AN EVALUATION OF POTENTIAL INFECTIOUS HEALTH
EFFECTS FROM SPRINKLER APPLICATION OF
WASTEWATER TO LAND: LUBBOCK, TEXAS
SECOND INTERIM REPORT
EPA Cooperative Agreement CR 807501
SwRI Project01-6097
LCCIWR Subcontract on
EPA Grant CR 806204
SwRI Project 01-6001
Prepared by
D. E. Camann,1 R. L. Northrop,2 P. J. Graham,2
M. N. Guentzel,3 H. J. Harding,1 K. T. Kimball,1
R. L. Mason,1 B. E. Moore,4 C. A. Sorber,4
C. M. Becker,2and W. Jakubowski6
Southwest Research Institute,1 San Antonio, Texas, 78284
University of Illinois of Chicago,2 Chicago, Illinois, 60680
University of Texas at San Antonio,3 San Antonio, Texas, 78285
University of Texas at Austin,4 Austin, Texas, 78712
Environmental Protection Agency,5 Cincinnati, Ohio, 45268
Prepared under EPA Cooperative Agreement for:
Walter Jakubowski, Project Officer
U.S. Environmental Protection Agency
Health Effects Research Laboratory
Cincinnati, Ohio 45268
Prepared under LCCIWR Subcontract for:
Dennis B. George, Project Director
LCC Institute of Water Research
Lubbock, Texas 79407
Jack L. Witherow, Project Officer
U.S. Environmental Protection Agency
Kerr Environmental Research Laboratory
Ada, Oklahoma 74820
May 1983
SOUTHWEST RESEARCH INSTITUTE
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AN EVALUATION OF POTENTIAL INFECTIOUS HEALTH
EFFECTS FROM SPRINKLER APPLICATION OF
WASTEWATER TO LAND: LUBBOCK, TEXAS
SECOND INTERIM REPORT
EPA Cooperative Agreement CR 807501
SwR I Project 01-6097
LCCIWR Subcontract on
EPA Grant CR 806204
SwRI Project 01-6001
Prepared by
D. E. Camann,1 R. L. Northrop,2 P. J. Graham,2
M. N. Guentzel,3 H. J. Harding,1 K. T. Kimball,1
R. L. Mason,1 B. E. Moore,4 C. A. Sorber,4
C. M. Becker,2 and W. Jakubowski5
Southwest Research Institute,1 San Antonio, Texas, 78284
University of Illinois of Chicago,2 Chicago, Illinois, 60680
University of Texas at San Antonio,3 San Antonio, Texas, 78285
University of Texas at Austin,4 Austin, Texas, 78712
Environmental Protection Agency,5 Cincinnati, Ohio, 45268
Prepared under EPA Cooperative Agreement for:
Walter Jakubowski, Project Officer
U.S. Environmental Protection Agency
Health Effects Research Laboratory
Cincinnati, Ohio 45268
Prepared under LCCIWR Subcontract for:
Dennis B. George, Project Director
LCC Institute of Water Research
Lubbock, Texas 79407
Jack L. Witherow, Project Officer
U.S. Environmental Protection Agency
Kerr Environmental Research Laboratory
Ada, Oklahoma 74820
May 1983
APPROVED:
Donald E. Johnson, Director
Department of Environmental Sciences
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SOUTHWESTRESEARCH INSTITUTE
POST OFFICE DRAWER 28510 • 6220 CULEBflA ROAD • SAN ANTONIO, TEXAS. USA 78284 • (512) 684-5111'TELEX 76-7357
DIVISION OF CHEMISTRY
AND CHEMICAL ENGINEERING
June 20, 1983
TO: Lubbock Land Treatment Project Advisory Committee Distribution
FROM: David E. Camann, Staff Scientist
Southwest Research Institute
SUBJECT: Second Interim Report, Lubbock Health Effects Study
SwRI Projects 01-6097 and 01-6001
EPA Cooperative Agreement CR 807501
LCCIWR Subcontract on EPA Grant S 806204
Enclosed is the subject report, which is a separate volume of the
Second Interim Progress Report of the Lubbock Land Treatment Research and
Development Project. The subject report presents methods, results, and
findings through 1982, in the format of our future final report. I look
forward to discussing the highlights with you in Lubbock on July 11.
£.
Distribution:
Robert G. Fleming
Charlene Foushee
Dennis B. George (unbound and 4 bound)
Clint Hall
Walter Jakubowski (unbound and 4 bound)
Ancil Jones
H . George Keeler
Jack W. Keeley
Myron Knudson
William A. Rosenkranz
Richard E. Thomas
Richard Whittington
Jack L . Witherow
cc: Curtis C. Harlin, Jr.
T. A. Hicks .
Raymond Mittel
Bob Sweazy
Wade Talbot
Sam Wahl
SAN ANTONIO, TEXAS
WITH OFFICES IN HOUSTON. TEXAS. AND WASHINGTON. DC
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PREFACE
The LCC Institute of WatervResearch (LCCIWR), Lubbock, Texas, is
conducting a five-year (1979-t983) research and demonstration program
entitled the Lubbock Land Treatment Project to expand and study Lubbock's
municipal wastewater land treatment system. A pipeline, storage
reservoirs, distribution system, and spray irrigation equipment have been
installed at the Hancock farm site, located about 15 miles southeast of the
sewage treatment plant and the edge of Lubbock. The research programs of
the Lubbock-Landf-reatment-Project include ground water recovery studies at
a farm practicing land application of wastewater for over 40 years (the
Gray site), a health effects study at the Hancock site, and impact studies
on crops, spil, and ground water.
The five-yea'r study, "An Evaluation of Potential Infectious Health
Effects from Sprinkler Application of Wastewater to Land: Lubbock, Texas,"
is being conducted by Southwest Research Institute (SwRI), the University
of Illinois (UI), and the University of Texas at San Antonio (UTSA) and
Austin (UTA). This Lubbock Health Effects Study (LHES), as it is referred
to throughout the report, has been funded primarily through a cooperative
agreement with the U.S. Environmental Protection Agency, Health Effects
Research Laboratory (EPA-HERL) and a subcontract from LCCIWR.
m
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CONTENTS
Preface iii
Figures ix
Tables xi
Abbreviations xvii
1. Introduction 1
Background 1
Land application and potential infectious hazards 1
Recent literature . 2
The Lubbock Health Effects Study (LHES) 3
Study objective 4
Study organization 5
2. Conclusions 9
3. Recommendations 10
4. Methods and Materials 11
Study design 11
Principles of design 11
Approach 14
Monitoring schedule 15
Health watch 18
Environmental monitoring 18
Study site 21
Study area 21
General climatology 21
City of Wilson 25
Rural area 25
Lubbock sewage treatment plants 26
Lubbock land treatment system 26
Study population 29
Sampling 29
Health interview and recruitment 31
Poliovirus immunization 33
Health watch 35
Serosurvey 35
Tuberculin skin testing 35
Household health diary 36
Illness specimens 36
Illness surveillance 38
Fecal specimens 38
Activity diary 39
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Contents (Cont'd)
Environmental sampling 41
Wastewater 41
Wastewater pathogen screens 41
Wastewater sampling collection in 1981 41
Wastewater sample collection in 1982 42
Wastewater aerosol 42
Background runs—1980 baseline year 42
Wastewater aerosol monitoring—1982.Irrigation Year 47
Microorganism runs 47
Quality assurance runs 48
Enterovirus runs 51
Dye runs 51
Particle size runs 54
Dust storm runs 57
Calculation of microorganism density in air 57
Flies 60
Meteorological data 62
Background aerosol runs 62
General climatology 62
Meteorological measurements during aerosol runs 63
Laboratory analysis 71
Clinical specimens 71
Serology 71
Serum processing and storage . 71
Selection of serologic antigens 71
Serologic methods 77
Clinical bacteriology 87
Clinical virology 91
Electron microscopy of fecal specimens 94
Environmental samples 95
Wastewater samples 95
Microbiological screens 95
Routine wastewater samples 110
Enterovirus identification samples 110
Limited bacterial screen samples 110
Legionella samples 111
Aerosol samples 112
Fly samples 114
Data management 117
Sample labels 117
Reporting forms 121
Data processing 121
Data verification 123
Data summaries 123
Quality assurance 125
Health watch 125
Aerosol measurement precision 125
VI
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Contents (Cont'd)
Page
Laboratory analysis 129
Serology (hepatitis A) 129
Virus serology 132
Clinical bacteriology 136
Clinical virology 141
Electron microscopy 141
Environmental samples 143
Data management 143
Archiving of clinical specimens 146
Data analysis 147
Describe pattern of infections 147
Association of infection with exposure 147
Exposure estimation 148
Identification of infection episodes 153
Statistical approach 155
Serology 155
Fecal specimens 163
Tuberculin test 165
Health diaries 166
Interpretation of the statistical results 167
Results and Discussion 171
Health data 171
Description of the study population 171
Health watch sampling 171
Health diary data 180
Illness specimens 180
Clinical bacteriology 191
Data 191
Patterns of infection 195
Interpretation of fecal and illness specimen bacterial
data 199
Clinical virology • 203
Electron microscopy of fecal specimens 209
Tuberculin test data 209
Serologic data 209
Serum neutralization serology 209
Reoviruses 215
Hepatitis A 215
Environmental data 219
Microorganism levels in wastewater 219
24-Hour composite samples 219
Search for Legionella isolates from 24-hour composite
samples • 235
30-Minute composite samples 242
Vll
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Contents (Cont'd)
Microorganism levels in air 247
Aerosolization efficiency 247
Aerosol viable particle size 247
Background runs 252
Microorganism runs 255
Virus runs 261
Summary of microorganism data 264
Aerosol exposure 264
Microorganism levels on flies 267
Microorganism levels in drinking water 267
Activity patterns 267
Exposure estimates and groups 272
References 279
Appendixes
A. Personal Interview for Health Watch A-l
B. Personal Interview Update B-l
C. Informed and Parental Consent Forms C-l
D. Activity Diaries and Maps D-l
E. Procedure for Wastewater Sample Collection, Lubbock
Southeast Water Reclamation Plant E-l
F. Procedure for Wastewater Sample Collection, Wilson
Imhoff Tank Effluent F-l
G. Description of Litton Model M High-Volume Aerosol
Sampler G-l
H. Decontamination Procedure for Model M Samplers H-l
I. Collection Efficiency of Litton Model M Large Volume
Samplers 1-1
J. Data Reporting Forms J-l
Glossary
Cross Reference Index
vi ii
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FIGURES
Number Page
1.1 Principal investigators and functional areas 6
4.1 LHES monitoring schedule, June 1980-October 1983 16
4.2 Regional map of the study area 22
4.3 Wind frequencies for the two-month period of March-April,
Lubbock, Texas 23
4.4 Wind frequencies for the two-month period of July-August,
Lubbock, Texas 24
4.5 Map of the Hancock site 28
4.6 Sampling zones comprising the study area 30
4.7 Sampler locations for background runs 46
4.8 Wind frequencies for the 1982 spring irrigation period:
Hancock farm meteorological station 64
4.9 Wind frequencies for the 1982 summer irrigation period:
Hancock farm meteorological station 65
4.10 Competitive binding of anti-HAV in serum with radio-
actively tagged anti-HAV to HAV coated on a solid
phase 78
4.11 Titration of anti-HAV in serum by the HAVAB® test 80
4.12 Development of anti-HAV in subject with hepatitis A 81
4.13 Isolation and identification of selected organisms from
feces 88
4.14 Isolation and identification of organisms from throat
swabs 89
4.15 Illness specimen log 92
4.16 Viral isolation from clinical specimens 93
4.17 Isolation of Gram-negative enteric bacteria from
wastewater 105
4.18 Analyses of insect vectors 115
4.19 Data flowchart for LHES 122
4.20 Flow diagram for specific objective 3, association of
infection with exposure 149
4.21 Relation of activity diary collection weeks to periods
of wastewater irrigation 152
5.1 Time series of fecal coliform and corrected enterovirus
densities in Lubbock pipeline, Hancock reservoir, and
Wilson wastewater 231
IX
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Figures (Cont'd)
5.2 Time series of physical analyses of Lubbock pipeline,
Hancock reservoir, and Wilson wastewater 232
5.3 Particle sizes of the Andersen sampler stages are designed
to simulate deposition in the human respiratory system 249
5.4 Drinking water sampling locations 269
5.5 Location of home of activity diary respondents 275
5.6 Time spent in Lubbock by activity diary respondents 276
5.7 Distribution of preliminary exposure index for the 1982
preplanting irrigation period 277
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TABLES
Number Page
1.1 Principal Participating Personnel and Areas of Activity 7
4.1 Suggested Prevalence of Antibody and Seasonal Occurrence of
Infection for Agents Potentially Present tn Wastewater 12
4.2 Irrigation Plan for Hancock Farm in 1983 17
4.3 Measurement in Wastewater of Interpretable Infectious Agents
Monitored in the Health Watch 19
4.4 Comparison of Study Populations in 1980, 1981 and 1982 32
4.5 Summary of Participant Poliovirus Protection Status 34
4.6 Comparison of Sentinel Population to Original Population 37
4.7 Wastewater Sampling Dates, 1980-81 43
4.8 Wastewater Sampling and Assay Schedule: 1982 44
4.9 Summary of Sampling Conditions—Aerosol Runs—Operational
Year 1982 49
4.10 Sampler Operating Voltage on the Microorganism Aerosol Runs 50
4.11 Summary of Sampling Conditions—Quality Assurance Runs--
Operational Year 1982 52
4.12 Summary of Sampling Conditions—Virus Runs--0perational
Year 1982 53
4.13 Summary of Sampling Conditions—Dye Runs--0perational
Year 1982 55
4.14 Summary of Sampling Conditions—Particle Size Runs--
Operational Year 1982 56
4.15 Correction Factor for LVS Operating Voltage 59
4.16 Summary of Meteorological Conditions—Aerosol Runs--
Operational Year 1982 66
4.17 Summary of Meteorological Conditions--Quality Assurance
Runs—Operational Year 1982 67
4.18 Summary of Meteorological Conditions—Virus Runs--
Operational Year 1982 68
4.19 Summary of Meteorological Conditions--Dye Runs—Operational
Year 1982 69
4.20 Summary of Meteorological Conditions—Particle Size Runs--
Operational Year 1982 70
4.21 Virus Types 73
4.22 Incidence of Anti-HAV in Specimens from Different
Populations as Determined by the HAVAB® Test 79
4.23 Semiquantitative Reporting of Growth by Four Quadrant
Plating Method 90
xi
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Tables (Cont'd)
Page
4.24 Recovery of Salmonella from Wastewater Samples Using
Two Procedures 97
4.25 Comparison of Procedures for Recovery of Yersinia
enterocolitica--Unseeded Samples 100
4.26 Comparison of Procedures for Recovery of Yersinia
enterocolitica—Seeded Samples 101
4.27 Parallel Testing of Clostridium perfringens Assays:
Comparison of Multiple Tube Inoculation and Membrane
Filtration Techniques 103
4.28 Viral Types Recovered from Wastewater by the Bentonite
Adsorption Procedure 106
4.29 Viral Isolates Recovered from the Same Wastewater Samples
by Various Assay Procedures 108
4.30 Enterovirus Assay Matrix for Wastewater Samples 107
4.31 Concentration Efficiency of Organic Flocculation and
Two-Phase Separation 113
4.32 LHES Health Data Processing Status Report 118
4.33 LHES Serology Data Processing Status Report 120
4.34 Sampled Microorganism Densities on the Quality Assurance
Aerosol Runs 126
4.35 Consistency of Aerosol Measurement Precision Over Density
Range 128
4.36 Estimated Magnitude of Sources of Precision Variation 130
4.37 Quality Assurance Testing of Unknown Sera Using the
HAVAB® Competitive Binding Assay 131
4.38 HAVAB® Results for Replicate Sera Shipped Under Three-
Digit Code by Northrop's Laboratory 133
4.39 Quality Assurance, Clinical Bacteriology 137
4.40 Clinical Bacteriology Quality Assurance Unknowns 139
4.41 Quantisation of Growth by the Four Quadrant Method 140
4.42 Viral Quality Assurance Testing 142
4.43 Quality Assurance, Replicate Environmental Analyses 144
4.44 Comparison of Bacterial Indicator Values Reported by
Separate Laboratories 145
4.45 Number of Cases (bg,) Required for Rejection of PI=P? in
Favor of PiPi in
Binomial Populations 159
4.48 Sample Size Required for Testing Pi=P2 Versus Pi
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Tables (Cont'd)
4.50 Criteria for Judging Quality of Wastewater Evidence for
Each Microorganism 168
5.1 Initial Interview: Demographic Characteristics of
Households and Individuals 172
5.2 Initial Interview: Dwellings 173
5.3 Initial Interview: Crops and Livestock 174
5.4 Initial Interview: Exposure to Wastewater 175
5.5 Initial Interview: Health History 177
5.6 Samples Collected for Health Watch Activities 178
5.7 LHES Blood Donor Status for Participants Currently in Study 181
5.8 Summary of Fecal Donor Information for Participants During
1982 182
5.9 Activity Diary Compliance for Current Population 183
5.10 Comparison of Total Acute Illness Incidence Rates for First
Three Years of Study 184
5.11 Incidence of Self-Reported Acute Illnesses in Study
Population 185
5.12 Comparison of Total Acute Illness Prevalence Rates for
First Three Years of Study 186
5.13 Prevalence of Self-Reported Acute Illnesses in Study
Population 187
5.14 Summary of Clinical Bacteriology Results for Illness
Specimen Throat Swabs 188
5.15 Microorganisms Found in the Oropharynx 189
5.16 Summary of Clinical Bacteriology Results for 23 Requested
Throat Swabs from Well Participants 190
5.17 Organisms Isolated from Fecal Specimens in Sampling Period
201 192
5.18 Organisms Isolated from Fecal Specimens During all
Preirrigation Periods 193
5.19 Organisms Isolated from Fecal Specimens During all
Post-irrigation Periods in 1982 194
5.20 Comparison of Clinical Bacteriological Analyses of Fecal
Specimens Between Preirrigation and Post-irrigation 196
5.21 Possible Episode of Bacterial Infection in June 1982
Determined from Scheduled Fecal Specimens 197
5.22 Possible Episode of Bacterial Infection in August and
September 1982 Determined from Scheduled Fecal Specimens 198
5.23 Viral Isolates Recovered from Scheduled Fecal Specimens 204
5.24 Viral Isolates Recovered from Individuals During Baseline
Monitoring 205
5.25 Viral Recoveries and New Viral Infections from Scheduled
Fecal Specimens in 1982 207
5.26 Summary of New Viral Infections (Events) in Scheduled Fecal
Specimens During Irrigation Periods in 1982 208
xiii
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Tables (Cont'd)
5.27 EM Analysis of Fecal Specimens 210
5.28 Tuberculosis Skin Test Results 213
5.29 Summary of Serum Neutralization Serology for LHES Study
Participants During 1982 214
5.30 Household Seroconversion Rate by Zone During Post-
irrigation Period 216
5.31 Distribution of Hepatitis A Antibody (IgG) as Determined
by an RIA Test 217
5.32 Conversion from Negative to HAVAB® Positive 217
5.33 Microorganism Concentrations in Lubbock Wastewater 220
5.34 Microorganism Concentrations in Hancock Reservoir 225
5.35 Microorganism Concentrations in Wilson Wastewater 226
5.36 Bacterial Screen—Lubbock, Texas 233
5.37 Bacterial Screen—Hancock Reservoir 234
5.38 Bacterial Screen—Wilson, Texas 236
5.39 Viruses Isolated from Lubbock Effluent During Baseline
Years 237
5.40 Viruses Isolated from Lubbock Pipeline Effluent During 1982 238
5.41 Viruses Isolated from Wilson Effluent During Baseline Years 239
5.42 Viruses Isolated from Wilson Effluent During 1982 240
5.43 Species of Legionella Detected in Wastewater Samples by
Direct Fluorescent Antibody Staining of the Original Samples
or Tissues from Guinea Pigs Inoculated with Those Samples 241
5.44 Wastewter Samples Collected During 1982 Aerosol Monitoring
(30 Minute Composites) Wastewater from Pipeline During
Preplanting Irrigation 243
5.45 Wastewater Samples Collected During 1982 Aerosol Monitoring
(30 Minute Composites) Wastewater from Pipeline during
Summer Crop Irrigation 244
5.46 Wastewater Samples Collected During 1982 Aerosol Monitoring
(30 Minute Composites) Wastewater from Reservoir During
Summer Crop Irrigation 246
5.47 Source Strength of Rhodamine in Wastewater During Dye Runs 248
5.48 Rhodamine Aerosol Concentration During Dye Runs 248
5.49 Sampled Standard Plate Count in Air by Particle Size 250
5.50 Microorganism Densities in Air on Background Air Runs 253
5.51 Geometric Mean Air Levels Sampled on Background Runs 254
5.52 Sampled Fecal Coliform Densities on the Microorganism Aerosol
Runs 256
5.53 Sampled Fecal Streptococcus Densities on the Microorganism
Aerosol Runs 257
5.54 Sampled Mycobacteria Densities on the Microorganism Aerosol
. Runs 258
5.55 Sampled Clostridium perfringens Densities on the Micro-
organism Aerosol Runs 259
5.56 Sampled Coliphage Densities on the Microorganism Aerosol
Runs 260
xiv
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Tables (Cont'd)
Page
5.57 Viruses Recovered from Aerosol Samples During Virus Runs 262
5.58 Sampled Enterovirus Densities on Virus Runs 262
5.59 Identification of Viral Isolates Recovered During Virus
Runs 263
5.60 Estimated Densities Sampled on Microorganism and Virus
Aerosol Runs 265
5.61 Estimated Microorganism Densities in Air Downwind of
Irrigation Relative to Ambient Background Levels Near
Homes and in Fields 266
5.62 Bacterial Isolates from Flies 268
5.63 Analysis of Drinking Water Wells on and Around the Hancock
Farm 270
5.64 Activity Diary Participants 274
5.65 Percent of Time Spent at Home 274
5.66 Frequency of Direct Wastewater Contact 274
xv
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LIST OF ABBREVIATIONS
ARD -- acute respiratory disease
ATCC — American Type Culture Collection
BGM -- buffalo green monkey kidney cells
BHI — brain heart infusion
BSA -- bovine serum albumin
CAL -- cellulose arginine lysine agar
CDAS — cassette data acquisition system (Climatronics Corp.)
