phages and enteric'viruses is
suggested.
The monitoring wells yielded total coliforms, fecal coliforms, and
fecal streptococci with relatively high frequency. Median fecal coliform
levels were from 2.4x10 to 3^7x10 cfu/100 ml while median fecal strepto-
cocci values were from 6.7x10 to 7.7x10 cfu/100 ml. No monitoring well had
fewer than 40% of samples positive for fecal coliform, or 39% positive for
fecal streptococci. No confirmed virus isolations were made from any of the
monitoring wells.
It was not possible to calculate a mass balance of indigenous enter-
viruses at the site due to low numbers of enteroviruses observed at most
of the sampling points. In general, the Kerrville site represents a well-
operated, biologically and chemically efficient treatment system (including
the land application component). In some respects, the efficiency of this
system precluded achieving this particular stated objective of the study.
These studies of wastewater treatment plants processing dilute to mo-
derate strength sewage in efficient treatment schemes represent a ''best
-------
possible case" for the use of undisinfected, domestic wastewater effluents
for irrigation. The isolation of enteroviruses in water from lysimeters but
not from the monitoring wells suggests that depth to groundwater should be
a critical factor in the selection of irrigation sites. From data developed
in this study, it appears that a depth of 4.5 ft is not sufficient for effective
viral attenuation in soils such as those described in this report.
In general, the land application of wastewaters with as low an entero-
virus density as that observed at the study site onto soils such as those
described for the Kerrville site appears to provide adequate attenuation of
enteroviruses, and should not represent an undue hazard to any potential, bene-
ficial uses of groundwater or surface wajzer.
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SECTION 3
RECOMMENDATIONS
In general, the land application of wastewaters with as low an enterovirus
density as that observed at the study site onto soils such as those described
for the Kerrville site appears to provide adequate attenuation of enteroviruses,
and should not represent an undue hazard to any potential, beneficial uses of
groundwater or surface water.
The isolation of enteroviruses from lysimeters remains a disturbing find-
ing, however. In particular, the recovery of polioviruses lacking temperature
sensitivity (ts) is of some concern. It would be useful, in assessing the
meaning of such data, if the proportion of poliovirus isolates lacking this ts
marker were known for virions found in raw influent and in treated effluents.
One would wish to determine whether vaccine strains and those lacking the ts
marker survive the irrigation process and soil percolation equally. Further,
more critical genetic identification of isolated viral strains should be made.
It is essential that field isolates be shown incontrovertibly to be different
from reference strains used in laboratories carrying out such studies. Thus,
it is recommended that such techniques as RNA fingerprinting be developed for
representative enteroviruses and applied to the differentiation of field iso-
lates from laboratory strains.
In addition, because soil samples yielded fewer positive viral isolations
than did lysimeter water samples, the suspicion remains that the existing
methodology for viral recovery from soils is simply inadequate. More effort
should be devoted to improving recovery methodology from field samples; espe-
cially from soils, pond sediments, and algal-rich waters.
Other investigations of this nature should be conducted at field sites
which are significantly different in terms of wastewater quality, soils and
groundwater hydrology. It certainly would appear important and prudent to
evaluate a "worst case" site in terms of the results reported herein. However,
it will be of utmost importance that similar methodologies be used in order to
make a comparative analysis of the data.
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SECTION 4
BACKGROUND
INTRODUCTION
The first organized efforts at sewage farming in England and in continental
Europe date from the mid-nineteenth century. Sanitary conditions deteriorated
rapidly as the Industrial Revolution brought large numbers of people from sparsely
settled rural regions into densely populated and often-times ill-planned urban
areas. The most critical problems of waste purification first developed in
England. The dilution and purification capacities of her smaller rivers were
exceeded as increasing amounts of sewage reached these waterways.
The first Royal Commission on Sewage Disposal in England was appointed to
study and report on sewage problems in 1857 (Chase, 1964). Their report, filed
in 1865, recommended that distribution of sewage on land was a proper method
of purification and the only means of preventing pollution of rivers. Subse-
quent commissions appointed in 1868 and 1882 further defined the options of
sewage collection and disposal and returned similar recommendations.
The practical application of land treatment proved to be difficult due to
the nonporous nature of certain soils and the relatively large amounts of land
necessary for the spreading of sewage. Sewage farms in Paris in 1924 covered
12,584 acres (Chase, 1964), while a similar operation for the City of Berlin
utilized 56,800 acres in 1934 (Gray, 1968).
The direct application of sewage to land for the fertilization of crops
was never carried out with continuous success in the United States. However,
sewage irrigation with effluent from wastewater treatment plants has been
practiced in arid and semiarid regions of the country. In 1968, approximately
60 municipalities on the High Plains of West Texas were diverting treated
effluent to the land (Wells, 1968). Ideally, the applic'.tion of wastewaters
in this manner serves the multiple purposes of crop irrigation, effluent dis-
posal, and groundwater recharge in areas where surface water is scarce.
Surface spreading in basins or ditches, irrigation of land, and stream im-
poundment is most successful (in terms of amount of water recharged) where
water table aquifers exist. However, for the same reasons, these unconfined
aquifers are more susceptible to surface pollution, especially in those regions
where the water table is near ground level. Mechanically, surface spreading
is accepted as most feasible where lower quality water is available, as
suspended solids can be removed by filtration through the bottom of the
spreading area. Other methods of application of wastewater include spray
-------
or trickle irrigation and overland flow. Operation and design of these
type systems have been described previously (Pound and Crites, 1973; and
Reed et al, 1972).
Regardless of the methods chosen to treat and dispose of effluents, the
persistent question of water quality arises. It is an inevitable fact that
possible contamination of groundwater is inherent in any recharge system. The
probability of introducing pollutants is increased if wastewater in some form
is used for recharge purposes. The consequences of groundwater contamination
can be damaging because of the possibility of long-term persistence even after
the contaminating source has been eliminated.
An optimized sewage irrigation system would allow the continuous applica-
tion of wastewater to a relatively small surface area, with only minimal pre-
treatment necessary to alleviate aesthetic problems. After percolating through
the unsaturated soil zone, the resulting groundwater would be suitable for re-
use without further treatment (McGauhey and Krone, 1967). Unfortunately,
nature has placed physical, chemical, and biological constraints upon such an
idealized system. Detailed discussions in this regard have been provided by
Sepp (1971) and Sorber and Guter (1975).
Physical Limitations
Physical clogging of the soil pore space results in the loss of infiltra-
tion rates. Such "sewage-sick" soils have led to the failure of many waste-
water irrigation systems. Some of the factors related to the cause of clogging
have been identified as compaction of soil, deflocculation of soils (resulting
in decreased permeability when sodium represents a high percentage of the
cationic content of the water), deposition of suspended solids, and growth of
bacterial slimes especially under anaerobic conditions which allow the preci-
pitation of ferrous sulfide (McGauhey and. Krone, 1967) . Experience has shown
that.the most severe clogging problems can be eliminated by restoring aerobic
conditions within the soil system by controlling the frequency of effluent
application (Thomas and Law, 1968).
Chemical Limitations
The behavior of various chemicals prevalent in domestic wastewaters has
been studied at various irrigation sites (Pound and Crites, 1973; Sorber and
Guter, 1975). Traditionally, the presence of heavy metals and synthetic or-
ganic chemicals is attributable to industrial discharges and urban runoff.
This report will not emphasize such studies, concentrating instead on biologi-
cal hazards. The literature documenting removal of either of these chemical
classes by secondary treatment is .limited. However, trace metals are associ-
ated with sludge solids. Nomura and Young (1974) reported that 90% of aluminum,
iron, mercury, lead, and zinc settled with the biomass when suspended solids
removals were 90-95%. Under the same conditions, chromium (VI) and nickel had
median removals of 77% and 50%, respectively. Conversely, no significant
removal of a wide variety of potentially carcinogenic organic compounds at
three activated sludge treatment plants was observed (Malaney et al, 1967) .
Using routine analytical procedures, however, all chlorinated organics
examined for in dry sludges were found (see Table 1).
6
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Clearly, the concentration of trace metals in sludges and effluents is
a primary consideration in the land disposal of these residuals. While arsenic,
copper, lead, mercury, nickel, selenium, and zinc are of concern, cadmium poses
the greatest human health problem because of its accumulation in kidneys and
liver and its subsequent toxic effects beyond a threshold level. As trace
elements are retained in soils, their entry into the food chain and their long-
term accumulation in soil systems present potential health risks. Hinesly
et al, (1977) have discussed the effects of sewage sludge applications on
assimilation of zinc and cadmium by a typical feed crop, corn. Differential
levels of cadmium and zinc have been shown in fruit and leaves of corn, soy
beans and tomatoes (Kirkham, 1974; Table 2). At this time, however, no apparent
consensus exists among technical experts and regulatory agencies as to what the
toxicity levels are. These problems are compounded further by inadequate
methodology for the evaluation of toxicological and epidemiological effects of
heavy metals in the environment.
Much the same argument can be made for the ultimate fate of a seemingly
endless number of synthetic organic chemicals found in wastewater effluents
and sludges. Studies have shown translocation of chlordane, heptachlor,
and dieldrin from soil into the oil of soybean seeds (Moore et al, 1976b).
While the amounts detected were low, these findings demonstrate that sludge
can serve to recycle organic contaminants back into the food chain.
TABLE 1. Pesticide and PCB Content, Dry Sludge
Contaminant
Range
Min.
(ppm)
Max.
Sludges
Examined
Aldrin*
Dieldrin*
Chlordane*
DDT+DDD*
PCB'S
ND
0.08
3.0
0.1
ND
16.2
1.4
32.2
0.1
352.0
5
7
7
7
69
a From Jelinek et al, 1976.
* Examined, 1971.
+ Examined, 1971, 1973, 1975.
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TABLE 2. Concentration of Trace Elements in Plants*
Cd (ppm) Zn (ppm)
Plant Fruit Leaves Fruit Leaves
Corn
Soybeans
Tomatoes
1.
2.
0.
03
40
50
11.
10.
6.
6
2
1
152.
80.
29.
3
0
0
212
249
153
* From Kirkham, 1974.
Studies in Lubbock, Texas, showed a marked deterioration of chemical quality
in the aquifer after sustained irrigation with effluent (Wells, 1968) . While
POD and COD were eliminated, calcium, magnesium, chloride, sulfate, and nitrates
increased significantly. Nitrate levels in the water beneath the storage pond
on the Texas Tech University farm were increasing at a rate of approximately
1.0 mg/I/month.
More detailed information regarding the interaction of vegetation in a
soil system was reported from the Penn State Wastewater Renovation and
Conservation Project where municipal effluent was applied to both forest and
cropland. Phosphorus was retained effectively within the first six to 24-inch
soil horizon. Calculations based on experimental data showed that only 1.7
percent of the phosphorus added yearly passed the four-foot depth (Hook, et
al, 1973). The remaining 98.3% was removed either by the harvest of reed
canary-grass, by fixation in the soil, or by surface runoff. The cycling of
nitrogen applied in effluent within the soil system led to increased nitrate
levels. The concentration of nitrate-nitrogen was held below 10 mg/1 (USEPA
limit for drinking water) at the four-foot soil depth by grass-legume hays and
reed canary-grass. Forested areas rapidly exceeded USEPA drinking water
standards reaching 25 to 40 irg/1 nitrate-nitrogen at four feet below the surface
with the application of two inches of effluent per week (Kardos and Sopper,
1973a). It is apparent, therefore, that the potential for microbial denitri-
fication must be maximized in any continuous irrigation project.
Changes in other chemical constituents at the Penn State irrigation site
demonstrated the removal of potassium, calcium, magnesium, sodium, manganese,
boron, and chlorides from the wastewater effluent at the four-foot depth. In
the crop rotation plots, increases in the soil content of sodium, magnesium,
boron and chlorides were observed (Kardos and Sopper, 1973b). Such accumu-
lations of soluble salts are much more likely to occur in those regions
where effluent provides the major source of irrigation water and rainfall is
relatively low. Accumulations of salts and heavy metals could present the
dual problems of reducing soil permeability and of increasing toxicity towards
plants.
8
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Biological Limitations
Perhaps one of the greatest constraints placed upon sewage irrigation
is the survival and movement of pathogens. Even before the final acceptance
of the germ theory of disease, John Snow published results linking London's
outbreaks of Asiatic cholera with an underground contaminated water supply
(Burrows, 1968). In like fashion, the classic work of Neefe and Stokes
(1945) on infectious hepatitis was founded initially on epidemiological
evidence which implicated sewage-contaminated well water as the source of
infection.
The concentration of biological contaminants in domestic sewage is in-
fluenced by several complex factors including the age and health of the •>
contributing population as well as the season of year. Species of potentially
pathogenic microorganisms may be categorized conveniently as bacteria,
helminthic parasites, protozoa, and viruses. The presence and survival of
these major groups of pathogens in wastewater were reviewed by Foster and
Engelbrecht (1973) and Akin, e_t al_ (1978). While certain pathogenic organisms
such as Salmonella typhi have a relatively brief survival time in waste-
water, other pathogens (including mycobacteria, Ascaris ova, and certain
enteric viruses) are highly resistant to many environmental stresses.
The levels of microbial pathogens ultimately present in sewage effluents
or sludges depends upon the degree of removal achieved by the treatment train
employed. Results given in Table 3 indicate that primary treatment alone does
not remove the pathogen load in domestic wastewater, although primary sludges
may contain large numbers of parasite eggs. On the other hand, the sludge
biomass generated by conventional secondary treatment may be expected to con-
tain a large portion of that microbial population which has been removed from
incoming wastewater.
TABLE 3. Pathogen Removal by Conventional Wastewater Treatment (%)..*
Organism
So, Imone 1 la
Mycobacteyiwn
Amoebic Cysts
Helminth Ova
Enteric Viruses
Primary
Sedimentation
15
48-57
No Reduction (3 hr)
72-98
3-extensive
Trickling
Filters
84-99+
69-99
11-99+
62-76
0-84
Activated
Sludge
96-99
Slight-87
No Apparent Removal
No Apparent Removal
76-99
* From Foster and Engelbrecht, 1973.
-------
Generally, disinfection (specifically chlorination) of effluents before
discharge provides the last step in the treatment scheme. Difficulties arise
in comparing results of chlorination studies because of a lack of specific
information on (1) initial chlorine dosage, (2) amount and form of chlorine
residual, (3) temperature, (4) pH, and (5) the presence of organic or inorganic
nitrogenous compounds. However, certain microorganisms including mycobacteria,
amoebic cysts, some enteric viruses, and the viral agent of waterborne hepatitis,
may be more chlorine-resistant than are indicator coliform organisms. Neverthe-
less, chlorination of effluents can provide an additional removal of certain
potential pathogens as illustrated by field studies in Table 4.
TABLE 4. Effect of Wastewater Treatment on Various Organisms*
Total Reduction Reduction by
in Treatment Chlorination
Plant (Log ) Only (Log )
Plant #1 (Standard-rate trickling filter,
chlorine residual 1.5 mg/1)
Fecal coliform 3.7 2.]
Fecal streptococci 2.4 2.0
Salmonella 1.4 0.5
Enteric viruses 0.4 0.3
Plant #2 (High-rate trickling filter,
chlorine residual 4 mg/1 w/improved mixing)
Total coliform
Fecal coliform
Klebs~iella
6.8
5.8
5.2
5.5
3.6
3.7
* From Sorber et al, 1974.
+ Presumptive, M-bismuth sulfite broth with MF procedure
a Three days on BGM cells after concentration
10
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A typical microbial analysis of a sample from the aeration chamber of an
urban treatment plant (Table 5) yielded confirmed isolates of species of Clostridia,
Enterobacter, H s-producing Escherichia, fecal coliform, Klebsiella, Leptospira,
mycobacteria, Providencia, staphylococci; total coliforms in the sample were
1.1x10 /100 ml and total plate count was 5.8x10 /100 ml (Guentzel, 1978). Con-
centration of the microbial population into the biomass-makes it essential,
therefore, to implement sludge processing procedures which can diminish the
concentrations of bacteria, helminth ova, and viruses.
TABLE 5. Results of Wastewater Microbiological Screen
Bacteria: cfu/lOC ml_
Citrobacter <5.0xlo't (ND)
Clostridiwn 2.8xlo2
Edwardsiella <5.0xlou (ND)
Enterobacter 3.OxlO6
Esaheriahia (H S ) l.OxlO6
Fecal Coliform l.OxlO7
Klebsiella 6.OxlO6
Leptospira 4.6xlo3
Mycobacteria 7.0x10
Providenaia l.OxlO6
Serratia <5.0xlo" (ND)
Staphyloeoccus 3.OxlO5
Total coliform l.lxlO8
Total Plate Count 5.8xl08
Yersinia * (ND)
Of the total number of colonies which were randomly picked and biochemically
tested for Enterobacteriaceae:
Oxidase positive 44.9%
Enterobaater 14.4%
Klebsiella 14.4%
Proteus 1.4%
Provideneia 1.4%
Escheriohia 1.4%
No growth 4.3%
Not identified 17.3%
Note: ND - None Detected
* - Nonquantitative Procedure
+ - From Guentzel, 1978
11
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Anaerobic sludge digestion, aerobic sludge stabilization and sludge
lagooning are among the most economically popular methods for treating
waste sludges prior to land disposal. The EPA report (Process Manual, 1974)
on microbial survival during anaerobic digestion showed that while coliform
populations are reduced greatly, other pathogens such as Mycobacteria and
As earn, s ova can withstand prolonged digestion. Recently, several laboratory
studies have reported the fate of selected viruses during anaerobic digestion.
Using a fill-and-draw model digester at 35C, Bertucci and coworkers (1975) ob-
served inactivation rates ranging from 74.9% per day for Echovirus II to 97%
per day for Coxsackievirus A-9. Ward and Ashley (1976) added poliovirus to
digested sludge at 28C and observed an inactivation rate of one log. per day.
In contrast to these findings, Sanders et al, (1979), have reported much slower
rates of 0.3 log per day using solids-incorporated poliovirus (an important
methodological difference) in a 34C reactor. While significant pathogen re-
ductions can be achieved by anaerobic digestion, actual field digesters may be
less efficient due to the continuous input of contaminated raw sludaes coupled
with probable short-circuiting and incomplete mixing.
Aerobic sludge digestion may be viewed as a continuation of the extended
aeration process. To date, little information is available dealing directly
with pathogen removal by aerobic digestion. It is obvious that some degree
of pathogen inactivation will occur during the 15 to 20 day period required
for endogenous respiration. Comprehensive studies on the fate of microbial
pathogens in sludge lagoon systems also are lacking. Until such *ime as
more definitive data are available, one should assume conservatively that
wasted sludges from these systems also will contain environmentally-resistant
microbial forms such as mycobacteria, helminth ova, and viruses.
LAND APPLICATION OF WASTEWATER
If domestic sewage is subjected to adequate secondary treatment followed
by disinfection, the resultant effluent may be expected to contain on a per
unit basis low levels of potential human pathogens. The continual application
of effluents to soils may allow the retention and accumulation of such organisms
within a given soil profile. The dimensions of the problems inherent in land
disposal of effluents have been suggested by Foster and Engelbrecht (1973) as
they attempted to relate treatment effectiveness to application rate (organisms/
acre/day) for land disposal systems (Table 6).
By using estimated pathogen removal efficiencies of primary and secondary
treatment followed by disinfection, Foster and Engelbrecht (1973) computed the
number of organisms applied per acre per day at an irrigation rate of two
inches per week. Under the stated conditions, 1.6x10 viruses, 3.9x10
Salmonella, 1.2x10 mycobacteria, 9.3x10 E. h-lstolytica, and 3.9x10 helminth
ova per acre would be introduced into the soil by effluent irrigation. The
ultimate fate of these pathogens then would depend upon their survival and
movement in any given terrestrial system.
While their calculations1 included disinfection as an integral part of
the treatment train, it should be noted that operational land disposal sites
12
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13
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may use unchlorinated effluents pumped directly from holding ponds, especially
in small communities with older installations. These effluents may carry an
even larger pathogen load into a soil system.
In similar fashion, residual sludges can transport large quantities of
potentially pathogenic organisms onto a land disposal site. Assuming 1x10
enteric viruses per liter MLSS and a solids level of 0.2 to 0.4%, one can
estimate the potential transport of human enteric viruses to land disposal
sites. Even with a hypothetical 99% reduction in virus level in the anaerobic
digestion process, the use of 10 dry tons/acre/year as soil additive implies
the addition of more than 2x10 pfu/acre (or about 1x10 pfu/ft, assuming
injection or plowing to a six inch depth). The ultimate public health impor-
tance of these and other organisms in the soil environment would depend
both upon their survival rate and their potential for movement to surface^
or groundwaters.
Microbial Survival and Transport in Soil Systems
In a recent review, Gerba e_t al, (1975) , listed the factors affecting
the survival of enteric bacteria in soil as moisture content, moisture-holding
capacity, temperature, pH, sunlight, organic matter, and antagonism from soil
microflora. These parameters should be remembered when comparing microbial
survival data among various studies. Early studies by Beard (1938, 1940)
demonstrated that Salmonella ti/phi- could be recovered from loam and peat
soils for periods up to 85 days, while survival of this organism in drying
sand was only 4 to 7 days. Additionally, -5. typhi may survive as long as two
years at freezing temperatures. Mycobacteria, because of their high content
of waxy substances, can survive even dry conditions for long periods of time.
Greenberg and Kupka (1975) in a review of available literature cited survival
times ranging from 150 days to 15 months for Mycobacteria in soil.
Survival of viruses in soils are influenced by many of the same parameters
described above, although at this time little direct evidence supports viral
inactivation by antagonistic microorganisms. The effect of temperature on
the survival of poliovirus 1 (Chat) is shown in Table 7. As expected, lower
temperatures favor longer survival times. Observation of a one log.- loss of
viral titer required approximately 3 months at 4C, 1 month at 20C, and less
than one week at 30C. In like fashion, an optimal soil moisture content favors
poliovirus survival in soil, while dessication results in a more rapid loss
of virus recoverability (Table 8). Bagdasaryan (1964), working with a wide
variety of human enteroviruses including polioviruses, Coxsackieviruses, and
Echoviruses, reported survival times ranging from 110 days to 170 days at a
soil pH of 7.5 .and a soil temperature of 3 to IOC.
Removal of bacteria from liquid percolating through a soil is due to both
mechanical removal (straining or sieving at the soil surface) and adsorption
to soil particulates. Studies in Rumania (recently reviewed by Gerba et al,
1975) using coliform bacteria labeled with radioactive phosphorus demonstrated
that 92 to 97% of the bacteria were retained in the first centimeter of
soil, while 3 to 5% were detected at depths between 1 and 5 cm. The direct
relationship of coliform removal from percolating water to increasing cation
concentration and decreasing pH are consistent with classical adsorption
theory.
14
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TABLE 7. Effect of"Temperature on the Survival of Poliovirus I
in Soils at 15% Moisture Content
Days
1
3
8
10
14
21
28
42
49
80
100
134
Virus
4C
74
68
48
68
47
45
33
22
13
12
8
5
Recovered (%)
20C
99
139
44
40
53
24
12
9
5
0.7
0.4
0.2
30C
33
17
2
1
0.5
0.1
0.01
0.006
ND+
ND
ND
ND
* From Duboise et al, 1976a.
+ No virus detected in eluate.
15
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TABLE 8. Effect of Soil Moisture on the Survival of Poliovirus I at 20C*
Days
1
3
8
10
14
21
28
42
49
80
100
134
% Virus Recovered
25%
69
41
22
17
13
10
5
2
1
0.2
0.07
0.004
at Various
15%
99
138
44
40
53
24
12
9
5
0.7
0.4
0.2
Moisture Contents
Drying
74
35 (10
0.3 (6
0.08 (5
0.02 (4
NDa
0.003 (4
ND
0.002 (4
ND
ND
ND
.9)
-2)
.5)
-6)
.6)
.6)
* From Duboise et al, 1976a.