CDC -- Centers for Disease Control
CF -- complement fixation
cfu -- colony-forming unit
CPE — cytopathic effect
CPM -- counts per minute
CYE — charcoal-yeast extract
DCP — data collection period
DE -- diatomaceous earth
DFA -- direct fluorescent antibody
DRCM -- differential reinforced Clostridia medium
ELISA -- enzyme-linked immunosorbent assay
EM -- electron microscope
EMB -- eosin methylene blue
EWS -- electronic weather station (Climatronics Corp.)
FITC -- fluorescein isothiocyanate
GI -- gastrointestinal illness
GMT -- geometric mean titer
GPRBC -- guinea pig red blood cells (erythrocytes)
HAV -- hepatitis A virus
HI — hemagglutination inhibition
IFA -- indirect fluorescent antibody
IgG — immunoglobulin G
IPV -- inactivated polio vaccine (Salk)
LDB -- Legionnaire's disease bacterium
LIA -- lysine-iron agar
MIO -- motility-indole-ornithine
MPN — most probable number
OPV -- oral polio vaccine (Sabin)
PBS -- phosphate buffered saline
pfu -- plaque-forming unit
PTA — phosphotungstic acid
QA -- quality assurance
RD — rhabdomyosarcoma
RDE — receptor destroying enzyme
xvii
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List of Abbreviations (Cont'd)
RIA -- radioimmimoassay
SDA -- Sabouraud dextrose agar
SS — Salmonella-Shigella
TOC -- total organic carbon
TPB — tryptose-phosphate broth
TSA -- trypticase soy agar
TSI -- triple sugar iron
TSS -- total suspended solids
TVSS -- total volatile suspended solids
XLD -- xylose-lysine-deoxycholate
XVTM
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1. INTRODUCTION
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SECTION 1
INTRODUCTION
BACKGROUND
Land Application and.Potential Infectious Hazards
Land application of wastewater can be an attractive alternative to
traditional waste disposal practices. It avoids contamination of surface
waters, provides additional waste treatment, returns nutrients to the soil,
and reuses the water. The policy of the U.S. Environmental Protection
Agency (EPA) is to "press vigorously for publicly-owned treatment works to
utilize land treatment processes to reclaim and recycle municipal
wastewater" (Costle, 1977). Applicants for federal construction grants
(Section 201) must show in their requests that they have considered the
application of wastewater to land as an alternative. Financial incentives
are provided to encourage land application (Clean Water Act of 1977). Slow
rate application of wastewater to land by sprinkler irrigation has been and
continues to be one of the most popular application methods. With EPA
encouragement, it is likely that the practice of applying wastewater to
land by sprinkler irrigation according to EPA design criteria (USEPA, 1977)
will become more prevalent as a means of final treatment and disposal.
The wastewater and the aerosol produced by its sprinkler application
contain viable potentially pathogenic bacterial and viral agents. There
are various environmental pathways by which these agents might be
introduced and initiate infection in susceptible exposed individuals.
Agents in the wastewater aerosol can be transported by the wind and might
be inhaled or ingested in exposed food while still viable and infective.
Other potential environmental pathways include: 1) ingestion of
wastewater-contaminated ground water used as the domestic water supply; 2)
dust storms in which wastewater-irrigated surface soils are entrained by
strong winds; 3) insect vectors (e.g., flies attracted by the wastewater
lagoons); 4) rodents (e.g., feed or food stuffs contaminated by fecal
droppings or urine from field mice, infected by wastewater spray, which may
be spending the winter in farmhouses and barns); and 5) fomites (e.g.,
wastewater-contaminated work shoes, clothing, hands, or doorknobs). Once
introduced into the local population, the infectious agents might be
transmitted by contact between infected and susceptible individuals.
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Recent Literature
Katzenelson et al. (1976) observed retrospectively that the incidence
of reported cases of shigellosis, salmonellosis, infectious hepatitis, and
typhoid fever during the summer irrigation season were each two to four
times higher in 77 Israeli kibbutzim practicing spray irrigation of
partially-treated undisinfected wastewater than in 130 control kibbutzim.
Because of the serious methodological constraint of relying solely on
official communicable disease reports, the investigators cautioned that "no
conclusive findings may be based on this report" (Shuval and Fattal, 1980).
They were unable to corroborate these findings in a later study using
primary medical and environmental data collected at each kibbutz (Shuval
et al., in preparation).
Two prospective epidemiologic studies were recently conducted among
residents around activated sludge sewage treatment plants near Chicago,
Illinois using the family-based virus watch approach developed by Frost
(1941a,b,c) and Fox (1957, 1966, 1972, 1974). Both studies included a
health watch of participating households that involved health diaries,
serology, and clinical specimen isolations. Neither Johnson et al. (1978,
1980) nor Northrop and coworkers (1979, 1980, 1981) detected any obvious
adverse health effects on residents potentially exposed to wastewater
aerosols from aeration basins.
Occupational health effects of wastewater and wastewater aerosols have
also been investigated. A prospective seroepidemiologic investigation
(Clark et al., 1980, 1981a) did not detect any significant health effects
of occupational exposure in American sewer and sewage treatment plant
workers when compared to control groups. However in Sweden, Rylander and
Lundholm (1980) found increased incidence of acute febrile illness among
workers exposed to sludge dust (probably due to endotoxins) and also
increased incidence of gastrointestinal symptoms among sewage treatment
workers.
A clinical and viral serologic evaluation of workers at the Muskegon
County (Michigan) Wastewater Management System was conducted in 1979 to
assess the potential for health risks from sprinkler irrigation of
wastewater (Clark et al., 1981b). Illness and virus isolation rates and
antibody titers to six enterovirus serotypes did not differ between
irrigation workers and a control group of highway workers. However,
initial antibody titers to coxsackievirus B5 were significantly higher for
six sprinkler irrigation nozzle cleaners who were frequently soaked with
wastewater. This observation may indicate an increased risk of viral
infection only in workers with the greatest and most direct exposure to
wastewater.
No prior study has been conducted of the effects on nearby residents'
health from sprinkler systems th
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EPA design criteria. The Lubbock Health Effects Study (LHES) has been
designed to investigate the potential infectious health effects.
The Lubbock Health Effects Study (LHES)
The LHES is seeking to determine the relationship between (sprinkler)
land application of wastewater which may contain potentially pathogenic
microorganisms and the incidence of infection and illness in the nearby
population. The initial two years of operation of the Lubbock Land
Treatment Demonstration Project at the Hancock farm near Wilson, Texas is
being investigated. The study involves a four-year health watch of nearby
residents and monitoring of the wastewater and its aerosol. This site is
unique in that a typical rural community with no prior wastewater exposure
was challenged by the enteric agents active in a much larger urban
community (Lubbock). Thus, persons residing around the Hancock site may be
exposed to infectious agents indigenous in the Lubbock population to which
many may be susceptible. A health watch of the rural community is being
maintained before, during, and after periods of wastewater sprinkler
irrigation. The health watch focuses on infections detected serologically
and through isolates recovered from scheduled fecal specimens. Hence, the
study maximizes the opportunity to detect any adverse infectious health
effect which might occur. The site and study design also enhance the
likelihood of interpreting observed episodes of infection by monitoring
likely routes of introduction and transmission.
The recent studies of health effects of wastewater and its aerosol
suggest there are unlikely to be significant health hazards from minimal
exposure to properly treated municipal wastewater, as planned at the
Hancock site. However, scientific data from the LHES is needed to verify
this impression and to foster public acceptance of land application of
wastewater at other potential sites.
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STUDY OBJECTIVE
The general objective of the Lubbock Health Effects Study (LHES) is to
identify possible adverse effects on human health from slow rate
(sprinkler) land application of wastewater which may contain potentially
pathogenic microorganisms.
This general objective will be achieved by addressing three specific
objectives:
1) to maintain surveillance of the health status of the study
population;
2) to describe the distribution of infections in the study
population;
3) to determine if the incidence of infections to agents found (or
presumed to be prevalent) in the wastewater is associated with
exposure to sprinkler irrigation of wastewater.
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STUDY ORGANIZATION
The LHES involves five major functional activities: project
management, a health watch, environmental sampling, microbiological assay
of clinical specimens and environmental samples, and data analysis. The
field activities (i.e., health watch, environmental sampling, and their
management) are funded by a subcontract to SwRI from LCCIWR (SwRI Project
01-6001). The other activities (i.e., laboratory analysis, data analysis,
and their management) are funded by a cooperative agreement between EPA-
HERL and SwRI (SwRI Project 01-6097).
The LHES is being conducted by Southwest Research Institute, the
University of Illinois at Chicago, and the University of Texas at San
Antonio and Austin. The following is a listing of participating
organizations:
Southwest Research Institute (SwRI) Lubbock Christian College
Department of Environmental Sciences Institute of Water Research (LCCIWR)
San Antonio, Texas Lubbock, Texas
University of Illinois at Chicago (UI) University of Texas
School of Public Health School of Public Health (UTSPH)
Chicago, Illinois Houston, Texas
University of Texas at San Antonio Naval Biosciences Laboratory (NBL)
(UTSA) Oakland, California
Center for Applied Research and
Technology (CART) H. E. Cramer Company (HEC)
San Antonio, Texas Salt Lake City, Utah
University of Texas at Austin (UTA) Texas Department of Health
Department of Civil Engineering Public Health Region 2
Austin, Texas Lubbock, Texas
U.S. Environmental Protection Agency
Health Effects Research Laboratory
(EPA-HERL)
Cincinnati, Ohio
The project manager for the LHES is Mr. David E. Camann, SwRI. Each
of the functional activities is directed by a principal investigator who
reports to Mr. Camann as shown in Figure 1.1 Details regarding principal
participating personnel, participating organizations, and areas of specific
activity are presented in Table 1.1 for each functional activity area.
-------
PROJECT MANAGEMENT
David E. Camann
SwRI
HEALTH WATCH
Robert L. Northrop, Ph.D.
UI
ENVIRONMENTAL SAMPLING
H. Jac Harding
SwRI
UT LABORATORY ANALYSIS
Charles A. Sorber, Ph.D.
UTSA/UTA
UI LABORATORY ANALYSIS
Robert L. Northrop, Ph.D.
UI
DATA ANALYSIS
David E. Camann
SwRI
Figure 1.1. Principal investigators and functional areas
-------
TABLE 1.1. PRINCIPAL PARTICIPATING PERSONNEL AND AREAS OF ACTIVITY
Personnel
Organization
Specific activity areas
PROJECT MANAGEMENT (D. E. Camann, SwRI)
D. E. Camann
R. J. Prevost
H. J. Harding
A. Holguln
HEALTH WATCH (R.
P. J. Graham/
C. M. Becker
SwRI
SwRI
SwRI
UTSPH
L. Northrop, Ph.D., Ul)
UIMC
I. Smlth/S. Stabeno
ENVIRONMENTAL SAMPLING (H.
H. J. Harding
M. A. Chatlgny
D. B. Leftwich
Planning, technical and financial status, meetings,
reports
Administration of subcontracts
Annual reports
Consultant (epidemiology)
Recruitment, health surveillance, serum and
specimen collect'on, household health
and activity diary collection
On-slte coordinator, Wilson, Texas
1ABORATORY ANALYSIS (C. A. Sorber, Ph.D
Environmental Samples
Harding, SwRI)
SwRI Wastewater aerosol sample collection,
and meteorological sampling
NBL Loan and calibration of LVA samplers
LCCIWR Sample col lection
UTSA/UTA, R. L. Northrop, Ph.D., Ul)
wastewater
B. E. Moore/C. A. Turk/
M. Ibarra
D. B. Leftwich
R. L. Northrop/
R. Cordel I
B. P. Sag Ik
Clinical Specimens
P. J. Graham
R. L. Northrop/
R. Cordel I
B. E. Moore
B. E. Moore/C. A. Turk
M. N. Guentzel/
C. Herrera
W. Jakubowsk!
UTSA/UTA
LCCIWR
Ul
EPA-HERL
DATA ANALYSIS (David E. Camann, SwRI)
K. T. Klmball
R. L. Mason/J. Garza
D. E. Camann/
M. C. Marshall
R. Harrist '
J. Stober
A. Anderson
Analysis of wastewater samples
(microbiological screens, routine wastewater
assays; enterovlrus Identification)
Analysis of aerosol and fly samples
Analysis of drinking water
Analysis of Leglonella In wastewater
Drexel Univ. Consultant (virology)
Serology
Ul Poliovlrus, coxsacklevirus, echovlrus,
adenovlrus
Ul Reovirus, Norwalk virus, rotavirus,
Leg Ione I la bacl11 us
UTSA Hepatitis A
UTSA/UTA Clinical virology
UTSA Clinical bacteriology
Electron microscopy of fecal specimens
SwRI Health data analysis
SwRI Data management
SwRI Aerosol exposure
UTSPH Consultant (statistical methods)
EPA-HERL Consultant (statistical methods)
HEC Dispersion modeling
-------
2. CONCLUSIONS
3. RECOMMENDATIONS
-------
SECTION 2
CONCLUSIONS
The wastewater utilized at the Hancock site contains a broad spectrum
of enteric bacteria and viruses. Sprinkler irrigation of wastewater
directly from the pipeline was found to be a substantial aerosol source of
each monitored microorganism (i.e., fecal coliforms, fecal streptococci,
mycobacteria, Clostridium perfringens, coliphage, and enteroviruses) under
most conditions of actual operation of the irrigation system. Under some
conditions, particularly at night or with high winds, pipeline irrigation
appeared to elevate the ambient (i.e., upwind) density in air of fecal
coliforms, fecal streptococci,^, perfringens, and coliphage to at least
400 meters downwind and of mycobacteria to about 300 meters downwind.
Sprinkler irrigation of reservoir wastewater was also found to be a source
of aerosolized fecal coliforms, fecal streptococci, and coliphage,
sometimes to downwind distances of at least 125 meters. Geometric mean
microorganism densities in air at the specified distances exceeded the
ambient background levels in fields and outside the homes of study
participants.
Surveillance of illness in the study population is continuing.
Apparent episodes of infection have been observed during the initial year
of irrigation. Preliminary characterizations of the infection episodes
have been made as a surveillance measure. No obvious significant
connection between health effects and wastewater exposure has been
observed. Conclusions with respect to possible association of infection
with wastewater exposure must await verification of processed data,
description of patterns of infection, calculation of exposure estimates,
statistical analysis, and epidemiologic interpretation.
-------
SECTION 3
RECOMMENDATIONS
No recommendations concerning health status or irrigation practices
are indicated at this time.
10
-------
4. METHODS AND
MATERIALS
-------
STUDY DESIGN
-------
SECTION 4
METHODS AND MATERIALS
STUDY DESIGN
Principles of Design
To determine if there is a relationship between land application of
wastewater and the incidence of infection and illness in the nearby
population, the following principles of design have been incorporated in
the LHES:
1) The study will use epidemiologic approaches in attempting both a)
to detect the occurrence of communicable disease and episodes of
infection in the study population and b) if detected, to
investigate the probable cause.
2) While both illness and infection will be monitored, primary
emphasis will be placed upon infection monitoring. Clinically
apparent disease may only constitute a small part of the total
number of infections that occur during the period of monitoring.
Furthermore, the literature offers little evidence that clinical
disease if likely to result from the anticipated level of
wastewater exposure at the Hancock site.
3) Because the wastewater will be introduced into the study area
from another community (Lubbock) and will represent the enteric
illnesses prevalent there, the clinical and environmental
monitoring and the data analysis will all focus on the specific
infectious agent in order to permit interpretation of the
resultant data.
4) The study population will be monitored to determine the incidence
of a spectrum of infections whose etiologic agents might be
present or prevalent in the sprayed wastewater. The diseases,
estimated susceptibility, and periods of prevalence of the human
pathogens potentially present in wastewater are summarized in
Table 4.1. The infections of interest will depend on which
agents are passing through the study community and are present in
the sprayed wastewater during periods of wastewater irrigation.
The particular infections of interest as dependent variables
cannot be specified in advance of the monitoring.
11
-------
TABLE 4.1. SUGGESTED PREVALENCE OF ANTIBODY AND SEASONAL OCCURRENCE OF
INFECTION FOR AGENTS POTENTIALLY PRESENT IN WASTEWATER
Agent (human pathogens
potentially present
in wastewater)
Occurrence
Types
Disease
Susceptible JFMAMJJASOND
Viral
Poliovirus
Coxsackievlrus
Echovirus
Reovirus
Adenovirus
Hepatitis A virus
Rotavirus
Norwalk virus
Coronavirus
Bacterial
Salmonella sp.
ro Shigella sp.
Echerichia col i,
enteropathogen i c
Mycobacteria, atypical
Klebsiella pneumoniae
Yersinia enterocolitica
Campylobacter, "related"
Leg i one I I a pneumoph iI a
Staphylococcus aureus
Streptococcus beta,
hemolytic
Pseudomonas sp.
Proteus sp.
Fungal
Candida albtcans
1-3; wild and vaccine
A 1-24, B1-6
1-33
1-3
1-9,11,19,21
1
1-4
1-3
2
5 groups
4 groups
Serotype 0 and other
4 groups
>24
4 biotypes
Unknown, 4 or 7
Unknown, 5 or 7
Groups
4 of 15 candidates
7
3 or 7
A, B groups
Enteritis, meningitis, paralysis <10? child
Enteritis, meningitis, respiratory, rash >50$
Meningitis, conjunctivitis >50%
Unknown >40?
Respiratory >50?
Systemic >70$
Enteritis
Enteritis
Uncertain, enteritis
>50%
?
>75?
>75$
>75%
Enteritis, systemic
Enteritis
Enteritis
Respiratory, adenitis, granuloma
5? respiratory, enteritis >75|
Enteritis, cutaneous >75J5
Enteritis, systemic ?
Respiratory, renal, other >90%
Respiratory, enteric, cutaneous >75?
Respiratory, enteric >75?
Cutaneous, respiratory, other <25$
Cutaneous, respiratory, other <25?
Cutaneous, respiratory, other <25%
-------
5) An infection episode will be defined as the observation in the
study population of a number of similar infection events (either
serologically or in serial clinical specimens) within a
restricted interval of time. Episodes will be statistically
analyzed for association with wastewater exposure when the
infectious agent(s) was(were) found (or can be presumed) to be
present in the wastewater that was sprayed during that period.
6) A study population of approximately 450 residents living on and
within 4.8 kilometers (3 miles) of the perimeter of the Hancock
site will be recruited and monitored through a health watch. The
susceptible population will be stratified by degree of exposure
to the wastewater and its aerosol when investigating possible
wastewater-associated episodes of infection.
7) The health watch and environmental monitoring will be more
thorough during those seasons of the year when heaviest
wastewater irrigation is planned and when feces-transmitted
infections are more prevalent. These seasons are summer
(covering the cotton crop irrigation) and spring (covering the
cotton preplanting irrigation). Infections from nearly all of
the human pathogens potentially present in wastewater have high
seasonal prevalence during summer or spring (see Table 4.1).
8) Baseline health and environmental monitoring will be conducted
for at least one year prior to the commencement of wastewater
irrigation so the data from periods of wastewater irrigation can
be analyzed and interpreted in relation to previously existing
conditions and patterns.
9) The health of the study population and its environment will be
monitored, as a minimum, through the first full year of normal
wastewater irrigation at the Hancock farm.
10) It will be important to distinguish whether the route of
introduction of infection into the study population was
wastewater-associated or a "normal" route. Plausible wastewater-
associated routes of introduction will be investigated. The
comparability the exposure strata with respect to normal routes
will be assessed.
11) The study design is primarily descriptive rather than analytic in
nature since it involves a single population and since a
particular infection of interest cannot be specified during
design. However, the study has analytic aspects because the
hypothesis of no association with exposure will be tested in
exposure subgroups using statistical and epidemiologic methods of
analysis.
13
-------
12) It is unlikely that conclusive findings about the health effects
of land application of wastewater will result from this or any
other single study of the process. However, this study of
initial wastewater irrigation at the Hancock farm provides the
best opportunity to develop definitive findings at the current
state-of-the-science.
Approach
A prospective descriptive study was conducted on the incidence of
infection and illness in a rural American community in relation to
occupational and residential exposure to wastewater applied by sprinkler to
land. The study population resides on and within 5 kilometers of the
periphery of wastewater irrigation at the Hancock farm. This study area
includes the City of Wilson and the surrounding rural area.
The approach involved the following activities:
1) recruit and obtain medical histories for a population of 450
residents from 150 households for a health watch;
2) conduct a health watch of the infections, illnesses, and
activities of participants during two baseline years and the
initial two years of wastewater spray irrigation using the most
sensitive and practicable routine health monitoring measures:
• infections via
semiannual serosurvey
- fecal specimens from donors monthly
spanning the irrigation periods
- annual tuberculin skin tests
• reported illnesses via household health diaries
and confirmation via illness specimens
• activity diaries during representative weeks of the
wastewater irrigation periods;
3) assay sera for antibody titers to certain meaningful microbial
agents;
4) isolate bacterial and viral pathogens from fecal specimens;
5) monitor wastewater:
• periodically to screen for all microbial pathogens
• regularly to assay for selected bacteria and
enteroviruses
• as required to identify and determine the
distribution of enteroviral types;
14
-------
6) sample wastewater aerosols for indicator bacteria and
enteroviruses;
7) estimate participant exposure to the wastewater and its aerosol
from the activity diary and dispersion modeling;
8) maintain surveillance of the participants' health status and
promptly report health problems;
9) determine the distribution of infections and illnesses in the
participants and donors from self-reported diaries, serologic
data, pathogen isolates, and skin test data;
10) retrospectively group the participants according to their level
of wastewater aerosol exposure over observed episodes of
infection;
11) determine if observed episodes of infection are temporally
associated with degree of exposure to the wastewater aerosol;
12) investigate alternative routes of introduction of infection,
besides the aerosol, both these related (direct wastewater
contact, time spent in application areas, flies, and dust
storms), and probably unrelated (person-to-person spread,
contaminated drinking water, and time spent in Lubbock) to
wastewater irrigation.
Monitoring Schedule
The schedule of LHES monitoring was dictated by the schedule of
wastewater irrigation at the Hancock farm. Sprinkler irrigation of
wastewater commenced on February 16, 1982, instead of March 1981 as
initially planned, because of delays in construction and in obtaining the
discharge permit.
The wastewater irrigation schedule was tailored to meet the water and
nutrient of the crops and to prevent contamination of the ground water. To
prevent crop damage, scheduled irrigation was not conducted during periods
of excessive precipitation. Actual periods and degree of irrigation during
1982 are indicated at the top of Figure 4.1. The irrigation scheduled for
1983 is presented in Table 4.2.
The LHES monitoring plans, especially the scheduled fecal collections,
were revised and refined several times to optimize health monitoring over
the actual periods of irrigation. The actual LHES monitoring schedule
through 1982 and the planned schedule for 1983 are presented in Figure 4.1.
15
-------
^ J5 Heavy -
H rr
V) a: Very Light -
LHES MONITORING
Serosurvey
Scheduled Fecal Specimens
Illness Specimens
Skin Test
Activity Diary
Aerosol Sampling
Wastewater Sampling
Drinking Water Sampling
1
^ - —
•
•
1 f S 7 j
^^^M ^^H \S / /
III!)
1
I960
M|J
A
A
A
J|A
AA
M^^M
^^^™
A
A
S|0
A
•
^^m
i
N|D
A
1
I i i i I i i i i i I il
1981
J|F
M|A
A
^m
^
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A
A A
1^^^^
^^^^
A
J|A
k A
.^ ....
^ ^
slo
i
A
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H , I//:
1982
J|F
:
A
1.
M|A
-
AA
A
M|J
A
A
A
J|A
A
A
AAA
s|o
A
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A
A
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L
,
'//,
/ / / /
^/%Xj
7
/
'///
///
///
///
'///
///
7/ / /
s / /
V/A
\ i i
1983
J|F
A
LEGEND
Planned Activity /\ I
l,Ac
tual Activity A ^MHl , Planned Irrigation /
^
M|A
A
A
AA
M|v.
i
A
A
A
PLAN DATE
J|A
A,
A
AA
s|o
A
A
'
N|D
= OCTOBER 1982
/ , Actual Irrigation ^^^H
Figure 4.1. LHES monitoring schedule, June 1980-October 1983
-------
TABLE 4.2. IRRIGATION PLAN FOR HANCOCK FARM IN 1983a
Wastewater pumped for irrigation, inches/month
Month
January
February
March
April
May
June
July
August
September
October
November
December
Annual
1880 Acres in
single crop
0
1.5
3.5
1.0
0
0
4.5
5.0
1.0
0
0
0
16.5
600 Acres in
double crop
1.0
1.5
3.0
4.0
2.5
0
4.5
4.0
5.0
0
1.0
1.0
27.5
Hancock farm
weighted average
0.2
1.5
3.4
1.7
0.6
0
4.5
4.8
2.0
0
0.2
0.2
19.2
a. Source: N. Klein, Tentative Irrigation Plan for 1983,
December 1, 1982
17
-------
Health Watch—
The schedule of health watch activities is indicated in Figure 4.1.
From 1980 through 1982, all study participants were asked to provide blood
samples semiannually (generally in June and December) for serologic
analysis to determine the incidence of seroconversions to specific viral
serotypes. Sera were assayed for titers to enteroviruses found in the
sprayed wastewater, to hepatitis A virus, adenovirus 7, reoviruses 1 to 3,
rotavirus, and Legionella, and to influenza as a control. Participants
received tuberculin tests annually to assess atypical mycobacteria
infections. Participants gave self-reports of illness weekly through the
household health diary and were asked to provide appropriate clinical
specimens for assay when ill. Health diaries and illness specimens were
collected over the entire period of irrigation (January 1982 to October
1983) and over appropriate baseline periods (July to September 1980 and
April to September 1981).
Eligible donors (i.e., all children 12 years of age and less, plus the
next oldest participant in families with one such child) were requested to
provide a fecal specimen every four weeks during the baseline health watch.
Fecal specimens were also requested from one adult participant per
household in 1982 and 1983 to obtain specimens from a sufficient number of
households. The one-week fecal collections in 1982 and 1983 were scheduled
before and at monthly intervals during the two heavier periods of
irrigation (mid-February to April and Ju;ly to August). A spectrum of
enteric bacterial and viral isolates were sought from the approximately 120
fecal specimens received during each one-week fecal collection period (cf
Table 4.3).
To permit assessment of exposure to wastewater and the wastewater
aerosol, each participant kept an activity diary of the pattern of his
activities as they related to the Hancock site. The activity diary was
kept during a representative week each season to characterize the activity
pattern during the school year and during the summer.
Environmental Monitoring--
The schedule of environmental monitoring activities is shown in Figure
4.1. The correspondence of infectious agents being monitored in wastewater
to those monitored in the health watch is indicated in Table 4.3.
Wastewater samples of the sprayed effluent from the pipeline and
reservoirs and of the Wilson effluent were obtained biweekly to span the
heavier irrigation periods; corresponding baseline samples were obtained
with the same frequency in 1981 and at lesser frequency to characterize the
effluents in 1980. These samples were assayed for total enteroviruses,
fecal coliforms, total suspended solids (TSS), total volatile suspended
solids (TVSS), and total organic carbon (TOO. A limited screen for
bacterial pathogens and an assiy for the same microbiological indicator
18
-------
TABLE 4.3. MEASUREMENT IN WASTEWATER OF INTERPRETABLE INFECTIOUS
AGENTS MONITORED IN THE HEALTH WATCH
Procedure
Serology
viruses:
bacteria:
Skin Test
Agents monitored in health watch
Infectious agents
(serotypes potentially
present in wastewater)
(total enterovi ruses:
coxsackie, echo, polio)
Coxsackie A virus (1-24)
Coxsackie B virus (1-6)
Echovirus (1-33)
Adenovirus (1-9, 11, 19, 21)
Reovirus (1-3)
Hepatitis A virus
Rota virus (1-4)
Norwalk virus (1-2)
Leg i one 1 la pneumophila
Mycobacteria (tubercu-
Measurement In
Sprayed Wi (son
wastewater effluent
R R
R R
R R
R R
1
R R
wastewater
Data type
Q
S (by ID)
S (by ID)
S (by ID)
+/- (will
Q
ID)
losis + atypical )
Clinical Bacteriology
bacteria:
fungus:
Clinical Virology
Salmonel la sp.
Shigel la sp.
Yersinia enterocol itlca
Campy lobacter fetus
Staphy lococcus aureus ft
Fluorescent Pseudomonast
Klebsiellatt
Proteus tt
Serratia and otherstt
Aeromonas hydroph i 1 a t
Candida al bicans tt
Coxsackie A virus (1-24)
Coxsackie B virus (1-6)
Echovi ruses (1-33)
t - elevated to moderate or heavy level
tt - markedly elevated
to heavy level
R
R
R
R
1
R
R (Kl-like)
1
1
1
R
R
R
R
R - regu lar
1 - infrequent
R
R
R
R
1
R
R
1
1
1
R
R
R
R
0
S
+/-
+/-
+/-
+/- «? if high)
+/-
0
Q
Q
Q
Q
S
0
S (by ID)
S (by ID)
S (by ID)
- quantlatlve
- semlquantitatlve
- present/absent
19
-------
organisms sought in the aerosol sampling runs were conducted on most of
these samples. The distribution of enteroviral types within a sample was
determined by identifying about 50 viral isolates from about half of these
samples. The purpose of these samples was to determine the presence,
prevalence, and longitudinal pattern of viral and bacterial pathogens
possibly introduced by the wastewater and their passage through the study
community.
Microbiological screens were conducted on one sample from each
location per irrigation season. The microbiological screens provided the
relative densities and seasonal variability of a wide range of indigenous
enteric bacteria in some of the same samples for which enteroviral types
were identified.
Aerosol sampling was conducted to characterize the aerosol density of
microorganisms produced by the sprinkler irrigation. Twenty microorganism
runs were conducted each using eight large volume aerosol samplers to
measure levels of indicator microorganisms upwind and from 50 meters to 400
meters downwind of operating sprinkler rigs. Four background runs were
conducted in August 1980 to measure ambient levels of the same indicator
microorganisms near participant households before irrigation commenced.
Two quality assurance runs were conducted to estimate the variability in
sampled microorganism levels associated with the field sampling, shipping,
and laboratory procedures. Four virus runs were conducted to measure the
density of aerosolized enteroviruses emanating from a sprinkler rig. Four
dye runs were conducted to determine the aerosolization efficiency (i.e.,
the fraction of the sprayed wastewater that is carried off by the wind as
an aerosol) of the sprinkler rigs at the Hancock site. Four particle size
runs were performed to determine the distribution of aerosolized
microorganism colony forming units (cfu) by the size of the carrying
particle.
Environmental monitoring also evaluated other means of introduction of
wastewater microorganisms into the study population besides direct
wastewater contact (determined from the health diary) and aerosol exposure
(determined from the aerosol sampling and an exposure index based on
activity patterns). Dust storms, houseflies, and drinking water were
examined as alternative means of introduction. Drinking water samples
obtained quarterly from over 20 residences throughout the study area were
assayed for fecal coliforms, fecal streptococci, and Salmonella to assess
drinking water as a source of infection.
20
-------
STUDY SITE
-------
STUDY SITE
Study Area
The study area involved in the Lubbock Land Treatment System lies in
northwestern Texas in Lynn County and Lubbock County. The source of
wastewater for this irrigation project is the Lubbock Southeast Water
Reclamation Plant (SeWRP), situated in the southeast portion of the city of
Lubbock. The storage and irrigation facilities are located at the Hancock
farm in the north central portion of Lynn County, 30 km (18 miles) south of
Lubbock. Both counties are located in a plateau area, the South Plains
Region of the Llano Estacado of the High Plains. A regional map of the
study area is shown in Figure 4.2.
The Lubbock area is the center of the largest cotton producing section
of Texas. Other segments of the agroeconomy of the area include grain
sorghum production and cattle feeding. The Ogallala aquifer, an extensive
unconfined aquifer system stretching from western Nebraska and eastern
Colorado south to the Texas panhandle and eastern New Mexico, is used for
irrigation purposes as a supplement to natural rainfall to improve crop
yields. Withdrawal of ground water from the Ogallala aquifer has greatly
exceeded the natural recharge. In the Lubbock area, the aquifer is
approaching depletion; in 20 years it may no longer be economical to
produce irrigation water from this source.
General Climatology--
The South Plains Region is semiarid, transitional between the desert
conditions to the west and the humid climate to the east and southeast.
The average annual precipitation is 46.8 cm (18.4 inches), most of which
occurs from May through September, usually as moderate to heavy afternoon
and evening thunderstorms which may be accompanied by hail. Snow may occur
from late October until April, but is generally light and seldom remains on
the ground for more than two or three days at any one period.
For the eight-month period from March through October, winds are
predominantly from the south. However, during the late winter and
springtime, winds in excess of 11 meters/second (25 MPH) occur for periods
of 12 hours or longer from a westerly direction with the passage of low
pressure centers. These strong winds bring widespread dust, the quantity
and amount of which is influenced by the precipitation patterns of the
previous few days and the agricultural practices of the area (NOAA, 1977).
To show variation in wind direction, wind roses were constructed for the
two high level periods of irrigation planned for 1982 and 1983. Figure 4.3
displays the wind direction pattern for March and April corresponding to
the spring irrigation period, while a wind rose for the July to August
period (summer irrigation) is shown in Figure 4.4. The wind roses are
based on data from the five-year oeriod, 1969 to 1973.
21
-------
-u_- f -4 \ V
>~^« I- !• • %«
I • I' 1_ ;JkJ, J 5
—-I! J j-T^, T^ 1
Figure 4.2. Regional map of the study area (showing components
of the Lubbock Land Treatment Project)
22.
-------
NW
315
WNW
292.
WS
247.5°
NNW
337.5°
N
0/360°
NNE
22.5°
SW
225
SSW
202.5°
135C
ENE
67.5°
ESE
112.5
NOTE: Three-hour observations are from the five-year period, 1969-1973.
Radiating-bar lengths indicate the percent of the period that
winds blow from the indicated directions
Figure 4.3. Wind frequencies for the two-month period of
March-April, Lubbock, Texas
23
-------
WNW
292.5°'
WS
247.5°
SW
225°
NNW
337.5°
0°/360<
NNE
22.5°
ssw
202.5°
SSE
157.5°
ENE
67.5°
NOTE: Three-hour observations are from the five year period, 1969-1973.
Radiating-bar lengths indicate the percent of the periods that
winds blow from the indicated directions.
Figure 4.4. Wind frequencies for the two-month .period of
July-August, Lubbock, Texas
24
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City of Wilson--
The city of Wilson is the nearest community to the Hancock farm. It
is situated at the southern boundary of the farm. The population of 576
(1980 census) occupy 181 residences ranging from small two bedroom stucco
or frame bungalows to large all-brick homes. Local commerce is based
primarily on agriculture. Support facilities located in Wilson include
three cotton gins, one grain elevator, a welding and machine shop, a pump
service facility, and a combined lumber, hardware and feed store. Other
businesses within Wilson include a bank, two cafes, two service stations,
and a grocery store. During 1982 the grocery store ceased to do business
and one service station was converted into a convenience store. A
municipal building, a school complex for grades 1 through 12, a municipal
park, a post office and six churches are also located within the city
1imits.
The municipal water supply for city residents is obtained from the
Ogallala aquifer. Six wells tap this source, and a water tower and
underground tank provide storage facilities where the water is periodically
chlorinated manually prior to distribution.
All but ten of the households within the city limits are serviced by a
municipal wastewater collection and treatment system. The treatment plant
consists of an Imhoff tank preceded by a bar screen. Plant effluent is
allowed to evaporate from a series of lagoons while the settleable solids
are removed from the tank on a monthly basis and placed in an adjacent
drying bed. Those households not connected to the municipal system have
septic tanks.
Rural Area--
The rural area within 4.8 km (3 miles) of the Hancock farm lies
primarily in Lynn County (1977 estimated population, 8,900) with a small
portion above the northern boundary in Lubbock County. Approximately 130
households are located in this area with an estimated population of 450.
Rural residents obtain domestic water from wells which tap the
Ogallala aquifer. Treatment of domestic wastewaters is accomplished by
septic tank systems in half of the rural houses while the other half,
typically the older homes, utilize cesspools.
In the predominantly agricultural economy of this region, an annual
income of $60 million (Lynn County) is derived from a primary crop of
cotton and secondary crops of winter wheat, grain sorghum, sunflowers and
soybeans. Livestock is kept primarily for owner use, though some pasture
land is dedicated to grazing of livestock for market. There is some
production of oil and gas, and some exploration, with attendant drilling
activity, is occurring in the area. The value of these mineral resources
and those of a stone quarry amounted to $2 million during 1977 for Lynn
County (Texas Almanac, 1980).
25
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Lubbock Sewage Treatment Plants
The City of Lubbock operates two wastewater treatment plants: the
Southeast Wastewater Reclamation Plant (SeWRP) and the Northwest Wastewater
Reclamation Plant. The SeWRP is in reality three separate systems: two
trickling filter plants and an activated sludge plant. It treats the
majority of wastewater generated in the city. One of the trickling filter
plants is currently not operational since it is being upgraded.
The second trickling filter plant has a hydraulic loading of 30,000 to
49,000 cubic meters/day (8 to 13 MGD), consisting of 25 to 30% industrial
waste. The majority of industrial wastes are from cotton gin operations
and industrial plating operations. Effluent quality from this plant is
mediocre with an average five-day biochemical oxygen demand (8005) of 103
mg/L and total suspended solids (TSS) of 118 mg/L for the period October
1979 through September 1980.
The activated sludge plant has an average daily flow of 34,000 cubic
meters/day (9 MGD). During the period October 1979 through September 1980,
this plant discharged a final effluent of good quality with a BODs of 25
mg/L and TSS of 18 mg/L. An average of 25,000 cubic meters/day (6.5 MGD)
of the activated sludge effluent is dosed at about 12 mg/L chlorine prior
to transfer to the Southwestern Public Service Company's Jones Power Plant
where it is used as cooling and boiler makeup water.
The remaining effluent from the activated sludge plant (ASP) and all
the unchlorinated effluent from the trickling filter plant (TFP) are
currently pumped to one of three lagoons at the Frank Gray farm for
irrigation purposes. The maximum contribution of ASP effluent is no more
than 20% at any given time, and when averaged on a daily basis, the overall
contribution by ASP effluent to irrigation is less than 5%. This same
combined effluent stream will provide the wastewater to be utilized at the
Hancock farm for irrigation.
The Northwest Wastewater Reclamation Plant treats wastewater generated
mainly from the extreme northwest portion of Lubbock and from Texas Tech
University. The 4,000 cubic meters/day (1 MGD) effluent from this plant is
used by Texas Tech University for irrigation studies on the Tech farm.
Lubbock Land Treatment System
The land treatment system for the Lubbock Land Treatment Project is
located at the Gray farm and the Hancock farm. Both farms will receive
wastewater from the Lubbock Southeast Water Reclamation Plant. The Gray
farm site consists of a 1,210 hectares (2,990 acres) near the SeWRP where
Lubbock wastewater has been land treated and disposed since 1938.
Currently, the system is hydraulically overloaded. The existing irrigation
26
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system at the Gray site will be modified. The completely new treatment and
disposal system for the Hancock farm consists of wastewater conveyance,
storage and irrigation facilities. The Hancock site is a 1,600-hectare
(3,900-acre) farm 27 km (17 miles) south of the Lubbock treatment plant and
just north of Wilson.
A pipeline will convey half the available irrigation wastewater,
approximately 13,000 to 15,000 cubic meters/day (3.5 to 4.0 MGD) of
secondary effluent, from the Lubbock SeWRP to the Hancock site as required
by the project. The pipeline system consists of a three-pump pumping
station at the SeWRP and 25,030 meters (82,120 feet) of 0.69-meter (27-
inch) diameter effluent force main. Three natural playas on the Hancock
farm have been modified to serve as storage reservoirs, with a total
capacity of 2.6 x 10? cubic meters (24,000 acre-feet). The irrigation
system will cover a total of 1,150 hectares (2,850 acres) at the Hancock
farm: 970 hectares (2,400 acres) irrigated by 22 electric-drive center
pivot sprinkler systems and 180 hectares (450 acres) irrigated by the
furrow flooding technique to maximize land use in areas not accessible to
the center pivot system. A schematic of the Hancock site showing the
irrigation pivot rigs is shown in Figure 4.5. The choice of sprinkler
types for the spray irrigation laterals were low pressure Nelsons, which
provide a 360° umbrella pattern with an effective wetted diameter of 8.5 to
9.1 m (28 to 30 ft) to allow for the greatest application intensity. The
spray nozzles are situated on drops 3.2 m (10.5 ft) apart on 52.1 to 54.3 m
(171 to 178 ft) spans between towers. Nozzle heights are 1.5 m (5 ft) to
2.1 m (7 ft) above ground, while nozzle diameters range from 2.4 mm (3/32
in.) up to 7.1 mm (9/32 in.) with the smaller nozzles located near the
pivot and the larger ones at the end of the lateral.
The end gun of each lateral is a Rainbird® type which can be activated
to irrigate all or some of the corners. The height of the end sprinklers
is from 3.0 m (10 ft) to 4.6 m (15 ft) depending upon the terrain. When
the end guns are activated, their effective wetted diameter is 18.3 m (60
ft).
The laterals vary in length from 307 m (1007 ft) to 476 m (1562 ft)
with six to eight towers per pivot. The speed of traverse of each lateral
is variable, and at maximum speed a pivot will complete a full cycle in 13
or 14 hours (Basis of Design Report, Sheaffer and Roland, Inc. and
Engineering Enterprises, Inc., 1980).
27
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t— —. —, -- EXHIBft- I -
Figure 4.5. Map of the Hancock site
28
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STUDY POPULATION
AND HEALTH WATCH
-------
STUDY POPULATION
Sampli ng
An area within 4.8 kilometers (3 miles) of the perimeter of the
Hancock site was designated as the study area. This area includes the
small city of Wilson, Texas and the rural area north, northwest, and
northeast of Wilson (Figure 4.6). The prevailing winds are from the south
during the planned summer crop irrigation, but the wind shifts to blow from
the north after frontal passages which occur regularly.
The rectangular Zone 1 includes all rural households located on the
Hancock farm and within 0.5 miles of its perimeter. Zone 2 represents the
households located within 0.5 miles of the Hancock site boundary within
Wilson. Included in Zone 3 are all rural residences located from 0.5 to
1.0 (E and W) or 1.5 (N or S) miles from the Hancock farm. Zone 4 consists
of the Wilson households which are located 0.5 to 1.0 miles from the site.
Zone 5 contains the rural households which are from 1.0 or 1.5 to 2 miles
(E and W), 2.5 miles (S) and 3 miles (N) of the Hancock farm boundary.
Zone 5 extends to approximately 3 miles north of the farm because the
prevailing winds are from the south. The households of the small number of
Hancock farm workers who resided outside the study area were placed in Zone
6.
Due to the limiting number of residences in the rural area
(approximately 130), the sampling plan was to invite all households within
Zones 1, 3, 5, and 6 to participate in the study. Special emphasis was
placed on recruiting all households located in Zone 1 in order to maximize
the amount of information from individuals who, presumably, would be most
highly exposed to wastewater aerosols.
There were approximately 172 households located within Wilson, and
one-half of these were selected for recruitment into the study. Thus,
every other Wilson household was designated a part of the sample. When a
refusal was obtained, the next available house on the block was contacted,
according to a standardized selection procedure.
One hundred fifty-six households with 439 participants were originally
recruited for the study in May and June 1980. A 20% decrease in the number
of participating households occurred between the onset of the health watch
and the end of 1982.
The study population is not considered to be a transient population,
but in the interval of time since the initial recruitment, several families
have moved out of the study area, a few relocated in the area, and
occasionally a family would leave and reenter the area some time later. In
any of these cases, a new or returning family rehabiting a vacant house was
29
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Figure 4.6. Sampling zones comprising the study area
30
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recruited as a replacement or continuing household. Families or
individuals lost through attrition were replaced, when possible, with
households in the same area. Selecting a new sample of households for
replacements has not been necessary.
The number of households and participants participating in the study
for three yearly periods, 1980 to 1982, is given in Table 4.4.