+ Sample permitted to dry. Actual moisture content shown in parenthesis.
a No virus detected in eluate.
16
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Bacterial movement through soils has been demonstrated at several field
sites. Reporting from the available literature, Gerba et al, (1975) noted
coliform movements in a variety of soils for distances ranging from 3 to 1500
feet. Release and movement of microorganisms would be expected since physical
adsorption of particulates is a reversible phenomenon and, in part, ion-dependent.
Duboise (personal communication, Samuel Monroe Duboise, The University of
Texas at Austin, 1977) has monitored the movement of a genetically-distinguishable
coliform organism through soil cores during cyclic applications of secondary
effluent followed by distilled water. The release and subsequent movement of
this organism was consistant with decreasing conductivity of the core effluents.
Changes in the ionic nature of percolate waters would be expected to have the
same effect in field situations.
In contrast to the assembled information on bacterial contamination, the
survival of enteric viruses in natural water systems is ill-defined. This
is due largely to the technical problem of monitoring their relatively low
concentration in the environment and the expense incurred in carrying out
investigations involving tissue culture and animals. Once again, epidemio-
logical studies have implicated viral movement in groundwater in a number of
infectious hepatitis outbreaks. Specifically, Clark and Chang (1959) list
five outbreaks involving 538 cases of hepatitis in which viral-contaminated
water traveled 50 to 75 feet through the soil.
A number of laboratory studies have shown that certain bacteriophages
and viruses tend to adsorb to soil particles. Carlson et al (1968) ,
reported effective adsorption of phage T2 and poliovirus to kaolinite,
montmorilionite, and illite in the presence of electrolytes. In their
system they also were able to elute free infectious virus from the clays.
In 1968, Drewry and Eliassen found that phages Tl, T2, and f2 adsorbed to
nine different soil types taken from California and Arkansas. Recently,
Schaub and Sagik (1975) and Moore
-------
conductance, was repeated through three cycles with 22.4% of the total virus
applied being recovered in the core effluents. Additionally, these authors
found the capacity of surviving virions to migrate through the soil columns
during an 84-day period (during which time the natural soil moisture was
maintained) was unchanged. Similar movement of poliovirus in 250 cm columns
packed with calcareous sand was reported by Lance et al (1976). While most
of the virus inoculum applied to the column surface in secondary effluent
was adsorbed in the top 5 cm of soil, subsequent application of deionized
water resulted in virus desorption and movement to a depth of 160 cm. In
this study, drying for 1 day between viral application and flooding with
deionized water reportedly prevented desorption (or enhanced viral inactiva-
tion).
Both laboratory and field data indicate that microbial pathogens, including
viruses, can be retained in soil systems. It is equally apparent that surviving
pathogens can be desorbed and moved through soil profiles given conducive ionic
conditions.
Data from Operational Land Application Sites
In recent years, many municipalities practicing land application of
wastewater residuals have undertaken studies of various aspects of their soil
disposal systems. A brief review of selected operations may serve as a
realistic starting point in assessing some of the public health aspects of
land disposal.
One of the largest land disposal sites for the recycling of anaerobically
digested sludges is located in Fulton County, Illinois, and operated by the
Metropolitan Sanitary District of Greater Chicago. An extensive environmental
monitoring system was developed in conjunction with the Illinois EPA to evalu-
ate long-term effects of sludge disposal on land. Biological testing included
fecal coliform and virus monitoring at selected surface water sites (Zenz et
al, 1976). The greatest increases in fecal coliform levels due to sludge dis-
posal could be seen in the minimal to maximal counts in field runoff water.
While geometric mean values differed little, the maximal fecal coliform counts
per 100 ml volumes-before and after the application of digested sludge were
2.3x10 and 1.2x10 , respectively. Other bacterial and viral data reported for
surface streams and reservoirs showed no dramatic increases at points downstream
from the disposal site when compared to upstream values. Unfortunately, insuf-
ficient information was provided in this publication to allow critical evaluation
of the sensitivity of the viral recovery methods used. Additionally, no bio-
logical parameters were reported for the extensive groundwater monitoring pro-
gram at the site.
A sludge disposal site operated by the East Bay Municipal Utility District
is located in Solano County, California (Hyde, 1976). Anaerobically digested
sludge is sprayed onto both row crop test plots and irrigated and dryland
pasture at application rates ranging from 3.3 dry tons/acre to 32.3 dry tons/
acre. In most instances, the applied sludges are then plowed into the upper
soil layer. Three parasitic helminths (Ascaris lumbricoides, Strongylo-ides
, and HymanoZepsis nancC) were found represented in the soil-sludge
18
-------
samples from the row crop plots. The high sludge application fields had the
highest percentage of positive parasite samples. Helminth ova densities ranged
from 1 to 50 per gram of soil-sludge. Significant numbers of total and fecal
coliform, fecal streptococci, Salmonella, and Shi-gella survived for as long as
seven months. Similar microbial prof iles were reported for pastureland, with dryland
pasture having the lowest percentage of positive parasite samples. Streptococcus
jecal'ls, Clostrid'Lwn tetan-i, dostr-idi-ion perfringens, and butyl-butyric Clostridia
were found in small numbers on both irrigated and dryland pasture seven months
after sludge application during the winter season. Clostri-d'iim botul-lnwn was
isolated at the same time from the dryland pasture.
Isolation of enteric viruses from the soils and sludges at sludge injection
sites located in Butte, Montana and Boulder, Colorado was reported by Moore et al
(1978) . These field studies support the thesis that if viruses reach the sludge-
soil matrix, extended survival can be expected, especially during extended low
temperature periods.
Additional reports of pathogen isolation from wasted sludge has been pub-
lished by Wellings et al (1976), in Florida. One poliovirus type 3 isolate was
recovered from 500 ml of sludge after 48 hours on a spray field. Twenty-four
isolates identified as Echovirus-7 were recovered from 250 grams of sludge after
a 13-day period on a sludge-drying bed.
In a study using anaerobically-digested sludge to recover a forest clear-
cut area in northwest Washington, Edmonds (1976) monitored the survival and
movement of indigenous coliform bacteria. Fecal coliform counts in sludge ap-
52
plied in summer decreased from 1.1x10 to 3.6x10 /gram in 204 days and were unde-
tectable after 267 days. Coliform bacteria also moved out of the sludge layer
into underlying soils. Although few bacteria moved past the first 5 cm depth,
fecal coliform were recovered from ceramic lysimeters at a depth of 180 cm.
Fecal coliform also were isolated from both a spring draining beneath the sludge
application site and a groundwater well (52 fecal coliform/100 ml), the depth of
which was not reported.
Several recent reports have considered public health aspects of land dis-
posal of treated effluents. The Flushing Meadows Wastewater Renovation Project
near Phoenix, Arizona, ha-s been operational since 1967. Research initiated in
1974 sought to ascertain the fate of fecal coliforms, fecal streptococci,
Salmonella, and enteric viruses (Gilbert et al, 1976). Both sewage effluent
applied to the infiltration basins and renovated well water taken from depths of
6 to 9 meters were screened for these microorganisms. No viruses or Salmonella
were detected in well samples; fecal coliform and fecal streptococci levels
were diminished by 99.9%. However, difficulties attributable to the viral con-
centration methodology used (filter clogging and precipitate formation during
reconcentration of eluates) were noted.
In contrast to these findings, Wellings et al (1975) repeatedly have isolat-
ed enteric viruses from groundwater. Monitoring a wastewater spray irrigation
site at St. Petersburg, Florida, this group showed virus to have moved through 5
feet of sandy soil. Following heavy rains, viruses were isolated at this site
from wells 20 to 30 feet deep. Similar isolations were made during a study of a
19
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cypress dome receiving secondary effluents. Waters from 10-foot deep monitoring
wells were shown to contain virus. Two of the three positive isolations reported
coincided with a period of heavy rainfall 28 days after the last application of
sewage effluent.
In a study using f bacteriophage as a tracer, phages were observed to
travel distances in excess of 600 feet in the groundwater at a rapid infiltration
site. Similar observations we.re made with indigenous enteroviruses (Schaub and
Sorber, 1977).
POTENTIAL USE OF LAND APPLICATION SITES
The application of wastewater residuals to food crops, specifically to fruits
and vegetables which may be eaten raw, raises the obvious question of possible
ingestion of surviving pathogenic microorganisms. No conclusive data exist to
support the uptake and subsequent translocation of pathogens from contaminated
soil into non-traumatized edible plant tissues. However, various studies have
shown survival times ranging from hours to months for microbial pathogens applied
to the surface of fruits and leafy vegetables (Dunlop, 1968). Rudolfs et al
(1951a, b, c) in a series of papers cited 7-day survivals for Salmonella and
Shigella on tomatoes, 3 days of dry weather survival for E. histolyt-laa cysts on
lettuce and tomatoes, and 35 days for immature Ascafis ova.
Bagdasaryan (1964) reported survival of enteroviruses on artificially
contaminated tomatoes and radishes over a period of two weeks under household
storage conditions. Larkin et al (1976) added poliovirus I to wasted sludge
and secondary effluent used to spray irrigate a series a test plots planted
with lettuce and radishes. Test plots were exposed to prevailing environmental
conditions including soil-surface temperatures reaching 45C and rainfall. As
expected, greater viral numbers were observed on the sludge-irrigated plants
due to retention of particulates. A two logjo loss of detectable virus during
the first 5 to 6 days was reported for the first study from August through
October, 1973. In the 1974 study, conducted from June through August, heavy
rains fell immediately after the last spray irrigation; most of the poliovirus
applied apparently was flushed from the plants and subsequently detected in
runoff at a concentration of 500 pfu/ml.
Common sense suggests that non-agricultural uses of treatment plant resi-
duals could gain wide acceptance. Enhancement of parks and forests and
reclamation of marginal and damaged soils are apparent appropriate applications.
There is some question as to whether such sites ought to be used for the growth
of human food crops eaten raw. Forage and pasture crops may not need the same
degree of caution.
20
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SECTION 5
OBJECTIVES
The overall objective of this study was to evaluate the survival and
transport of enteric viruses at land application sites for wastewater
following conventional treatment. Specific, detailed objectives were:
(1) To determine the survival of enteroviruses distributed to the
soil following wastewater irrigation.
(2) To follow the movement of indigenous enteroviruses and bac-
teriophages specific to Escheri,ch'ia. aoli, through the unsaturated
soil zone at a wastewater irrigation site, using lysimeters
placed at depths to 4.5 feet at several locations within that
s i te.
(3) To ascertain possible movement of indigenous enteroviruses and
bacteriophages to the groundwater beneath a wastewater land
application site by regular sampling of monitoring wells in-
stalled at various locations on the site.
(4) To conduct a mass balance of indigenous enteroviruses entering
the site by wastewater irrigation and remaining in the soils on
the site or leaving the site through transport in the groundwater
or by surface run-off.
(5) To determine the role of pond sediments as a potential virus
reservoir in irrigation with wastewaters.
(6) To initiate the study of the presence in wastewater of specific
bacterial pathogens at the irrigation site under study.
21
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SECTION 6
DESCRIPTION OF STUDY SITES
Several wastewater land application sites were considered for
inclusion in this study. The selection of study sites was based on the
following criteria:
1. The availability of at least 1.0 mgd of wastewater, primarily
domestic in origin, which is subjected to conventional wastewater
treatment;
2. The existence of a land application operation of sufficient
size so that research could be conducted on an uninterrupted
basis;
3. The willingness of municipal officials to cooperate in the
research project; and,
4. The proximity of the site to The University of Texas at San
Antonio to facilitate sampling and analysis.
Although several potential sites were considered, most were eliminated
as they did not satisfy at least two of the first three stated criteria.
Through field investigations and preliminary analysis, the number of
sites selected for study was reduced to two. A detailed description of
these sites follows.
KERRVILLE, TEXAS
Kerrville's wastewater (an average of approximately 1.4 mgd) is
generated from a population of approximately 15,500 and enters the
treatment plant where it is divided between two essentially separate
treatment systems (see Figure 1). The first 800,000 gpd is diverted to a
new oxidation ditch system and the remainder is divided equally between
the oxidation ditch and a trickling filter system. Each system has its
own clarifiers. From the final clarifiers all wastewater is discharged
to an irrigation pond with a storage capacity of approximately 75 acre-
ft. The oxidation ditch became completely operational in 1977. Although
completed in early 1976, initial problems (including sludge handling)
precluded its normal operation until early in 1977.
An aerial photograph presented as Figure 2 shows the location of
both the treatment plant and the land application site, including the
irrigation and tailwater ponds. The irrigation system is of the fixed
22
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. jlrrigation Pond
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FIGURE 2. Aerial View of the Kerrville Site.
24
-------
type covering an area of approximately 135 acres. Laterals (4 inches and 6
inches) are trenched to 3.0 ft. and spaced at a nominal 200 ft. along a 12-
inch irrigation main fed by the system pumps. Impact sprinkler heads (Buckner
No. 250G) are fixed on 18-inch risers above the ground and are rated at 140
gm at 80 pse. They are spaced at 100 ft. intervals staggered at adjacent lat-
erals. The sprinkler system ws designed and installed as a fully automated
system. Current operaton (1.4 mgd) results in a mean application rate of 3
inches/week at a 90% operating factor. Design flow (2.25 mgd), when reached
will result in a mean application rate of 4.8 inches/week.
Soils at this site vary in type from loams to clays over dstinctly class-
ified segments of the site (see Figure 3). Prior to the installation of the
present system (1976) approximately 50 acres were never under irrigation, while
other portions of the site have been flood irrigated since 1930. Other sections
have been flood irrigated since 1950 and some areas were irrigated between 1930
and 1960 only. Consequently, a variety of soil types and prior irrigation sit-
uations exist at one site. Coastal Bermuda grass is the present crop on the
site and it is harvested 2 to 3 times each year. Further, the site is used as
pasture for a limited number of cattle.
Site Geology
The study site is located in the Edwards Plateau physiographic province.
The plateau region is strongly dissected by stream erosion yielding a rugged
topography refered to as the Texas Hill Country.
The streams in the area have eroded headward to form narrow valleys with
steep walls of limestone and linestone marls of the Upper Glen Rose Formation.
Outcrops of carbonate strata are present but are limited to the walls of the
valley. Third Creek is incised some 160-170 feet below the interstream divides.
Valley slopes are concave with slope breaks formed by resistant limestone beds
within the Upper Glan Rose.
Third Creek is a perennial stram and is spring fed in the upper and middle
reaches. Drainage to the creek is well integrated with no observed swales or
depressions. Alluvial materials in the valley are mixtures of flood plain
terrace and alluvial fan deposits. The deep alluvial soils in the valley are
developed on these alluvial materials.
The Upper Glen Rose Formation is composed of alternating beds of
limestone, dolomite and marly limestone. In the study area the Upper
Glen Rose is approximately 350 feet -thick. Erosion of the Glen Rose and
the overlying Edwards Formation on the interstream divides has produced
the alluvial materials in the Third Creek Valley.
Soil of the Study Site
The Kerrville site is located in an area of thick alluvial soil and
thin upland soils. Soil series as mapped by the U.S. Department of
Agriculture Soil Conservation Service are shown in Figure 3 and include
the Brackett, Frio, Krumm, Orif, Denton, Doss and Lewisville Series.
25
-------
ft
10
0
CO
26
-------
LEGEND FOR FIGURE: 3
Prior
Area Irrigation*
1 None
a.
b.
c.
Type Area
Burn and cover
garbage area
Soil Classification
and Description **
HIe-5. Denton Clay, shallow,
1-5% slope
Flood Irri-
gated since
1930
Cropland
Flood Irri-
gated between
1930 and 1960
Sanitary landfill Shallow, moderate to highly
area erodible, gently sloping to
sloping clays or silty clays
Cropland 10-20 inches deep.
and
1-1. Lewisvilie Clay Loam,
0-1% slope
Deep, nearly level, dark grayish
brown and pale; brown silty clay
loam arid clay soils which take
water readily and have adequate
water and fertility storage
capacity.
Ilc-1. Lewisville Clay Loam,
0-1% si ope
Catalpa Clay Loam,
high bottom, 0-1%
slope
Catalpa fine sandy
loam, 0-1% slope
Deep nearly level soils with
moderate permeable subsoils.
Ilc-1. Lewisville Clay Loam,
0-1% slope
Catalpa Clay Loam,
high bottom, 0-1% slope
Catalpa fine sandy
loam, 0-1% slope
Deep nearly level soils with
moderately permeable subsoils.
Cropland
27
-------
LEGEND FOR FIGURE 3 (Continued)
None
Pasture
Flood Irri-
gated since
1950
Cropland
IIIe-5. Denton Clay, shallow,
1-5% slope
Shallow, moderate to highly
erodible, gently sloping to
sloping clays or silty clays
10-20 inches deep
and
IIIe-3. Krum Clay, 3-5% slope
Denton Clay, 3-5% slope
Deep, highly erodible, sloping
clay and silty clay loam soils
with moderately permeable sub-
soils.
IIc-1. Lewisville Clay Loam,
0-1% slope
Catalpa Clay Loam,
high bottom, 0-1%
slope
Catalpa fine sandy
loam, 0-1% slope
Deep nearly level soils with
moderately permeable subsoils.
* Prior to installation/operation of the present sprinkler installation
system (February, 1976)
** U.S. Department of Agriculture, Soil Conservation Service, Kerrville,
Texas
28
-------
The Orif Series is-confined to a narrow flood plain adjacent to
Third Creek. The irrigated area does not include this soil series. In
addition, the Krumm Series occupies a very small portion of the upland
area and is not included in the irrigated area.
Lewisville Series (13A1)—
The Lewisville Series is a member of the fine-silty mixed, thermic
family of Typic Calclustolls. A representative profile is given below
(1):
Ap 0-6" — Dark grayish brown (10YR 4/2) silty clay; very
dark grayish brown (10YR 3/2) moist; moderate very fine
subangular blocky and granular structure; hard, friable;
contains a few strongly cemented CaCO concretions;
calcareous; abrupt smooth boundary. (0 to 7 inches
thick)
Al 6-16" — Dark grayish brown (10YR 4/2) silty clay, very
dark grayish brown (10YR 3/2) moist; moderate fine suban-
gular blocky structure; hard, firm; few root channels;
common strongly cemented CaCO concretions about 2 to
5 mm in diameter; calcareous; gradual smooth boundary.
(7 to 15 inches thick)
B21ca 16*-34" — Grayish brown (10YR 5/2) silty clay, dark
grayish brown (10YR 4/2) moist; moderate' fine subangular
blocky structure; very hard, firm; common strongly cemented
CaCO concretions 2 to 5 mm in diameter; a few threads of
soft CaCO ; calcareous; gradual smooth boundary. (13 to
30 inches thick)
B22ca 34-62" — Pale brown (10YR 6/3) silty clay; brown (10YR
5/3) moist; weak subangular blocky structure; hard, firm;
common soft masses of segregated CaCO , few small, strongly
cemented CaCO concretions; calcareous.
The Lewisville Series is typically well drained, moderately permeable,
calcareous soils. The soils occupy level to sloping terrace deposits,
with a slope range of 0 to 10%. Soil permeability is moderate, ranging
from 0.6 - 2.0 in/hr. The soil is well drained with slow to medium
runoff.
Frio Series (26A1)—
The Frio Series is a member of the fine, mixed thermic family of
Cumulic Haplustolls. A representative profile is given below (1):
All 0-22" — Very dark grayish brown (10YR 3/2) silty clay,
very dark brown (10YR 2/2) moist; strong fine granular
structure in the upper 4 inches and moderate fine' sub-
angular blocky structure below; hard, firm, sticky,
plastic, COLE is .065; many fine tree and grass roots;
29
-------
few snail shells; 22 percent calcium carbonate equiva-
lent; few films and threads of CaCO visible in lower
part when dry; calcareous, moderately alkaline; diffuse
smooth boundary. (12 to 28 inches thick)
A12 22-40" — Very dark grayish brown (10YR 3/2) silty clay,
very dark brown (10YR 2/2) moist; moderate fine and
medium subangular blocky structure; hard, firm, sticky,
plastic; COLE is .060; few fine tree roots; 27 percent
calcium carbonate equivalent; many threads and films of
CaCO visible when dry; calcareous; moderately alkaline;
gradual smooth boundary. (10 to 30 inches thick)
Cca 40-62" -- Dark grayish brown (10YR 4/2) silty clay, very
dark grayish brown (10YR 3/2) moist; massive; hard, firm,
sticky, plastic; few thin strata of very dark grayish
brown silty clay; a few thin bedding planes in lower
part; 29 percent calcium carbonate equivalent; many films
and threads of CaCO and a few soft powdery masses of
CaCO ; calcareous; moderately alkaline.
The Frio Series consists of well-drained, level soils of the bottom
lands. The soil is formed from calcareous alluvial materials. The soil
is well-drained with slow runoff and has a moderately slow permeability,
ranging from 0.2-0.6 in/hr.
Denton Series
The Denton Series is a member of the fine, montmorillonitic, thermic
family of Vertic Calclustolls. A representative profile is given below
(1):
Ap 0-6" — Dark grayish brown (10YR 4/2) silty clay, very
dark grayish brown (10YR 3/2) moist; moderate medium and
fine granular and subangular blocky structure; hard,
firm, sticky and plastic; many roots; few fine fragments
of limestone; calcareous; moderately alkaline; clear
smooth boundary. (4 to 8 inches thick)
All 6-14" — Brown (7.SYR 4/2) silty clay, dark brown (7.SYR
3/2) moist; moderate medium and fine subangular blocky
structure; very hard, firm, sticky and plastic; many
roots; common very fine pores; few partially sealed
cracks filled with material from above; few fine fragments
of limestone; calcareous; moderately alkaline; gradual
wavy boundary. (5 to 15 inches thick)
A12 14-26" — Brown (7.SYR 4/3) silty clay, dark brown (7.SYR
3/3) moist; moderate medium and fine angular and subangular
blocky structure; very hard, firm, sticky and plastic;
many roots; common very fine pores; common shiny pressure
30
-------
faces; vertical cracks' filled with dark grayish brown
(10YR 4/2) silty clay; few fine fragments of limestone;
calcareous; moderately alkaline; gradual wavy boundary.
(7 to 16 inches thick)
Bca 26-34" — Brown (7.SYR 5/4) silty clay, brown (7.SYR 4/4)
moist; moderate medium and fine angular and subangular
blocky structure; hard, firm, sticky and plastic; few
roots; few dark streaks; common limestone fragments; few
fine weakly cemented CaCO concretions; calcareous;
moderately alkaline; abrupt irregular boundary. (0 to 19
inches thick)
Cca 34-38" — Mixture of about 80 percent flaggy limestone
fragments, some of which can be cut with a spade and 20
percent brown (7.5YR 5/4) silty clay; massive soil is
hard, firm, sticky and plastic; few roots; limestone
flags are up to 2 inches thick and 12 inches across the
long axis; common soft masses of CaCO ; calcareous;
moderately alkaline; abrupt irregular boundary. (0 to 20
inches thick)
R 38-60" — Fractured limestone that cannot be cut with a
spade, interbedded with calcareous clayey marl.
The Denton Series occurs on level to very gently sloping uplands.
The soil is formed from a mantle of marly materials over weakly cemented
to fractured limestones and marly limestones. The soil is typically
well-drained with rapid runoff. Soil permeability is slow-ranging from
0.06 - 0.2 in/hr.