Health Interview and Recruitment
A team of interviewers-recruiters was trained and obtained the medical
history of each family member in the sample households. Each interviewer
received an instruction manual describing methods for conducting the
interview and recording illness history. They were instructed in methods
of recruiting residents to participate, in maintaining health diaries, in
submitting to tuberculin testing, and in providing stool, illness, and
blood specimens. The purpose, duration, and incentives for participation
in this study were explained to each interviewer to enable them to respond
to questions from interviewees during the recruitment period. The
incentives included: 1) continuing information about the health status of
each participant; 2) laboratory information regarding infectious agents
recovered from specimens collected during an illness; 3) a brief layman's
version of the findings from the study; 4) a small monetary reward at the
end of each study year for the inconvenience imposed on each participant
for cooperating in the health watch, and 5) small payments for each fecal
specimen provided.
A questionnaire was developed to record information on the number of
members in each family, their age, level of education, occupation, income,
chronic health conditions, and relevant medications. This form is
presented in Appendix A. A pretest of the instrument was done to evaluate
the interviewee's understanding of and responses to the questions being
asked. The interview required 15 minutes of participant time.
An update questionnaire of all participating families was administered
the week of January 31, 1982. An adult member of each household was
contacted by telephone either by interviewers in the UI staff office or by
the field representative (in those cases where telephone contact was not
possible).
The abbreviated questionnaire, Appendix B, was designed to update
information concerning chronic health conditions, occupation, extended
leaves from the study area, etc., in order to document what and when
changes have occurred since the original interview, maximally two years
earlier.
31
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TABLE 4.4. COMPARISON OF STUDY POPULATIONS IN 1980, 1981 and 1982
NOV iy»u
Zone
1
2
3
4
5
6
Total
Households
22
32
14
39
44
-
151
Adults
45
61
31
59
76
-
272
Children
13
39
13
51
45
-
161
oct iybi
Households
22
33
12
34
38
2
141
Adults
39
59
27
58
72
4
259
Children
14
46
11
36
33
-
140
Dec lybZ
Households
22
33
9
33
31
4
132
Adults
37
57
20
55
61
4
234
Children
13
37
6
35
30
3
124
GO
ro
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Polioviriis Immunization
Based on serological analysis, a significant proportion of the study
population appeared to be susceptible to at least one of the three
poliovirus serotypes. Because poliovirus was found in the Lubbock
effluent, prophylactic immunization of susceptible residents (particularly
those within 400 m of a spray rig) was recommended and implemented.
All participants who gave a blood sample were notified by mail or
telephone of their poliovirus immune status and as to whether immunization
was recommended. (A susceptible individual was defined as someone who has
a serum titer of less than 1:8 against one or more of the poliovirus
serotypes by serum neutralization. Individuals with titers greater than 4
for all three serotypes were considered immune.)
Special immunization clinics were conducted at the Wilson City Hall by
the Texas State Department of Health, and all susceptible participants were
invited to attend. The first clinic was held in early April 1981 in order
to allow time for immunity to develop before the initiation of irrigation.
Subsequent clinics were conducted in May and June and in January 1982.
Study participants could also receive immunization at the Health Department
clinics in Lubbock or Tahoka if they preferred.
According to the Texas Department of Health's recommendations,
susceptible adults (18 years or over) received four doses of the Salk
inactivated polio vaccine (IPV). Injections were given monthly from April
through June 1981, with a booster shot to be administered in January 1982.
Susceptible children received the Sabin oral polio vaccine (OPV).
They began their series of immunizations in May 1981, in order to minimize
the risk of infecting a susceptible adult with the vaccine strain virus.
All individuals submitting to the immunization signed the informed
consent form which is used by the Health Department. (Parents signed for
minors.) A copy of this form is presented in Appendix C. All individuals
attending the clinic also received a short polio immunization history
questionnaire. The questionnaire was administered by the field
representatives by telephone to individuals who did not attend the clinic.
When an individual was deemed susceptible by serological analysis but
presented proof of immunization, a booster immunization was recommended.
A summary of the poliovirus protection status of participants is
listed in Table 4.5.
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TABLE 4.5. SUMMARY OF PARTICIPANT POLIOVIRUS PROTECTION STATUS
(January 1983)
Study Population
Total number tested
Number recommended for immunization
Number receiving complete immuniza-
tion series
Number receiving incomplete immuni-
zation series
Number refusing immunization
Number current study participants
who have not given blood
Children
158
71
63a
0
8
10
Adults
274
123
61
46
16
8
Total
432
194
124
46
24
18
a All children who were recommended for immunization had a previous
history of immunization. Therefore, only a booster dose was
administered.
34
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HEALTH WATCH
Serosurvey
Little information was available from the literature regarding the
serologic status of persons working or living near the production of
wastewater aerosols from sprinkler irrigation. This was especially true
with respect to their immunologic history against enteric viruses.
A serologic study was conducted because it provides useful and
important information with regard to microbial infection in or near areas
of wastewater aerosol production. Many, if not most, infections by enteric
viruses produce little or no clinical illness. Therefore, any reported
increase in infection and carrier rates may represent only a small portion
of the actual infection rate. Seroconversion rates and significant titer
elevations should provide much more evidence as to the true extent of
health risk.
Twice each year during the field study period, each participant was
instructed to report to a bleeding station at the Wilson community hall to
provide a blood sample for the serosurvey. An experienced phlebotomist
obtained blood either by venipuncture or finger-stick depending upon
volunteer's age or preference. Approximately 30 mL of blood was obtained
at each bleeding from each participant. .Finger-stick blood was collected
in nonheparinized capillary tubes while venous blood was allowed to clot in
vacutainer tubes at room temperature. A sufficient volume of blood was
collected to ensure that adequate material was left for cataloguing for
retroactive analyses. Blood specimens were then transported in ice chests
to the serology laboratory (UTSA in 1980 and 1981; UI in 1982 and 1983).
Informed and parental consent forms (Appendix C) were signed prior to
collecting these samples.
Tuberculin Skin Testing
Tuberculin skin tests were administered in June 1980, June 1981 and
December 1982 when the blood samples were obtained in order to monitor
possible infection with mycobacteria.
The intradermal Mantoux test is performed by the Texas Department of
Health using five tuberculin units (5 TU of PPD-S) introduced into the skin
of the volar surface of the forearm by syringe and needle. Participants
were asked to return in 48 to 72 hours to report positive or negative
reactions as defined in specific instructions. Those reporting positive
tests will be referred to the Texas State Department of Health to confirm
the reading and for clinical evaluation.
35
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Household Health Diary
Records of self-reported illness were maintained by each family member
to monitor health status during the pre- and post-irrigation periods during
the four years of field study. Records of self-reported illnesses were
maintained during all months of irrigation. Diaries were kept for about
one month after an irrigation season to record illnesses and infections
that may have begun during irrigation but were not apparent until weeks
later.
During 1980, diaries were collected on a biweekly basis from
participating households. They were then sent to the University of
Illinois for review and coding before being sent to SwRI for data entry.
Due to logistical problems involved in contacting the families, collection
of all diaries within a reasonable period of time was not always possible.
This not only presented problems in the review and coding process, but also
probably contributed to a reduction in completeness and accuracy of diary
entries, as participants often neglected to record illness information
until they were prodded by the field representative's visit. Consequently,
for the 1981-1983 health watch periods, there were some modifications in
the diary collection procedure.
Instead of personally visiting every household, the field
representatives telephoned each household once a week to obtain diary
information during 1981 and 1982. Also, by contacting families once a
week, better recall of illness information!was expected.
Beginning October 24, 1982, the number of families that were contacted
on a weekly basis was reduced by approximately half. The distribution of
households which were included as "sentinel families" is listed by zone in
Table 4.6. All households which have members that have exposure to
wastewater are included in the sentinel family list. The remainder of the
families were selected on the basis of geographic distribution and on the
family's past record of participation.
Illness Specimens
Field representatives were instructed to request permission to collect
an illness specimen from a study participant whenever the participant
reported the recent onset of an illness. Throat swabs were collected
within a three-day period after a participant reported the onset of a
respiratory illness. Stool specimens were collected within a ten-day
period after a participant reported the onset of GI or respiratory
symptoms. Study participants were also actively encouraged to contact the
field representatives immediately after the onset of a respiratory or GI
illness to request that illness specimens be collected.
36
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TABLE 4.6. COMPARISON OF SENTINEL POPULATION TO ORIGINAL POPULATION
Original population
Zone
Rural
Wilson
Total
1
3
5
2
4
6
Households
22
9
31
33
33
4
132
Adults
37
20
61
57
55
4
234
Children
13
6
30
37
35
3
124
Sentinel popu
Total Households
50
26
91
94
90
7
358
22
6
12
11
13
2
66
Adults
37
12
23
19
25
4
120
lation
Children
13
3
15
11
16
3
61
Total
50
15
38
30
41
7
181
CO
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The procedure fpr collection of throat swabs was taught to the field
representatives by personnel at' the Texas Department of Health. The Marion
Culturette II swabs were used for collection and preservation. In most
instances, two swabs were used for each illness specimen. All specimens
were kept on wet ice and shipped to UTSA laboratories as quickly as
possible.
Illness Surveillance
Since April 1982, UI has contacted the field representatives on a
weekly basis for the following health diary information:
• study participants who reported an illness,
• type of illness,
• dates of onset and conclusion of illness,
• study participants who could not be contacted,
• study participants who were out of town for more than two days.
This information was used to compile a weekly summary which listed the
number of participants who were contacted and the number of new acute
illnesses (by type) which were reported by the study participants. All
illnesses reported from Zone 1 were also npted in this report in order to
provide a rapid method for comparing illnes(s rates of participants who live
in the high exposure zone to the illness rates for all study participants.
:]
Illness information was also reviewed on a weekly basis to determine
if any unusual patterns of illness had developed. Patterns of interest
included geographic distribution of illnesses, age distribution of
illnesses, unexpected increases in respiratory or GI illness, and
households with unexpected recurrences of illnesses.
Fecal Specimens
During 1980 and 1981 regularly scheduled fecal specimens were
requested only for children age 12 or under. In cases where the household
had only one child in the age group, the next oldest household member was
also recruited as a donor. Due to the fact that only two households on the
Hancock farm regularly provided specimens in 1981, one adult was randomly
selected from each household and asked to provide a specimen in 1982. If
the selected adult was not willing to provide the specimens, then another
family member was given the option of providing specimens for the
household. In households which had been previous specimen providers, the
same family members were encouraged to continue providing samples. In order
to obtain a maximum amount of information during periods of irrigation
three consecutive specimens were solicited. A $5 subject fee was offered
38
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for each specimen and a $15 bonus was given to participants who provided
specimens all three times (one preirrigation and two post-irrigation
specimens).
Collection of the children's specimens took place over three two-week
periods in 1980 and six two-weeks periods in 1981. In 1982, each of the
six collections took place over a one-week period which was coordinated
with the irrigation schedule.
The fecal specimens were collected in the Sage stool specimen system
and processed by transferring approximately 10 g to each of two
appropriately labeled vials. Ten ml of a phosphate-glycerol buffer (pH 7)
was added to one vial to preserve bacterial viability. The remaining fecal
specimen (Vial 2) was shipped without addition of any preservative. Fecal
specimens were stored at 4°C and shipped on wet ice to arrive at the
laboratory within 24 to 36 hours after actual specimen collection.
The purpose of collecting and analyzing these specimens was to
determine whether there were any changes in the types and frequencies of
bacterial and viral agents recovered from children and adults relative to
their being exposed to wastewater aerosols. The routine collection of
stool specimens provided an unusual opportunity to determine any such
changes. Children were initially selected as the age subgroup to monitor
because being still susceptible they have higher infection rates and
because they are more readily available for specimen collection. The point
that the specimen collection was routine and not necessarily associated
with episodes of acute illness is also important. This is because any
pathogenic or unusual agents present in aerosols will usually produce
inapparent (subclinical) infection. By cross-referencing the health diary
information on self-reported illnesses with the laboratory findings, the
frequencies and types of clinical or subclinical infection can be
determined. Then these can be compared with the degree of exposure to
viable organisms in irrigation aerosols.
Activity Diary
An activity diary was distributed to each household participating in
the health watch in March, April, August and December 1982 and in April and
July 1983. The diary was maintained during weeks in which sprinkler
irrigation with wastewater occurred. The purpose of the activity diary was
to obtain information regarding the exposure of each participant to:
1) wastewater aerosols at the Hancock site (airborne transmission);
2) the wastewater at the Hancock site (contact transmission);
3) Lubbock (the ultimate source of agents transmitted via the
wastewater).
39
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The activity diaries which were sent to the households in March and
April 1982 were returned to UI in the self-addressed, stamped envelopes
which were included with the diaries. Due to the low compliance rate (55%
in March, 41% in April) and the high number of incorrectly completed
diaries, subsequent activity diary periods were scheduled for the week
prior to another health watch activity (either fecal collection or blood
drawing). The schedule permitted the health watch manager or field
representatives to be available to help the participants correctly complete
the activity diary. It was also possible with this schedule to follow up
participants who did not respond to the request for activity diary
information. This modification in activity diary collection procedures
resulted in a marked improvement (80 to 90%) in the response rate. The
activity diary form and the maps which accompanied these forms are included
in Appendix D.
40
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ENVIRONMENTAL SAMPLING
-------
ENVIROWENTAL SAMPLING
Wastewater
Wastewater Pathogen Screens--
A total of five large volume composite samples of wastewater were
collected and analyzed during the 1980 calendar year to determine the
relative densities of a wide range of enteric bacteria and viruses from two
sources: the Lubbock Southeast Trickling Filter Plant (LTFP) effluent and
the Wilson Imhoff Tank (WIT) effluent. Three of these samples for the
wastewater pathogen screen were from the Lubbock source, whereas the
remaining two were from the Wilson source. The basic purpose of these
pathogen screens was to identify organisms to which individuals within the
study population at the Hancock site presumably have not been exposed as
determined by baseline monitoring studies of antibody status. Furthermore,
the viruses isolated from these samples of wastewater can be used to
determine the selection of the most meaningful antigens for viral serology.
The three samples for the wastewater pathogen screen from the final
effluent of the LTFP (Plant 2) were collected at approximately three-month
intervals (June 3 and 4, July 28 and 29, and November 3 and 4, 1980). To
accomplish this sample collection, a 24-hour flow-weighted composite was
obtained by collecting six consecutive four-hour time-weighted samples of
effluent with an ISCO Model 1580 automatic sampler followed by flow-
weighted compositing based on plant flow data for each four-hour period.
During collection each four-hour sample was cooled to 4°C, and after
compositing the final large volume sample was transferred to sterile
bottles and shipped in a 4°C environment to the UTSA-CART laboratories via
airline parcel service for analysis within 24 hours. A complete
description of equipment used, sampling procedure, and compositing
calculation are shown in Appendix E.
The two wastewater pathogen screens from the WIT effluent were
collected concurrently with the first and second LTFP sample collections
(June 3 and 4 and July 28 and 29, 1980). To collect this sample, an ISCO
Model 1580 automatic sampler was used in a time-weighted mode over a 24-
hour collection since no flow measuring device was available. During
collection the sampled wastewater was cooled to 4°C and at the conclusion
of the 24-hour sampling period was transferred to sterile bottles and
shipped with the LTFP effluent samples. A complete description of
equipment used and sampling procedure is given in Appendix F.
Wastewater Sampling Collection in 1981—
In 1981, a total of ten wastewater composite samples were collected
from the LTFP effluent and 13 from the Wilson Imhoff tank effluent. These
24-hour wastewater composites were collected using the same methods as
41
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described in Appendixes E and F with the exception of the time-weighing
periods for composite preparation of the LTFP effluent being changed from
four hours to eight hours. A summary of wastewater sampling dates and
microbiological assay groups for 1980-81 is presented in Table 4.7.
Wastewater Sample Collection in 1982—
A total of 20 sampling periods occurred in the 1982 monitoring year.
Composite samples were collected from the pipeline, the reservoir and the
Wilson Imhoff tank according to the schedule shown in Table 4.8.
Compositing was accomplished by using an ISCO Model 1580 automatic sampler
in a time-proportioned operational mode. The pipeline sample location at
the northern boundary of the Hancock farm (i.e., "can" 4) replaced the
sampling location previously used at the LTFP when the pipeline became
operational in February 1982. Compositing for the pipeline sample was
accomplished by a time-weighing method rather than the flow-weighing method
previously used due primarily to the expectation that flows in the pipeline
would be more uniform than the effluent flows experienced at the LTFP.
Also, when the pipeline became operational, a new sampling location was
added at the Hancock storage lagoons. Since only Reservoir 1 was approved
to receive wastewater during the 1982 irrigation year, samples for this
location were collected either as a composite of grabs from various depths
in the lagoon or as a time-weighted composite from Can 1 when irrigation
from reservoir was occurring. A summary o:f wastewater sampling dates and
microbiological assay groups for the 1982 molnitoring year is presented in
Table 4.8. I
Wastewater Aerosol
Background Runs--1980 Baseline Year--
Four background air sampling runs were performed in August 1980 before
commencement of any spray irrigation at the Hancock farm. The objectives
of these runs were two-fold: to estimate the air concentrations of the
microorganisms of concern which residents in the area typically breathe
when outdoors and to identify whether there are any significant aerosol
sources of these microorganisms besides the irrigation system planned for
the Hancock site (e.g., the Wilson effluent pond). The first objective
includes determining background air concentration estimates both for Wilson
and for the rural area. The information collected from these runs aided in
the selection of microorganism groups to monitor on the other types of
aerosol runs. Additionally, background exposure information is an
important component of a balanced overall assessment of the significance of
participant exposure to a given microorganism concentration due to
wastewater aerosol sources.
These runs were conducted on four consecutive days during the period
August 5 through 8, 1980. Aerosol samples were collected by operating nine
42
-------
TABLE 4.7. WASTEWATER SAMPLING DATES, 1980-81
co
- . - _ — •• ~ • - -
Sampling dates
1980
6-3/6-4
7-28/7-29
11-3/11-4
1981
1-19/1-20
2-16/2-17
3-9/3-10
3-23/3-24
4-20/4-21
5-4/5-5
5-18/5-19
6-1/6-2
6-15/6-16
6-29/6-30
7-20/7-21
8-17/8-18
9-14/9-15
11-17/11-18
x - wastewater sample col
0 - viral identification
EV - enterovirus assay
FC - fecal col i form assay
Lubbock trickli
Full
microbiological
screen
X
X
X
X
X
ng filter eff
Limited
bacterial
screen
X
X
X
X
X
X
X
X
X
X
X
luent
ET
and
FC
®
®
®
X
X
X
X
®
X
®
X
®
®
X
Wilson Imhoff tank effluent
Full Limited FV
microbiological bacterial and
screen screen FC
x ®
x ®
x
x
x
x
x
x
x
x
®
X
X X
X ®
X X
X X
lected for indicated assay
performed on thi
s sample
-------
TABLE 4.8. WASTEWATER SAMPLING AND ASSAY SCHEDULE: 1982
Col lection
date
2-15/2-16
3-1/3-2
3-8/3-9
3-15/3-16
3-22/3-23
3-29/3-30
4-5/4-6
4-19/4-20
4-26/4-27
5-2/5-3
5-17/5-18
6-14/6-15
6-29/6-30
7-19/7-20
7-26/7-27
8-9/8-10
8-30/8-3 1b
9-13/9-14
9-27/9-28
10-11/10-12
Pipel ine ef f luent
Full Limited
microbiological bacterial Routine
screen screen assay3
xL
x
x
xL
x
X
xL
XL
X
X
X
x - wastewater sample collected for indicated
. - assay performed as subset of another assay
0 - viral identification scheduled on this sam
.
X
X
X
.
X
X
X
X
X
X
•
X
X
assay
p le
EV
and
FC
.
.
©
*
0
*
0
0
•
•
0
-0
X
•
0
Reservoir effluent Wilson effluent
Full Limited EV Limited EV
microbiological bacterial Routine and bacterial and
screen screen assay3 FC screen FC
x x
X
x 0
X X
x 0
X
X
X
X X
x^ x 0 x (x)
X X
-"- xL 0
X XX 0
x'- x • x x
X X • X 0
X X
X X
EV - enterovirus assay
FC - fecal col I form assay
x<- - I eg lone I la assay scheduled In addition to regular assay
a same organisms monitored on aerosol runs (fecal collform, fecal streptococci, coliphage, total enterovlruses, and c.
perfr1ngens/mycobacterI a)
b chlorlnation of pipeline effluent at Lubbock wastewater treatment plant
-------
Litton Model M large volume samplers (LVS) simultaneously for 30 minutes
before sunrise (0630 to 0700) at nine locations in or near the Hancock
farm. Locations for samplers included three within the city limits of
Wilson, one downwind of the Wilson effluent pond, one at a farm household
near the center of the Hancock farm, and the remaining four at farm
households in quadrants of the study area. Specifically, the sampler
locations as shown in Figure 4.7 were as follows:
Wilson: Three samplers were placed in fixed predetermined locations (A, B,
C) in the backyards of three Wilson families in the health watch.
These samplers were 400 meters apart, with residences in all
directions from each sampler location.
Wilson effluent pond: One sampler was located downwind from the middle of
the first effluent pond, 13 meters from the pond edge (Location D).
Rural area: Five samplers were placed in fixed predetermined locations
near the homes of five rural families participating in the health
watch:
E - farm near center of Hancock site
F - farm in northeast quadrant (4 km NE of Hancock site)
G - farm 0.7 km south of Wilson (upwind)
H - farm in southwest quadrant (<1 km SW of Hancock site)
I - farm in northwest quadrant (3.5 km NW of Hancock site).
Each sampler location was in an open area at least ten meters from any
house, farm, or lane. No obvious sources of microorganism aerosols were
located near or upwind of any selected locations near homes. Cotton was
growing on all nearby farmland. There were no cattle or horses at or
within a kilometer upwind of any sampler location. There were hogs near
locations D and H, but they were never upwind during sampling. A few
household and farmyard animals (dogs, cats, chickens, etc.) were observed
at nearly all sampling locations. Sampler operators wore surgical masks
and usually stayed downwind during the air sampling to minimize their
effect.
A grab sample of wastewater was taken near the middle of the large,
shallow Wilson effluent pond after each run. During the week of sampling,
the effluent was being diverted to an adjacent pond along a ditch about 12
meters upwind of the air sampler locations. The fecal microorganism levels
were much lower in the pond than in the Imhoff tank effluent.
The wind was from the south-southeast (160° to 168°) on all four
background runs. Winds were fairly strong on Run 2 (5.8 m/sec), but light
on the other runs. Solar radiation was nil (<15 W/m^) since sunrise was at
0703. Temperature ranged from 19°C to 23°C, while the relative humidity
varied from 69 to 76%.