Doss Series
The Doss Series is a member of the loamy, carbonatic, thermic,
shallow family of Typic Calclustolls. A representative profile is given
below (1):
Al 0-8" — Dark grayish brown (10YR 4/2) silty clay, very
dark grayish brown (10YR 3/2) moist; moderate fine and
medium subangular blocky structure; very hard, very firm,
very sticky and plastic; many fine and medium grass
roots; common fine pores; common very fine soft bodies of
CaCO ; about 3 percent weakly cemented fragments of CaCO
about 1/4-inch across the long axis; calcareous; moderately
alkaline; clear smooth boundary. (7 to 12 inches thick)
B2ca 8-19" -- Brown (10YR 5/3) silty clay, dark brown (10YR
4/3) moist; moderate fine and medium subangular blocky
structure; very hard, very firm, very sticky and plastic;
31
-------
common fine and few CaCO ; few angular fragments of
weakly cemented limestone up to 1/4-inch across the long
axis; calcareous, moderately alkaline; clear smooth
boundary. (4 to 13 inches thick)
Cca 19-48" — Very pale brown (10YR 8/4) weakly cemented
marlaceous limestone interbedded with silty clay, very
pale brown (10YR 7/4) moist; platy in the upper 3 inches
with hardness of 2.0 on Mohs scale, massive below and
hardness of about 1 on Mohs scale; many veins and bodies
of CaCO ; calcareous, moderately alkaline.
The Doss Series is typically a shallow, upland soil formed on marly
limestone and weakly cemented limestone. The soils occupy nearly level
to moderate slopes. The soils are well-drained with medium runoff.
Soil permeability is moderately slow-ranging from 0.2 - 0.6 in/hr.
Brackett Series (2EG)
The Brackett Series is a member of the loamy, carbonatic, thermic,
shallow family of Typic UstY>oc~h.Y>epts. A representative profile is given
below (1):
Al 0-6" — Light brownish gray (2.SYR 6/2) loam, grayish
brown (2.5Y 5/2) moist; moderate fine and very fine
granular and subangular blocky structure; hard, firm;
many grass roots; many worm casts of lighter colored
material from horizon below; about 3 percent fragments of
limestone mostly 5 to 15 mm in diameter; the bulk of
these are on the surface as a "pavement"; CaCO equi-
valent is about 55 percent; calcareous; moderately alka-
line; clear wavy boundary. (3 to 12 inches thick)
B2 6-16" — Pale yellow (2.5Y 8/4) loam, pale yellow (2.5Y
7/4) moist; moderate very fine subangular blocky structure;
hard, friable; many roots; about 5 percent, by volume of
subrounded weakly and strongly fragments of limestone,
mostly 2 to 15 mm in diameter; common tongues of darker
soil from layer above in old root channels or cracks; a
few soft bodies"of CaCO ; CaCO equivalent is about 65
percent; calcareous; moderately alkaline; clear boundary.
(4 to 16 inches thick)
16-50" — Thinly interbedded weakly and strongly cemented
platy limestone and pale yellow calcareous clay loam,
cleavage planes of rock structure are evident in both the
limestone and in the clay loam; few roots in the upper
part in vertical crevices and between the horizontal
plates of the limestone.
32
-------
The Brackett Series occurs on the uplands and is formed from inter-
bedded limestone and marly limestone. The soils are well-drained with
rapid runoff. Soil permeability is moderately slow, 0.2 - 0.6 in/hr.
UVALDE, TEXAS
The treatment facility operating at the Uvalde site is shown in
Figure 4. Approximately 3.0 mgd of domestic wastewater generated from a
population of approximately 9,000 is treated by preaeration, primary
settling and trickling filtration prior to discharge to a series of six
holding ponds having a maximal capacity of about 96 acre-ft. Treated
wastewater then flows by gravity to another pond adjacent to Cook's
Slough from which it can be pumped to irrigate approximately 41 acres of
cropland and 22 acres of pasture land. Two additional ponds collect some
of the irrigation run-off prior to entry to Cook's Slough.
In practice, the municipality operates the land application project
by periodically irrigating the cropland, although only part of the
wastewater is used. For study purposes, selected areas can be isolated
for controlled application. At present, the irrigation operation is not
highly structured.
33
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SECTION 7
METHODS AND MATERIALS
LYSIMETERS
Although relatively simple, the lysimeters designed for use in this
study appear to serve adequately for sampling water from the percolation
zone when saturated. They are constructed of a single piece of 3/4-inch
schedule 40 PVC pipe 11 inches long. The PVC pipe is capped at one end
and a threaded hose fitting is attached to the other end. Twelve 60-
gauge diameter holes are drilled into the lower portion of the pipe.
The pipe is mounted in a shallow polyethylene pan 7 inches wide X 10
inches long X 5 inches deep with the hose fitting extended through one
end of the pan. The pan is filled with washed gravel graded from 1/4-
inch diameter surrounding the pipe to fine washed sand at the edges and
top of the pan. An evacuation hose, 1/4-inch rigid polyethylene, is
attached to the hose fitting and brought to the surface. Details of
lysimeter construction can be found in Figure 5.
Three lysimeter locations were selected at the Kerrville site (see
Figure 3). A trench approximately 2 feet wide and 6 feet deep was dug
at each location. The lysimeters were installed at depths of 1.5, 3.0
and 4.5 feet beneath the surface. By burrowing into the side walls of
the trenches, horizontal staggering between lysimeters was maintained.
In addition, a soil moisture probe was installed adjacent to each lysi-
meter. The trenches were backfilled following installation of lysimeters
and soil moisture probes.
MONITORING WELLS
As previously discussed, Third Creek (Kerrville site) is located in
a valley formed on the Upper Glen Rose Formation. Using a characteristic
cross section of stream valleys in the Edwards Plateau region, a general-
ized geologic cross-section is given in Figure 6.
Groundwater movement in the irrigated field area should follow the
topography of the area — that is, down-slope towards Third Creek and,
once within the thick alluvial sequence, down the stream valley. Numerous
small, shallow water wells have been completed in these alluvial materials
in Kerr County. However, all major, large water wells near this site
are more than 600 feet in depth and have been completed in the Sligo and
Hosston Formations.
Well #1 was located to permit groundwater sampling in an area of
35
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Cap
0.25" Washed Gravel
0.75" OD PVC Pipe
7"
5"
Section A-A
FIGURE 5. Details of Polypropylene Pan Lysimeter.
36
-------
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37
-------
shallow bedrock. Samples taken at this location should indicate any
movement of the effluent down into the limestone bedrock and subsequent
lateral movement. Also, this location would allow the sampling of any
leachate from the up-slope solid waste disposal site. If no anomalous
water quality concentrations were encountered at this site, it would be
reasonable to assume that there were no complicating factors as a result
of leachate.
Two sampling wells (#2 and #3) were located along the western
border of the irrigation field and designed to intercept the shallow
groundwater in the alluvial materials. Well #2 was placed immediately
downslope from the irrigation pond. If there was continuous downward
percolation and lateral movement of effluent from the pond, this well
location was in an excellent position to sample the resulting groundwater
quality. Well #3 was designed to sample groundwater quality in an area
that would have effluent application at intermittent intervals rather
than continuously as at Well #2. Again, the well sampled water from
the shallow groundwater in the alluvial materials.
Well #4 was located down valley from the tailwater pond. Groundwater
quality in this location should reflect the contribution from the spray
irrigated area, downward percolation of run-off, and down valley movement
of groundwater from areas upstream from the irrigation site.
The fifth well (Well #5) , as with Well #1, sampled groundwater in
an area of relatively thin soil cover, no underlying alluvial materials
with limestone and marl bedrock. The downward percolation and lateral
movement of effluent and the resultant water quality could be monitored
without the potential complications of the solid waste leachate at Well
#1.
Special care was taken in the construction of the monitoring wells
to preclude direct contamination by irrigation wastewater (see below) .
For specific well locations, see Figure 3.
Well Installation
The monitoring wells were drilled with an 8-inch diameter bit to
total depth. Natural mud and water were used as drilling fluids in
Wells #1 and f4 to prevent contamination of the surrounding formations
by exotic drilling muds. Wells #2, #3 and tt5 were drilled with natural
fluids and a coicnercial biodegradable detergent. The detergent was used
to increase the viscosity and hence the cutting return capability of the
drilling fluid. Wells completed with the soaping agent showed no mea-
surable detergent prior to the initiation of routine sampling.
Each monitoring well was completed with a 5-foot long PVC-covered
well screen equipped with a check valve in the base. Well screen opening
diameters were 0.01-inch. Production casing was 5-inch schedule 40 PVC
pipe. In order to prevent any contamination from surface run-off moving
down the annular space, each well was completed with a PVC sheet wrap
packer set below the base of the soil zone and cemented back to the
38
-------
surface (see Figure 7). Each well was tested for packer leaks by utiliz-
ing pH measurements to detect the presence of cement in the well water.
Sampling of the monitoring wells was by submersible centrifugal pumps
mounted at the well screen. Each well was equipped with a 1/3 horsepower,
115 VAC pump rated at 12.0 gpm at a depth of 40 ft. with a discharge
pressure of 20 psi.
Well Drilling Sample Study
Samples were collected from drilling operations at each monitoring
well site at 1 to 2-foot intervals. The samples were examined in the
laboratory for mineralogical content, porosity and textural features
such as grain size, rounding, sorting and degree of cementation. A
generalized well log for each well can be found in Figure 8.
Well Development
After the observation wells were drilled and completed, each well was
tested for yield and chemical quality. Difficulties were encountered
with Wells #2 and #5. Both wells had a very low yield and water from
Well #5 had an abnormally high pH (pH > 12). The high pH was indicative
of a cement leak past the packer. In order to improve the yield and to
remove any cement from the well annulus below the packer, the well was
acidized with 150 gallons of 30% hydrochloric acid (HC1). After one
hour, the well was flushed with fresh water, bailed and pumped. After
development, the pH in the well dropped to a normal range of groundwater
in this region and the yield was substantially improved.
Well #2 was acidized solely to improve the yield, utilizing 150
gallons of 30% HC1. The yield of the well failed to improve. It was then
surged and air shocked using both high pressure water and air. Unfor-
tunately, despite this extensive development, the yield of Well #2 re-
mained minimal.
Direction of Groundwater Flow
The elevation of each monitoring well was determined. Water levels
in each monitoring well were measured over a sufficient period of time
to determine the configuration of the piezometric surface at the Kerrville
site. The relationship of groundwater flow direction and the movement
of various wastewater contaminants in the subsurface can be determined
through the construction of groundwater contour maps.
SURFACE WATER MEASUREMENTS
Precipitation and Irrigation
Initially, gauges to measure rainfall and irrigation were constructed
of modified Buchner funnels (9 cm diameter) affixed to either one or 5
liter plastic bottles. These gauges were mounted 1.5 ft (irrigation
39
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SOIL ZONE
Annular seal. Cement
Packer. PVC sheet wrap
•Casino. 5" PVC
Screen. PVC coated brass
#10 slots
FIGURE 7. Monitoring Well Completion Design
Wells #1,5
0
Wells #2, 3, 4
0
35'
> p-
1 0-
<>• 0
O'o.o
P.O
O'.o
I '0'
Frio silty clay
loam with occ.
calcareous nodules
Silty gravel. l/4"-3"
Chert and limestone.
Polished and rounded.
Fair to poor sorting.
Water saturated
Shale. Blue-gray
impermeable. Glen
Rose fm
65-
Den'ton clay
calcareous clay
with silt
Marl, clayey,
yellow. Glen
— Rose fm
Marl, clayey,
yellow, inter-
bedded thin
limestone. Glen
Rose fm.
Shale. Blue-gray.
Impermeable. Glen
Rose fm
FIGURE 8. Generalized Well Log for Monitoring Wells at Kerrville Site
40
-------
sampling) to five feet (rainfall sampling) above the field surface.
Volumes in the collection vessels were recorded at regular intervals
(generally one week) to allow cumulative measurement of precipitation
over a period of time. The gauges worked very effectively for the mea-
surement of irrigation but proved inadequate for rainfall. Evaporative
loss from the one liter bottles and damage caused by roosting birds
necessitated an alternate approach.
A remote recording rain gauge (Weather Measure Corp. Model 5501-1)
was installed on the site late in the study. The gauge has a 20 cm
diameter orifice with a tipping bucket mechanism calibrated to tip after
each 0.01 inch of rainfall. The rain gauge was coupled electronically
to a self-contained, battery-operated event recorder (Weather Measure
Corp. P520) equipped with a seven-day chart recorder. The rain gauge
was secured to a concrete pad within a fenced enclosure, and the recorder
was installed inside an existing building approximately 45 feet away.
Pond Volumes
The ponds were surveyed and measured for depth utilizing standard
surveying techniques. The perimeter of each pond was measured with
steel tape and compass at a known water level. Approximate pond bottom
contours were measured utilizing a rigid depth staff from a boat. Depth
measurement locations were recorded by triangulation from fixed points
on shore.
Pond volumes were calculated from these measurements and pond level
readings regularly taken from calibrated staves fixed on piers in both
ponds.
Creek Flow
Flow in Third Creek at the Kerrville site was established using
90 V-notch weirs and level recorders. Two 90 V-Notch weirs were
installed on Third Creek as indicated on Figure 3. Continuous level
recorders were installed in still-wells behind each of the weirs to
record fluctuations in the flow of the creek.
The upstream weir was installed in a concrete double-piped culvert.
A circular plywood plug was placed in one of the concrete pipes and
caulked in place. The other pipe was fitted with a notched circular
piece of plywood onto which was mounted the 90 V-notch weir. A staff
was fixed to the solid plywood plug. The still-well and recorder were
mounted on a sandbag supported wooden platform.
The downstream recording device consisted of a 4 ft x 8 ft plywood
dam onto which was mounted the 90 V-notch weir. The plywood dam was
placed in a trench dug across the stream bed and then sandbagged in
place providing structural support for the dam. The still-well and
recorder were placed about 8 feet upstream from the weir.
41
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SAMPLING
Water
Sampling at the Kerrville and Uvalde plant sites was planned ini-
tially to produce results that would be average in nature and would not
represent extremes. This is particularly important when dealing with
wastewater treatment facilities which commonly are subjected to extreme
loading variations. Thus, a combination of flow composite, time composite
and grab sampling techniques were employed. Table 9 provides a summary
of the sampling methods employed.
Flow Composite Samples—
Twenty-four hour flow composite samples were constructed by col-
lecting fixed volumes at 4-hour intervals and recording total flow for
the sum of the 2 hours before and the 2 hours after the sampling time.
At the end of the 24-hour period, the individual samples were composited
into a larger sample as a direct proportion to the flow which occurred
during the sampling period. For example, samples could be collected at
12, 4, 8pm and 12, 4 and Sam. Total flow was determined for the 24-hour
period as well as for the periods 10am - 2pm, 2-6pm, 6-10pm and so
forth. A fixed volume of composite sample (say 10 liters) was made up
from the respective individual samples as a percentage of the total flow
which occurred during the sampling period (i.e., if 20% of the total
daily flow occurred between 10am and 2pm, the 10-liter composite sample
would contain 2.0 liters of sample collected at 12pm). Experience has
shown that this method provides a representative daily composite sample.
Time Composite Samples—
" For those sampling points which were expected to show dampened
pollutant variation and minimal flow variation throughout a 24-hour
period, a time composite sample was used. This procedure usually in-
volved collecting samples at fixed time intervals (say every 8 hours).
When all samples were collected, they were formed into a composite
sample by taking equal volumes.
Grab Samples—
Collection of a single grab sample was used when there was little
chance of significant flow or pollutant characteristic changes throughout
any 24-hour period.
Soils
Soils were collected from six sampling locations at the Kerrville
site and from four sampling locations at the Uvalde site. Soil to a
depth of approximately three centimeters from the surface was collected
and placed in appropriate containers, depending on the analysis to be
performed.
42
-------
TABLE 9. Sampling Points and Sampling Method
Sampling Point Identification
Sampling Method
24-Hour Composite
Flow Time Grab
Kerrville
Influent (Raw Wastewater)
Oxidation Ditch Secondary
Clarifier Effluent
Trickling Filter Secondary
Clarifier Effluent
Irrigation Pond
Tailwater Pond
Third Creek, Upstream of Site
Third Creek, Downstream of Site
Lysimeters
Monitoring Wells
Soils
Uvalde
Influent (Raw Wastewater)
Trickling Filter Secondary
Clarifier Effluent
Ponds #1 and #2 Effluent
Ponds #3 - #6 Effluent
Cook's Slough, Upstream of Site
Cook's Slough, Downstream of Site
Soils
X
X
X
X
X
X
X
X
X
X
X
X
X
43
-------
Sediments
Pond sediments were collected with a coring device which was driven
manually into the sediment layer and capped on the upper end. The
sample was raised to the pond surface and transferred as a slurry to
sterile sample bottles. Sediments were held in wet ice during transport
to the laboratory.
WASTEWATER ANALYSES
Virus Concentration Procedures
Laboratory Method—
Volumes of up to 10 liters were handled readily in the laboratory.
Wastewater was placed in a vessel of convenient size and 100 mg/1 of
expanded bentonite was added, along with sufficient CaCl to bring the
wastewater to approximately 0.01M CaCl . The pH was adjusted to 6.0
with HC1 and mixed for 30 minutes. After mixing, the virus-solids-
bentonite complex was sedimented by low speed centrifugation and 10 to
60 ml-of tryptose phosphate broth (TPO ) added to the pellet for elution.
Elution was accomplished by vortexing £he TPO solids-virus suspension
for 5 minutes. The suspension was separated by centrifugation (3000 xg)
and the TPO containing the eluted viruses was assayed (see below).
The range of viral recovery for more than 55 separate field samples
using this methodology was from 21 to 100%, and the mean was 53%. These
were separate daily samples over a consecutive 49-day period. In a
recent experiment, the recovery variability for one series of samples of
oxidation ditch and trickling filter effluents ranged from 91% to 97%,
well within expected variability. In two other studies (not Kerrville)
identical samples processed in our Austin laboratory (now closed) and in
our San Antonio laboratory resulted in similar corrected data, despite a
considerable difference in recovery efficiency as described below (see Table
10).
TABLE 10. Comparison of Bentonite Concentration Technique
Recovery Efficiency
Sample ttl Sample #2
Austin San Antonio Mean Austin San Antonio Mean
Corrected Virus 35
(3d), pfu/1
Corrected Virus 51
(5d), pfu/1
46
51
41
51
263
345
243
307
253
326
Concentration
Efficiency, %
74
31
100
31
44
-------
These results emphasize the importance of an efficiency of recovery
control with seeded virus being a part of every assay, every day. The
control seed of poliovirus is mixed into the water sample to be tested
at approximately 10 pfu/ml, a dilution from stocks of at least 1:1000.
At these viral input levels aggregation is not considered a problem.
(Or, rather, verification of monodispersed titers is not justified.) It
is likely that if a particular ionic milieu tends towards viral clumping,
it will do so for both naturally - occurring and seeded virions.
After a 15-minute mixing period which allows dispersion of the
poliovirus seed, the control sample is concentrated as described above.
The total virus recovered from the eluate compared to the known amount
of virus in the original sample allows calculation of the recovery
efficiency.
Differences in reported recovery efficiencies between laboratories
may be attributed to any of several variables including handling of the
bentonite (expanded vs. nonexpanded clay) , age and viral sensitivity of
monolayer cultures employed for assay, and technician variability.
Field Batch System --
Prototype 1
In order to adapt .the bentonite concentration procedure to a
flow-through system, diatomaceous earth (Celite*%35 or 512) was added to
retain the clay used as a viral adsorbent. The batch filtration system
was prepared by placing a 15 cm diameter Buchner funnel on a five-gallon
carboy. A #4 Whatman filter was placed on the funnel. Diatomaceous
earth was employed.at the precoat rate of 0.25 Ib/ft and body feed at
rates up to 450 mg/1. Adequacy of filtration was determined by the
observation of breakthrough (turbidity) of bentonite in the filtrate.
After filtration of the test water, the filter was lifted from the
funnel and the filter mat scraped into a 250 ml plastic bottle. A
sufficient volume of tryptose phosphate broth was added to the bottle to
completely saturate the diatomaceous earth-bentonite-solids mass. The
entire mixture was shaken vigorously for 5 minutes and packed in wet ice
for shipment to-the laboratory for assay. Separation of solids prior to
assay was by low-speed centrifugation as described above.
Prototype 2
Subsequent efforts to increase the recovery of indigenous
viruses from surface waters led to the modification of the first field
batch system.- Not only were flow rates limited by the presence of
bentonite clay, but viral recoveries also were decreased due to in-
creased amounts of diatomaceous earth used to maintain flux in the
system.
In order to remove bentonite from the batch procedure, an alternate
viral adsorbent was required. Developmental work on another study (EPA
45
-------
Grant R-804474-02-0) had indicated the validity of viral concentration
by retention of viruses on diatomaceous earth in the presence of divalent
cation. Using the physical system described above, Celite 512 was
applied as a precoat to a #4 Whatman filter at a rate of 0.03 gm/cm .
Routinely, 20 liters of surface water (specifically pond water with high
algal content) were concentrated through this precoat with the addition
of body feed at a rate of 600 mg/1. Viruses were recovered from the
diatomaceous earth-solids complex by elution in tryptose phosphate broth
as described.
Standard Methods
Virus Concentrator—
A portable virus concentrator developed by personnel of the USEPA
Environmental Research Center, Cincinnati, was constructed. The viral
adsorbent system utilized the Balston Grade C, 8 ym porosity filter
cartridges. Viral concentration procedures outlined in Standard Methods
fcr the Examination of Water and Wastewater, 14th edition (1976) , were
employed.
Using this field concentrator, minimally 100 gallon samples were
processed routinely from wells having adequate flow. The relatively
high particulate content of certain surface waters, especially ponds,
limited volumes processed from these sources to 20 to 100 gallons.
Virus Assay
Initially, virus-containing concentrates had been assayed on HeLa
cell monolayers for plaque-forming ability. Because poliovirus plaques
increased in size rapidly in this system, it was difficult to determine the
presence of other enteric viruses. Therefore, samples were divided into
two portions, with two-thirds of the concentrate assayed for plaque
enumeration after 3 days' incubation. The remaining one-third of the
concentrate was mixed with pooled commercial poliovirus antisera (types
1, 2, 3) so as to make the concentrate 2% antisera. (This level of
antiserum was sufficient to result in the neutralization of 95-99% of
laboratory seed poliovirus.) After incubation at 37C.for 30 minutes,
the entire sample was plated onto HeLa monolayers, overlayered and
incubated for 5 days before staining.
Final reported titers were calculated as given in the following
example:
46
-------
2.0 1 Wastewater
(a)
10 ml Concentrate
(b)
7 ml concentrate plated onto 7 plates,
incubated 3 days, stained; 21 plaques
counted in all
3 ml concentrate incubated with antisera
and plated onto 3 plates, incubated 5
days, stained; 9 plaques counted in all
I
I Calculations
7 ml /10 ml = 21 pfu/x pfu 3 ml/10 ml = 9 pfu/x pfu
7x = 210 3x = 90
x = 30 pfu/10 ml concentrate (3-day) x = 30 pfu/10 ml concentrate
(15 pfu/1 wastewater - 3-day)
60 pfu/10 ml concentrate (5-day)
or 60 pfu/210 1 wastewater (5-day)
(30 pfu/liter wastewater - 5-day)
Beginning in January, 1978, viral assays were performed by the
plaque assay method utilizing HeLa and/or BGM monolayers on 100 mm
plates. Samples were evenly divided between the two cell lines when
both HeLa and BGM were used for viral detection. The growth medium was
aspirated from the plates and the inoculum added, generally 1.0 ml per
plate. Infected plates were rocked continuously on a Bellco Glass, Inc.
rocker platform at room temperature for one hour. The plates were then
washed with Hanks' Balanced Salt Solution containing penicillin G (500
units/ml) and streptomycin (250 yg/ml) for 20 minutes. The agar-based
overlay media consisted of Eagle's Minimal Essential Media without
phenol red containing 8% calf serum, 100 units of penicillin G per ml,
50 yg of streptomycin per mi, 25 yg of GentamicirS- per ml, and 0.5
yg of Fungizone^ per ml. Three days post-inoculation a second agar
overlay containing 30 yg/ml neutral red was applied to plates. The
plates then were read each succeeding day and scored for plaques through
5 days. Care was taken not to expose the plates to light for extended
periods of time.