45
-------
4 MILES
6 KM
Figure 4.7. Sampler locations for background runs
46
-------
Litton Model M large volume samplers were selected for performing both
the background runs and microorganism runs, primarily because the large
volumes of air which can be sampled provide sensitivity to detect low
microorganism levels in the air. These samplers are designed to collect
airborne particles by electrostatic attraction to a rotating disk on which
they are concentrated into a thin, moving film of collection media. A
complete description of the sampler is provided in Appendix G. Collection
efficiencies for electrostatic precipitators depend on the operating high
voltage. Sufficient voltage must be supplied to produce a particle charge;
the greater the voltage, the greater the driving force (particle charge) to
effect particle separation from air. However, very high voltages produce
sparking which in turn disrupts the electrical equipment and electrodes,
reducing the effective voltage.
Field operation of the samplers first required that an effective
decontamination be performed followed by suitable storage in this sterile
state. This was accomplished by a cleanup procedure using both absolute
ethanol and a buffered Clorox® solution, followed by sealing all sampler
openings. All decontamination procedures, both before commencement of any
aerosol run attempts and at the conclusion of each aerosol run, were
performed in a laboratory at LCCIWR. A copy of the step-by-step cleanup
procedure can be found in Appendix H.
Sampler runs were initiated by placing the necessary equipment with an
operator at each sampling site prior to the preagreed start time of 0630.
At each site the operator placed the LVS on a table which was leveled by
means of adjustable legs and connected an extension cord to a nearby power
source. At all sampling sites except the Wilson effluent pond where a
gasoline-powered alternator was used, arrangements were made to operate
samplers from a local power outlet. By a predetermined arrangement and
synchronization of watches, all operators started sampler operation at
0630. During these runs sampler operational parameters included an air
flow rate of 1000 liters per minute (1.0 m^/min), a high voltage setting of
12 to 15 kV (highest voltage obtainable without significant sparking) and a
minimum recirculation rate of brain-heart infusion (BHI) broth, the
collection media, of 10 mL/min. BHI with 0.1-% Tween 80® to prevent
foaming was selected as the collection and transfer medium, which was shown
to be adequate for sample concentration and for preservation and assay of
the microorganisms (Johnson et al., 1979). At the conclusion of each
sampling run, media containers were tightly capped, appropriately labeled,
cooled to 4°C, and immediately shipped to San Antonio via commercial
airline counter-to-counter parcel service. Sample analyses were initiated
the same day as sample collection.
Wastewater Aerosol Monitoring--1982 Irrigation Year--
Microorganism runs—A total of 20 microorganism runs similar to those
conducted at the Pleasanton, California spray irrigation site (Johnson
47
-------
et al., 1979) were completed during the preplanting and summer 1982
irrigation periods at the Hancock farm to characterize the wastewater
aerosol. Results from these runs characterized microorganism densities in
air under various conditions at the Hancock site at distances up to 400
meters downwind of the irrigation rig.
To conduct these runs, ten large volume aerosol samplers (Litton Model
M) as used on the background runs were loaned by the Naval Biosciences
Laboratory to SwRI under a subcontract. These were deployed at various
downwind distances up to 400 meters from the rig sampled and upwind of the
primary aerosol source sampled. Initially, samplers were located at
nominal downwind distances of 50 m, 75 m, 150 m (paired), 200 m (paired)
and at an upwind location (paired). Nominal downwind sampler distances
were subsequently adjusted for some microorganism runs to 125 m, 175 m,
300 m (paired) and 400 m (paired) to determine microorganism aerosol levels
out to the 400-m buffer zone boundary.
Model M samplers were decontaminated utilizing the same procedure used
for the background runs. Brain-heart infusion (BHI) plus 0.1% Tween
80® was again used as the sampling fluid. All runs consisted of a
simultaneous 30-minute sampling time with sampler operation at 1.0 m^/min
air flow and maximum high voltage obtainable with minimal plate sparking.
Field conditions occasionally required LVS operation below 12 kV to
eliminate sparking. It was often difficult for field operators to maintain
an average air intake flow rate for a run at 1.0 nvVmin, since sporadic
wind gusts would temporarily alter the air flow rate.
During the time of aerosol sampling, a simultaneous wastewater
composite sample was collected from the irrigation spray rig being
monitored. At the completion of each run, samples were labeled, cooled to
4°C, and shipped to the UTSA-CART laboratories for analysis on the
following day.
Sampling conditions for the microorganism runs are summarized in Table
4.9. The operating voltages of the large volume samplers during these runs
are provided in Table 4.10.
Quality assurance runs—Two quality assurance (QA) runs were conducted
as in the Pleasanton study (Johnson et al., 1979) to determine assay
variability between samplers, between aliquots of BHI from the same sampler
and between replicates split by the receiving laboratory. These runs
consisted of the same cleanup and operational protocols utilized for the
microorganism runs with the exception that all operational samplers were
lined up in a row (2-meter separation) equidistant and parallel to the
orientation of the spray irrigation rig. For QA Run 1, conducted during
spring irrigation at a time of blowing dust, the nozzle line to sampler
line distance was 50 meters, whereas for QA Run 2, conducted during the
48
-------
TABLE 4.9. SUMMARY OF SAMPLING CONDITIONS—AEROSOL RUNS—OPERATIONAL YEAR 1982
Aerosol sampler location
Sampled rig
Run
no. No.
Ml 9
M2 2
M3 15
M4 12
M5 15
M6 3
M7 11
M8 15
M9 15
M10 4
Mil 4
M12 8
M13 8
M14 7
M15 10
M16 12
M17 14
M18 14
M19 9
M20 10
a 6S| -
b 9* -
Orien- End gun
tat ion status
315°
130°
290°
315°
230°
50°
325°
70°
70°
330°
280°
80°
80°
55°
125°
300°
30°
20°
90"
130°
On
Off
Off
On
Off
Off
Off
Off
Off
Off
Off
Off
Off
Off
Off
On
On
Off
Off
Off
angle of sampler
mean angle
Line
Position/ Angle
tower 0S |a
Outer/6 35°
Center/3 80°
Center/4 60°
Outer/5 30°
Outer/5 80°
Inner/3 45°
Outer/6 50°
Inner/3 75°
Inner/3 75°
Outer/6 70°
Center/4 85°
Center/4 90°
Center/4 90°
Center/3 75°
Center/4 65°
Center/4 65°
Center/4 90°
Center/4 90°
Outer/5 65°
Outer/5 85°
line with rig (0°
Rig
Other rigs In
movement Mean wind
Distance to
rig, (m)
Single Single Pair Pair
39
35
49
55
64
50
61
50
55
50
125
125
125
125
125
50
125
125
23
80
<6s1
64
60
80
80
115
75
87
75
80
75
175
175
175
175
175
75
175
175
23
130
_^ 90°)
of wind with the rig during the run,
139 214
135 210
140 203
148 225
174 288
125 200
155 236
125 200
130 205
125 200
300 400
300 375
300 375
300 365
300 400
125 200
290 400
275 400
48 98
255 323
measured in
during direction
run (m) G^b
+2
-2
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
4
90
same
25°
100°
75°
23°
113°
60°
60°
50°
go-
so0
so-
los'
90°
80°
60°
65°
90°
85°
50°d
110°
direction from
operation
Possibly Not
upwind
3
None
None
2,7
2,7
12,19
None
None
None
None
None
None
None
4
6,7,8
22
7
None
21C
6,7
rig as i
upwind
15
3,5
3,5,8,11
6
None
None
None
None
None
None
None
6,11,12,13
17,19
11,13,17,
19,2QC
7,17,19,
20C
2,4,8,9,
12,18,c
20,C21C
3,4,7,8,
9,11,12,
18,C20,C 21C
2,8,11,15,
18,19
15,18,19
9s 1
Wastewater
Temp
Source (°C)
Pi pel Ine
Pipel ine
Pipeline
Pipeline
Pipeline
Pipeline
Reservoir -
Pipeline
Reservoir
Reservoir
Pipeline 27
Pipeline
Reservoir
Pipeline 27
Pipel ine
Reservoir
Pipeline 26
Pipeline 24
Reservoir
Pipeline 28
c Rig with drops
d From Cl 1 matron ics
Weather Station at the
tech
plot
-------
TABLE 4.10. SAMPLER OPERATING VOLTAGE ON THE MICROORGANISM AEROSOL RUNS
en
o
Operating high voltage of large volume samplers (kV)
Aerosol Upwind of
run irrigation
number rig 20-39 m
PREPLANTING IRRIGATION
Ml ? ? 14
M2 13 11.5 11
M3 12 9
M4 14 14
M5 13 13
M6 12 14
SUMMER CROP PIPELINE IRRIGATION
M7 12.7 10.7
M8 14 12
Mil 15 12.5
M12 12 12
M14 " 13 13.5
M15 13 13
M17
M18
M20
SIMMER
M9
M10
M13
M16
M19
12
12
12
12
12
12
40-59 m
10
11
10
12
12
Downwind of irrigation nozzle line
60-89 m 90-149 m
14 12.
11 12.
6 12.
10 12
12
11 12
12 12
12 12.
2
8
5
9
8
12
12
10
12
13
14.
14
13.8
12.8
12.5
12
13
12
12.4
5
150-249 m
14
14
10
12.5
12
12
11
12
12
12
12
12
12.
16.
14
15
12.5
12.5
12
12.5
12
12
5
5
250-349 m 350-409 m
12.5 13
12 11 14 13
12.8 12.5 13 12
13.2 12.8 11.5 12
12.5 13 12.8 11
12.8 12.8 13 13
12.8 14 13
16.4 11 14 14
CROP RESERVOIR IRRIGATION
15
13
12
12.5
12
12
13
11.5
13.5
12 14 14.5
12
12
12
12 14.5
12 - 11.
12 12.
12 12.
14
5
8
12
6
12.4
12.5
13.2
14
12.7
12.5
11
12.5
12.7
12
12.8
12.5 12.8 12 13
? - Voltage not recorded.
-------
summer irrigation period, the distance was 75 meters. Sampling conditions
for the quality assurance runs are shown in Table 4.11. After aerosol
collection, but prior to shipment to the CART laboratories, the 100-mL BHI
aliquots from each sampler were split into four equal aliquots to achieve a
blind distribution of "identical" samples for a predetermined sequence of
microorganism assays.
Enterovirus runs—Since the wastewater contains a high enough level of
enteroviruses for the microbiological dispersion model to predict their
probable detection in aerosols, four special aerosol runs similar to those
performed at Pleasanton, California (Johnson et al., 1979) will be
conducted to estimate the enterovirus aerosol concentration. To conduct
this type of run, all functional large volume samplers were operated
simultaneously at a downwind distance of about 50 meters from the nozzle
line for five consecutive 30-minute sampling periods. The samplers were
aligned parallel to the nozzle line with a sampler spacing of 1.5 m. The
irrigation rig was operated at a reduced travel setting so it progressed on
the dry side of the field (i.e., toward the samplers) at some minimal rate,
typically 5 to 10 m/hr at the tower used for alignment of the sampler line.
At the end of every two 30-minute segments, the sampler position was
adjusted to compensate for the rig movement. The initial and final
distances from sampler line to nozzle line are shown for each segment on
the sampling conditions summary presented in Table 4.12. The BHI
collection fluid was changed after each sampling segment, and all BHI was
pooled at the conclusion of the run. After transport to the UTSA-CART
laboratory, the BHI was concentrated and plaque-assayed for human enteric
viruses.
Dye runs—Four dye aerosol runs were conducted using the procedures of
the Pleasanton study (Johnson et al., 1979) to estimate the aerosolization
efficiency (i.e., the fraction of sprayed wastewater that becomes
aerosolized) of the spray irrigation rigs at the Hancock site. Because the
rigs direct a fairly fine spray downward toward the ground, it was
anticipated that this aerosol ization efficiency may differ substantially
from the 0.33% (geometric mean) aerosolization efficiency of the impact
sprayers used for wastewater irrigation at Pleasanton, California.
One dye run was conducted in March 1982 while the last three were
completed in July 1982. To perform these runs, a 20% solution of Rhodamine
WT dye mixed with glycerol was injected at a constant rate into the
pipeline supplying the sprayers of the irrigation rig being sampled. The
dye was injected with a Zenith Constant Torque Unit Type ZM coupled with a
No. 11 Zenith Metering Gear Pump.
Aerosols were collected using 500-mL graduated all-glass impinger
(AGI) samplers connected to a vacuum pump as indicated in the following
schematic:
51
-------
TABLE 4.1). SUMMARY OF SAMPLING CONDITIONS—QUALITY ASSURANCE RUNS—OPERATIONAL YEAR 1982
Sampled riq
Run
no.
01
Q2
Or i en-
No, tat ion
11 340°
15 65°
End gun
status
Off
On
Aerosol sampler location
Position/
tower
Right/4-5
Center/3
Distance to
rig (m)
75
50
Rig
movement
during
run (m)
0
0
Mean wind
direction
9wa
110°
75°
Other rigs in
operation
Poss i b 1 y
upw ind
None
None
Not
upw ind
None
None
Wastewater
Source
Pipe! ine
Pi pel !ne
a 6W - mean angle of wind with the rig during the run, measured in same direction from rig as Qs\
en
ro
-------
TABLE 4.12. SUMMARY OF SAMPLING CONDITIONS—VIRUS RUNS--OPERATIONAL YEAR 1982
tn
CO
Aerosol sampler location
Run
No.
VI
V2
V3
V4
Segment
No. No.
1 4
2 "
3 »
4 "
5
1 17
2 "
3 "
4 i.
5 »
1 14
2 ..
3 "
4 ii
5 "
1 14
2 ii
3 »
4 »
5 "
Samp led
Orien-
tation
320°
320°
325°
325°
325°
60°
60°
58°
58°
56°
70°
70"
68°
68°
66°
35°
32°
30°
27 o
25°
rig
End gun
status
Off
It
II
II
II
Off
It
It
It
"
Off
II
II
II
II
On
ii
ti
ii
ii
Position/
tower
Right/4-5
M
II
II
II
Center/5
ii
n
ii
ii
Center/4
n
n
"
n
Center/5
n
ii
n
n
Distance
to rig (m)
Start
60
60
55
55
55
50
45
50
45
52
47
44
50
47
50
60
51
55
46
50
Finish
60
60
55
55
55
47
42
47
42
49
44
41
47
44
47
54
45
49
40
44
Rig movement
during
segment (m)
0
0
0
0
0
3
3
3
3
3
3
3
3
3
3
6
6
6
6
6
Mean wind
direction
6W
30°
50°
105°
110°
110°
105°
110°
105°
110°
115°
80°
85°
55"
80"
55°
-
45°
75°
60°
35°
Other rigs in
operation
Possibly
upwind
None
n
M
n
n
None
n
n
n
n
4,7
M
M
»
n
7
ft
It
It
It
Not
upwi nd
None
ti
it
tt
ti
2,4,6,7,
11,12,13
17,19
M
it
it
ii
6,11,13,
17,20
It
It
It
II
2,4,8,9,
12,18,20,
21
ii
tt
n
n
Wastewater
Source
Pi pel ine
it
n
it
n
Pipe! ine
n
n
it
n
Pipel Ine
ti
ti
ti
M
Pipel ine
(27eC)
n
ii
n
n
-------
Rotameter
0-2 cfm
AGI
Trap
Critical
Orifice
Pump
The rotameter was used only for calibrating the system in the laboratory.
With the critical orifice in line and a pump vacuum of at least 15 inches
mercury, the nominal flow rate through AGI sampler was 1.0 cfm (cubic feet
per minute).
To perform a dye run, AGI samplers containing 100-mL deionized water
as collection media were set up in pairs at four locations: two pairs at
25 m and two pairs at 75 m downwind of the monitored rig. One sample set
(i.e., 25-m and 75-m pairs) was aligned with a tower near the center of the
irrigation rig while the other sampler set was aligned at the same
orientation but displaced to the right or left of the center line set
depending upon wind direction by two rig spans. When all equipment was in
place, the Zenith gear pump began injection of dye into the irrigation
system, and when the dye was visible in front of a sampler set, the AGI
samplers commenced operation. Samplers were operated for a 6- or 7-minute
period until dye was no longer visible at the nozzles directly in front of
the sampling station. At the conclusion of the sampling period, the water
media was transferred to glass bottles for storage until analysis. As soon
as dye was visible in the wastewater at the nozzle closest to the injection
pump, grab samples were obtained at 1-minute intervals for as long as dye
was visible to determine source strength. Dye concentrations in both the
aerosol samples and wastewater samples were determined using a Turner
Spectrofluorometer Model 430, Sampling conditions for the dye runs are
displayed in Table 4.13.
Particle size runs—Five particle size runs were performed using
Andersen 1 ACFM six-stage particle samplers to determine the concentration
and particle size distribution of the wastewater aerosol microorganisms.
The samplers were connected to the orifice system and vacuum pump that was
utilized on the dye runs to maintain a nominal flow rate of 1 cubic foot
per minute (CFM) through the sampler. Each run was made with eight
samplers deployed in pairs, one upwind of the sampled source and the
remaining three at nominal downwind distances of 25, 50 and 75 meters.
Sampling times ranged from 8 to 10 minutes. A summary of sampling
conditions during each of the particle size runs is shown in Table 4.14. A
composite wastewater sample was collected simultaneously from the rig being
sampled to determine source strength.
Standard plate count agar was used as the collection medium in these
samplers. After sample collection, plates were incubated at the LCCIWR
laboratories for 24 hours at 35 ± 0.5°C and counted for colonies.
54
-------
TABLE 4.13. SUMMARY OF SAMPLING CONDITIONS—DYE RUNS—OPERATIONAL YEAR 1982
Sampled rig
Run
No.
D1
D2
D3
D4
a 9S|
b 9W
No.
15
4
4
15
- angle
- mean
Orien-
tation
230°
330°
330°
65°
of sampler
End gun
status
Off
Off
Off
On
Tower
Left
pos i t i on
3
6
6
3
line with rig (0°
angle of wind with
Right
position
5
4
4
5
< 9s 1 < 90°)
the rig during the run.
Aerosol samp ler
Li ne
angle
9s|a
65°
70°
70°
90°
location
Distance to rig
Left
pos i t i on
25 75
25 75
25 75
40 80
measured in same
(m)
Right
Mean wind
direction
position 9w
25
25
25
40
75
75
75
80
direction
80°
90°
80°
90°
from rig as 9S j
Wastewater
Source Temp (°C)
Pipeline
Pipel ine
Pipel Ine
Pipeline 25.5
en
en
-------
TABLE 4.14. SUMMARY OF SAMPLING CONDITIONS—PARTICLE SIZE RUNS—OPERATIONAL YEAR 1982
Aerosol sampler location
Run
no.
PI
P2
P3
P4
P5
No.
2
11
15
4
14
Sampled
Or i en-
tat ion
130°
330°
70"
280"
30°
rig
End gun
status
Off
Off
Off
Off
Off
Position/
tower
Center /3
Right/6
Inner/3
Center/4
Center/5
Line
Angle
es|3
80°
85°
75°
85°
60°
Distance to
Pair
36
33
20
35
35
Pair
61
58
45
60
60
rig, (m)
Pair
86
83
70
85
85
Rig
movement
during
run (m)
0
0
0
0
0
Mean wind
direction
9wb
70°
30°
60°
125°
70°
Other
rigs in
operation
Possibly
upwind
None
None
None
None
None
Not
upwind
3,5
None
None
None
3,4,7,8,9
11,12,18,
20,21
Wastewater
Source
Pipel ine
Pipel ine
Pipel ine
Pipel ine
Pipel ine
- angle of sampler I ine with rig (0° _<_ 9s 1 _<_ 90°)
- mean angle of wind with the rig during the run, measured in same direction from rig as 9S|
01
-------
Dust storm runs—Dust storms that could entrain many sprayed
microorganisms from the spray fields as a particulate aerosol are common in
the Lubbock area, especially in spring. These dust storms may be another
wastewater-associated pathway of infection in addition to the wastewater
aerosol. If dust storms occurred during aerosol monitoring periods,
special dust storm sampling runs were to be attempted. These runs would
have been performed by utilizing AGI samplers with BHI collection medium
operated for a brief period (about 15 minutes). Samplers would have been
located both upwind and downwind of the spray fields on each dust storm
run. No localized dust storms occurred during any of the monitoring
periods. However, QA Run 1 took place during a time of blowing dust.
Calculation of Microorganism Density in Air
The microorganism density sampled in air was calculated from the
assayed microorganism concentration in the sampler's collection fluid. For
an individual LVS, the equation is
c- Axv
F x R x D
where C - concentration of detectable microorganism units/m^ of air
(e.g., cfu/nP)
A - concentration of detectable microorganism units assayed in the
collection fluid (cfu/mL)
V - final volume of collection fluid (usually 100 ml)
R - air sampling rate (usually 1.0 nvVmin)
D - sampling duration (usually 30 min)
F - correction factor for LVS operating voltage (reference basis of
12 kV)
LVS are not as efficient as impinger samplers in the collection of
microorganisms in air, and the efficiency varies with the LVS operating
voltage. The collection efficiency of the LVS units employed in the field
sampling was determined relative to AGI samplers in wind tunnel experiments
performed in July 1980 and October 1982. The relative collection
efficiencies (mean ± standard error) of the LVS were found in the 1982
tests to be 0.29 ± 0.017 in 18 tests at 12 kV and 0.68 ± 0.022 in 29 tests
at 13 to 18 kV.
No attempt was made to adjust the aerosol concentration to the AGI
collection efficiency since there is no standard aerosol sampling method
57
-------
and since the absolute collection efficiency of AGI samplers was not
determined. Rather,-the"LVS-data were corrected for operating voltage to
render these data as internally consistent as possible.
The applied correction factors F for various operating voltages are
presented in Table 4.15. These correction factors are the minimum expected
correction. Appendix I presents the details on the calibration studies and
evaluation of four candidate operating voltage correction factors. While
other environmental factors such as particle concentration in air and
relative humidity may also influence collection efficiency, no corrections
have been applied to the aerosol data for such factors because the
experimental data were insufficient to develop calibration curves.