BSC-1 tube cultures also were used during portions of the study in
an attempt to recover viruses from well concentrates. These cells were
grown in 16x25 mm glass tubes. Each tube was inoculated with 0.1
ml of suspect viral concentrate and subsequently monitored for a period
of 21 days. The maintenance medium was changed approximately every
fourth day.
All plates and tubes were incubated at 37C in a humidified atmosphere
of 5% CO in-air.
47
-------
Viral Confirmation, Identification and Characterization
Possible viral isolates were picked from areas exhibiting cytopathic
effect based on microscopic examination of the stained monolayer. The
removal of plaque-like areas was accomplished by first removing the
second overlay above the area of CPE. The entire plaque then was aseptically
collected using a spatula. The sample was placed in 0.5 ml of Medium
199 containing 200 units of penicillin G per ml and 100 yg of streptomycin
per ml and held at -76C to -80C until confirmation.
Confirmation of potential viral isolates was performed in homologous
tube culture systems. Culture tubes were grown out to 50-75% confluence
and inoculated with 0.1 ml of sample. After 48 hours, tubes were observed
daily for evidence of CPE. At such time as CPE was observed, the sample
was removed and frozen at -76C until the second tube culture passage.
After seven days, all samples not showing CPE were harvested and passaged
blind. Those isolates that demonstrated CPE after a second passage were
reported as positive viruses.
Isolates were identified using the Lim-Benyesh-Melnick enterovirus
typing pools. In those cases where viral breakthrough was observed at
five days, identifications were confirmed using neutralization testing
with monospecific antisera.
Because of the large number of viral isolates identified as polio-
virus 1, further characterization of selected isolates were done using
the reproductive capacity temperature (ret) marker. The limited-thermal
exposure test presented by Carp and Koprowski (1962) was followed. Polio-
virus 1 field isolates were dilution plated on HeLa monolayers as pre-
viously described. One half of the plates at each dilution were incubated
at 37C ± 0.5C in a humidified atmosphere of 5% CO . The remaining
plates were placed in a 40.1C ± 0.2C humidified CO incubator for 18
hours before transfer to the 37C incubator. Laboratory stocks of poliovirus
1 (Chat) and poliovirus 1 (Mahoney) were run as standard controls.
An r value for both laboratory attenuated and wild type virus strains
and field isolates was calculated where:
= total number of plaques observed at 40C
total number of plaques observed at 37C
Bacteriophage Assay
Samples to be assayed for coliphage content were plated using
Escherichia coli K 13 as the host of choice. This organism was found to
be the most sensitive in terms of total numbers of indigenous bacterial
viruses detected in a variety of wastewater samples. Other coliform
strains evaluated were E. coli B, E. coli F , and E. coli K 12.
— 100/CoOO
Appropriate sample volumes (0.1 - 1.0 ml) and 0.5 ml of an 18-hour
broth culture of E. coli K 13 were added to 3.5 ml of liquified 1%
48
-------
tryptose phosphate agar at 45C ± 1.5C. This inoculated volume was
poured over a pre-formed tryptose phosphate 1.5% agar basal layer in a
100 mm petri dish. When firm, the plates were inverted and incubated at
35C for 8-18 hours, depending upon sample source. Soil and sediment
analyses for coliphage were counted after 8 hours to avoid overgrowth of
indigenous bacteria. Plaques were observed on an illuminated colony
counter under 5X magnification.
Total and Fecal Coliform Assay
For coliform densities where samples greater than 0.1 ml were
required, membrane filtration procedures using Gelman GN-6 filters as
described in Standard Methods for the Examination of Water and Wastewater
(1976), were utilized. However, for samples with high solids content
where dilutions were required, a spread plating technique in either m-
Endo agar LES (total coliforms) or m-FC agar (fecal coliforms) was
utilized.
Results presented in Table 11 show representative results from
field samples collected at the Kerrville site. Spread plating appeared
to be comparable to membrane filtration for the recovery of total coli-
forms. Only slightly improved fecal coliform recoveries were observed
using spread plating, perhaps due to a detrimental effect of filtration
through the membrane matrix on these organisms.
Fecal Streptococci Assay
During the last year of the field study, fecal streptococci were
added as a routine microbiological indicator of fecal pollution at the
Kerrville irrigation site. A basis for the calculation and use of a
fecal coliform to fecal streptococci ratio was set forth by Geldreich
and Kenner (1969) and Feachem (1975). Subsequently, several investigators
have used this measurement (or ratio) in evaluating environmental fecal
pollution (DeMichele et al, 1974; Sayler et al, 1975; Davenport et al,
1975). As the field site at Kerrville was used as pasture for cattle
grazing, the use of FC:FS ratios was necessary to differentiate pollution
attributable to domestic wastewater irrigation from that due to animal
wastes.
KF Streptococcus agar, prescribed by Standard Methods for the
enumeration of fecal streptococci by plating procedures, and m-Entero-
coccus agar, suggested by M. Neal Guentzel, The University of Texas at
San Antonio, were compared using identical field samples. Appropriate
dilutions of water samples from three different sources were spread
plated in triplicate. After incubation at 35C for 48 hours, plate
counts were taken, and selected random colonies were inoculated onto
bile esculin slants. (Bile esculin is a differential biochemical that
confirms streptococci as being of fecal origin.) Table 12 shows the
results of these efforts.
M-Enterococcus agar yielded superior plate counts and a higher
percentage of confirmed fecal streptococci from all water sources.
49
-------
Therefore, this medium was chosen for routine enumeration of fecal
streptococci. As described above, for organism densities requiring
samples greater.than 0.1 ml, membrane filtration procedures were em-
ployed. However, for samples with fecal streptococci densities requiring
less than 0.1 ml, spread plating was used as the plating method.
TABLE 11. . Comparison of Membrane Filtration and Spread Plating Techniques
Sample
1
2
3
4
5
Total Coliform
Membrane
Filtration
1.7x10
1.9xl06
B.OxlO5
5.7x10
3.6xl05
(cfu/100 ml)
Spread
Plating
6
1.4x10
2.0xl06
8.9x10
S.lxlO5
3.8x10
Fecal Coliform
Membrane
Filtration
2.0x10
2.1x10
7.0xl04
(cfu/100 ml)
Spread
Plating
2.5x10
2.7x10
9.5xl04
TABLE 12. Comparison of Fecal Streptococci Isolations on Selective Media
Fecal Streptococci # colonies tested % confirmed
Media & Sample counts (cfu/100 ml) on bile esculin fecal streptococci
KF Streptococcus
Agar a
tail water 3.0x10
pond ^
lysimeter 2.4x10'
water .
well water 8.0x10
m-Enterococcus
Agar ^
tail water 8.3x10
pond f
lysimeter- 1.8x10
water
well water 2.6x10'
8
42
9
20
37
28
100
95
88
100
95
100
50
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Bacteriological Screen
During the course of this study, a bacterial screen for overt as
well as opportunistic human pathogens was conducted on wastewater samples
at both Kerrville and Uvalde. In addition to indicator organisms,
screening procedures were run to optimize recovery of a variety of gram-
negative enteric bacteria.
Total Plate Count--
Serial dilutions of the sample were prepared in sterile phosphate
buffered saline. A soft-agar overlay (tryptose-phosphate with 0.3%
Agar) and 1 ml of the appropriate dilution of sample were pour-plated
over five plates of plate count agar. Incubation was at 35C for incuba-
tion period of 24-48 hours. Only plates showing 30-300 colonies were
counted.
Mycobacteria—
Samples were treated for 20-35 minutes with 500 ppm benzalkonium
chloride (Zephiran), diluted and plated to the surface of Middlebrook's
7H10 agar plates. Plates were incubated at 37C in a 5% CO^ atmosphere
and examined over a period of one month for the appearance of typical
Mycobacteria colonies. Suspect colonies were identified by examination
of stained (Ziehl-Neelson) smears for acid-fast bacilli.
Staphylococci—
Appropriate dilutions of the wastewater sample were spread plated
onto Mannitol Salt Agar. Typical staphylococcal colonies showing a
yellow zone of mannitol fermentation were isolated for confirmation of
identity by observation of gram-positive cocci.
Fluorescent Pseudomonads—
Appropriate dilutions of sample were plated onto Pseudomonas Agar F
(Difco) and Cetrimide Agar (Difco). Following incubation at 35C for 24
hours and subsequent exposure to fluorescent light for 24 hours, the
number of fluorescent pseudomonads was determined by counting the number
of fluorescing colonies observed under long-wave ultraviolet light in
the dark.
Enterobacteriaceae--
Salmonella species: Enrichment for Salmonellae was done by inoculating
Selenite and Tetrathionate broths with various sample volumes (10-
25 ml). The broths were incubated at 35C for 24 hours. After enrichment,
aliquots were streaked for isolated colonies onto XLD, Hektoen, and SS
Agars. Identification of suspect colonies was accomplished by use of
the Enterotube®, a biochemical and computer-coded identification system
for Enterobacteviaeeae.
Shigella species: Enrichment for Shigallae was done by inoculating
GN broth (BBL) with various volumes of sample (10-25 ml). The GN Broth
was incubated at 35C for 24 hours. After enrichment, aliquots were
plated for isolated colonies onto XLD agar. Identification of suspect
51
-------
colonies was accomplished by use of the Enterotube®, a biochemical and
computer-coded identification system for Enterobacteriaceae .
Yersinia enterocolitioa: isolation of Yers-inia was attempted by
selective enrichment by inoculation of sample into isotonic saline containing
25 mg/1 of potassium tellurite. Enrichments were incubated at SC for 7 days.
Aliquots from enrichment media were streaked onto SS agar and incubated at
room temperature (25C) for 2 days. Colonies were identified using the
©
Enterotube system.
Other Enterobacteriaceae (i.e., Escherichia, Citrobaeter, Klebsiella,,
Enterobacter, Serratia, Proteus, and Providenai-a) •. These organisms were
enumerated by isolating and subculturing colonies from samples dilution
plated to moderately selective (XLD & Hektoen) and highly selective
(Bismuth Sulfite & SS) enteric plating media. Two to three representative
of every colony type observed were subcultured and subsequently identified
using the Enterotube® system.
Other Gram-Negative Enteric Bacteria (Aeromonas,
Plesiomonas 3 and Pseudomonas sp.): Colonies isolated from
the non-selective enteric plating media (i.e., EMB & MacConkeys agars)
were identified using the Oxi/Ferm© tube, a standardized system speci-
ficially designed for the identification of oxidative-fermentative gram-
negative rods. This identification scheme was utilized only at the
Kerrville site.
Chemical and Physical Analyses
All samples for wastewater analyses were stored at 4C and handled
in accordance with guidelines shown in Table 13. Chemical analyses were
performed within time degradation limits established by studies per-
formed on selected samples from the Kerrville and Uvalde sites. Addi-
tionally, recovery tests utilizing various sample dilutions and additions
of known standards to samples were conducted routinely to eliminate
erroneous data due to interferences.
Analyses of field samples for five-day biological oxygen demand
(BOD ) , pH, turbidity, specific conductance, total suspended solids
(TSS) and volatile suspended solids (VSS.) were conducted on unpreserved
samples in accordance with the Environmental Protection Agency's Methods
for Chemical Analyses of Water and Wastes (1974) and Standard Methods
for the Examination of Water and Wastewater (1976) . Samples for total
organic carbon (TOC) analysis were acidified with either hydrochloric or
sulfuric acid and subsequently measured using methodology specified by
the EPA.
Samples for other chemical analyses were preserved by the addition
of 40 mg/1 of mercuric chloride. These tests included manual procedures
for total and "reactive" (soluble) phosphorus and automated procedures
for determination of nitrite nitrogen, ammonia nitrogen, and total
Kjeldahl nitrogen. Nutrient analyses were performed utilizing procedures
52
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TABLE 13. Holding Procedures for Routine Chemical/Physical Analysis
Analysis Remarks
pH No hold time, field measurement
turbidity Measure within 4 days, hold @ 4C
conductance Relatively stable, read at room temperature
solids Measure within 4 days, hold @ 4C
BOD 5 Hold @ 4C, set up within 24 hours
TOG Hold @ 4C for 2 weeks with acidification
Nitrogen Hold time of 2 weeks with preservation
(HgCl ) @ 4C
TKN
NH3
NO3/NO2
Phosphates Hold time of 2 weeks with preservation
(HgCl ) @ 4C
Total P
Soluble P
reportedly Fruh e_L a_L (1975) a'dapted from EPA and Technicon Auto-
Analyzer methodologies.
Additionally, samples of groundwater collected from each of the
monitoring wells at the Kerrville site were analyzed for Pb, Cd, Fe, Cr,
Cu, Mn, Ni, Na, K, Ca, Mg, and Zn. Atomic absorption analysis was
performed using a Perkin-Elmer Model 460 dual-beam, background corrector-
equipped atomic absorption spectrophotometer.
Groundwater samples collected in the field were fortified immediately
with concentrated nitric acid to reduce analytic loss due to adsorption
on the collection container walls. Laboratory preparation included
additional acidification and dilution or concentration depending on
concentrations of metals present. Samples analyzed for Na and K were
treated additionally to control ionization interferences.
53
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SOIL/SEDIMENT ANALYSES
Bacteriophage Assay
Approximately 50 gm of each soil sample were placed in a 250 ml
centrifuge bottle with 100 ml tryptose phosphate broth and mixed in a
controlled environment incubator-shaker at 300 rpm for 30 minutes. The
samples then were centrifuged at 1500 xg for 5 minutes. A supernatant
volume was removed for bacteriophage assay as described above. The
pellet was collected in deionized water and dried at 103C. The dried
soil weight was used for reporting the number of viruses recovered per
gram of soil. Alternately, soil moisture values were used to calculate
phage recoveries.
Enteric Virus Recovery
Initial attempts to recover animal viruses from soil used the proto-
col described above for bacteriophage work. The procedure was modified
in that samples were centrifuged at 10,000 xg for 30 minutes to pack soil
solids. During subsequent field studies, it became obvious that larger
amounts of soil should be sampled in an effort to detect enteric viruses.
Therefore, an alternate elution and reconcentration procedure was developed.
Viral elution from Kerrville soils was optimized to allow reconcentration
of soil eluates. Poliovirus 1 (Chat) suspended in secondary effluent was
introduced into replicate 50 gm soil samples contained in 7 cm Buchner
funnel "bins". After the bins had drained, each filtrate volume was
assayed for viral infectivity to allow calculation of the viral retention
by the soil samples. Each soil sample was then subjected to a combination
of elution-reconcentration schemes.
Two general classes of eluting media were compared: high pH glycine
buffer and high soluble organic medium as typified by beef extract and
tryptose phosphate broth. Reconcentration procedures were dictated by
the type of elution medium employed. Glycine buffer eluates were con-
centrated by adsorption on Cox filters followed by reelution of viruses
as outlined in the reconcentration procedure for microporous filter
concentration of enteric viruses (Standard Methods, 14th edition).
Organic flocculation was done as described by Katzenelson, et al (1976).
Hydroextraction of samples was accomplished according to the method of
Wellings, et al (1975) .
Results from this experiment (Table 14) indicated that elution with
3% beef extract, pH 9, followed by organic flocculation resulted in the
best overall recovery of poliovirus from this soil. Therefore, this
technique was utilized in attempts to recover viruses from 500 gm soil
samples.
During soil sampling, ten 50 gm samples were processed. Each sample
was homogenized in a Waring blender with 150 ml of 3% beef extract
for 3 minutes. Samples then were centrifuged at 1500 xg for 10 minutes
54
-------
TABLE 14. Poliovirus Recovery from Kerrville Soil.
Elution -Reconcentration Virus „ Virus Total «
System Eluted (%) Reconcentrated (%) Recovery (%)
1. Distilled HO
Bentonite 23 100 23
2. 0.05 M Glycine, pH 11.5 -
Cox filter adsorption 65 58 38
3. 0.25 M Glycine, pH 11.5 -
Cox filter adsorption 61 63 38
4. 3% beef extract, pH 9 -
organic flocculation 73 70 51
5. Tryptose phosphate broth -
PEG hydroextraction 56 48 27
C6
Soil + eluate homogenized in a blender for 1 minute, followed
by low speed centrifugation to remove solids.
o
% of pfu retained soil bin.
v
% of pfu recovered in soil eluate.
and supernatants pooled. The eluate volume was concentrated by organic
flocculation. Final volumes for assay ranged from 50 - 80 mis.
Sediment samples were treated as soil samples. Sediments, taken as
a slurry, were homogenized with equal volumes of 3% beef extract for
three minutes. Samples were centrifuged by 1500 xg for 10 minutes. The
supernatant was reconcentrated by organic flocculation as described by
Katzenelson (1976). Final volumes ranged from 30-50 mis.
Total and Fecal Coliform Assay
The methodology initially used to assay for coliform levels in
soils depended upon elution of these organisms from the soil sample. In
addition to highly variable elution, low-speed centrifugation (necessary
to remove particulates prior to membrane filtration) introduced yet
another variable. Total bacterial counts based on direct plating of
soil slurries and post-centrifugation counts based on plating of soil
eluates attest to this variability (see Table 15). -For this reason,
55
-------
standard multiple tube fermentation was used as a more reproducible
method to enumerate coliform organisms in soil and sediment samples.
An aqueous suspension of either soil or sediment was mixed in a
laboratory shaker at 300 rpm for 30 minutes. Appropriate dilutions
then were inoculated into the presumptive MPN test medium (Lauryl Tryptose
Broth) using the procedures described in Standard Methods (1976). Sub-
sequently, the samples were transferred into confirmation test media,
Brilliant Green Bile, 2% and EC Medium, for total and fecal coliform
MPN determinations, respectively.
Reported values were calculated from MPN tables and dry weight
determinations of soil or sediment taken at 103C.
TABLE 15. Enumeration of Total Aerobic Bacteria from Soils
Soil
Area
Sample
Bacterial Colonies/gin of Soil
Direct Plate Eluted % Eluted
1 A
1 B
2 A
2 A
3 A
3 B
8
2
2
1
2
1
.4
.4
.2
.5
.0
.0
X
X
X
X
X
X
10
10
10
10
10
10
6
6
7
7
7
7
1
1
8
2
1
1
.8
.9
.6
.2
.9
.8
X
X
X
X
X
X
10
10
10
10
10
10
6
5
6
6
5
5
21
8
39
15
1
2
Fecal Streptococci Assay
The multiple-tube technique for the enumeration of fecal streptococci
as described in Standard Methods (1976) was used during this study.
Verification of the confirmed test procedure was done by transferring a
loopful of growth from positive EVA tubes to Bile Esculin slants.
Results of this testing from three separate soil samples are presented
in Table 16. Based on these observations, the MPN procedure as used
probably results in somewhat lower numbers. However, for the routine
detection of fecal streptococci in samples with a high solids content
and low indicator organism density, the multiple-tube technique was the
most consistent procedure available.
Samples were handled as aqueous suspensions (as described for
coliform indicator organisms).
56
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TABLE 16. Enumeration of Fecal Streptococci in Soil Samples
Fecal Streptococci (MPN/100 ml)
Soil Sample EVA broth Bile Esculin
1
2
3
1
1
3
.7
.6
.5
x 10
x 10
x 10
5
6
3
2
> 2
7
.2
.4
.0
X 10
x 10
x 10
5
6
4
Chemical/Physical Analyses
Sieve Analysis—
Fractionation of soils was by standard, mechanical sieve analysis
employing sieve's sized from 0.05 to 1.4 mm. From these data, soil
aggregate distribution curves were prepared.
Moisture Content—
Moisture content was determined in the laboratory by oven drying to
103C. Soil moisture cells (Soil Test, Inc.) manufactured as two metal
plates separated by a fiberglass binding were installed adjacent to each
of the lysimeters. The design of these soil moisture cells provided a
coupling so that resistance increased as ohms varied with soil moisture
content. The soil moisture cells must be calibrated for each soil on
the basis of a curve relating soil moisture to resistance.
Chemical Analysis—
Preparatory to analyses- for cation exchange capacity (CEC) and
organic carbon content, soils were dried overnight at 103C and passed
through a 16 mesh (1 mm) sieve.
CEC determinations were performed using the method outlined by
Jackson, 1958.
The percentage of organic carbon in soils was determined according to
Hesse (1971) except that the procedure was modified by using a phenan-
throline indicator titrating to a turquoise blue end point.
Measurements of pH were taken on wet samples diluted 50% wt/wt with
distilled water and equilibrated for 30-60 minutes.
Physical Properties—
Soil particles are discrete units of diverse composition, size,
and shape. Soil particles may be classified by determining their size
(generally the width of the smallest square opening through which the
particles can pass) and applying the relative proportions of the particle
57
-------
sizes present to a textural classification such as sand, silt and clay.
Soil particle sizes are classified by the U.S.D.A. as >2 mm=gravel,
0.5-2 mm=sand, 0.001-0.05 mm=silt, <0.002 mm=clay. Soil samples from
the field were analyzed by standard dry sieving methods. Organic material
was removed mechanically and by oxidation with hydrogen peroxide. Clay
fractionation was performed by pipette analysis, utilizing a modification
of Stokes principle.
Atomic Adsorption Analyses—
Soil samples were collected from each major soil series persent at
the Kerrville site. The soil samples were analyzed using the Perkin-
Elmer Model 460 atomic absorption spectrophotometer for Pb, Co, Fe, Cr,
Cu, Mn, Ni, Na, K, Cd, Mg and Zn.
Soil samples were collected from the upper 0.2M of the soil profile
at each sampling site. Each sample was thoroughly mixed and stored in
air-tight containers for transport to the laboratory. In the laboratory
the soil samples were sieved to remove large rocks and the organic
debris was removed by mechanical extraction. The samples were ground
and crushed using a morter and pestle and then oven-dried at 60C.
Samples were digested with 5N nitric acid, centrifuged and the leachate
analyzed.
X-Ray Diffraction, Qualitative Analyses—
Each major soil series at the Kerrville site was analyzed for
mineralogical composition of the silt and clay size fraction. As the
silt and clay size fraction present in a soil plays a major role in
determining the physical and chemical characteristics of that soil, a
knowledge of the mineralogical composition is necessary to assist in
evaluating the soil for wastewater disposal purposes.
The term "clay" has no generic significance. Clays are materials
which are produced by the weathering of other geologic materials and by
deposition as sediment. Silt and clay are primarily particle size
terms. The size range used in this report is silt (0.05-0.002 mm) and
clay (finer than 0.002 mm).
Soil samples were dispersed by sonication in distilled water. Each
sample was wet-sieved through a 325 mesh sieve. The portion of the
sample passing through the sieve then was sonicated and centrifuged at
10,000 rpm (7800x g) for 5 minutes. Samples then were redispersed by
sonication in distilled water and centrifuged for 90 sec at 1000 rpm
(approximately 80xg). At this point, the separation of silt from clay
was made, with the clays being decanted. The sediment remaining was
applied to glass slides and air-dried. The decanted volume was sonicated
and centrifuged at 10,000 rpm for 5 minutes. The clay sediment was
applied to glass slides and air-dried.
A Siemens X-ray diffractometer was used for all samples. The
scanning speed was 2.5° 20 per minute and chart speed was 2 inches per
minute. All samples were run from 2° to 64° 20. After obtaining a
58
-------
diffraction pattern for each sample each peak was indexed and converted
to the appropriate d sparings. ASTM powder data file cards then were
referenced to identify the minerals present.
SPECIAL IRRIGATION STUDY
Studies of the effect of intensive irrigation were undertaken at
the Kerrville field site. Arrangements were made with local personnel
to manage the area around selected lysimeters for ten-day study periods.
All irrigation was withheld from the area for 72 hours prior to the
collection of background soil samples. At this time lysimeters also
were evacuated. Normal cycles or irrigation performed on a 12-hour
spray period followed by a 36-hour drying period then were initiated.
Samples for laboratory analyses were obtained on a daily basis. Ly-
simeter samples were collected after approximately 6 hours of irrigation.
Grab samples from the effluent line at the irrigation pond also were
taken at this time. Soil samples were collected after approximately 18
hours of drying.
59
-------
SECTION 8
RESULTS AND DISCUSSION
Early second year study efforts focused on developing a data base
at both the Kerrville and Uvalde sites. The research plan was designed
to accumulate significant data on treatment effectiveness prior to and
during lysimetex and monitoring veil installation, thus permitting a
shift of emphasis to studies of the lysimeters and wells during the
final study year. Further, it was thought important to develop the same
types of data for the soils prior to initiation of a regular irrigation
schedule as would be obtained in the final year.
WASTEWATER ANALYSES
Chemical and Physical Analyses
Kerrville—
Table 17 presents the mean results of wastewater chemical and
physical analyses at all sampling points at the Kerrville site. The
wastewater is of moderate strength (BOD = 175 mg/1) and the treatment
quite effective as shown by 92% removal of BOD by both the oxidation
ditch system and the trickling filter system. These results were not
unexpected if one considers that the treatment plant currently is operating
at approximately 70% of its design capacity of 2.25 mgd. The average
solids carry-over from the oxidation ditch system was comparatively high
but this was.due to sludge handling difficulties at the plant during the
Winter of 1976-77.
Total organic carbon, BOD and solids concentrations found in the
irrigation pond were in keeping with the effectiveness of treatment.
Considerable variation in these parameters, however, was observed in the
tailwater pond. The tailwater pond variations were attributed to the
periodic presence of extensive duck and algal populations.
Statistically significant changes in Third Creek's chemical proper-
ties, such as increases in orthophosphate and nitrate concentrations,
were seen downstream of the irrigation site. These changes may be the
result of minor seepage of wastewater through the dike protecting the
creek and/or from groundwater discharges to the creek. However, the
observed increases do not represent a marked degradation of water quality.
Uvalde—
Table 18 presents the mean results of wastewater chemical and
physical analyses for all sampling points at the Uvalde site. Here the
60
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wastewater was relatively weak as reflected by the BOD (79 mg/1), total
suspended solids (73 mg/1), the orthophosphate (3.1 mg/1) and nitrogen
forms. Local observations indicate that considerable infiltration
exists in the Uvalde sewage collection system. This would provide some
basis for the observed low strength of the wastewater. The fact that
only 56% of the BOD,, and 51% of the total suspended solids were removed
by the time the wastewater passed the trickling filter was most likely a
direct result of the extremely high hydraulic loading on the unit pro-
cesses in the treatment train. It should be noted, however, that the six
ponds are a part of the total treatment system and that by the time the
wastewater passes through the sixth pond, 77% of the BOD has been removed.
The collected data do not reflect any major negative impact of the
wastewater treatment plant discharge on Cook's Slough. In fact, except
in the case of the nitrogen forms, the water quality as measured by
these parameters exhibited no significant changes as a result of the
treatment facility.
Bacteriological Assay
Kerrville—
Table 19 contains the mean results of total and fecal coliform
determinations for all sampling points at the Kerrville site. These
data were consistent with the chemical data in terms of treatment effec-
tiveness throughout the system.
There was one interesting observation, however. Although the total
coliform and fecal streptococci concentrations downstream of the treatment
facility were higher than they were upstream on Third Creek, the fecal
coliform concentrations were not. In fact, there was probably no signi-
ficant difference between the fecal coliform concentrations upstream and
downstream of the treatment facility on Third Creek.
When examining the fecal coliform-fecal streptococci ratios (FC:FS)
shown in Table 19, one should not conclude that the majority of human
fecal material in Third Creek was present prior to the stream entering
the site, although Feachem (1975) has suggested that due to differential
die-off, such a falling FC:FS ratio indicates fecal material of human
origin. In this situation, however, the falling ratios can be attributed
to a significant increase in the absolute numbers of fecal streptococci
al'ong the stretch of Third Creek studied. The exact source of this
increase is not known, but may be associated with either wastewater from
the irrigation pond or the treatment plant effluent which is piped
beneath the creek under a significant hydraulic head. If the latter
were the source, however, one would expect higher concentrations of both
total coliform and fecal coliform at the downstream sampling point.
Uvalde—
Table 20 includes the mean results of total and fecal coliform
determinations for all sampling points at the Uvalde site. These data
are reflective of the chemical data, also.
It is interesting to note that the total and fecal coliform concen-
63
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trations of the Uvalde raw wastewater were lower than those observed at
Kerrville. Again, this reflects the lower strength of the Uvalde wastewater.
Bacteriological Screen
Table 21 presents the data from a single bacteriological screen of
the raw wastewater and irrigation pond sampling sites at Kerrville as
well as the raw wastewater at Uvalde. As the bacteriological screen
used at each site differed in scope, the results are not directly com-
parable. However, the organisms detected by these screens attest to the
presence of a wide variety of potential-pathogenic and/or opportunistic
bacterial species at each site.
Virus Assay
Kerrville—
Table 19 includes a summary of results of analyses for coliphage for
most sampling points at the Kerrville site.
Net bacteriophage reductions throughout the treatment system were
not as dramatic as were the total and fecal coliform reductions. In
part, this was because the oxidation ditch was much more efficient in
coliphage removal than was the trickling filter. The minor increase in
bacteriophage concentrations in Third Creek may be the result of small
amounts of wastewater penetration through the dike. This observation
is consistent with the data obtained for selected chemical parameters.
Enterovirus concentrations observed in the raw wastewater have been
extremely variable throughout the study period, (Tables 22 and 23). The
initial low levels observed were thought to be related to some component
in the wastewater which was either virucidal or interfered with the
recovery and/or assay of indigenous virions by bentonite concentration.
Experimentation with stock virus did not support this theory. A more
plausible explanation involves the demography of Kerrville. The commu-
nity has a large contingent of retired individuals, who are not likely
to be shedding poliovirus, usually the most common enterovirus isolated.
Reduction of enteroviruses through the oxidation ditch system
ranged from 61% to 99.9%. Reduction through the trickling filter system
ranged from 59% to 95%, which appears to be somewhat high but is con-
sistent with mean bacteriophage reductions (see Table 19).
Virus levels observed in the effluent from the ox.idation ditch
system were always low (see Table. 23). Further, virus levels observed
in the trickling filter system effluent paralleled the variability
observed in raw wastewater levels. Removal efficiencies were high,
generally, in both systems. It is probable that this was due to both
hydraulic and organic underloading of the facilities. As can be seen in
Table 23, there does not appear to be any correlation between virus
levels observed and flow or virus concentration efficiency.
66
-------
TABLE 21. Bacteriological Screen, Kerrville and Uvalde Sites
Colony-Forming Units/100 ml
Organism
Total coliform
Fecal coliform
Fecal streptococci
Fluorescent Pseudomonads
Mycobacteria
Staphylococci
ENTEROBACTERIACEAE
Citrobactei* diversus
Citrobaoter freundii
Enterobacter aerogenes
Enterobacter agglomerans
Enterobacter cloacae
Enterobacter hafniae
Escherichia coli
Klebs-iella ozaenae
Klebs-iella pneumoniae
Proteus mirabalis
Proteus morganii
Proteus rettgeri
Providenaia alcalifaoiens
Providencia stuarti.i
Salmonella sp.
Serratia liquefaciens
Serratia marceaaena
OTHER GRAM-NEGATIVE BACTERIA
Aeromonaa hydrophilia
Flavobacteria
Pasteurella sp.
Pleeiomonas shigelloides
Pseudomonas aemginosa
Pseudomonas fluoreacens
Pseudomonas put-ida
Pseudomonas stutzeri
Kerrville Site
Raw Wastewater
(24-hr Composite)
6.8xl07
2.3X107
9.2xl05
7.8X106
1.2X105
1.2xl05
1.3xl06
S.OxlO6
ND
2.7xl06
l.SxlO7
ND
6 . OxlO6
8.3X106
l.SxlO7
> 6. 7x10° (E)
ND
8.3xl06
> 3. 3x10° (E)
> 3. 3x10° (E)
ND
1 . 3X106
ND
Kerrville Site
Irrigation Pond
(Grab Sample)
1.4xl05
S.lxlO4
2.1xl03
3. OxlO3
l.OxlO2
8.7xl03
ND+
6.7xl02
0
> 3.3x10 (E)
2. OxlO3
l.lxlO4
> 3.3x10° (E)
5. 3x10 3
6. 7x10 2
1.3xl04
6.7x10°
3.3x10°
ND
ND
ND
> 6.7x10° (n)
6.7xl02
> 3.3x10° (r)
Uvalde Site
Raw Wastewater
(24-hr Composite)
1.4xl08
4.2xl06
1.4xl05
1.7xl06
CS
9.3xl04
ND
l.OxlO5
5
3.3x10
•l.OxlO6
1.4xl06
ND
2. OxlO6
ND
6.6xl05
ND
ND
ND
ND
ND
3.3xl04
ND
ND
(Kerrville Site Only)
1.7xl08
6.7X105
8.3xl06
3 . 3xl06
6.7X105
1.3xl06
1.3xl06
1 . 7x10 7
1.3xl04
ND
ND
4. OxlO3
ND
1.3xl03
ND
ND
CS - Contaminated Sample
ND = None Detected
(E) Isolated from Enrichment Sample
67
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Uvalde—
Table 20 includes a summary of results of analyses for bacterio-
phage for most sampling points at the Uvalde site.
Bacteriophage concentrations found in the raw wastewater, like the
total and fecal coliform concentrations, were somewhat below those
observed at Kerrville. Net reductions throughout the treatment system
appear to parallel those found for total and fecal coliform. The sig-
nificance of the minor increase in bacteriophage concentrations in
Cook's Slough is attributed to the impact of the wastewater treatment
facility discharge.
Concentrations of enteroviruses observed in the raw wastewater were
consistent despite the relatively dilute sewage (see Tables 24 and 25).
The lack of removal of enteroviruses in the hydraulically overloaded
trickling filter was anticipated (see Table 25). These results were in
contrast with those obtained at the Kerrville site, where hydraulic
loading is not a problem.
The observed increase in virus concentrations downstream of the
site in Cook's Slough is consistent with the virus levels found in the
effluent of Pond #6. Unfortunately, the virus isolates detected in
Cook's Slough were not retained for confirmation and identification.
IRRIGATION POND STUDIES
Kerrville
A more detailed study of the Kerrville irrigation pond was undertaken
because of the limited number of isolations of indigenous enteric viruses
in irrigated effluent. As previous research in a model-pond system had
documented viral deposition with particulate materials (Funderburg et al,
1978), a program of sediment sampling was initiated. Water column
samples taken at approximately mid-depth and corresponding sediment
samples were obtained on a line from the pond influent to the pond
effluent as illustrated in Figure 9. Both water and sediment samples
were analyzed for total and fecal coliforms, fecal streptococci, coliphage
and enteric viruses.
Results presented in Table 26 show representative results from
three separate sampling times. From an analysis of water column data
collected in March and April, it appears that mixing within the pond was
reasonably good. In general, variation in microbial concentrations at
the three sampling points was insignificant. Levels of fecal strepto-
cocci and bacteriophage in pond effluent was lowest in August, perhaps
due to higher summer temperatures and increased algal activity. During
these special studies, no indigenous enteric viruses were recovered from
20-liter water samples concentrated at each point utilizing the Prototype
2 procedure as described in METHODS AND MATERIALS.
Not surprisingly, the levels of indicator organisms per equivalent
unit (ml vs. gin) showed an enrichment of viable microorganisms in the
70
-------
TABLE 24. Results of Wastewater Enterovirus Assay, Uvalde Site*
Sample Point
Range of Enterovirus Concentrations
Recovered (pfu/1)
Effluent
Pond #1
Pond #2
Pond #3
Pond #4
Pond #5
Pond #6
Cook's Slough,
Upstream
Cook's Slough,
Downstream
Remarks
3-Day
5-Day
Raw Wastewater
Trickling Filter
59-880
21-460
64-1340
28-980
Chemical data indicate th.
12-240
1.8-160
6.0-172
12-860
1.8-620
7.6-355
<1.1-21
<0.1=5.1
<0.5-4.6
<0.5-1.5
6.5-257
<0.09-21
<0.09-]2
<0.09-8.5
<0.09-14
trickling filter is biolog-
ically underloaded, though
hydraulically overloaded.
Early in the study there was
an indication that short
circuiting of raw wastewater
existed to Pond #3.
* Values derived from ten samples; in two cases grab samples were collected for
all sample points.
71
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72
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TRKKUKG .—.
FILTER —(J
FIGURE 9. Water and Sediment Sampling Points,
Kerrville and Uvalde Ponds.
73
-------
TABLE 26. Organism Distribution in the Kerrville Irrigation Pond
Sampling Point
March, 1978
Influent
Middle
Effluent
April, 1978
Influent
Middle
Effluent
August, 1978
Effluent
March, 1978*
Influent
Middle
Effluent
April, 1978°
Influent
Middle
Effluent
August, 1978*
Influent
Effluent
Fecal Coliform
(cfu/100 ml)
7. 6x10 3
7. 6x10 3
3.6xl04
l.OxlO5
1.2xl05
7.0xl04
1.7xl04
(cfu/qm)
2.8xl04
1.4xl04
l.SxlO3
9.3xl03
7.5xl04
2.8xl03
l.lxlO4
6. 8x10 3
WATER COLUMN
Fecal Streptococci
(cfu/100 ml)
6. 0x10 2
l.lxlO3
3. 0x10 3
1.2xl03
3. 3x10 3
3. 7x10 3
2.7xl02
SEDIMENTS
(cfu/gm)
9. 7x10 3
2.5xl04
2.2xl04
4-lxlO5
6.7xl05
5.7xl04
2 . 3xl03
8.6xl03
Bacteriophage
(pfu/1)
9 . 3xl04
1.3X105
S.SxlO4
5.4xl04
2.5xl04
S.SxlO4
2. 0x10 3
(pfu/gm)
4. 3x10 3
3. 6x10 3
i.sxio2
1.2X103
l.SxlO4
4.2xl02
S.OxlO2
8. 4x10 2
t E. coli K13 as host organism.
a MPN procedure for bacterial indicators.
* Samples for bacterial analysis were spread plated onto appropriate selective
media.
74
-------
sediments. However, organism distribution at the sediment sampling
points did not follow a consistent pattern. As indicated in Table 26,
March values for fecal coliform and bacteriophage reflected an anticipated
distribution with the highest concentrations detected at the influent
sampling point where maximal sedimentation would be expected. On the
other hand, fecal streptococci showed a relatively even distribution at
all three sampling points. In April, the greatest concentration of all
indicator organisms appeared at the middle sampling point. By August,
little, if any difference in bacterial or coliphage densities could be
ascertained between influent and effluent points. As with the water
samples, fecal streptococci and bacteriophage levels in sediments were
lowest in August.
The only positive enteric virus sediment sample obtained was taken
in August at the effluent sampling point. Subsequent handling of the
isolate resulted in its identification as a Coxsackievirus B5. It
should be noted, however, that sample toxicity toward cell monolayers
was a recurring problem in plating sediment concentrates.
Uvalde
In June, 1978, both pond waters and sediments were sampled at
points indicated on Figure 9. As noted earlier, a reduction in micro-
bial numbers was observed throughout the series of six ponds. Represen-
tative results showing fecal coliform and coliphage removals are presented
graphically in Figure 10. An anomaly in the treatment effectiveness was
noted in pond 3. Previous monitoring at the Uvalde site had suggested a
possible source of infiltration into this pond. The aerial photograph
presented as Figure 4 lends further credence to the probability of an
inflow at the northeast corner of pond 3. In this picture, the visible
turbidity throughout the third pond exceeded that present at the trickling
filter outflow into pond 1. Nonetheless, the remaining ponds in series
(4, 5 and 6) achieved bacterial reductions of three to four log, as
shown in Table 27. During the June sampling, fecal coliform levels,
discharged to Cook's Slough, did not exceed 300 .cfu/100 ml. In like
fashion, coliphage concentrations in the final pond effluent were reduced
to 400 pfu/liter. By concentrating a maximum of 20 liters of water,
indigenous enteric viruses were detected only through pond 4 in the
series.
An important mechanism of microbial removal during pond treatment
appears to be deposition with particulate matter into bottom sediments.
As shown in Table 27, the highest concentrations of all indicator or-
ganisms in pond sediments were observed at the influent end on pond 1.
Additionally, higher numbers of total coliform, coliphage and enteric
viruses were recovered from sediments located at the effluent end of
pond 3. These findings further implicate an additional source of waste-
water entering the third pond.
In general, sediment samples through pond 4 were enriched in the
number of organisms recovered-when compared to overlaying water. This
phenomenon was particularly pronounced for fecal streptococci in ponds
1, 2, 3 and 4 and suggests prolonged survival of these enteric organisms
75
-------
I.OT
•o'-f-
CCUFORM
• FECAL COUFORM
X COLIPHAQE
INFLUENT PI
n PS P4
POMO EFFLUENTS
P«
FIGURE 10. Fecal Coliform and Coliphage Removals
in Uvalde Ponds
76
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77
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in pond sediments. While this study was limited in that no attempts
were made to measure quantitatively the amount of solids deposited in
the ponds, the number of viable organisms in the sediment sink, through
pond 4, could constitute a large microbial reservoir.
IRRIGATION AND PRECIPITATION
Table-28 contains a summary of wastewater flow, irrigation and
precipitation data for the period approximating the final year of the
study. Wastewater flow over the period was relatively stable except
immediately after major periods of precipitation (note 10.4 inches of
precipitation during the period July 24-August 1, 1978), which resulted
in significant infiltration. Therefore, wastewater available for irri-
gation was relatively constant ranging from 1.7 to 4.7 inches/week.
More often than not, however, it was close to the mean rate of 2.7
inches/week. Actual irrigation rates ranged from zero to 4.2 inches/week
with a mean of 2.4 inches/week. This is a direct result of the operation
of an irrigation system as a wastewater treatment (control) process with
interest in irrigation being secondary. This is not a negative state-
ment but rather a fact of life. Under these circumstances, irrigation
is practiced when wastewater begins to accumulate normally during periods
of high precipitation and low evaporation. As indicated in Table 28,
the highest irrigation rates occurred in November 1977, following signi-
ficant precipitation (5.2 inches in less than three weeks).
SOIL ANALYSES
Chemical Properties
Table 29 contains the results of selected chemical and physical
analyses of surface soil samples. The pH analyses of the soils at the
Kerrville and Uvalde sites indicate that these are intermediate to basic
range soils. Relatively high soil pH is to be expected in light of the
basic calcium carbonate parent rock in the area. The cation exchange
capacity (CEC) of the soils from both sites is in the medium to high range,
again indicating similar soil material and, therefore, similar exchange
capacity. The organic content of the soils from the two sites differs
substantially, however. The Uvalde soils fall within the normal range
in organic content, while the Kerrville soils are somewhat high. The
higher organic content of some of the soils at the Kerrville site is
related primarily to the long-term use of the site for irrigation by
the application of wastewater.
Soil samples were collected from the upper 0.2M of the soil profile
from each major soil series present at the Kerrville site. The soil
samples were analyzed for Pb, Cd, Fe, Cr, Cu, Mn, Ni, Na, K, Ca, Mg and
Zn. Results of these analyses are shown in Table 30. The metallic ion
concentration in a soil is the result of a complex series of geochemical
reactions involving mineral weathering and decomposition, plant activity
and animal activity. The total concentrations of metallic ions in soils
usually relates to the concentrations of the metallic ions present in
78
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81
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the parent materials. The high calcium content (17,000-21,000 ug/5
grams) of the Kerrville site soils reflects the parent calcium carbonate
bedrock.
The metallic ion concentrations in the Kerrville site soils, in
particular those soils in the upland areas (Brackett series) which have
not been subjected to intensive irrigation with wastewater, are probably
representative of the natural soil metallic ion concentrations prior to
irrigation. The soil samples from the lowland, level Denton soil series
show an increase in cadmium, zinc and copper concentrations when compared
with the concentration present in the other soil series. This increase
in these ions indicates the more intense utilization of these lowland
sites for wastewater application.
Mineralogy
Silt Size Range—
Table 31 shows the analyses of the silt size range for each major
soil type at the Kerrville site. Minerals present in the silt size
range of the soil samples were calcite (CaCO ), quartz (SiO ) and gypsum
(CaSO ) as determined by X-ray diffraction. These minerals are the
result of weathering and erosion of the Cretaceous carbonate strata in
the drainage basin of Third Creek.
TABLE 31. Minerals Present in the Silt Size Range
Dominant Mineral Present Listed First
Denton Series Lewisville Series
Calcite Calcite
Quartz Gypsum
Gypsum Quartz
Frio Series Brackett Series
Quartz Quartz
Calcite Calcite
Gypsum Gypsum
Clay Size Range—
Table 32 lists the mineral content of clay size particles present
in the soils at the Kerrville site. The minerals present include mont-
morillite, kaolinite, calcite, quartz and gypsum.
82
-------
Montmorillite is a term used to describe a group of hydrated silicates
with the general formula (OH) Si Al O .xH O where x molecules of water
can be driven off at low temperatures. Montmorillite is composed of a
single sheet of silica tetrahedrons arranged as octahedrons enclosed by
two silica tetrahedral sheets (Figure 11). The oxygen atom layers at
the base of the tetrahedron are adjacent to each other, causing a weak
bond and facilitating excellent cleavage. The tetrahedron layers carry
a negative charge on their surfaces which attracts varying amounts of
water. Calcium and sodium cations can enter the layered silicate struc-
ture and there is some substitution within the layers. Montmorillite is
therefore a clay mineral with a high cation exchange capacity.
Kaolinite is a clay mineral composed of a single octahedral sheet
and a single tetrahedron sheet. The sheets of octahedrons and tetrahedrons
TABLE 32. Minerals Present in the Clay Size Fraction;
Dominant Mineral Present Listed First
Denton Series Lewisville Series
Montmorillite Montmorillite
Kaolinite Kaolinite
Calcite Calcite
Gypsum
Quartz
Frio Series Brackett Series
Montmorillite Montmorillite
Kaolinite Kaolinite
Calcite
Quartz
Gypsum
are combined or stacked and tied together with hydrogen bonds. The
formula of kaolinite is (OH) jSi Al O . The atomic arrangement of
kaolinite allows little substitution within the kaolinite lattice and
kaolinite has a limited cation exchange capacity (Figure 11). Because
of the relatively weak hydrogen bond, kaolinite can be split into very
thin platelets which are negatively charged on their flat surfaces and
will attract thick layers of water. These water layers in kaolinite
produce the characteristic plasticity exhibited when kaolinite is mixed
with water.
Soil Particle Size Distribution
The size distribution of the soil particles at the Kerrville site
83
-------
wcrttr
o
©
KAOUNITE
wattr
krycn
MONTMORILLONITE
(HydraUd)
O Oxygen © tOH) • Silicon • Si-AI o Aluminium
FIGURE 11. Diagrammatic Representation of the Succession of Layers
in Some Layer Lattice Silicates. (Brown, 1961)
84
-------
is important in the determination of the dimensions of available pore
space in the soils. The size of the pores determines the movement and
distribution of water and gases in the soil. Soil aggregate size distri-
bution will vary in a given irrigation site dependent on the amount of
water applied and duration of irrigation. Figure 12 presents the soil
aggregate distribution curves for soil samples from the Kerrville site.