The enterovirus density in air was determined during virus runs in
which the collection fluid from many LVS was pooled and all except 100 mL
of the fluid was concentrated prior to assay for enteroviruses. The
enterovirus density in air equation is
C= BxU
(V-100 ml) n
v 1J1 Fl * RI x DI
where B - concentration of detectable enterovirus units in concentrated
collection fluid, pfu/mL
U - final volume of concentrated collection fluid, mL
'V - final volume of pooled collection fluid, mL
n - number of LVS samplings pooled.
For particle size aerosol runs the number of viable aerobic particles
per unit-volume of air for each stage in the sampler was calculated using
the formula
C =
R0 x T x 0.0283
where C - concentration in air
R0 - sampling rate for system from calibrated orifice in ft-Vmin
T - sampling time in minutes
0.0283 - conversion factor for ft3 to m3.
58
-------
TABLE 4.15. CORRECTION FACTOR FOR LVS OPERATING VOLTAGE
(Referenced Basis of 12 kV)
Operating voltage (kV) Correction factor (F)
6 0.33
8 0.36
9 0.38
10 0.42
11 0.57
11.5 0.80
12 1.00
12.5 1.15
13 1.25
13.5 1.32
14 1.33
14.5 1.32
15 1.29
16 1.24
17 1.22
18 1.21
59
-------
Results for each stage were reported as cfu/m3 which represents the mean
number of viable particles.detected on ..stajidard plate count agar per cubic
meter of air sampled.
The concentration of Rhodamine dye in the aerosol collected in each
downwind .impinger during the dye runs was calculated using the formula
c. C'XV
R x T x 103
where C - concentration in air (yg/m3)
Cj - Rhodamine concentration in impinger
V - volume of impinger solution (usually 100 ml)
R - air sampling rate in L3/min
T - sampling time in minutes
103 - conversion factor for L3 to m3.
The geometric mean of all applicable aerosol density values was used
to estimate the middle of the aerosol density distribution in summary
tables. When all values were below the detection limit, the estimate
reported in place of the geometric mean' was less than the cumulative
detection limit obtained by pooling the total volume of air sampled.
Sometimes the set of aerosol data included some measured values and some
values below the detection limit. In such cases 1) the geometric mean was
calculated with x/2 substituted for
-------
wastewater lagoons); 3) rodents (e.g., feed or food stuffs contaminated by
fecal droppings or urine from field mice infected by wastewater spray which
may be spending the winter in farmhouses and barns); and 4) fbmites (e.g.,
wastewater-contaminated work clothes, hands, or doorknobs). Since the
possibility of a fly-insect vector pathway of infection is frequently cited
and the cost is low, a small pilot study was initiated to investigate this
potential route of transmitting infectious agents. However, lacking an
illness/infection distance pattern, the cost of investigating such other
pathways of infection as rodents and fomites could not be justified at this
time.
Houseflies and other flies were trapped at the farmhouses and at
effluent ponds. It was not necessary for the flies to be houseflies
(Musca domestica L.); in fact the predominant scavenger-type muscoid
Diptera at the sites were preferred. Using baited traps, flies were
collected at the Wilson effluent pond and at the several farmhouses in 1980
and collection attempts were made at the reservoirs and at farmhouses in
1982. An effort was made to isolate and quantitate the level of enteric
bacteria and viruses in these fly samples. A target number of at least 200
flies per sample were sought (100 for bacterial analyses and 100 for viral
analyses).
To collect flies, a stationary, bait-type trap was located and
anchored i.n a potentially fly-prone area protected from wind, direct
sunlight, children, animals and other potential disturbances. These traps
were baited with a nonpoisonous bait such as canned cat food and milk. The
cat food provided a perch for the fly to light on and the milk kept it
moist longer since dried-up bait does not attract flies. The traps were
checked every 24 hours at which time the bait was changed since fermented
bait (with only milk added each day) may be harmful to farm pets.
When at least 200 flies are in the trap, it was placed in a large
garbage bag and returned to the lab at LCCIWR. Initially, flies were
killed by using ether, but since this procedure could be lethal to the
bacteria and viruses of interest, it was discontinued. Thereafter the
entire garbage bag and trap were chilled in a cold room (4°C) for at least
one hour. The contents of each trap were emptied on paper, odd species of
flies were discarded, and a maximum of 200 flies was counted out from each
trap. The flies were transferred to a sterile container, appropriately
labeled, and maintained at 4°C until arrival in the CART laboratory.
During the baseline year, the first attempt to collect flies occurred
concurrently with the background aerosol runs, August 4 through 8, 1980.
These attempts were initially directed at locations adjacent to the Wilson
effluent pond and at farmhouses on the Hancock property which were later
surrounded by wastewater sprinklers. Since collection attempts at these
locations were unsuccessful, the effluent pond traps were moved 100 meters
to a location adjacent to pig pens. Also, the traps located near
61
-------
farmhouses were moved to locations which had livestock. Subsequently, 200
flies were collected at the pig-pen-locations. and no flies were collected
at any of the farmhouse locations.
With the occurrence of rain in early September and the following
evolution of a fly population, a second fly collection attempt was
performed September 15 and 16 with traps located near the Wilson effluent
pond, two farms within the study site, and a school trash can. No flies
were collected during this attempt.
During a subseqent third attempt (October 15 to 17) with traps at four
locations, approximately 1,200 flies were collected near the pig pens
adjacent to the Wilson wastewater treatment facility, and approximately 65
flies were collected from barns at farmhouses located near the reservoirs
under construction on the Hancock farm.
Fly collection during the operational year was scheduled to be
conducted concurrently with the aerosol monitoring tasks in July and August
1982. These attempts were performed utilizing baited fly traps in the same
manner as during the baseline year at locations adjacent to the lagoons on
the Hancock farm and the Wilson treatment facility. A fly collection
attempt in August 1982 yielded insufficient flies for laboratory analysis.
Observations for a significant increase in fly population were made until
the first freeze, but conditions never warranted another attempt at fly
collection. •
ti
Meteorological Data :
Background Aerosol Runs--
Various meteorological parameters were observed and recorded during
the four background runs conducted August 4 to 8, 1980 to quantify
background air levels of microorganisms and to identify potential aerosol
sources other than the spray irrigation system. These parameters included
wind direction and wind speed at a two-meter height utilizing a Meteorology
Research, Incorporated (MRI) Model IM-5810 Mechanical Weather Station,
temperature and relative humidity using a Bendix Psychron Model 566-2
psychrometer, and solar radiation using a Bel fort Pryhel iograph 5-3850.
All of these parameters were measured at the research plot near the center
of the Hancock farm during the actual run time. Additional parameters
obtained from the National Weather Service at Lubbock included time of
sunrise, wind speed, wind direction, cloud cover, cloud type, and minimum
height.
General Cl imato.logy--
An electronic weather station (EWS) and cassette data acquisition
system (CDAS) from Climatronics Corporation were installed at the research
62
-------
plot in March 1981 to measure and record general climatological parameters
on the Hancock farm. Sensors to measure wind speed and wind direction were
mounted on a 10-meter telescoping tower while sensors for measuring
temperature, dew point, and solar radiation were located on a 2-meter
tripod adjacent to the tower. These parameters were recorded continuously
on a 5-inch wide chart moving at 1 inch per hour (l"/nr). Additional,
instantaneous values of these parameters were recorded every five minutes
on a magnetic cassette tape in the CDAS unit. These tapes allowed cost-
effective digitizing of meteorological data for the irrigation periods.
For example, tables of hourly averages for all parameters plus wind rose
plots were obtained. Also, they provide a means for input of
meteorological data for computer modeling after transferring data from the
cassette tape to a nine-track tape.
The meteorological data accumulated in 1982 on the CDAS was processed
for the irrigation periods by Envirodata Corporation to produce hard copy
outputs of hourly averages, daily averages, and daily high and low values
for all parameters (wind speed, wind direction, solar radiation,
temperature, and dew point. Wind rose plots were generated for both the
spring (February 16 to May 4) and summer (July 26 to September 17)
irrigation periods. These plots are shown in Figures 4.8 and 4.9,
respectively. No wind speed data for the summer period is plotted due to a
malfunctioning anemometer translater board during most of this period.
These plots are in general agreement with the wind frequency plots based on
5-year historical data presented in Figures 4.3 and 4.4 which were employed
in exposure estimation.
Meteorological Measurements During Aerosol Runs—
During aerosol runs, meteorological parameters were measured about 100
meters downwind of the sampled rig to complement measurements made at the
research plot at the Climatronics EWS/CDAS units. Field measurements
included wind speed and wind direction at the 2-meter level, ambient
temperature and relative humidity, and solar radiation by the same
instrumentation utilized on the background runs. Visual observations were
made for cloud type (to determine minimum cloud height) and eights of the
sky with cloud cover. The Climatronics CDAS unit was programmed to scan
and record at 1-minute intervals during periods of aerosol sample
collection.
Summaries of meteorological conditions for the different types of runs
are presented in Tables 4.16 through 4.20. Values for the EWS are averages
obtained from the strip chart for the run period.
63
-------
JJBBOCX HERLTH EFFECTS STUDY
£afl!- CLIMRTOLOGY
HRNCCCK FRRM
WIND ROSE OBSERVED WIND FREQUENCY FOR Z/16/82 TO 5/04/82
N
ilNW
NNN
NNE
P05SI3LE nCUPJ 1372 j
NW2ER CF HOURS 1427 i
ZfCT~ CRPTijRE 8S.3ZS
ENE
ESE
PLOT LEGEND
« «im SPEED
PERCENT WIND
2-2.92
8 A O*
. '•O-.
3-5.92
44.7:
6-9.90
30.3X
s.sa:
.799S
25.:- 99
31
Figure 4.8. Wind frequencies for the 1982 spring irrigation period:
Hancock farm meteorological station
64
-------
LUBBOCK HEHLTH EFFECTS STUDY
iENEHRL CL1M9TOLOSY
HRNCOCK FRRM
WIND ROSE OBSERVED WIND FREQUENCY FOR 7/26/8Z TO 9/17/82
N
MNM
MSH
NKH
SH
HISSINS 11.57:
VfiRIflBLE it
POSSIBLE HOURS 1296
NUMBER OF HOURS 1U6
3RTS caPTURE 38.43;
NNE
NE
ENE
ESE
SE
m
PtOT LEGEND
PERCENT HIND
Figure 4.9. Wind frequencies for the 1982 summer irrigation period:
Hancock farm meteorological station
65
-------
TABLE 4.16. SUMMARY OF METEOROLOGICAL CONDITIONS—AEROSOL RUNS—OPERATIONAL YEAR 1982
<7>
Mean wind
direction (°)
Run no.
Run date
Run time
M 1/2-22-82
1850-1920
M2/2-23-82
1650-1720
M3/2-24-82
1400-1430
M4/3-17-82
1535-1605
M5/3- 18-82
1230-1300
M6/3- 19-82
1148-1218
M7/7-7-82
1620-1650
M8/7-8-82
1353-1423
M9/7-9-82
1331-1401
Ml 0/7- 11 -82
1530-1600
Mil/7-14-82
1350-1420
Ml 2/7- 15-82
1114-1144
Ml 3/7 -16-82
1025-1055
M14/8-3-82
1327-1357
M 1 5/8-5-82
1211-1241
M16/8-6-82
1210-1240
Ml 7/8-23-82
2030-2100
Ml 8/8-25-82
2125-2155
Ml 9/8-26-82
1422-1452
M2 0/8-27-82
1320-1350
A i r temp
At run
1 ocat i on
16
26
10
24
24
17
29
31
32
28
31
28
27
33
34
31
24
22
32
35
(°C)
EWSa
19.5
29
12.5
26.5
26
19
30
33
35
32
32
30
29
33
32
33
28
25
35
35
At run
location
(2 m)
160
200
35
145
155
240
85
120
160
50
150
155
170
155
185
235
120
115
b
200
EWS
(10 m)
170
205
_
40
155
160
255
110
140
160
65
180
185
180
210
170
240
120
140
220
235
Wind speed (m/sec)
At run
1 ocat i on
(2 m)
9.6
6.7
10.3
2.4
7.9
6.6
11.4
6.9
4.2
7.7
6.4
8.0
7.3
4.3
4.9
3.3
0.9
0.6
b
2.5
EWS
(10 m)
5.5
7.0
11.5
2.5
9.0
8.0
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
1.25
3.0
2.5
Humidity
at run
location
46
50
49
34
66
21
59
54
29
51
40
51
54
37
24
39
59
77
51
31
Radiation at run location
Dewpoint
at EWS
-12
-4
-17
-5
-7
-12.5
-4
-2
-1
-3
-2.5
-4
-4.5
-1.5
-2.5
-1.5
-6
-9
0
0
Cloud
cover
(8ths)
3
<1
7
6
4
<1
4
<1
<1
2
2
<1
0
0
0
0
<]
3
1
<]
Cloud
height
NA
_
High
High
High
_
Middle
_
—
High
High
_
_
_
_
_
_
High
Middle
& High
_
Solar radiation
gca 1 /cm^/m 1 n
0
0.73
0.90
0.93
0.95
1.23
0.51
1.25'
1
1.26,
0.15
1.35!
1.10'-'
1.05
1.20
1.15
1.17
After sunset
After sunset
0.63
1.14
NA - not aval(able
a MeteoroIogIcaI data co11ected
b Field met system malfunction
from Climatronics Electronic Weather Station (EWS) at research plot
-------
TABLE 4.17. SUMMARY OF METEOROLOGICAL CONDITIONS—QUALITY ASSURANCE RUNS—OPERATIONAL YEAR 1982
Mean wind
direction (°)
Run no.
Run date
Run time
01/3-15-82
1543-1613
02/7- 13-82
1359-1429
Air temp (°C)
At run
location EWSa
19 11
29 30
At run
location
(2 m)
230
170
EWS
(10m)
250
190
Wind speed (m/sec)
At run
location
(2 m)
9.4
3.8
EWS
(10m)
11.5
NA
Humidity
at run
location
(*)
30
49
Radiation at run location
Dewpoint Cloud
at EWS cover Cloud
(°C) (8ths) height
-10.5 Blowing dust
-4 <1
Solar radiation
gcal/cm^/min
0.44
1.34
NA - not aval I able
a Meteorological data collected from Cl imatronics Electronic Weather Station (EWS) at research plot.
01
-------
TABLE 4.18. SUMMARY OF METEOROLOGICAL CONDITIONS—VIRUS RUNS--OPERATIONAL YEAR 1982
CO
Run no.
Run date
Run time
V 1/3- 16-82
1027-1057
1109-1 139
1204-1234
1246-1316
1349-1419
V2/8-2-82
1431-1501
1509-1539
1600-1630
1637-1707
1733-1803
V3/8-4-82
1121-1151
1200-1230
1247-1317
1326-1356
1414-1444
V4/8-24-82
1113-1143
1153-1223
1246-1316
1326-1356
1426-1456
Segment
no.
1
2
3
4
5
Avg
1
2
3
4
5
Avg
1
2
3
4
5
Avg
1
2
3
4
5
Avq
Air temp (°C)
At run
location EWSa
14
-
17
19
22
18 18
31
31
31
31
31
31 33.5
29
30
32
32
33
31 32
29
30
31
32
33
31 33
Mean wind
direction (°)
At run
location EWS
(2m) (10m)
290
270
215
210
210
239 260
155
155
150
150
155
153 170
150
155
125
150
125
141 170
NA
170
140
155
180
161 180
Wind speed
(m/sec)
At run
location EWS
(2m) (10m)
6.0
3.5
4.6
4.5
5.8
4.9 4.0
4.8
5.2
5.1
5.0
5.9
5.2 NA
4.5
4.7
3.4
2.9
4.0
3.9 NA
NA
3.6
4.6
3.1
2.3
3.4 NA
Humid ity
at run
location
(?)
41
-
42
40
27
38
51
51
40
42
40
45
53
50
43
52
40
48
41
44
40
44
42
42
Radiation at run location
Dewpoint Cloud
at EWS cover Cloud Solar radiation
(°C) (Sths) height gca l/cn^/min
0.93
.12
.20
.12
.14
-13 6 High .10
1.24
1.15
1.05
0.95
0.69
-1 <1 High 1.02
1.08
1.15
1.20
1.15
1.18
-2.5 0 - 1.15
.02
.09
.15
.12
.10
-2 0 - .10
NA - not available
a Meteorological data collected fcom Climatronics Electronic Weather Station (EWS) at research plot.
-------
TABLE 4.19. SUMMARY OF METEOROLOGICAL CONDITIONS--DYE RUNS—OPERATIONAL YEAR 1982
Mean wind
direction (°)
Run no.
Run date
Run time
D 1/3- 18-82
1455-1502
D2/7-11-82
1733-1740
D3/7-11-82
1752-1758
D4/7-13-82
1533-1539
Air temp (°C)
At run
location EWSa
25 28
26 28.5
25 28
30 31.5
At run
location
(2 m)
NA
60
50
155
EWS
(10m)
160
65
60
180
Wind speed
At run
location
(2 m)
NA
7.9
7.9
3.6
(m/sec) Humidity
at run
EWS location
(10m) (?)
9.5 59
NA 63
NA 63
NA 50
Radiation at run location
Dewpoi nt Cloud
at EWS cover Cloud
(°C) (8ths) height
-6 4 High
-5 2 High
-5.5 2 High
-2 <1
Solar radiation
gcal/cm^/min
0.55
<0.05
<0. 05
1.34
cr>
NA - not available
a Meteorological data collected from Climatronics Electronic Weather Station (EWS) at research plot.
-------
TABLE 4.20. SUMMARY OF METEOROLOGICAL CONDITIONS—PARTICLE SIZE RUNS—OPERATIONAL YEAR 1982
Mean wind
direction (°)
Run no.
Run date
Run time
P 1/2-23-82
1609-1619
P2/3- 16-82
1539-1549
P3/7-8-82
1510-1518
P4/7- 14-82
1519-1527
P5/8-25-82
1730-1738
A i r temp
At run
location
28
22
31
29
29
(°C)
EWSa
29.5
13.5
33.5
32.5
31.5
At run
location
(2 m)
200
180
130
155
100
EWS
(10 m)
210
210
150
185
120
Wind speed (m/sec)
At run
location
(2 m)
7.8
6.7
7.6
6.7
2.5
EWS
(10 m)
7.2
7.0
NA
NA
NA
Humid ity
at run
location
(?)
20
21
46
43
49
Radiation at run location
Dewpoint Cloud
, at EWS cover
(°C) (8ths)
-4 <1
-8 6
-1.5 <1
-2 2
-1 5
Cloud
height
_
High
_
High
High
Solar radiation
qca l/crrr/mi n
0.73
0.61
1.21
1.15
NA
a Meteorological data collected from Climatronics
Weather Station (EWS)
-------
LABORATORY ANALYSIS
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LABORATORY ANALYSIS
Clinical Specimens
Serology--
Serum processing and storage—Upon arrival of blood samples at the
laboratory, the serum was separated from the clot, dispensed into four
(UTSA) or five (UI) vials, and catalogued. All but one vial was stored at
-70°C. The remaining (UI) vial was heat-activated and stored at -20°C for
use in enterovirus serology.
Selection of serologic antigens--It was originally proposed in
developing this study that viruses known or suspected of being present in
wastewater would be those used in serological testing of the study
population's blood samples. Human viruses that may be present in
wastewater are those infecting the gastrointestinal tract, excreted in
feces and able to survive in the wastewater; hepatitis A, the
enteroviruses, adenoviruses, reoviruses, rotaviruses and Norwalk virus are
such virus groups. Within these groups are three types of polioviruses, 29
coxsackieviruses, 31 types of echoviruses, 41 human adenoviruses, three
reoviruses, one or more rotaviruses and three Norwalk virus types for a
total of at least 111 specific types that could be considered as candidates
for the serological study. Obviously not all can, or need to, be included
from a financial, technical, and epidemiological aspect.
a. Criteria for selection—In order to determine which types of a
particular group would be candidates for this study, the basis for
selection should be defined. It seems that minimally the candidates should
have the following characteristics:
1) The virus is found in Lubbock wastewater. COMMENT: This
pertains only to those virus groups to which the tissue culture
system used is known to be sensitive. It does not address
viruses that cannot be recovered by in vitro techniques.
2) From 50 to 74% of the study population is expected to be
susceptible to the virus. COMMENT: The infectious disease
literature gives some information on which to estimate the
expected prevalence of antibody to a given virus in defined
populations. Confirmation of this judgment will be possible
after antibody titrations are done on the preirrigation blood
samples collected from the study population.
3) The virus is known to be present in stools of individuals during
acute infection. COMMENT: Most of viruses infecting the
gastrointestinal tract are spread by the fecal oral route. The
71
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acute infection usually begins in the oral pharynx and later in
the intestinal tract. Virus shedding from the pharynx is only
discernible for a few days, if at all, whereas the infection in
the intestine persists for weeks or months. The candidate virus
should cause an extensive intestinal infection to produce a
clear-cut seroconversion when infection does occur.
4) The natural occurrence of the virus is not rare or geographically
restricted. COMMENT: The prevalence of antibody is expected to
be more than a few percent in the study population. This can be
specifically shown after the preirrigation sera are tested with
the candidate virus and the prevalence of antibody determined.
5) The peak seasonal occurrence of the virus should be known so the
total battery of viruses chosen occur at different seasons and
not all in just one season. COMMENT: The serological search for
infections should be for viruses occurring in the winter and
spring and not just the summer and fall in order to obtain a
better understanding of infection rates relative to the times and
quantity of wastewater irrigation which is expected to vary
during the study period.
6) The virus must produce a clear cytopathic effect (CPE) in the
cell culture system chosen.
b. Selected agents—Table 4.21 is, a list of virus types in the
groups named showing the characteristics relative to the criteria stated
above. From this list of candidates, 35 have been selected for the
serology study. Influenza virus is included although it does not fulfill
the characteristics listed above. The incidence of seroconversions for
influenza serve as a "control" since the incidence should not be related to
level of exposure to wastewater or its aerosols. A few additional types
can ultimately be included and they may be chosen after wastewater analysis
for virus during the study period is complete.
Serum neutralizing antibody titers will be done for the polio-,
coxsackie-, echo-, and adenoviruses. Hemagglutination-inhibition (HI)
antibody tests will be used for the reoviruses and influenza.
Radioimmunoassay (RIA) will be performed for hepatitis A and rota- and
Norwalk viruses.
c. Justification for selecting the viral agents--
(1) Hepatitis A--The hepatitis A virus is the'agent of
infectious hepatitis. The illness involves fever and gastrointestinal
symptoms at onset and proceeds to jaundice, often involving the liver. It
varies from a mild illness lasting a few weeks to a severe disabling
disease lasting several months. Inapparent infection is common. The agent
is excreted in feces and urine and is presumably present in the Lubbock
wastewater. Immunity is presumed to be long lasting.
72
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TABLE 4.21. VIRUS TYPES
Virus and type
Hepatitis A
Pol iovirus 1
2
3
Coxsackie Al
A5
A7
A9
A10
A16
B1
B2
83
B4
B5
B6
Echo 1
5
6
7
9
11
12
13
14
15
17
19
20
21
24
25
27
29
30
31
33
Adenovirus 1
2
3
4
5
6
7
12
14
Reovlrus 1
2
3
Rota virus 1-4
Norwalk 1
Leg 1 one! la 1
Inr Inan7a 1
% Antibody
prevalence
40
95
95
95
40
25
15
15
40
30
15
Sporadic
15
15
10
15
15
15
15
15
15
15
15
10
15
15
15
15
15
5
15
15
5
15
60
60
60
60
60
60
25-75 (inc.
with age)
75 (adults)
50
50
50
50 by age 6
30 by adult
2-5
25-75
Type of disease
Inapparent, hepatitis
Inapparent, polio
Inapparent, polio
Inapparent, pol io
Inapparent, orphan
Rash, Gl
Rash, Gl
Rash, Gl
Rash
Rash
Colds, Gl
Colds, Gl, rash
Colds, Gl, rash
Colds, Gl, rash
Inapparent
Meningitis
Meningitis
Gl, meningitis
Meningitis
Gl, pneumonia
61, cold
61
Gl
Meningitis
Gl, pneumonia
Gl, pneumonia
Gl
Meningitis
Meningitis
Meningitis
61
Gl
Pharyngitis
ARD
Pharyngitis, 61
61
ARD
Inapparent
Orphan
Orphan
Orphan
Gl
61
Resp i ratory
Ra<;p 1 ra-f-ory
Isolated
from stool
Yes
Yes
Yes
Common
Yes, sporadic
Seldom
Common
6-year epidemic
Common
Rare
Rare
Rare, epidemic
Sporadic
Common, epidemic
Common
Most common, no epidemic
Common, no epidemic
Rare, epidemic
Rare, epidemic
5-year cycles
Sporadic
Frequent
Frequent
Frequent
Rare
Frequent
Rare, epidemic
Rare
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
Occurrence in
wastewater
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes-common
Yes-common
Yes
Yes-common
Yes
No
Yes-common
Yes
Yes
Yes
Frequent
Yes
Yes
Yes
Frequent
Yes
Yes
Yes
Yes
Frequent
ND
ND
ND
ND
ND
ND
ND
ND
ND
Yes
Yes
Yes
Yes
Yes
No
Seasonal Lubbock-WI Ison
occurrence wastewater Selected
Fal 1 /Winter
Al 1 year
Al 1 year
Al 1 year
Fal
Fal
Fal
Fal
Fal
Fal
Fal
Fal
Fal
Fal
Fal
Fall
Summer
Al 1 year
Fal
Winter
Spring/Summer /Fal 1
Fal 1 /Winter
Summer
Summer
Summer
Al 1 year
Summer
Summer
Winter
Al 1 year
Winter
Winter
Winter
Winter
Summer
Summer
Wintnr
ND
Yes
Yes
Yes
Yes
No
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
Yes
Yes
Yes
Yes
Yes
No
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
No
No
Hep A
P-1
P-2
P-3
CA9
CA16
CB2
CB3
CB4
CB5
El
E3
E5
E9
Ell
E12
E13
E17
E19
E20
E21
E24
E25
E27
E30
E31
E33
Ad7
Reol
Reo2
Reo3
Rota
Nor
Leg
In?
ND - not detectable
ARD - acute respiratory disease
Gl - gastrointestinal illness
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Screening for hepatitis A virus antibody was performed on
the initial sera from all participants. Subsequent sera from susceptible
participants are being screened after each period of collection both for
surveillance and to determine the distribution of new infections and their
possible association with exposure.
(2) Polioviruses—The antibody titers of study participants in
the blood samples obtained in June or December 1980 have been determined.
Individuals having low titers (<8) to any one of the three poliovirus types
were recommended for immunization. Immunization clinics were held in
Wilson, Texas in April, May, and June 1981 when adults were given the Salk,
inactivated vaccine. In May children (<18 years old) were given the Sabin,
live vaccine. Blood samples were collected in June at the same time the
third adult vaccine was given. Adults were given a booster immunization in
January 1982, when blood samples were obtained.
Polio antibody titrations will be done on bloods of only
those participants recommended to be immunized. It is not known what
percentage of the immunized will have protective levels of antibody after
only two doses (June 1981 blood samples), after three doses (January 1982
blood samples), or after the booster (June 1982 blood).
(3) Coxsackieviruses--A subgroup of the large group of
enteroviruses are the coxsackieviruses,. Although most infections are
subclinical, they can cause a variety of respiratory, gastrointestinal and
cutaneous illnesses, and in rare instances more severe manifestations such
as meningitis and heart disease. There are two groups of coxsackieviruses,
A and B, based on the disease caused in newborn mice. After exposure,
these viruses multiply sequentially in the oropharynx, the intestinal
tract, and briefly, systemically. The virus is shed in feces for 13 to 60
days and can be recovered in wastewater. This pattern occurs in both
clinical and subclinical infections. Immunity is probably of long duration
although reinfections may occur. However, immunity to one type probably
does not provide cross-protection to other types.
For this serological study a search for infection by the
types in Group A will be limited. The reasons for this include:
1) There is no one cell line that is regularly susceptible for all
types of Group A viruses. Maintaining three to five cell lines
for neutralizing antibody titrations would be costly and would
add more time than if one cell line could be used.
2) Facilities are not available to work with mice in either the
UISPH or IDPH laboratories; using mice for neutralizing antibody
titrations would be very expensive.
74
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3) Mainly due to points 1 and 2 above, the IDPH laboratory has never
performed serology for the Group A virus types and does not feel
competent to oversee and evaluate these titrations if they were
to be done. However, coxsackie A9 virus does cause distinct CPE
in cell culture and it is the only recommended candidate for
serology.
There are six types of Group B coxsackieviruses, all of
which grow in one cell line (Vero). Bl through B5 are commonly found in
populations at endemic or epidemic proportions. B6 rarely occurs and would
not be a good candidate. Since Bl through B5 types occur mainly in the
fall, they cause similar illnesses and there is a better understanding of
the prevalence of antibody expected compared with the As and B6. Since B2
through B5 occur in Lubbock wastewater and were frequent isolates, they
were recommended as candidates for the serology.
(4) Echoviruses--There is the third subgroup of enteroviruses
consisting of 31 types. Unlike the coxsackieviruses, the infection is
mainly intestinal (very brief viremia) and most infections cause little
illness. Protective antibody develops post-infection and persists. Cross-
protection between different types, which can occur with coxsackie Bl
through B5, has not been found to occur with the echoviruses. Echoviruses
1, 5, 9, 11, 17, 20 and 25 are recommended for testing. All are commonly
isolated from stools. Echo 5 and 11 have frequently been recovered in the
Lubbock wastewater while Echo 9 has not.
(5) Adenoviruses--0ne adenovirus type is recommended for this
serological study. These agents are associated with three clinical
syndromes: pharyngitis, acute respiratory disease (ARD), and enteritis.
Adenoviruses can be expected to be present in the Lubbock wastewater. The
virus is recovered in stool during any clinical or inapparent infection and
in urine when there is cystitis. In children the infection may persist for
up to one year with exacerbations of illness. Immunity is specific but
reinfections are common. Recently adenovirus-like particles have been seen
in stools of children with diarrhea, but these enteric adenovirus are not
cultivatable by the usual isolation techniques and serology is not possible
because of this.
In order to obtain information on adenovirus infections in
general, one candidate type is recommended. The selection of type 7 is
based on the fact that it is associated with enteritis, conjunctivitis, and
pharyngitis in children, it spreads in families, and the prevalence of
antibody increases with age.
(6) Reoviruses--As the name implies, these are respiratory-
enteric orphan viruses. They are frequent causes of infection since over
75
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50% of adults have specific antibody. Colds, diarrhea and rash are the
most frequent associated illnesses. Of all the animal viruses known to
occur in wastewater, they are the most common and in highest titers. Their
seasonal occurrence is highest in the winter.
All three serotypes are recommended for the serology test.
The HI test will be performed since it is cost effective compared with the
neutralization test and both tests measure the same antibody.
(7) Rotaviruses--These are reo-like viruses associated with
diarrhea in children causing both in sporadic and epidemic outbreaks of
enteritis in infants and children. Subclinical infections occur in adults.
These agents are noncul tivatible in cell cultures; thus, antibody
titrations are performed by RIA or other techniques. The potential role of
environmental sources (wastewater aerosols) is not known, but these are
good candidates for the serology study. Titrations would be limited to
bloods obtained from children under age 18 to determine infection rates in
this high risk group and not for reinfection rates in adults.
(8) Norwalk virus—A parvovirus-like agent is the possible cause
of enteritis in children. Three antigenically distinguishable types were
recovered in outbreaks in the U.S. Protection to one does not necessarily
result in protection to another type. JThe attack rate can reach 501 in
outbreaks. There appear to be two types of immunity, long term and short
term, that can develop after infection. Whether this is due to difference
in the agent and infection or different cohorts is unknown. Contaminated
water has been implicated as the source of some outbreaks. It is
recommended for this study performing the titrations only in children under
age 18 using the RIA test.
(9) Influenza virus—The lipoprotein envelope of influenza virus
is essential for its infectivity, and it does not survive in the intestinal
trace because bile and lipases in the gut readily destroy the envelope.
Since it is not found in wastewater, the occurrence of new infections in
the study population should not be related to exposure to wastewater or its
aerosols. The failure to find such an association would strengthen the
interpretation of the relationship between rate of seroconversions and
exposure to any enteric virus studied. Obviously, the opposite finding
would weaken any association observed.
Three distinct types of influenza virus are expected to
occur in the 1981-1982 winter season. When the Texas Health Department
determines which type is the most prevalent in Lubbock, that type will be
chosen for this serological determination.
The HI test will be used because it is type specific, in
contrast to the C-F test, and will give the most clear interpretation of
the roles of new infections for the prevailing agent.
76
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(10) Further candidate viruses—As wastewater samples for Lubbock
and Wilson are analyzed for viruses, additional serological testing can be
performed to incorporate those specific agents to search for serological
evidence of infections by any of them. Such an approach optimizes the
chance of determining any health effect of the wastewater on the exposed
population. A few additional viral agents can be added to this serological
survey as will be justified during the progress of this study.
(11) Legionella bacilli—This is the only bacterial antigen
recommended for this survey. Legionella organisms occur in the environment
and cause epidemic and sporadic cases of legionellosis in man. The disease
most commonly occurs as a pneumonia, with the majority of cases reported in
clusters. Its presence in wastewaters is unclear, but it may be present
from either or both human and environmental sources. Of particular
interest is the fact that large volumes of wastewater will be stored in
lagoons and that algae will grow in these lagoons. It is known that
Legionella organisms utilize algae as a natural medium and it is of
interest therefore to monitor for serological evidence of infection by this
agent since it could be abundant in aerosols when the stored water is
applied to the land. This, in addition to a search for Legionella bacteria
in the Lubbock wastewater and in the lagoon water, constitutes another
measure of the health effects of using wastewater for land application.
For all selected agents except hepatitis A and the
polioviruses, serologic testing will be performed on the paired first and
last baseline sera provided by each participant. The June 1982, December
1983 and October 1983 sera of susceptible participants (children under 18
for rotavirus and Norwalk virus) will be screened.
Serologic methods—
a. Hepatitis A--The analysis of sera for the presence of hepatitis A
virus (HAV) antibodies was performed with a commercially available RIA
system marketed by Abbott La-fcjljr-a-tories under the trade name of HAVAB®. The
HAVAB® test is based on the principle of competitive binding of anti-HAV in
serum with radioactively""Tagged anti-HAV to HAV coated on a solid phase
(see Figure 4.10).
If anti-HAV is present in the serum at an equal concentration to a
predetermined amount in the radioactively labeled sample, each antibody has
an equal chance of binding to the HAV on the solid phase. As a result, one
half of the radioactive counts in the labeled sample would be bound to the
solid phase. If anti-HAV is present in the serum at a higher
concentration, less radioactively tagged antibody will be bound to the
bead. The greater the amount of anti-HAV in the specimen, the fewer
radioactive counts will be b'ound to the solid phase. Conversely, the less
anti-HAV in the serum, the greater the number of radioactive counts which
77
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-•J
00
o
Sol id phase
bead coated
with HAV
V
Ant i- HAV
in test
serum
Anti-HAV
labeled
with 125
2nd wash
Competitive
binding
Figure 4.10. Competitive binding of anti-HAV in serum with radioactively tagged
anti-HAV to HAV coated on a solid phase
-------
will be bound to the solid phase. By the use of proper controls (known
positive and negative sera provided with the HAVAB® kit), it can be
determined whether an individual possesses anti-HAV.
The HAVAB® test is limited to the detection of anti-HAV in serum or
plasma. To determine the occurrence of anti-HAV expected in a population,
serum specimens from five eastern U.S. cities were screened using the
HAVAB® test (Abbott Laboratories, 1980, Table 4.22).
TABLE 4.22.
INCIDENCE OF ANTI-HAV IN SPECIMENS FROM DIFFERENT POPULATIONS
AS DETERMINED BY THE HAVAB® TEST
(Abbott Laboratories, 1980)
Commercial blood bank donors
Volunteer blood bank donors
Patients of a city hospital
Suburban high school students
Medical house staff (NYC)
Number
tested
150
300
447
100
80
Anti-HAV positive
No %
76 50
71 24
311 69
4 4
18 22
The prevalence of anti-HAV also was documented in a study reported by
Szmuness and associates (1976) where 45% of an adult population (n=947) in
New York City was hepatitis A positive using an immune adherence
hemagglutination test. Antibody was detected in a larger proportion of
lower class participants (72% to 80%) than in the middle and upper classes
(18% to 30%). Study results showed hepatitis A antibody prevalence was
closely related to age also. In middle class whites and blacks, the rate
was two to four times higher in those 50 or more years old than in 18 to 19
year olds. Further, in samples of healthy children from the same area, the
rate of hepatitis A antibody detection varied between 10% and 20%. A more
recent report by Snydam and co-workers (1981) noted that of 73 people
tested as part of a control group, 32% had IgG antibody to hepatitis A
virus as detected by solid-phase RIA.
The performance characteristics of the HAVAB® test were determined by
Abbott also. With regard to sensitivity, the amount of anti-HAV in a
specimen was found to be inversely proportional to the resulting CPM of the
test as shown in Figure 4.11.
By monitoring an individual with a known exposure to HAV, Abbott
Laboratories also concluded that the HAVAB® was highly specific for the
detection of anti-HAV. As shown in Figure 4.12, the individual became
reactive in the HAVAB® system by the fifth day of clinical illness and the
79
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cpm
Positive
Control
Cutoff
Negative
Control
4 8 16 32 64 128 256 512 1028
Reciprocal Dilution (x 10'2)
Figure 4.11. Titration of anti-HAV in serum by the HAVAB® test
(Abbott Laboratories, 1980)
80
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Positive
Control
cpn
Cutoff
Negative
Control
-20 -10 0 10 20 30 40
Days from Onset of Symptoms
50
Figure 4.12. Development of anti-HAV in subject with Hepatitis A
(Abbott Laboratories, 1980)
81
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anti-HAV serum passed the cutoff mark by day 8. Maximum serum levels of
anti-HAV were attained by day 15 (Abbott Laboratories, 1980).
The procedure shown below for the qualitative determination of
anti-HAV in serum is recommended by Abbott Laboratories and was followed by
the Serology Laboratory.
RECOMMENDED QUALITATIVE DETERMINATION OF ANTI-HAV IN SERUM
(Abbott Laboratories, 1980)
1. 10 pL of each serum specimen or control were placed into a well of the
reaction tray accompanying the kit.
2. 0.2-mL aliquots of 125I-labeled anti-HAV were placed into each well
containing a specimen or control.
3. One HAV-coated bead was added to each well containing either a control
or specimen ^^I-labeled anti-HAV mixture.
4. Each reaction tray was then gently shaken to ensure mixing of all
reagents, covered, and incubated on a level surface for 18 hours at
room temperature.
5. At the conclusion of incubation, the covers were removed and the
liquid contents of each well were aspirated into a container for
liquid radioactive waste.
6. Each bead was washed two times with 5'mL of distilled water.
7. The beads were then transferred to individual gamma-counting tubes,
capped, and placed in a gamma-scintillation counter. The amount of
radiation bound by the HAV on each bead was then determined by the
amount of radioactivity detected in one minute. Control samples and
unknowns were counted together.
8. For each test series, three negative and two positive controls were
run simultaneously as described above. The negative controls were
composed of recalcified human plasma nonreactive for anti-HAV or
hepatitis virus B surface antigen. The positive controls were
composed of recalcified human plasma reactive for anti-HAV but
nonreactive for hepatitis virus B surface antigen and adjusted to a
reactive titer of 1:200+2 Iog2 dilutions.
9. Following the determination of bound radioactivity, the test run was
evaluated for validity before the specimen results were determined.
The determination of validity was conducted in the following manner.
a. The negative control mean count rate (NCx) in counts per min
(CPM) was calculated from the individual net count rates obtained
for the negative controls.
82
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b. The positive control mean count rate (PCx) in CPM was calculated
from the individual net count rates obtained from the positive
controls.
c. The negative to positive ratio (N/P) was determined by dividing
the net NCx by the net PCx, e.g., N/P = NCx/PCx.
10. To calculate the cutoff value for the negativity and positivity of
each specimen, the sum of the NCx and PCx was divided by 2, e.g.,
NCx + PCx
2 = cutoff value
11. The results of the amount of radioactivity bound by each test sample
were then determined by comparing the amount of radioactivity bound to
each bead in CPM to the cutoff value.
12. The specimens with CPM less than the cutoff value were considered
reactive for the presence of anti-HAV.
13. Specimens with CPM greater than the cutoff rate were considered
negative for the presence of anti-HAV by the criteria of the
HAVAB®test.
Specimens which were repeatably reactive (minimum of two tests/sample)
in the screening procedure were considered positive for the presence of
anti-HAV by the criteria of the HAVAB® test.
To allow a comparison between tests conducted on separate days, a
"counts per minute (CPM) ratio" was calculated to reflect:
CPM of participant serum
cutoff value CPM
A value of less than one was interpreted as hepatitis A reactive serum
while a value greater than one indicated a negative serum. Where
available, all CPM ratios were reported.
b. Entero- and adenoviruses--The serum neutralization test is used
to determine antibody titers for the following viruses:
Polioviruses 1, 2, 3
Coxsackieviruses A9, B2, B3, B4, B5
Echoviruses 1, 5, 9, 11, 17, 20, 25
Adenovirus 7
The neutralization test is the procedure of choice since it is
considered to be the most sensitive and specific serological procedure for
these particular antibodies. In addition, the neutralization test is the
only reliable means for determining immune status for poliovirus.
83
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In this procedure, test sera (1:4 or 1:10 starting dilution) are
serially diluted in microtiter plates. A challenge dose of virus and a
suspension of Vero cells are added to each of the serum dilutions. The
antibody titer is determined as the highest initial dilution which inhibits
the CPE of the test virus dose. With paired sera, a fourfold or greater
increase in neutralizing antibody ttter for a particular virus indicates
that an infection with that virus type has occurred.
c. Reovirus types 1,2, and 3~The HI test is generally considered
to be the method of choice for measuring levels of serum antibody to
reovirus types 1, 2 and 3, because of its relative simplicity and its
sensitivity. The HI test is based on the fact that reoviruses have sites
on their surfaces (hemagglutinins) which attach to human erythrocytes (and
also bovine erythrocytes in the case of type 3) and that this
hemagglutination reaction is inhibited when specific antibody is combined
with virus. In this test, the sera are treated with kaolin, placed in V-
bottomed microtiter plates and serially diluted. A working dilution of
each reovirus antigen and a 0.5% human group 0 erythrocyte suspension is
added to the diluted sera.
The sensitivity of the HI technique is superior to that of either the
complement fixation or serum neutralization tests (Schmidt, 1980) although
specificity is dependent upon removal of nonspecific inhibitors of
hemagglutination from test sera by treatment with kaolin or other
extraction methods. Consultation with Dr. Leon Rosen indicated that kaolin
pretreatment of test sera is the standard method for removal of inhibitors;
this method will be used to pretreat all sera for reovirus antibody.
Antigens used in this test have been obtained from the Biologic
Products Division, Centers for Disease Control, Atlanta, Georgia, or will
be prepared by the University of Illinois from prototype strains obtained
from the American Type Culture Collection. Dr. Rosen emphasized the
necessity of using four complete hemagglutinating units of antigen (one
hemagglutinating unit is defined as the highest antigen dilution giving
complete agglutination of a standard erythrocyte suspension) in order to
assure reproducibility of results. Stock antigens will be titrated prior
to use and a back-titration included as a control in each test run in an
effort to follow this recommendation.
Each test will be interpreted as soon as cell controls indicate
complete settling of erythrocytes and before patterns begin to "collapse."
Each test will be assigned a numerical score based on the degree of
hemagglutination inhibition (4=75 to 100% inhibition; 3=50 to 74%
inhibition; 2=25 to 49% inhibition; l=less than 25% inhibition; and -=no
inhibition). An endpoint will be defined as the highest serum dilution
resulting in greater than 50% inhibition of hemagglutination. A fourfold
84
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or greater increase in HI antibody titer to a reovirus antigen is
considered evidence of a current reovirus infection.
d. Norwalk virus--Anti-Norwalk antibody is measured by a
modification of the RIA. Plates precoated with Norwal k antibody will be
inoculated with a 25-uL standard preparation of partially purified, Norwal k
antigen. The preparation is partially purified by isopyenic banding in
cesium chloride. This preparation contains approximately five "binding"
units of activity and gives a P/N ratio of greater than 4. After an
overnight incubation, the plates are washed and inoculated with 40-yL
samples of serial twofold or fivefold dilutions of the serum to be tested
for antibody. The plates are again incubated at room temperature overnight
and then 10 uL of ^"I-labeled anti-Norwalk IgG is added to each well and
incubated at 37°C. After receiving the IgG the plates are incubated at
37°C for four hours, again washed, and cut apart with scissors. The
individual wells are transferred to gamma-counting tubes for quantitation
of residual (bound) radioactivity in a gamma spectrometer. The residual
radioactivity (CPM) detected in the wells that receive test samples is
divided by the mean residual radioactivity in wells that received
phosphate-buffered saline (negative control). A 50% or greater reduction
in residual radioactivity produced by a serum compared to a buffer control
is taken as evidence of the presence of Norwalk antibody.