The clay minerals in the soils at the Kerrville site are predominantly
montmorillonitic. These soils have a cation exchange capacity (CEC) ranging
from 1.7 to 67 meq/100 gm. While not all of the CEC is directly attributable
to the type and quantity of clay mineral present in a given soil, the
predominance of one clay mineral over another will determine the suitability
of the soil as a wastewater renovation site.
Microbiological Assays
Table 33 illustrates the ranges of both total and fecal coliforms
enumerated in soils taken from the Kerrville site over a 16-month period
from October, 1976 through February, 1978. During the early stages of
this study, sample points 1 through 4 received considerably less waste-
water irrigation than did sampling points 5 and 6. Thus, variability
was observed in the early results, although differences lessened as
irrigation practices became more balanced over the site.
The fact that less irrigation was done in area 1 over the sanitary
landfill was reflected by overall lower levels of fecal coliform at soil
site number 4. Additionally, only one soil sample from this location
yielded a positive isolation of coliphage. in all other irrigated
4 o
areas, fecal coliform counts ranged as high as 10 -10 MPN/gm of dry
soil. In addition, multiple numbers of samples were positive for bac-
teriophage with levels ranging maximally to 5 pfu/gm and 16 pfu/gm.
Viewed on a monthly basis, the levels of both total and fecal
coliform isolated from the soils at Kerrville correlates reasonably well
with irrigation practices. For example, it is the practice of operating
personnel to irrigate more during periods of rainfall regardless of
season; less during dry cool periods and even less during dry hot periods.
This operating procedure insures maximal capacity in the ponds and
precludes direct discharge to Third Creek. The field data reflect these
conditions as the lowest values of both total and fecal coliform generally
were observed in December and again in July and August while the highest
values were recorded in May, June, September and October. Table 34
illustrates this observation more conveniently. High total coliform to
fecal coliform ratios were indicative of low irrigation periods.
Because of the overriding influence of irrigation at the Kerrville
site, no conclusions could be reached regarding organism survival as
influenced by seasonal conditions. It is interesting to note, however,
that bacteriophage were isolated from soils in all seasons.
In order to observe more directly the effect of irrigation on
indicator organism levels in soil, two special studies were undertaken.
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During January and February, 1978, and again in May, 1978, arrangements
were made with local personnel to manage the areas around selected
lysimeters over a 10-day study period.
Microbial levels observed during the first study in soil sampling
area 2 are presented in Table 35. Enumeration of indicator organisms in
grab samples taken at the irrigation pond outlet during spraying were 5
quite constant with mean values of 1.3x10 total coliform/100 ml, 4.3x10
fecal coliform/100 ml, and 5.4x10 fecal streptococci/100 ml. Soil
samples were taken approximately 18 hours after cessation of wastewater
application. While the levels of total coliform show an increase relative
to the controls during sustained irrigation, even greater rises in the
levels of both fecal coliforms and fecal streptococci were observed in
irrigated soils as compared to the non-irrigated controls. Bacterio-
phages plaquing on Escharichia coli K13 were observed at levels ranging
from 0.2 pfu/gm to > 10 pfu/gm by directly plating soils eluates.
Using an elution-reconcentration scheme for soil samples as described
previously, any areas showing cytopathic effects on either HeLa or BGM
monolayers were picked as potential viral isolates. However, subsequent
tube passage failed to confirm viral isolates.
The second controlled irrigation study was conducted in the area
around lysimeter 2. Results shown in Table 36 reflect similar bacterial
trends. Bacteriophage consistently were recovered from irrigated soils
at levels ranging from 1.0 pfu/gm to 4.5 pfu/gm of soil. During this
study, a single human enteric virus, subsequently identified as polio-
virus 1, was recovered from an irrigated soil.
It should be noted that only one other enteric virus (also poliovirus
1) was isolated from soil samples over the two-year study period. While
as many as 150 potential viral isolates (as defined by any evidence of
cpe) have been picked to tube cultures, only two could be confirmed.
The prevalence of soil microorganisms capable of cell destruction mi-
micking viral cpe has been noted previously in samples from sludge-
injected soils and serves to emphasize the importance of viral con-
firmation procedures.
Data summarizing the soil analyses conducted at the Uvalde site are
presented in Table 37. In comparison to the Kerrville data, it is
obvious that the levels of both bacterial indicators and coliphage are
significantly lower (several orders of magnitude). This, in part, may
be attributed to the less structured irrigation schedule at Uvalde.
It is interesting to note, however, that a similar pattern exists for
the total coliform to fecal coliform ratios. As can be seen in Table
38, these ratios .are in the same range as those at the Kerrville site
during periods of more intense irrigation.
LYSIMETERS
Soil Particle Size Analysis
The subsurface soils at the lysimeter sited are composed predominately
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Of silt size particles (0.0625 mm to 0.0039 mm). The soil samples ranged
from 90% to less than 20% silt size particles. Clay size fraction
(diameter less than 0.0039 mm) ranged from 30% to less than 1%.
The coarse fraction of sand (2.00 mm to 0.0625 mm) and granule size
particles (4 mm to 2 mm) were composed entirely of rounded limestone
fragments and some cemented CaCO concretions. The coarse fraction in
the lysimeter soils ranged from 80% to less than 10% of the total soil
sample.
The particle size distribution curves for the lysimeter sites
(Figures 13-15) at depths of 1.5 ft, 3.0 ft and 4.5 ft illustrate the
general pattern of soil particle size distribution. They provide more
detail regarding the sand, silt and clay size fraction distribution for
each of the lysimeter sites.
The vertical distribution of grain size at lysimeter sites #1 and #3
indicates a much lower clay size component in samples from the 4.5 foot
depth, with increasing clay size content in the upper soil layers. The in-
creased clay content in the upper soil layers is a result both of maximum
mineral weathering at or near the surface and of an influx of alluvial clay
materials. (Note, lysimeter site #2 does not follow this pattern; see Fig.14)
The silty clay and loam soils of the Kerrville site have a controlling
effect on the suitability of the site for wastewater renovation. As soil
particle size decreases, there is an exponential increase in soil adsorption
capacity and surface area. The soil grain size distribution is important in
determining the soil permeability and infiltration rate, also. The fine-
grained soils at the Kerrville site are classified as having moderate permea-
bility, ranging from 0.6 to 2.0 inches per hour. It should be noted that the
actual characteristics of the soils at the lysimeter sites differ from those
described for this general geographical area.
Chemical Properties of the Soil
Soil chemistry investigations at the Kerrville site involved the
determination of pH, organic carbon, and cation exchange capacity. Results
are shown in Table 39.
Soil pH—
The soil pH depends upon the amount and type of exchangeable ions
present in the soil. The soil pH can also be changed by biological
activity, by introduction of organic matter and, to a slight degree by
changes in water content. The pH of the soils at the lysimeter sites
ranged from 8.8 to 9^2 indicating a high concentration of the two
principal bases, Ca and Mg . The soil pH was not observed to vary
greatly with depth, indicating that the application of wastewater (pH
of 7.5-8.9) had not significantly reduced the soil pH.
Organic Carbon—
Organic carbon in the Kerrville site soils ranged from 0.19 to 0.97%
with a general trend toward an increase in organic carbon content in the
upper soil layer at the lysimeter sites.
94
-------
100 -•
Legend:
Lysimeter Depth
2.0 0.5 0.125 0.0310 0.0078 0.0020
1.0 0".25 0.0625 0.0156 0.0039
Particle Diameter (mm)
FIGURE 13. Soil Particle Size Distribution Curves for Lysimeter Site #1
95
-------
100
L«g«ndi
1.5'
3.0'
4.5'
2.0 0.5 O.j.25 ' 0.0310 0.0073 ' 0.3020
1.0 0.2S 0.0625 0.01SS 0.0039
Fardel* 3j.aaat.4r (inn)
71GCSZ 14. soil Particle Siz» Distribution Curv«»: Ly«ia«t»r Sit* "2
L«g«nd:
0«pth
100- •
SO..
•0 0.5 3.125 0.0310 0.0073 0.0020
1.0 0.25 O.C625 0.0156 0.0039
Particle Oi.ir.stsr (sal
15. Soil Pamela Size Cistribuiicr, Cur1/**: Lvsiir.ecer Sire *3
96
-------
Carbon is the chief component of soil organic matter and is derived
from plant and animal remains and decayed organic matter. The amount of
soil organic material plays an important role in the exchange capacity
of a soil, especially at high pH values. In addition, the soil organic
material has an effect on soil moisture retention and pH and in maintaining
soil structure. Long-term application of wastewater would tend to
increase the organic carbon content of the soil unless there was a
corresponding increase in soil microbial activity to utilize the addi-
tional carbon effectively.
Cation Exchange Capacity—
The cation exchange capacity (CEC) of a soil indicates the quantity
of ions held in an exchangeable form. Generally, CEC is highest for or- ,,
ganic matter, is intermediate for the expanding clay minerals and is
lowest in the clays with low expansion coefficients. The primary con-
trolling factors in soil CEC are mineral content and the amount of
organic material present. Additional factors contributing to the CEC
are pH and the size of the soil material present. CEC values in the
Kerrville site soils ranged from 21.2 meq/100 gm to 89.2 meq/100 gin.
The CEC generally increased with depth (Table 39).
The decrease in CEC in the upper layers, in spite of a higher clay
size fraction content, may be attributed to depletion of ion concentration
by leaching of water percolating through the soil.
TABLE 39. Chemistry - Kerrville Lysimeter Soils
LYSIMETER
DEPTH
(FT.)
pH
ORGANIC
CARBON
CATION EXCHANGE
CAPACITY
(meq/100 gm)
1.5
3.0
4.5
1.5
3.0
4.5
1.5
3.0
4.5
8.96±.25
8.89±.22
8.821.13
8.731.06
9.001.46
8.991.14
8.811.04
8.861.14
9.221.12
.271.17
.381.07
.291.00
.971.26
.161.05
.171.04
.441.04
.301.07
.261.08
21.21.7
67.5+3.4
41.212.0
36.81.7
76.614.4
89.2115.7
53.811.2
62.513.9
28.717.0
97
-------
Chemical Analysis of Lysimeter Extracts
As mentioned earlier, the lysimeters used in this study were de-
signed especially for the collection of microbiological data. As such,
they could not be operated under conditions of continuous tension.
Further, as relatively large samples were required for microbiological
analyses, sampling could occur only when irrigation rates were high
enough to saturate the soil in the vicinity of a particular lysimeter.
These restrictions limited the consistency of sample collection and the
amount of data available for analysis among lysimeters. For example,
samples were collected from lysimeter site #3 on only four occasions while
samples were available for analysis from lysimeter site #1 on as many as
15 occasions. Data collected from the soil moisture probes throughout
this study support this contention. Specifically, the soil adjacent to
the lysimeters at site #1 was saturated more frequently than were those
at sites 2 and 3. Further, the deeper lysimeters were saturated more
frequently than were the shallower ones.
Typical values for the analysis of lysimeter samples can be found
in Table 40. In general, these mean values indicate the chemical quality
of the lysimeter samples to be somewhat improved over the wastewater in
the irrigation pond. There are exceptions to this statement. For
example, the nitrate-nitrogen concentrations of the water from the
lysimeters was found to be consistently higher than that of the irrigation
pond water. This coupled with a decrease in total Kjeldahl nitrogen
should not be unexpected as such results have been observed at many
other sites. The higher solids levels found (particularly at the lysimeter
site £1) are not reflective of the wastewater applied but rather of the
soil conditions at the site, the lysimeter construction and the sampling
procedures.
Conclusions regarding wastewater renovation as a function of depth
cannot be drawn from these data. To evaluate these processes, it was
necessary to use data from the special irrigation studies only. Experi-
mental procedures .can be found in the METHODS AND MATERIALS section.
Tables 41 and 42 present the chemical and physical data developed
during both special irrigation studies. These data support the theory
of renovation of the wastewater applied to these soils with regard to
total phosphate, orthophosphate, ammonia-nitrogen and in most cases,
total Kjeldahl nitrogen. Under some conditions, depth appeared to play
a role. Little can be said regarding the effect of the soils or depth
on total organic carbon concentration. In the case of nitrate-nitrogen,
major increases were observed in the waters from the lysimeters. These
increased concentrations can be a direct result of the conversion (oxidation)
of other nitrogen forms as well as leaching of the soil. The latter
explanation is supported by the accompanying conductivity data.
Microbiological Analyses
The lysimeter microbiological data are of greater potential interest
than are the soil studies because of the possible opportunity they
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present for assessing the degree of movement of indicator bacteria,
coliphages, and enteric viruses from the soil surface. To this end,
the three lysimeter locations were chosen to represent distinctly dif-
ferent soil types at the Kerrville irrigation site. Unfortunately,
samples available from lysimeter #3 were sporadic and, therefore, of
limited interpretative value. Only lysimeters located at sites #1 and
#2 were amenable to regular sampling.
Prior to assessing field data, a laboratory experiment was conducted
to evaluate the potential for regrowth of indicator organisms in lysimeter
waters. Samples were collected aseptically from the 1.5 and 3.0 feet
depths at lysimeter #1. Immediately upon receipt in the laboratory,
indigenous levels of total coliforms and fecal streptococci were deter-
mined by appropriate membrane filtration techniques. Aliquots of the
original samples were held in.the dark at 4C, 20C, and 35C, and subse-
quently assayed at 2, 3, 4, 5, 6, 7 and 10 days. The data accrued from
this simulated testing are compiled in Appendix B. Representative
results, graphed as fecal streptococcal inactivation are shown in Figure
16. Organism die-away (as measured by recoverability) was observed at
all temperatures over the 10-day monitoring period. From these tests,
it was concluded that insufficient nutrients coupled with existing
environmental conditions were such that regrowth in lysimeters was not a
significant factor to consider in evaluating field data.
An overview of the lysimeter field data is presented in Table 43
as ranges of results for the bacterial and viral analyses completed
during an eighteen-month period. Generally, bacteria and coliphage
densities were highest in the water collected at lysimeter #1. Although
enteric virus isolations were made on at least one occasion at each
site, the most frequent recoveries occurred at lysimeter #2 (six water
samples yielding virus) and lysimeter #1 (3 water samples yielding
virus). These positive recoveries involved the concentration from volumes
ranging from 1 liter to 20 liters. Interestingly, viruses were detected
in water sampled to a depth of 3 feet at lysimeter #1 and 4.5 feet at
lysimeter #2. These results contrast with the poor rate of recovery
of viruses from soils in the lysimeter area and are remarkable in view
of the low levels of viruses reported for the irrigation pond (Table 22) .
Specific organism levels at each lysimeter for selected sampling
dates are shown in Table 44. The July, 1977 data were accumulated under
typical conditions of irrigation while the August, 1978 sampling was
done immediately after heavy rainfall and accompanying flooding. In
almost all cases, when samples were obtained at all three depths, little
attenuation of organism density was evident through 4.5 feet of soil.
An exception can be seen in the temporal sampling beginning on
August 2, 1978 at lysimeter #2. In the absence of site irrigation
during flooding, bacterial and coliphage counts were highest on the
first day of sampling with viruses being recovered at all three depths.
Over the following two days, organism concentrations decreased with both
depth and time. During the same period, bacteria and coliphage levels
in samples obtained at lysimeter #1 decreased with time, but no attenuation
through soil was observed at lysimeter #1 decreased with time, but no
102
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FIGURE 16. Fecal Streptococci Inactivation in Lysimeter Water
103
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6 Heavy rainfall beginr
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105
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attenuation through soil was observed. Once again, a positive viral
sample was obtained on the first day of sampling following the heavy
rainfall.
In an effort to assess more closely the effects of effluent irri-
gation at the Kerrville site, a series of coordinated studies involving
intensive sampling of lysimeters during scheduled irrigation were conducted.
The first study was done in an area around lysimeter #1 with water
samples from lysimeters and the irrigation pond being collected after
approximately six hours of irrigation. The irrigation schedule shown in
Table 45 reflects a total of 24.6 inches of secondary effluent applied
in the field during the study. Data presented in Table 46 profiles the
microbiological results from this first study. If one considers the
levels of organisms detected in irrigated effluent and water collected
from lysimeters, significant microbial penetration in the saturated soil
zone to 4.5 feet was observed. Using the level of these indicator
organisms in effluent grab samples from the irrigation pond as a general
measure of application, the bacterial removals observed ranged from no
reduction to 50% removal. Similarly, a comparison of coliphage levels
in irrigation pond effluent and lysimeter water samples show a maximal
reduction of 35% in water moving through the soil profile. Finally, al-
though no enteric viruses were recovered from irrigation pond grab'
samples, two confirmed viral isolates were detected at a lysimeter depth
of three feet in the January 31 sample. The viruses picked from HeLa
cell monolayers were identified as, ECHO 11 and ECHO 21.
A second intensive irrigation study was carried out at the Kerrville
site in the general area of lysimeter #2. Because of the interesting
findings of the first study, more emphasis was placed on lysimeter
sampling. Over the ten-day study period, a minimum of 18 inches of
secondary effluent and 1.9 inches of rainfall were measured in the study
area. Microbiological results are outlined in Table 47. Bacterial
retention within the soil profile in the second study area appeared to
be better than in the area of lysimeter #1, perhaps due in part to the
decreased hydraulic loading in the second study. Levels of fecal coli-
form and fecal streptococci generally were reduced by more than 90% in
lysimeter waters within the first 1.5 feet. However, attenuation within
the soil profile was sporadic. Interestingly, the levels of organisms
at all depths decreased with each sampling time even though levels in
the irrigation pond were relatively consistent
As in the previous special irrigation study, both bacteriophage and
enteric viruses were detected in water intercepted below 1.5 feet.
Enteric virus isolations (as plaque-forming units) were confirmed off
both HeLa and BGM cell lines. On-the first day of lysimeter sampling 32
pfu of poliovirus 1 were recovered from the irrigation pond in a 20-
liter grab sample. Additionally, one viral isolate was confirmed from
the 3.0-foot depth and 11 pfu from 4.5 feet. No enteric virus isolations
were obtained in subsequent testing. Further discussion of these isolates
can be found under VIRAL RECOVERIES, IDENTIFICATION AND CHARACTERIZATION.
Overall, microbial analyses of water collected from lysimeters
indicate a movement of both indicator organisms and enteric viruses
106
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to a sampling depth of at least 4.5 feet under an effluent irrigated surface
when relatively intensive irrigation schedules are maintained. Unfortunately,
these observations are not readily explained. Possibilities include the spe-
cific nature of the subsurface soils, cracking of the surface soils, or virus
adsorption/elution as a result of high hydraulic loading (both precipitation
and irrigation).
MONITORING WELLS
Well Cuttings Study
Samples were collected from all five monitoring wells at 2-foot intervals
during drilling operations. The wells were drilled with a rotary rig and a
natural drilling mud. Samples were collected from the drilling cuttings as
they returned to the surface. The cuttings were analyzed and a lithologic log
prepared for each well. Binocular microscope studies were made of the cuttings
to determine lithology, texture, and mineralogocal content. The alluvial ma-
terial in the well cuttings was generally shattered and broken by the rotary
drilling operations and no accurate dexcription of size, sorting or degree of
cementation could be made.
In addition to the cuttings analysis, an offset study of the alluvial
gravel in wells #2, 3, and 4 was performed using an exposed outcrop of alluvial
materials in the stream bed of Third Creek immediately to the west of well #3.
The alluvial gravels are widespread in the vally and the outcrop is considered
to be representative of the alluvial gravels in the wells. The results of this
study are included in the lothologic logs, Table 48.
Hydrogeology
Groundwater occurs under water table conditions throughout the
portion of the valley of Third Creek which contains alluvial deposits of
gravel, sand, silt and clay, Wastewater which is applied as irrigation
enters this shallow groundwater system and is confined to the alluvial
sediments. The "marls of the underlying Upper Glen Rose Formation act as
an aquiclude and effectively prevent downward migration of groundwater
from the alluvial sediments in the valley fill to the deeper limestone
formations. No structural faulting or jointing was evident in exposures
of the Upper Glen Rose Formation in the study area.
The alluvial materials are highly variable both horizontally and
vertically with interfingering, discontinuous leases of clay, silt, sand
and gravel. The thickness of the sand and gravel layers may vary from a
few centimeters to 40 or 50 centimeters. Because of the extreme varia-
bility of the alluvial sediments, these materials are anisotropic to
groundwater flow, resulting in a wide range of yields for wells completed
within the same interval.
Typical water level contours at the Kerrville site as shown in
Figure 17 indicate that the groundwater in the valley alluvium moves
in a west to southwesternly direction towards Third Creek. The water
level contours are based on water level measurements in the observation
wells.
109
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TABLE 48. Lithologic Logs of Monitoring Wells
Thickness Depth Lithology
(ft) (ft)
Well //I
1 1 Surface soil, light yellow to brown loam. Calcareous.
Small limestone fragments (10%) 2-7 mm diameter.
2 3 Loam, light yellow. Numerous limestone fragments (60%)
2-10 mm diameter. Calcareous.
5 9 Limestone, thin bedded, weakly cemented Biomicrite.
29 38 Marl (a soft, loose, earthy deposit consisting chiefly
of 35-65% carbonate and 36-65% clays). Yellow, plastic.
Some limestone fragments, especially in upper portions.
27 65 Marl, gray, plastic, low permeability.
Total Depth » 65 feet
Well 12
1 1 Surface soil. Dark gray, silty clay. Few CaCOo con-
cretions. Calcareous.
15 16 Gray to brown silty clay. Few limestone fragments
(
-------
TABLE 48. Llthologic Logs of Monitoring Wells (Continued)
Thickness Depth Lithology
(ft) (ft)
Well f4
10 1C Surface soil. Light brown to gray. Sandy loam. Calcar-
eous. Few (5%) rounded limestone pebbles 2-10 mm diam-
eter.
6 16 Light brown silty clay. Gravelly, numerous limestone
and calcite pebbles. Pebbles rounded 2-10 mm diameter.
24 40 Gravel, limestone (60%) and chert (40%) 2-20 mm diameter.
Rounded moderate to poor sorting. Some calcite cementa-
tion rinds. High porosity.
5 45 Marl, gray, very plastic. Low porosity.
Total Depth = 45 feet
Well #5
1 1 Surface soil light brownish gray loam. 2-5% small lime-
stone fragments.
19 19 Limestone, thinly bedded. Weakly cemented, interbedded
with marl and clay. Biomicrite. Moderate to low poros-
ity.
6 25 Marl, yellow, very plastic. Low porosity.
20 45 Marl, gray, very plastic. Low porosity.
Total Depth * 45 feet
111
-------
Legend:
• 1621.1 Monitoring Uell with Elevation of
Water Table above Mean Sea Level
—1600— Elevation of ','atBr Table Contour
1602.2
1590
FIGURE 17 Groundwatar Contours, Ke-rville Si-s
112
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Movement of the groundwater is generally toward Third Creek where
it is either discharged to the creek as seeps and springs or continues
as subsurface flow in the alluvium down-valley. Stream flow measurements
at the upstream and downstream limits of the site indicate that there is
an average increase in stream flow of 245 gpm (0.35 mgd) due to ground-
water being discharged to the stream. Additional groundwater is discharged
downstream from the site as indicated by the numerous springs and seeps
in the valley area below the downstream gauging station.
The water levels in the observation wells varied greatly in their
response to the application of irrigation water and rainfall. Figure 18
summarizes the precipitation and irrigation at the Kerrville site.
Wells #1 and 5, which were completed primarily in the Upper Glen Rose
Formation and upslope from the major irrigation areas displayed the
smallest change in water levels (see Figures 19 and 20). Wells #3 and 4
are completed entirely in the alluvial materials and display the greatest
changes in water levels in response to irrigation or rainfall as is
shown in Figure 19.
Comparison of the water levels in each well with the irrigation and
precipitation recorded during the study (Figure 18) indicate that the
response of Wells #3 and 4 is closely related to irrigation and/or
precipitation episodes. The water level contours indicate that a large
amount of the groundwater which originates as either irrigation or pre-
cipitation on the site will move in the alluvium past wells #3 and 4.
On the other hand, the response of Wells #1 and 2 is not correlated
with irrigation/precipitation episodes, thereby indicating that ground-
water flow to those wells is not directly related to precipitation or,
irrigation in the immediate vicinity of the wells.
Chemical and Physical Analysis
The hydraulic response of the monitoring wells completed in the
valley alluvium demonstrates that the groundwater system responds to the
application of wastewater as irrigation and to precipitation on the
site. The groundwater was sampled at intervals during the study to
determine the quality of the groundwater and to compare groundwater
quality with the wastewater applied as irrigation. Results of the
chemical and physical analyses of the groundwater can be found in Tables
49 and 50, These data indicate that significant renovation of the
wastewater has occurred after vertical infiltration through approxi-
mately 250 cm of soil and horizontal movement through the sand, silts
and gravels of the alluvial material.
That the wastewater renovation from a chemical viewpoint has been
relatively effective is clearly indicated by the levels of TOC, Kjeldahl
nitrogen, and ortho- and total phosphate found in the well waters. The
fact that higher levels of nitrate-nitrogen were found in wells #1 and 5
may be explained by the lack of a crop in these areas, thereby precluding
significant nitrogen uptake. It also should be noted that the ammonia-
nitrogen level was highest in well #1.
The solids and turbidity data of Table 49 are reflective of both
113
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FIGURE 20. Hydrographs of Wells 4 and 5.
116
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the material in which the wells are completed as well as the intermittent
pumping for sampling. In fact, individual samples could be found to be
as much as 100% in excess of the mean for both turbidity and total
suspended solids. Under these cases (as with the mean values) only a
small percentage of these solids were volatile.
Total organic carbon (TOG) levels were found to be highest in wells
#1, 3 and 4. In the case of well #1, the influence of the landfill may
be the cause of the elevated TOC (note this level was as high as that
found in the lysimeters at 4.5 ft.). In the case of wells #3 and 4, the
higher TOC levels can be attributed to the irrigation process and the
direction of groundwater flow.
The influence of the landfill on well #1 may be evident in the
metallic ion concentration data, also (see Table 50). Zinc and lead
levels were found to be significantly higher in well #1 than in the
other monitoring wells. The high levels of iron in well #3 were
observed indirectly as an interference to other analyses. The fact that
wells #2, 3 and 4 have higher iron concentrations is most likely re-
flective of the aquifer material in that part of alluvium. The calcium
levels reflect the type material into which each of the wells was drilled.
Bacteriological Assay
Wells #1-4 at the Kerrville irrigation site were monitored routinely
over a 13-month period beginning in July, 1977. Fewer samples were
collected at well #5 due to a cement leak which produced high water pH
(12-13). This problem was corrected in April, 1978, and regular sampling
was initiated at that time.
During initial sampling at well #1 in July, 1977, false positive
isolations for total coliform occurred on two occasions. Although
green-pigmented colonies were observed, no positive fecal coliforms were
detected in either sample. Subsequent biochemical characterizations
resulted in the identification of these isolates as Pseudomonas species,
possibly from the sanitary landfill located upslope of the well or from
the groundwater itself.
Because of these early observations, limited laboratory testing was
undertaken to verify the identity of organisms counted as fecal coliforms.
Colonies were isolated from m-FC agar plates onto Heart Infusion Agar.
Oxidase testing and a commercially-available identification system
(Enterotube™, Roche Diagnostics) were employed to complete the specia-
tion of the bacterial isolates. The results presented in Table 51
demonstrated the validity of the fecal coliform counts from well waters.
An overview of bacteriological results from all five monitoring
wells is presented in Tables 52 and 53 as percentage of positive isola-
tions and ranges of organism densities, respectively. As demonstrated
by these data, water samples from all five wells showed evidence of
fecal pollution. After its redevelopment (see above), well #5 consis-
tently yielded water samples positive for all three bacterial indicators
118
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TABLE 50. Groundwater Metallic Ion Concentration (mg/1)*
Well No.
1
2
3
4
5
Well No.
1
2
3
4
5
Sample
A
B
A
B
A
B
A
B
A
B
Sample
A
B
A
B
A
B
A
B
A
B
Fe_
" .43
.31
4.17
4.37
10.06
11.96
8.34
8.23
2.52
1.69
Cd
<0.027
<0.027
<0.027
<0.027
<0.027
<0.027
<0.027
<0.027
<0.027
<0.027
M2
10.20
9.69
57.0
58.0
48.0
47.25
39.25
40.00
14.79
15.81
Zn
0.39
0.12
0.021
0.025
0.083
0.029
0.020
0.023
0.12
0.12
_c§
124.0
118.0
97.50
97.50
96.5
95.0
97.50
98.50
234.0
216.0
Mn
0.02
<.02
0.267
0.273
<0.037
<0.037
0.277
0.027
.06
.04
K
12.51
12.21
14.49
14.48
9.42
9.15
8.17
7.97
14.49
14.48
Pb
1.73
1.12
<0.037
<0.037
<0.037
<0.037
<0.037
<0.09
<0.09
Na
50.0
48.0
132.0
132.0
140.0
145.0
156.25
156.25
132.0
132.0
Cu N
0.03 <0.04
<0.02 <0.04
0.009 <0.26
0.004 <0.26
0.004 <0.26
0.196 <0.26
0.007 <0.26
<0.02 <0.04
<0.02 <0.04
Cr
.01
.01
<0.01
<0.21
<0.21
<0.21
<0.21
<0.01
<0.01
*Notes: Values are % recovered adjusted, except Cu and N; values reported as
< are less than the detection limits based on three spiked distilled
water samples.
119
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TABLE 51. Identification of Fecal Coliform Isolates
t
Sampling Fecal Coliforms
Date (cfu/100 ml)
9/77 3.0 x 10°
1/78 8.7 x 10°
No. of
isolates
4
2
10
6
1
1
I den ti f ication
Klebsiella pneumoniae
Enterobacter cloacae
Escherichia coli
Klebsiella pneumoniae
Citrobacter freundii
Enterobacter cloacae
f Sample co'llected from Well #4, enumerated on M-FC agar as described
previously.
TABLE 52. Percentage of Positive Isolations of Indicator
Organisms from Kerrville Monitoring Wells
Well Positive Samples (%)
Number Total Coliform
1 46
2 82
3 91
4 92
5 100
Fecal Coliform
40
47
67
86
100
Fecal Streptococci
43
39
54
64
100
120
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I
tr
ca
S
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rd
a
g
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i
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-------
at densities exceeding those found in other wells. The upslope placement
and depth of wells #1 and 5 coupled with the relatively lower organism
densities in well #1 is consistent with a source of fecal pollution in
well #5 from outside the irrigation site.
pf the remaining wells, #3 and 4 showed the greatest frequency of
samples positive for fecal coliforms. Bacteriological data for total
and fecal coliforms at well #2 were comparable to those observed at
wells #3 and 4. Apparently, the effects of effluent irrigation on
groundwater quality were at least as pronounced as any seepage from the
irrigation pond (a result which would have been detected in well #2).
Results of well water analyses of samples taken with greater frequency
over a two-week period are presented in Table 54. During this period,
both wastewater irrigation and rainfall occurred. As noted earlier,
wells #3 and 4 showed the greatest increase in both total and fecal
coliforms with levels peaking on October 24 and 25, 1977. Within two
days, the levels of fecal coliform had fallen to nondetectable levels in
well #3 while densities in well #4 persisted at a low level of 2x10
fecal coliform/100 ml six days later.
Viral Assays
During initial sampling conducted in July, 1977, well water samples
were plated directly and after concentration (20-liter volumes) for
detection of bacteriophage. Over a three-week period, wells #1, 3 and
4 were each sampled three times as described. A single coliphage
plaque-forming unit was recovered from a concentrated sample collected
at well #3 and another from a sample collected at well #4.
Because of the interest in detection of human enteric viruses in
groundwater at an effluent irrigation site, the emphasis in field sampling
was shifted to concentrating large volumes of well water for assay in
primate cell cultures. Therefore, bacteriophage screening was discon-
tinued except for occasional plating of extra concentrated volumes. No
further coliphages were isolated. This was not unexpected as concen-
tration procedures were optimized for enterovirus, not phage, recovery.
As enterovirus levels were extremely low in the irrigated wastewater,
it was necessary to concentrate maximal volumes of well water. Wherever
possible, the portable virus concentrator as described in Standard
Methods, 14th edition, was utilized in monitoring well waters.
The greatest obstacle in routine viral testing was low water produc-
tivity in several wells. As indicated by sample sizes concentrated
(Table 55), the yields from wells #1 and 2 were severely limited. For-
tunately, both wells #3 and 4 which had shown the greatest influence of
irrigation (a statement based on the bacteriological results cited
earlier) were amenable to routine 100 gallon sampling.
Over a twelve-month study period (9/77-8/78), a total of nineteen
separate large-volume samples (- 380 liters) were concentrated from both
wells #3 and 4. Smaller volumes (~ 20 liters) from wells #1 and 2 were
122
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TABLE 54. Selected Bacteriological Results.
Kerrville Monitoring Wells
Well
Number
1
2
3
4
Well
Number
1
2
3
4
Well
Number
1
2
3
4
Total Coliforms (cfu/100 ml)
10/13
0.3X10"1
<3.3xlO~
1.0x10°
2.2x10
10/20
3.3X10"1
8.3x10
10/24
<3.3xlO~I
5.0x10°
2.6xl02
6.0x10
10/25
4.3x10
S.SxlO1
10/27
1.0x10°
3-OxlO1
10/31
<3.3xlO~1
<3.3xlO~1
1.0x10°
1.5x10
Fecal Coliforms (cfu/100 ml)
10/13
<3.3xlO~1
0.3X10"1
<3, 3X10"1
2.0x10°
10/20
<3.3xlO~1
2.0X101
10/24
<3.3xlO~
1.3x10°
1.0x10°
1.6X101
10/25
2.6X101
2.4x10
10/27
<3.3xlO~1
1.3X101
10/31
<3.3xlO~1
<3.3X10~1
<3.3xlO~1
2.0x10°
Fecal Streptococci (cfu/100 ml)
10/13
<1.6xlO~1
<1.6xlO~1
<1.6xlO~1
<1.6xlO~1
10/20
1.6xlO~
2.6x10°
10/24
<1.6xlO~1
<1.6xlO~1
7.0xlO~
6.0x10°
10/25
2.1x10°
3.8x10°
10/27
1.6xlO~X
3.0x10°
10/31
<1.6xlO~1
<1.6xlO~
1.6xlO~
3.3xlO~
Week of 10/20/77; 3.2" rain, 1.9" wastewater irrigation;
Week of 10/27/77; 0.3" rain, 0.1" wastewater irrigation.
123
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concentrated using diatomaceous earth, as described earlier, when water
was available. Four samples ranging in size from 150 to 210 liters were
concentrated from well #5.
TABLE 55. Volumes Routinely Concentrated for Enteioviruses from Wells
Volumes Concentrated (liters)
Well
Number Routinely Maximally
1 20 40
2 20 50
3 380 630
4 380 760
5 150 210
A total of 21 potential viral isolates (as evidenced by any dis-
cernible areas of cpe) were picked from either HeLa or BGM cells during
the course of this field monitoring program. Of this number, only two
potential isolates from well #4 were passaged with production of cpe in
homologous tube culture. However, the cpe attributable to both iso-
lates could not be neutralized using the Lim-Benyesh-Melnick enterovirus
pools (A-H).
An earlier passage of each isolate then was forwarded to a diagnostic
virology laboratory (Texas Department of Health Resources) for isolation
and identification. Although both in vivo and in vitTO testing, specific
for human enterovirus detection, were done, no viruses were isolated
from either sample. Subsequently, several frozen (-76C) passages of
both isolates were passaged again in the UTSA laboratory; however, no
evidence of viral cpe could be detected.
As neither a positive identification nor a second recovery of viral
infectivity could be achieved, no positive results for the well samples
can be reported. This statement must be interpreted in light of the
fact that positive viral isolations were made from water collected in
soil at a depth of 4.5 feet (see lysimeters).
VIRAL RECOVERIES, IDENTIFICATION AND CHARACTERIZATION
Comparative Recoveries of Enteric Viruses on Primate Cell Lines
During the last year of field monitoring, assay procedures were
modified in an attempt to increase viral detection sensitivity. Low
124
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passage Buffalo Green Monkey cells (BGM 110) were obtained from Dr.
Gerald Berg, USEPA, Cincinnati, Ohio. Screening procedures for myco-
plasma contamination were conducted routinely on this cell line with
negative results. BGM cells along with HeLa cells were used in parallel
plaque assay systems. In addition, BS-C-1 cells were used in a tube
culture assay system for groundwater samples.
Data collected from these assay systems have been compiled to allow
evaluation of relative viral detection. No positive viral isolations
were made on the BS-C-1 cultures from either lysimeters or wells. After
six weeks of intensive sampling at the Kerrville site, use of this
system was discontinued. Representative comparative recoveries of
indigenous viruses from different samples on the two cell lines used in
the plaque assay system are shown in Table 56. HeLa monolayers yielded
the highest number of isolates during this field study. On only two
occasions were confirmed isolations made on BGM cells without parallel
positive results on HeLa. On the other hand, it was not uncommon to
observe positive viral isolations on HeLa cells in the absence of any
recoveries in the BGM plaque assay system. Additionally, in the identi-
fication of isolates no viral types were unique to BGM cells, i.e.,
viruses isolated on ' BGM (polio 1) also were recovered on HeLa monolayers.
TBBLE 56. Comparison of Indigenous Virus Recoveries (pfu)
on HeLa and BGM Cells
Recovery
Sample Date of Total pfu Observed* Ratio
Source Sampling HeLa BGM HeLa: BGM
Kerrville,
Raw Wastewater 4/5/78 22 2 11
Raw Wastewater 8/14/78 6 8 0.8
Irrigation Pond 5/1/78 21 11 1.9
Lysimeter 5/1/78 7 4 1.8
Uvalde STP 6/14/78
Raw Wastewater 484 30 16
Trickling Filter
Effluent 485 24 20
Pond 1 Effluent 850 16 53
Pond 2 Effluent 588 14 42
* Equal volumes plated.
125
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Viral Identification and Characterization
After the first year of plant monitoring, it became apparent that
due to treatment effectiveness, only low levels of enteroviruses were
being discharged into the irrigation system. Therefore, rigorous proce-
dures were instituted for reporting the presence of viruses in pondwaters,
surface water, groundwater, and soils. Isolates which could not be
passaged in a homologous tube culture system were not reported. Further,
every attempt was made to identify confirmed viruses by conventional
serum neutralization procedures. In most cases, viruses were identified
successfully, although mixed samples containing more than one virus
undoubtedly contributed to the nonidentification of some isolates.
Results shown in Table 57 summarize the identified viral types
recovered on both HeLa and BGM cell lines from the Kerrville irrigation
site during the last two years. The predominant isolates were categorized
as type 1 poliovirus. Of the total number of confirmed viruses from the
irrigation pond and lysimeter waters, poliovirus 1 accounted for 87% and
67% of the isolates respectively. Coxsackievirus B and ECHO viruses
also were recovered from these sources.
Because of the large number of viral isolates identified as poliovirus
1 using the Lim-Benyesh-Melnick enterovirus typing pools, further charac-
terization of these isolates was undertaken. Representative results
from thermal exposure testing are presented in Table 58. Visual in-
spection of calculated r values indicate that of the field isolates
tested, 75% showed no reduction in titer as a result of incubation at
40C. Laboratory seed virus, poliovirus 1 (Chat), responded as expected
showing a 1 log,n reduction while a reference strain of wild type polio-
virus 1 (Mahoney) showed no plaque reduction at elevated incubation
temperature.
•Obviously, these results must be interpreted with care. No single
marker can be used to define a strain as the attenuated vaccine or wild-
type virus. In addition, reversion of the poliovirus temperature sensitivity
marker has been, noted. Nevertheless, these results from ret testing
strongly suggest that the isolates recovered from field samples were not
identical with the laboratory seed virus, poliovirus 1 (Chat).
The implications of the isolation from any single water sample of
virions whose ret behavior parallels a reference wild-type should not be
exaggerated. In no way does it constitute evidence of either a reser-
voir of wild-type poliovirus in the general population or selective
survival of wild-type over attenuated virions. The relative prevalence
of wild-type and attenuated poliovirus in the raw wastewater at Kerrville
was not determined. In the absence of this information and of more
definitive markers, these data can simply be construed as suggestive.
126
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TABLE 57. Identification of Viral Isolates from the Kerrville
Irrigation Site
Sample Source
Lysimeters
1.5 feet
3.0 feet
4.5 feet
Irrigation Pond
Soils
Third Creek,
Upstream
Date of
Isolation
2/23/77
8/2/78
10/26/77
1/3/78
5/1/78
8/2/78
2/18/77
5/1/78
8/2/78
5/9/77
11/3/77
5/1/78
2/28/77
5/2/78
5/9/77
Viral
Identification
polio 1
Coxsackie B4
Coxsackie B5
Coxsackie B5
ECHO 21
ECHO 11
polio 1
Coxsackie B4
polio 1
polio 1
Coxsackie B4
Coxsackie B3
polio 1
polio 1
Coxsackie B5
polio 1
polio 1
polio 1
polio 1
Number of
Isolates
1
1
2
1
1
1
1
1
5
11
1
1
4
1
4
21
1
1
3
127
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TABLE 58. Temperature Characterization of Poliovirus 1 Isolates,
Kerrville Irrigation Site
titer (pfu/ml) r
Sample Source 37C 40Ct Value*
Soil, 5/2/78 5.5xl07 G.OxlO7 1.09
Lysimeter, 5/1/78
K-314 5.3xl07 5.5xl07 1.04
K-315 6.6xl07 9.3x10 1.40
K-316 5.2xl07 6.0xl07 1.15
K-317 4.5xl07 4.7xl06 0.10
K-318 5.3xl07 7.2xl06 0.14
K-319 8.8xl07 9.8xl07 1.11
K-320 9.1xl07 9.0x10 0.99
Poliovirus 1
(Chat) 6.2xl08 6.9x10 0.11
Poliovirus 1
(Mahoney) 2.7xl08 3.3xl08 1.22
t 40.1 ± 0.2C
^ _ Total number of plaques observed at 40C
r Total number of plaques observed at 37C
128
-------
REFERENCES
Akin, E. W., H. P. Pahren, W. Jakubowski, and J. B. Lucas. 1978. Health
hazards associated with wastewater effluents and sludges: Microbio-
logical considerations. In Proceedings of the Conference on Risk
Assessment and Health Effects of Land Application of Municipal Waste-
water and Sludges, B. P. Sagik and C. A. Sorber, eds. Center for Applied
Research and Technology, The University of Texas at San Antonio, San
Antonio, Texas.
American Public Health Association, American Water Works Association and
Water Pollution Control Federation. 1976. Standard methods for the
examination of water and wastewater, 14th ed. American Public Health
Association, Washington, D. C.
Bagdasaryan, G. A. 1964. Survival of viruses of the enterovirus group
(Poliomyelitis, ECHO, Coxsackie) in soil and on vegetables. J. Hyg.
Epidemiol. Microbiol. Immunol. 8:497.
Beard, P. J. 1938. The survival of typhoid in nature. J. Am. Water Works
Assoc. 30:124.
Beard, P. J. 1940. Longevity of Eberfhella typhosus in various soils. Am.
J. Public Health. 30:1077.
Bertucci, J. L., C. Lue-Hing, D. R. Zenz, and S. J. Sedita. 1975. Studies on
the inactivation rates of five viruses during anaerobic sludge diges-
tion. Report No. 75-21. Department of Research and Development,
Metropolitan Sanitary District of Greater Chicago.
Brown, G. 1961. The x-ray identification and crystal structures of clay
minerals. Mineralogical Society, London.
Burrows, W. 1968. Textbook of Microbiology. W. B. Sanders Publishers, New
York, New York.
Carlson, G. P., F. E. Woodard, D. F. Wentworth, and O. J. Sproul. 1968.
Virus inactivation on clay particles in natural waters. J. Water Pollut.
Control Fed. 40:R89.
Carp, R. I. and H. Koprowski. 1962. A new test of the reproductive capacity
temperature marker of poliovirus: The limited thermal exposure test.
Virology. 16:71.
Chase, E. S. 1964. Nine decades of sanitary engineering. Part III. Back
to the land. Water-Works Wastes Eng. 1:48.
129
-------
Clark, N. A. and S. L. Chang. 1959. Enteric viruses in water. J. Am. Water
Works Assoc. 51:1299.
Davenport, C. V., E. B. Sparrow, and R. C. Gorden. 1976. Fecal indicator
bacteria persistence under natural conditions in an ice-covered river.
Appl. Environ. Microbiol. 32:527.
DeMichele, E. 1974. Pathogenic organisms in the Murderkill River estuary.
J. Water Pollut. Control Fed. 46:773.
Drewry, W. A. and R. Eliassen. 1968. Virus movement in groundwater. J.
Water Pollut. Control Fed. 40:R257.
Duboise, S. M., B. E. Moore, B. P. Sagik, and C. A. Sorber. 1976a. The
effects of temperature and specific conductance on poliovirus survival
and transport in soil. Abstract for the National Conference on Environ-
mental Research, Development, and Design. University of Washington,
Seattle, Washington.
Duboise, S. M., B. E. Moore, and B. P. Sagik. 1976b. Poliovirus survival and
movement in a sandy forest soil. Appl. Environ. Microbiol. 31:536.
Dunlop, S. G. 1968. Survival of pathogens and related disease hazards. In
Proceedings, Municipal Sewage Effluent for Irrigation, C. W. Wilson and
F. E. Beckett, eds. Louisiana Tech Alumni Foundation, Ruston, Louisi-
ana.
Edmonds, R. L. 1976. Survival of coliform bacteria in sewage sludge applied
to a. forest clearcut and potential movement into groundwater. Appl.
Environ. Microbiol. 32:537.
Feachem, R. 1975. An improved role for faecal colifonn to faecal strepto-
cocci ratios in the differentiation between human and non-human pollu-
tion sources. Water Res. 9:689.
Foster, D. H. and R. S. Engelbrecht. 1973. Microbial hazards in disposing
of wastewater on soil. In Recycling Treated Municipal Wastewater and
Sludge through Forest and Cropland, W. E. Sopper and L. T. Kardos, eds.
Pennsylvania State University Press, University Park, Pennsylvania.
Fruh, E. G., P. Schmidt and B. Maguire. 1975. Final report on the before
and after studies on the effects of a power plant installation on Lake
Lyndon B. Johnson. Environmental Health Engineering Research Laboratory
Technical Report EHE-75-04, 'The University of Texas, Austin, Texas.
Funderburg, S., B. E. Moore, C. A. Sorber, and B. P. Sagik. 1978. Survival
of poliovirus in model wastewater holding ponds. Prog. Wat. Tech.
10:(5/6)619.
Geldreich, E. E. and B. A. Kenner. 1969. Concepts of fecal streptococci in
stream pollution. J. Water Pollut. Control Fed. 41:R336.
130
-------
Gerba, C. P., C. Wallis, and J. L. Melnick, 1975. Fate of wastewater bac-
teria and viruses in soil. J. Irrig. Drain. Div., Am. Soc. Civ. Engrs.
101:IRS.
Gilbert, R. G. , C. P. Gerba, R. C. Rice, H. Bowver, C. Wallis, and J. L. Mel-
nick. Virus and Bacteria Removal from Wastewater by Land Treatment.
1976. Appl. Environ. Microbiol. 32:333.
Gray, J. F. 1968. Practical irrigation with sewage effluent. In Municipal
Sewage Effluent for Irrigation, C. W. Wilson and F. E. Beckett, eds.
Louisiana Tech Alumni Foundation, Ruston, Louisiana.
Greenberg, A. E. and E. Kupka. 1957. Tuberculosis transmission by waste-
waters - A review. Sewage Ind. Wastes. 29:524.
Guentzel, M. Neal. 1978. Potential impact on water resources of bacterial
pathogens in wastewater applied to land. In Proceedings of the Confer-
ence on Risk Assessment and Health Effects of Land Application of Muni-
cipal Wastewater and Sludges, B. P. Sagik and C. A. Sorber, eds. Center
for Applied Research arid Technology, The University of Texas at San
Antonio, San Antonio, Texas.
Hesse, P. R. 1971. A textbook of soil chemical analysis. First American
Edition, Chemical Publishing Company, New York, New York.
Hinesly, T. D., R. L. Jones, E. L. Ziegler, and J. J. Tyler. 1977. Effects
of annual and accumulative applications of sewage sludge on assimilation
of zinc and cadmium by corn (Zea mays L.). Environ. Sci. Technol.
11:182.
Hook, J. E., L. T. Kardos, and W. E. Sopper. 1973. Effect of land disposal
of wastewaters on soil phosphorus relations. In Recycling Treated
Municipal Wastewater and Sludge through Forest and Cropland, W. E.
Sopper and L. T. Kardos, eds. Pennsylvania State University Press,
University Park, Pennsylvania.
Hori, D. H., N. C. Burbank, Jr., R. H. F. Young, L. S. Lau, and H. W. Klemmer.
1970. Migration of poliovirus type 2 in percolating water through
selected Oahu soils. Water Resources Research Center Technical Report
No. 36, University of Hawaii, Honolulu, Hawaii.
Hyde, H. C. 1976. Utilization of wastewater sludge for agricultural soil
enrichment. J. Water Pollut. Control Fed. 48:77.
Jackson, M. L. 1958. Soil Chemical Analysis. Prentice Hall, Inc., Englewood
Cliffs, New Jersey.
Jelinek, C. F., G. L. Braude, and R. B. Read, Jr. 1976. Management of sludge
use on land, FDA considerations. Presented at AMSA Conference on Sludge
Management, Houston, Texas.
131
-------
Kardos, L. T. and W. E. Sopper. 1973a. Renovation of municipal wastewater
through land disposal by spray irrigation. In Recycling Treated Muni-
cipal Wastewater and Sludge through Forest and Cropland, W. E. Sopper
and L. T. Kardos, eds. Pennsylvania State University Press, University
Park, Pennsylvania.
Kardos, L. T. and W. E. Sopper. 1973b. Effect of land disposal of wastewater
on exchangeable cations and other chemical elements in the soil. In
Recycling Treated Municipal Wastewater and Sludge through Forest and
Cropland, W. E. Sopper and L. T. Kardos, eds. Pennsylvania State Uni-
versity Press, University Park, Pennsylvania.
Katzenelson, E., B. Fattal, T. Hostovesky. 1976. Organic flocculation: An
efficient second-step concentration method for the detection of viruses
in tap water. Appl. Environ. Microbiol. 32:638.
Kirkham, M. B. 1974. Trace elements in sludge. Science 184:1030.
Lance, J. C., C. P. Gerba, and J. L. Melnick. 1976. Virus movement in soil
columns flooded with secondary sewage effluent. Appl. Environ. Micro-
biol. 32:520.
Larkin, E. P., J. T. Tierney, and R. Sullivan. 1976. Persistence of virus on
sewage-irrigated vegetables. J. Environ. Eng. Div., Am. Soc. Civ. Engrs.
102:EE1:29.
Malaney, G. W., P. A. Lutin, J. J. Cibulka, and L. H. Hickerson. 1967. Re-
sistence of carcinogenic organic compounds to oxidation by activated
sludge. J. Water Pollut. Control Fed. 39:2020.
McGauhey, P. H. and R. B. Krone. 1967. Soil mantle as a wastewater treatment
system. SERL Report No. 67-11, University of California, Berkeley,
California.
Moore, B. E., B. P. Sagik, and C. A. Sorber. 1978. Land application of
sludges: Minimizing the impact of viruses on water resources. In Pro-
ceedings of the Conference on Risk Assessment and Health Effects of Land
Application of Municipal Wastewater and Sludges, B. P. Sagik and C. A.
Sorber, eds. Center for Applied Research and Technology, The University
of Texas at San Antonio, San Antonio, Texas.
Moore, B. E., B. P. Sagik, arid J. F. Malina, Jr. 1975. Viral association
with suspended solids. Water Res. 9:197.
Moore, S., H. B. Petty, and W. N. Bruce. 1976b. Insecticide residues in
soybeans in Illinois, 1965-1974. 28th Illinois Custom Spray Operators
Training School, Summary of Presentations: 226.
Neefe, J. R. and J. Stokes. 1945. An epidemic of infectious hepatitis
apparently due to a water-borne agent. J. Am. Med. Assoc. 128:1063.
132
-------
Nomura, M. M. and R. H. F. Young. 1974. Fate of heavy metals in the sewage
treatment process. Technical Report No. 82. Water Resources Research
Center, University of Hawaii, Honolulu, Hawaii.
Pound, C. E. and R. W. Crites. 1973. Wastewater Treatment and Reuse by Land
Application, Volume II. EPA-660/2-73-006b, U. S. Government Printing
Office, Washington, D. C.
Process Design Manual for Sludge Treatment and Disposal. 1974. EPA 625/1-74-
006.
Reed, S. C., P. Murrmann, F. Koutz, W. Rickard, P. Hunt, T. Buzzell, K. Carey,
M. Bibello, S. Buda, K. Guter, and C. Sorber. 1972. Wastewater manage-
ment by disposal on the land. Corps of Engineers, U. S. Army, Special
Report 171. Cold Regions Research and Engineering Laboratory, Hanover,
New Hampshire.
Robeck, G. G., N. A. Clarke, and K. A. Dostal. 1962. Effectiveness of water
treatment processes in virus removal. J. Am. Water Works Assoc. 54:
1275.
Rudolfs, W., L. L. Falk, and R. A. Ragotzkie. 1951a. Contamination of
vegetables grown on polluted soil, I: Bacterial contamination. Sewage
Works J. 23:253.
Rudolfs, W., L. L. Falk, and R. A. Ragotzkie. 1951b. Contamination of
vegetables grown on polluted soil, II: Field and laboratory studies on
Endamoeba cysts. Sewage Works J. 23:498.
Rudolfs, W., L. L. Falk, and R. A. Ragotzkie. i951c. Contamination of
vegetables grown on polluted soil. III: Field studies on Asoain-s eggs.
Sewage Works J. 23:656.
Sanders, D. A., J. F. Malina, Jr., B. E. Moore, B. P. Sagik, and C. A. Sorber.
1979. Fate of poliovirus during anaerobic digestion. J. Water Pollut.
Control Fed. 51:333.
Sayler, A. S., J. D. Nelson, Jr., A. Justice, and R. R. Colwell. 1975. Dis-
tribution and significance of fecal indicator organisms in the upper
Chesapeake Bay. Appl. Environ. Microbiol. 30:625.
Schaub, S. A. and B. P. Sagik. 1975. Association of enteroviruses with na-
tural and artificially introduced colloidal solids in water and infec-
tivity of solids-associated virions. Appl. Microbiol. 30:212.
Schaub, S. A. and C. A. Sorber. 1977. Virus and bacteria removal from waste-
water by rapid infiltration through soil. Appl. Environ. Microbiol.
33:609.
Sepp, E. 1971. The use of sewage for irrigation - A literature review. Cal-
ifornia Dept. of Public Health, Bureau of Sanitary Engineering, Berkeley,
California.
133
-------
Sorber, C. A. and K. J. Guter. 1975. Health and hygiene aspects of spray
irrigation. Am. J. Public Health. 65:1.
Sorber, C. A., S. A. Schaub, and H. T. Bausam. 1974. An assessment of a
potential virus hazard associated with spray irrigation of domestic
wastewaters. In Virus Survival in Water and Wastewater Systems, J. F.
Malina, Jr. and B. P. Sagik, eds. Center for Research in Water Re-
sources, The University of Texas, Austin, Texas.
Thomas, R. E. and J. P. Law, Jr. 1968. Soil response to sewage effluent
irrigation. In Municipal Sewage Effluent for Irrigation, C. W. Wilson
and F. E. Beckett, eds. Louisiana Tech Alumni Foundation, Ruston,
Louisiana.
U. S. Department of Agriculture, Soil Conservation Service, Kerrville, Texas
U. S. Environmental Protection Agency. 1974. Manual of Methods of Chemical
Analyses of Water and Wastes. Methods Development and Quality Assurance
Laboratory, National Environmental Research Center, Cincinnati, Ohio.
Ward, R. L. and C. J. Ashley. 1976. Inactivation of poliovirus in digested
sludge. Appl. Environ. Microbiol. 31:921.
Wellings, F. M., A. L. Lewis, and C. W. Mountain. 1976. Demonstration of
solids associated virus in wastewater and sludge. Appl. Environ. Micro-
biol. 31:354.
Wellings, F. M., A. L. Lewis, C. W. Mountain, and L. V. Pierce. 1975. Demon-
stration of virus in groundwater after effluent discharge onto soil.
Appl. Environ. Microbiol. 29:751.'
Wells, D. M. 1968. Groundwater recharge with treated municipal effluent. In
Municipal Sewage Effluent for Irrigation, C. W. Wilson and F. E. Beckett,
eds. Louisiana Tech Alumni Foundation, Ruston, Louisiana.
Zenz, D. R., J. R. Peterson, D. L. Brooman, and C. Lue-Hing. 1976. Environ-
mental impacts of land application of sludge. J. Water Pollut. Control
Fed. 48:2332.
134
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APPENDIX A
Viral Identification and Characterization
Isolate
No.
S-8
S-9
S-10
S-ll
S-12
S-13
S-14
S-18
K-9
K-43
K-45
K-46
K-306
K-307
K-308
K-309
K-50
K-52
K-54
K-55
K-56
K-67
K-68
K-310
K-314
K-315
K-316
K-317
K-318
K-319
K-320
K-347
K-349
K-350
K-351
K-352
K-321
K-322
K-323
K-324
K-325
K-326
K-327
K-328
K-329
Sample
Source
Lysimeter 2-B
Lysimeter 2-B
Lysimeter 2-B
Lysimeter 2-B
Lysimeter 2-B
Lysimeter 2-B
Lysimeter 3-T
Soil #1
Lysimeter 1-M
Third Creek Up
Third Creek Up
Third Creek Up
Irrigation Pond
Irrigation Pond
Irrigation Pond
Irrigation Pond
Irrigation Pond
Irrigation Pond
Irrigation Pond
Irrigation Pond
Irrigation Pond
Lysimeter 1-M
Lysimeter 1-M
Soil @ Lys. 2
Lysimeter 2-B
Lysimeter 2-B
Lysimeter 2-B
Lysimeter 2-B
Lysimeter 2-B
Lysimeter 2-B
Lysimeter 2-B
Lysimeter 2-M
Lysimeter 2-B
Lysimeter 2-B
Lysimeter 2-B
Lysimeter 2-B
Irrigation Pond
Irrigation Pond
Irrigation Pond
Irrigation Pond
Irrigation Pond
Irrigation Pond
Irrigation Pond
Irrigation Pond
Irrigation Pond
Date of
Isolation
2/18/77
2/18/77
2/18/77
2/18/77
2/18/77
2/18/77
2/23/77
2/28/77
10/26/77
5/9/77
5/9/77
5/9/77
5/9/77
5/9/77
5/9/77
5/9/77
11/3/77
11/3/77
11/3/77
11/3/77
U/3/77
1/31/78
1/31/78
5/2/78
5/1/78
5/1/78
5/1/78
5/1/78
5/1/78
5/1/78
5/1/78
5/1/78
5/1/78
5/1/78
5/1/78
5/1/78
5/1/78
5/1/78
5/1/78
5/1/78
5/1/78
5/1/78
5/1/78
5/1/78
5/1/78
Cell
Line
HeLa
ReLa
HeLa
HeLa
HeLa
Mixed
Culture
HeLa
HeLa
HeLa
HeLa
HeLa
HeLa
HeLa
HeLa
HeLa
HeLa
HeLa
HeLa
HeLa
HeLa
HeLa
HeLa
HeLa
HeLa
HeLa
.HeLa
HeLa
HeLa
HeLa
HeLa.
HeLa
BGM
BGM
BGM
BGM
BGM
HeLa
HeLa
HeLa
HeLa
HeLa
HeLa
HeLa
HeLa
HeLa
Poliovirus 1
Viral Characterization
Identification r#
Polio 1
Polio 1
Polio 1
Polio 1
Polio 1
Possibly Polio 1
+ ECHO 21
Polio 1
Polio 1
Coxsackie B5
Polio 1
Polio 1
Polio 1
Polio 1
Polio 1
Polio 1
Polio 1
Polio 1
Coxsackie B5
Coxsackie B5
Coxsackie B5
Coxsackie B5
ECHO 21
ECHO 11
Polio 1
Polio 1
Polio 1
Polio 1
Polio 1
Polio 1
Polio 1
Polio 1
Polio 1
Polio 1
Polio 1
Polio 1
Polio 1
Polio 1
Polio 1
Polio 1
Polio 1
Polio 1
Polio 1
Polio 1
Polio 1
•Polio 1
1.09
1.04
1.4
1.15
0.104
0.14
1.1
0.99
0.7
0.78
1.26
# r = total number of pfu observed at 40C: total number observed at 37C.
135
-------
APPENDIX A (continued)
Viral Identification and Characterization
Isolate
No.
K-330
K-331
K-332
K-333
K-334
K-335
K-336
K-337
K-338
K-339
K-340
K-341
K-353
K-354
K-355
K-356
K-357
K-358
K-359
K-360
K-362
K-363
K-364
K-371
K-373
K-374
K-375
K-377
K-378
K-379
K-385
K-47
Sample
Source
Irrigation Pond
Irrigation Pond
Irrigation Pond
Irrigation Pond
Irrigation Pond
Irrigation Pond
Irrigation Pond
Irrigation Pond
Irrigation Pond
Irrigation Pond
Irrigation Pond
Irrigation Pond
Irrigation Pond
Irrigation Pond
Irrigation Pond
Irrigation Pond
Irrigation Pond
Irrigation Pond
Irrigation Pond
Irrigation Pond
Irrigation Pond
Irrigation Pond
Irrigation Pond
Lysimeter 1-T
Lysiraeter 2-B
Lysimeter 2-M
Lysimeter 2-T
Lysimeter 2-T
Lysimeter 2-B
Lysimeter 2-T
Irrigation Pond
Sediment
Third Creek Down
Date of
Isolation
5/1/78
5/1/78
5/1/78
5/1/78
5/1/78
5/1/78
5/1/78
5/1/78
5/1/78
5/1/78
5/1/78
5/1/78
5/1/78
5/1/78
5/1/78
5/1/78
5/1/78
5/1/78
5/1/78
5/1/78
5/1/78
5/1/78
5/1/78
8/2/78
8/2/78
8/2/78
8/2/78
8/2/78
8/2/78
8/2/78
9/13/78
11/25/77
Cell
Line
HeLa
HeLa
HeLa
HeLa
HeLa
HeLa
HeLa
HeLa
HeLa
HeLa
HeLa
HeLa
B<2>..
BGM
BGM
BGM
BGM
BGM
BGM
BGM
BGM
BGM
BGM
BGM
HeLa
HeLa
HeLa
HeLa
HeLa
HeLa
HeLa
HeLa
Poliovirus 1
Viral Characterization
Identification r#
Polio 1 0.9
Polio 1
Polio 1 0.84
Polio 1 1.0
Polio 1 1.02
Polio 1
NI*
NX
NI
NI
NI
NI
Polio 1
NI
Polio 1
Polio 1
NI
Polio 1
NI
NI
Polio 1
Polio 1
NI
NI
Coxsackie B4
Coxsackie B4
Coxsackie B4
Coxsackie B5
Coxsackie B3
Coxsackie B5
Coxsackie B5
NI
#r = total number of pfu observed at 40C: total number observed at 37C.
*NI = virus confirmed by tube culture passage but not identified.
136
-------
APPENDIX B
Growth of Bacterial Indicators in Lysimeter Waters
Lysizneter, 1.5 feet (pH 7.4)
Sampling
Time (days)
Total Co li forms
1
2
3
4
5
6
7
10
Fecal Streptococci
1
2
3
4
5
6
7
10
Total Coliforms
1
2
3
4
5
6
7
10
Fecal Streptococci
1
2
3
4
5
6
7
10
Teitipe rature
4C
cfu/lOOml
6.1xl04
1.8xl04
6.7xl04
9.7xl04
8-lxlO4
7.2x104
8.7xl04
8.2xl04
4.5xl03
3. 0x10 3
3.5xl03
3.3xl03
2.6xl03
1.7xl03
1.3xl03
1.3xl03
3.2xl04
1.6xl04
l.SxlO4
3.8xl04
l.SxlO4
1.3xl04
l.lxlO4
9. 9x10 3
l.SxlO3
l.lxlO3
6.3xl02
3.1xl02
8.3X101
6-OxlO1
e.OxlO1
3.7xK>l
N/No
1.0x10°
3.0x10"!
1.1x10°
1.6x10°
1.3x10°
1.2x10°
1.4x10°
1.3x10°
1.0x10°
6.7x10-1
7.8x10-1
7.3x10-1
5.8x10"!
3.8x10"!
2.9x10"!
2.9X10"1
1.0x10°
5.0x10-1
4.7x10-1
1.2x10°
4.7x10"!
4.1x10"!
3. 4x10" !
3.1x10"!
1.0x10°
7.3x10-1
4.2x10"!
2.1x10-1
5.5xlO-2
4.0xlO-2
4.0xlO-2
2.5x10-2
20C
cfu/lOOml
6.1xl04
1.9xl04
3.2xl04
5 . 7xl04
3.2xl04
1.3xl04
8.8xl03
4.1xl03
N/No
1.0x10°
3.1x10"!
5 . 2x10-1
9.3x10"!
5.2x10"!
2.1x10-1
1.4x10"!
6.7xlO"2
4.5xl03 ! 1.0x10°
4.2xl03
1.2xl03
9.1xl02
3.5xl02
1.4xl02
6.7X101
1.7X101
3.2xl04
l.SxlO4
1.0 xlO4
2.3xl04
8. 4x10 3
6.3xl03
7.6xl03
4. 3x10 3
l.SxlO3
3.0xl02
l.SxlO2
l.OxlO2
8.7xlQl
8.7x101
G.OxlO1
l.OxlQl
9.3x10"!
2.7x10-1
2.0x10"!
7. 8x10 "2
3-lxlO"2
l.SxlO"3
3.8xlO~3
1.0x10°
4.7x10-1
3.1x10"!
7.2x10"!
2.6x10"!
2.0x10"!
2.4x10"!
l.SxlO"1
1.0x10°
2.0x10-1
1.2x10-1
6.7xlO-2
5.8xlO~2
5.8xlO-2
4. 0x10" 2
6.7xlO-3
35C
cfu/lOOml
6.1xl04
8.2xl04
3.3xl04
6.0xl03
1.4xl03
6.1xl02
4.1xl02
1.2xl02
4. 5x10 3
6.7xl03
7.8xl02
9-OxlO1
3.3X101
1.3x10
6 . 7x10°
6.7x10°
3.2xl04
3.7xl04
1.2xl04
7. 2x10 3
2. 7x10 3
7.3xl02
4. 1x10 2
7.7X101
l.SxlO3
9.8xl02
2.4xl02
1.7xlQl
3.3x10°
6.7x10°
1.3xlQl
6.7x10°
N/No
1.0x10°
1.3x10°
5.4x10"!
9.8xlO"2
2.3x10-2
l.OxlO"2
6.7xlO~3
2.0xlO~3
1.0x10°
1.5x10°
1.7x10"!
2.0xlO~2
7.3xlO~3
2.9xlO~3
1.5x10-3
1.5xlO~3
1.0x10°
1.2x10°
3.8x10"!
2.3x10"!
8.4xlO~2
2.3xlO~2
1.3xlO"2
2.4xlO~3
1.0x10°
6.5x10-1
1.6x10"!
l.lxlO"2
2.2x10-3
4.5xlO"3
8.6x10-3
4.5xlO-3
137
-------
TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing/
1. REPORT NO.
EPA-600/1-80-004
3. RECIPIENT'S ACCESSION NO.
4. TITLE AND SUBTITLE
Human Enteric Virus Survival in Soil Following
Irrigation With Sewage Plant Effluents
5. REPORT DATE
June 1980
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
Bernard P. Sagik, Barbara E. Moore, and
Charles A. Sorber
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Center for Applied Research and Technology
The University of Texas at San Antonio
San Antonio, Texas 78285
10. PROGRAM ELEMENT NO.
1BA607
11. CONTRACT/GRANT NO.
R-803844-03
12. SPONSORING AGENCY NAME AND ADDRESS
Health Effects Research Laboratory
Office of Research & Development
U. S. Environmental Protection Agency
Cincinnati. Ohio 45268
13. TYPE OF REPORT AND PERIOD COVERED
Final: 7/28/75-1/27/7Q
14. SPONSORING AGENCY CODE
EPA /600/10
15. SUPPLEMENTARY NOTES A report on this work presented at Water Pollution Control
Federation annual meeting, Houston, Texas, October 1979: "Viral Transport to Ground-
water and Operational Wastewater Land Application Sites" by Sagik, Sorber, & Moore.
16-ABSTfftSTwastewater "treatment processes at Kerrville and Uvalde, Texas, were evalua-
ted in terms of their efficacy in reducing human enteric viruses. (Data on the re-
duction of TOC, BOD , suspended solids, orthophosphate, nitrogenous compounds, total
coliform, fecal colxform, and bacteriophage were also obtained.) Enteric viruses
were reduced by greater than 99% at Kerrville and at least 99% at Uvalde. These
waters are used for irrigation without disinfection. Soil samples at the Kerrville
and Uvalde application sites yielded both fecal coliforms and bacteriophages. In
addition, two confirmed enterovirus isolations were made at the Kerrville site.
Lysimeters placed 1.5 ft, 3.0 ft, and 4.5 ft depths at the Kerrville site yielded
large numbers of bacteriophage isolates. In addition, ten lysimeter samples yielded
a total of 29 confirmed viral isolates. This is a strikingly high number of isola-
tions of indigenous enteric viruses, relative to the irrigation pond which was de-
monstrably low in viruses (when assayed on the same cell lines). Cell changes (CPE)
but no confirmed isolations were made from five monitoring wells.
These studies of wastewater treatment plants processing dilute to moderate
strength sewage in efficient treatment schemes represent a best possible case" for
the use of undisinfected, domestic wastewater effluents for irrigation. The isolation
of enteroviruses in water from lysimeters but not from the monitoring wells suggests
that depth to groundwater should be a critical factor in the selection of irrigation
sites. From data developed in this study, it appears that a depth of 4.5 ft is not
sufficient for effective viral attenuation in soils such as those described in this
report.
17.
KEY WORDS.AND DOCUMENT ANALYSIS
DESCRIPTORS
b.IDENTIFIERS/OPEN ENDED TERMS C. COS AT I Field/Group
enterovirus, waste disrosal water re-
sources, waste treatment public health
lysimeters, monitoring
wells, land disposal,
virus penitration througl
soil, southwestern USA
06M
68D
57K
57U
18, DISTRIBUTION STATEMENT
Release to public
19. SECURITY CLASS (ThisReport)
Unclassified
21. NO. OF PAGES
152
20. SECURITY CLASS (Thispage)
TTnclassif-jpd
22. PRICE
EPA Form 2220-1 (9-73)
138
1 POINTING OFHfE 1980-657-146/5689
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