The limitation in being able to conduct this portion of the serosurvey
is the nonavailability of Norwalk viral antigen and coating antibody for
the RIA test. Arrangements are being made to procure sufficient Norwalk
virus for this study. The basis of this problem is that Norwalk virus does
not reproduce in any known cell system in vitro. Human volunteer studies
are necessary wherein experimentally infected individuals may become
infected and produce sufficient viral antigen in stools for use in the RIA
assay.
e. Rotavirus--Rotavirus antibodies are measured by the enzyme linked
immunosorbent assay (ELISA). A supply of rotavirus stock antigen has been
prepared in MA-104 cells (obtained from M.A. Bioproducts) using the WA
strain of human rotavirus. The antigen is immobilized on wells of
polystyrene microtiter plates to which human serum samples, after serial
dilution, are added. An antihuman globulin conjugated with alkaline
phosphatase is added to the antigen-antibody complex fixed to the well to
react with the antibody on the antigen. The enzyme is then provided
substrate which after reaction forms a visible complex. The quantitative
measurement of the complex is obtained spectrophotometrically in an ELISA
plate reader. These measurements are compared with readings of
simultaneous reactions involving uninfected cell materials and rotavirus
antisera containing high titer and low titer antibody levels for
interpretation of specificity and quantity.
85
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f. Influenza--Al though not expected to be present in wastewater
samples, influenza virus has been included in the list of proposed antigens
to act as a control agent. The HI test has been selected for use in
quantitating levels of serum antibody to this agent. As with the reovirus
HI test, nonspecific inhibitors of hemaggluti nation affect specificity and
must be removed by pretreating test sera with receptor-destroying enzyme
(RDE) or other techniques such as CC>2 extraction. Influenza viruses
agglutinate erythrocytes from chickens or guinea pigs and either may be
used in the HI test. The Illinois Department of Public Health Virology
Laboratory's influenza HI test protocol is based on C02 extraction of test
sera and uses guinea pig erythrocytes. Pretreatment with RDE is the most
commonly used method for removing nonspecific inhibitors of
hemaggluti nation.
Several lots of RDE (Sigma Chemical Company, St. Louis, Missouri);
Centers for Disease Control, Biologic Products Division, Atlanta, Georgia)
have been tested using guinea pig erythrocytes and found to be unsuitable
due to low levels of activity. These lots will be retested using chicken
erythrocytes. If activity is improved, RDE will be used to pretreat sera
and chicken erythrocytes used in the test. If activity is not improved,
sera will be pretreated by C02 extraction and guinea pig erythrocytes used
in the test.
Antigen stocks have been received. Dr. Leffingwell (Texas Health
Department Virus Laboratory) provided an H3N2 isolate from the 1981-1982
influenza season. This strain was inoculated into the allantoic cavity of
embryonated hen eggs and harvested 72 hours post-inoculation. Allantoic
fluids from eggs inoculated with the H3N2 strains failed to agglutinate
guinea pig erythrocytes (indicating failure to pass). An isolate of egg-
passed A-England 333 (H^Ni) has been obtained from Dr. Allen P. Kendell
(WHO Reference Center, Centers for Disease Control, Atlanta, Georgia) who
has also agreed to provide a similar culture of A-Bangkok
Treated test sera (1:10 starting dilution) will be serially diluted in
a microtiter plate and allowed to react with a standardized amount of viral
antigen. A suspension of GPRBC will be used to detect antibody-antigen
reactions in that the absence of hemaggluti nation in a given well indicates
levels of specific antibody sufficient to block hemaggluti nating surface
antigem'c sites.
g . L e g i o n e 1 1 a b a c i 1 1 i - - T h e L e g i o n e 1 1 a IFA test is an
immunofluorescence procedure for detection of anti-Legionella antibodies in
human serum. At the present time the IFA has only been standardized for L_.
pneumophila serogroup 1, for which a sensitivity of 78% and specificity of
99% has been estimated (Wilkinson et al . , 1981). The specificity of this
research IFA test appears to be good when paired sera from patients with
86
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symptoms of Legionnaires' disease are tested; however, if possible, the
test should be used in conjunction with isolation of the organism from
biopsy or autopsy material or demonstration of the organisms in tissue
specimens. Recently several new serotypes of Legionella have been
identified. The species currently known are: Legionella pneumophila (six
serogroups) , L_. micdadei, L_. bozemanii, L_. dumoffii, L_. gormanii, L_.
1 ongbeachae (two serogroups), L_. jordanis, and L_. oakridgensi s (two
serogroups).
As with any serological test, the most convincing serological
evidence of a recent infection with the Legionella bacterium is a fourfold
rise in titer between the acute phase of illness (within the first week)
and convalescent phase (three to six weeks after onset). In the
Legionella IFA test, the rise in titer must be to at least 128 to be
considered positive. A single or standing titer of >256 is considered
presumptive evidence of Legionella infection at an undetermined time.
Current data indicate that titers of 32 and 64 in the absence of detectable
disease are common.
The IFA test is a "sandwich" immunofluorescence technique which
uses a two-stage reaction procedure. In the first stage, the Legionella
antigen of interest is overlaid with dilutions of animal antiserum or human
serum; the slides are incubated, washed and dried. In the second stage,
fluorescent dye-labeled antibody (to the IgG contained in the animal or
human serum which was applied in the first stage) is placed on the slide.
In this manner Legionel la antigens are rendered fluorescent by positive
sera which themselves are not labeled.
Legionella strains isolated from Lubbock wastewater were sought as
these would be the most likely Legionella infecting the study population as
a result of wastewater exposure. Attempts to isolate Legionella from
Lubbock wastewater samples have been unsuccessful although antigenic
evidence of Legionella has been found in most samples (see Microorganism
Levels in Wastewater, Section 5). Prototype strains of serogroups and
species antigenically demonstrated in Lubbock wastewater will be used in
lieu of wastewater isolates. All paired sera from Periods 025, 212, and
312 will be tested for antibody to L_. pneumophila 1 while a sample of 30 to
50 pairs of these sera will be screened for antibody to pools of species
and serogroups possibly present in Lubbock wastewater.
Clinical Bacteriology--
The primary isolation of overt and opportunistic pathogens followed
the schemes diagrammed in Figures 4.13 and 4.14. Fecal specimen
homogenates in buffered glycerol saline were plated directly onto selective
media. An unpreserved portion of each fecal specimen was used for
isolation of Campylobacter fetus subsp. jejuni only. Additionally,
portions of each preserved specimen were inoculated into enrichment media
prior to plating. Subsequent identification of representative Gram-
87
-------
FECES
Homogeneous Suspension
Transport Medium
CO
oo
1
(A) (B) (C)
STREAK PLATE ENRICHMENT
1 1
4 ; i
Selective Media, GN broth 0.067M Phosphate
- Cell obi ose Arginine 1 Buffered Saline
Lysine (CAL) Agar 4 1
- MacConkey Agar Streak to
- Hektoen Enteric Agar XLD Agar 1
- Bismuth Sulfite Agar 4
Alkali Treatment
I
J J
Select Representative Streak to CAL Agar
Colonies
1 Day 3^ Incubate
~^>- at Room
4 Day 7 Temperature
Gram Stain and Subculture
Gram Negative Organisms
i
1
4 Identification Using
Oxidase Test API 206^ Biochemical
1 Screen
Identification Using
API 20PB) Biochemical Screen
1
(D)
STREAK PLATE
i
Campy-BAP
i
GasPak Jar with
CampyPak 1 1
1
Select Typical
Colonies
1
4
Presumptive
Tests
1
4
Confirmation
(A) Salmonella, Shigella, Yersinia enterocolitica,
(B) enrichment for Shigella
(C) enrichment for Y. enterocolitica
(D) Campylobacter fetus subsp. jejuni
(E) Candida albicans
(F) Staphylococcus aureus
\
(E)
STREAK PLATE
i
Sabouraud Dextrose
Agar
(+ chloramphenicol )
1
4
Typical Colonies
I
Germ Tube Test
1
4
Tests for
Chlamydospores,
Sucrose
Ass i mi latioii
other enterics
|
(F)
STREAK PLATE
i
Mannitol Salt
Agar
1
1
4
Typical Colonies
J
Gram Stain,
Coagulase Test
Figure 4.13. Isolation and identification of selected organisms from feces
-------
THROAT SWABS
Place Swab into 1 ml of
Todd-Hewitt Broth for 2 Hours
FLUID
FA Screen for
Group A Streptococci
SWAB
MacConkey Agar
Sheep Blood Agar
I
Select Representative Colonies
Gram Stain and Subculture
I
Gram Positive
Organisms
Catala
Gram Negative
Organisms
se Test
I
Oxidase Test
I
Coagulase
Test
Additional
Tests as
Required, e.g.
Bacitracin
Phadebac$>
Identification Using
API 20E®Biochemical
Screen
Figure 4.14. Isolation and identification of organisms from
throat swabs
89
-------
negative bacteria was accomplished using a commercially available
biochemical identification system (API 20E®, Analytab Products).
Salmonella and Shi gel! a isolates were typed using commercially available
antisera (Difco). £. fetus subsp. jejuni was presumptively identified by
the following criteria: Gram-negative curved rods, characteristic darting
motility, oxidase +, catalase + . The organisms were confirmed by growth in
1 percent glycine, lack of growth at 25°C, and susceptibility to nalidixic
acid (30 ug disk). Staphylococcus aureus and Candida a 1bicans were
identified as indicated in Figure 4.13.
Screening for £. a 1 b i c a n s in stool specimens was initiated in
September 1980 while the £. fetus subsp. jejuni protocol was added in April
1981. The alkali treatment coupled with plating on CAL agar was
substituted for an existing procedure in April 1981 for the improved
detection of Yersinia enterocolitica. Prior to that time, fecal samples
were analyzed for Y_. enterocolitica by enrichment at 5°C in isotonic saline
containing 25 ug/mL of potassium tellurite with subsequent plating onto
Salmonella-Shi gel!a (SS) agar.
After processing, throat samples were plated onto 5% sheep blood agar
and MacConkey agar. Incubation of the first medium was at 37°C in an
atmosphere of 5% C02 to facilitate cultivation of Group A streptococci.
Representative colonies from each medium were identified using traditional
tests as described in Lennette et a1.,(1980) in conjunction with
commercially available testing systems. Beta-hemolytic streptococci were
grouped using the Phadebact® (Pharmacia) coagglutination test. Throat
samples also were screened for Group A streptococci using a fluorescent
antibody technique.
The prevalence of different microbial types in the clinical specimens
was determined in a semiquantitative manner. Plates were streaked by a
four quadrant method, and the amount of growth was reported (as shown in
Table 4.23) by determining the highest quadrant in which the organisms were
isolated as discrete colonies.
TABLE 4.23. SEMIQUANTITATIVE REPORTING OF GROWTH
BY THE FOUR QUADRANT PLATING METHOD
Terminology Amount of growth
Heavy (H) On three or all quadrants
Moderate (M) On first two quadrants
Light (L) On first quadrant
Very light (VL) 1 to 10 colonies on plate
Clinical bacteriology monitoring, particularly of illness specimens,
provides the most timely mechanism of surveillance for a possible health
90
-------
effect associated with irrigation operations. Isolation of a pathogen or
any other cause for concern during periods of scheduled samples was
reported by telephone to health watch investigators at the University of
Illinois within a week of receipt of the sample. The results of all
illness specimens were reported by telephone within a week of receipt of
the specimen. In addition, an illness specimen log (Figure 4.15), starting
with specimens collected during DCP 212, was updated on the last Friday of
each period and sent to the University of Illinois and the project manager.
The mechanism of surveillance reporting allowed feedback of results to the
participants and collection of follow-up specimens when the results
provided cause for concern.
Clinical Virology--
Appropriate enteric and respiratory viral agents were sought via
traditional diagnostic isolation schemes (as illustrated in Figure 4.16)
coupled with microidentification techniques. Fecal suspensions were
prepared by adding 10 ml of antibiotic diluent (Medium 199 containing
penicillin and streptomycin) to 1 to 2 g of stool sample. Sterile glass
beads were added, and the mixture was vortex-mixed for 1 minute. After
centrifugation (8,000 x g) for 10 minutes in a refrigerated centrifuge, the
supernatant fluid was recovered for inoculation of primate cells in tube
culture. Similarly, an antibiotic diluent was added to the fluid expressed
from the throat swab into the transport medium. If necessary, throat swab
eluates were centrifuged to remove gross particulates prior to inoculation
of cultures.
Cell cultures used were primary rhesus monkey kidney, human
rhabdomyosarcoma (RD), African green monkey kidney (BGM) and HeLa
(pretested for adenovirus sensitivity). A 0.1-mL aliquot of supernatant or
eluate was inoculated into two tubes of each cell line. Tubes were
observed microscopically over a 10- to 14-day period for viral cytopathic
effect (CPE). HeLa cell tube cultures were frozen and thawed prior to a
second blind passage to enhance detection of adenoviruses.
As a result of quality assurance testing conducted during 1981, it
became obvious that the likelihood of recovering viruses from nonillness
(routine) fecal specimens was low. Beginning with Period 201 sampling,
changes in the clinical assay procedures were made to enhance the
sensitivity of viral isolations from routine fecal specimens. The volume
of sample inoculated into each cell line was increased from 0.2 ml to 1.0
ml by inoculating two 60-mm plates when monolayers reached 50 to 75%
confluence (0.5 mL/plate). Primary rhesus monkey kidney cells obtained
from a commercial supplier continued to be used as tube cultures.
The identification and typing of viral isolates from clinical
specimens was performed by microneutralization using the Lim Benyesh-
Melnick enterovirus typing pools. Fluorescein conjugated antisera specific
91
-------
University of Texas at San Antonio
Center for Applied Research and Technology
Lubbock Health Effects Study
Illness Specimen Log
Starting Date Date
DCP Date Participant I.D. Number Type of Specimen Collected Received Results
Figure 4.15. Illness specimen log
-------
FECES
Prepare Fecal Suspension by
Adding Antibiotic Diluent to
Sample and Vortex Mixing with
Glass Beads .
Pellet Solids by
Centrifugation (8,000 x g)
Recover Supernatant Fluid
THROAT SWAB
J
Express Fluid, Add Antibiotics
As Necessary, Clarify Sample
by Centrifugation (8,000 x g)
Recover Supernatant Fluid
Inoculate Tube Cultures
of Appropriate Primate
Cells, e.g.,
- Primary Rhesus Monkey Kidney
- BGM
- RD
- HeLa (Pretested for Adenovirus
Sensitivity)
4
Observe for Cytopathic Effect
over 10-14 day period
Freeze Positive Samples, -76°C
Identify Isolates by Serological
Procedures
Figure 4.16. Viral isolation from clinical specimens
93
-------
for adenovirus group antigen was purchased from M.A. Bioproducts.
Preliminary testing showed that optimal fluorescence was obtained by using
a 1:5 dilution of the conjugate. Prior to use, the conjugate was
centrifuged at 2 x 103 RPM for 10 minutes in an IEC tabletop centrifuge to
remove any particulate contaminants.
Those clinical isolates exhibiting CPE characteristic of adenoviruses
and unidentified by enterovirus microneutralization procedures underwent
fluorescent antibody staining. HeLa cells were grown in 125-mm tissue
culture tubes to about 50% confluence and subsequently were inoculated with
0.1 ml of the virus suspension. The tubes were observes daily for evidence
of CPE. When 75% of the monolayer showed viral involvement, the tube was
vortexed to remove infected cells. In the case of negative controls
(uninfected cells) , the cells were scraped off of the glass with a rubber
policeman. The tubes were then centrifuged at 6 x 103 RPM in an IEC
centrifuge for 10 minutes. The supernatant was decanted and the pelleted
cells were washed three times with 5 nl of phosphate buffered saline (PBS)
(pH 7.6). After the last centrifugation the PBS was carefully decanted and
the cell pellet resuspended in a minimal volume of saline (0.1 ml). The
cell suspension was placed on a microscope slide, allowed to air dry, and
fixed in cold acetone (-20°C) for 10 minutes. At this point, slides could
be stored at -70°C to await further processing.
After warming to room temperature, fixed cells were covered with 0.05
mL of a 1:5 dilution of the adenovirus-specific fluorescein conjugate.
Slides were incubated in a moist chamber for 30 to 45 minutes followed by
two 10-minute rinses in PBS and a final distilled water wash. Cells were
scored for adenovirus antigen production by visually observing fluorescence
using a Zeiss Model 18 microscope equiped with an epiiluminator and a
fluorescein isothiocyanate (FITC) filter set.
Electron Microscopy of Fecal Specimens--
Electron microscopic (EM) examination of fecal material using negative
staining techniques has been used to distinguish an increasing number of
morphologically distinct viral agents which have been associated with
gastrointestinal illness. These agents include: rotavirus, astrovirus,
calicivirus, adenovirus, coronavirus, and Norwalk-like viruses. Routine
cell culture techniques cannot currently be used to isolate many of these
agents and specific immunoassays are only capable of detecting
antigenically related viruses. Using EM, the USEPA HERL-Cincinnati
laboratory has detected a variety of viral agents in acute illness
specimens. Stool specimens from the health effects study for the LHES
will similarly be examined for the presence of virus-like particles. Such
agents are frequently shed by infected individuals in large numbers (often
in excess of 10*° particles/g of stool) and thus are detectable by the
relatively insensitive EM procedure. Depending upon virus type, state of
94
-------
aggregation, adsorption to grids, background material and other factors, a
suspension titer of approximately 106 particl es/mL is required for
detection by EM.
Fecal specimens, labeled with the donor's name and code number, are
shipped by the University of Texas at San Antonio to the USEPA laboratory
in Cincinnati once per month during the intensive health watch. The
specimens are shipped in glass vials on dry ice in insulated containers.
Shipping time is generally less than 24 hours and samples are cold upon
receipt. All specimens are stored frozen at -70°C until processed as
follows:
1) The fecal specimen is thoroughly mixed on a vortex mixer or with
a glass rod or pipette.
2) A small amount is removed and enough distilled water added to
give a slightly turbid suspension.
3) A drop of the turbid suspension is placed on a copper EM grid
(carbon substrate) and allowed to stand one minute.
\
4) Excess sample is removed with filter paper and the grid rinsed
with 1 or 2 drops of distilled water.
5) The grid is dipped into 2% phosphotungstic acid (PTA), pH 7, and
then dried (negative staining).
6) The grid is examined at 80 kV on a JEOL 100CX transmission
electron microscope for the presence of virus-like particles.
Specific details of these procedures may be found in Kapikian et al.
(1975) and in Flewett (1978).
Specimens yielding rotavirus or Norwalk-like virus identifications are
sent to Dr. N. R. Blacklow's laboratory at the University of Massachusetts
for examination by RIA.
Environmental Samples
Wastewater Samples—
Microbiological screens--
a. Indicator bacteria—Indicator organisms enumerated include total
coliforms, fecal coliforms, and fecal streptococci. These bacterial groups
were detected using membrane filtration procedures as specified in Standard
Methods for the Examination of Water and Wastewater, 14th Edition (1975).
Note however that fecal streptococci were isolated on M-Enterococcus agar
instead of KF Streptococcus agar. Additionally, the standard plate count
as outlined in Standard Methods was used to determine the levels of aerobic
and facultatively anaerobic, heterotrophic bacteria in each sample.
95
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b. Other bacteria-
ID Salmonella—Prior to March 23, 1981, Salmonella screening
was accomplished by filtering a measured volume of wastewater through a
diatomaceous earth (DE) plug as described in Standard Methods (1975).
Portions of the DE plug as well as aliquots of wastewater (_<25 ml) were
placed in separate bottles of selenite and tetrathionate broths for
enrichment at 35°C. Aliquots from the broths were streaked for isolated
colonies onto brilliant green agar and incubated at 42°C.
In an attempt to improve detection sensitivity, an
alternative procedure described by Kaper and associates (1977) was tested.
As described above, portions of the DE plug (for volumes >25 mL) and
aliquots of wastewater were placed in ducitol broth and incubated at room
temperature for 4 hours followed by incubation at 35°C for an additional 18
to 20 hours. An aliquot from each primary enrichment volume was
transferred into selenite cystine broth and incubated for 24 hours at 42°C.
Subsequent plating was as described above.
Characteristic colonies were counted and tested for oxidase
reactivity. Oxidase-negative organisms were transferred to an appropriate
biochemical test screen: triple sugar iron (TSI) agar and lysine-iron agar
(LIA). Based on these results, presumptive Salmonellae were confirmed with
commercially available polyvalent and group-specific antisera.
As shown by results presented in Table 4.24, the double
enrichment procedure yielded better recoveries of Salmonella from Lubbock
wastewater. On this basis, this procedure was selected to replace the
standard selenite enrichment technique.
(2) Shi gel! a—A portion of a diatomaceous earth plug resulting
from filtration of wastewater as described under procedures for Salmonella
along with _<25-mL portions of the unconcentrated wastewater were used for
detection of Shi gel!a. Each of these samples was added to a separate
bottle of GN broth. After 18 to 24 hours of enrichment at 35°C, aliquots
from the bottles were dilution-plated onto xylose-lysine-deoxycholate (XLD)
agar and incubated at 35°C. Oxidase-negative colonies were inoculated to a
biochemical screen utilizing TSI and motility-indole-ornithine (MIO)
medium. Shi gel la isolates were confirmed using commercially available
polyvalent and group-specific antisera.
(3) Staphylococcus aureus—Al iquots of wastewater were spread-
plated onto plates of mannitol salt agar and incubated at 35°C. Typical
colonies showing a yellow zone of mannitol fermentation were counted and
identified by microscopic observation of Gram-positive cocci and by testing
for coagulase activity.
96
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TABLE 4.24. RECOVERY OF SALMONELLA FROM WASTEWATER SAMPLES
USING TWO PROCEDURES
Sample
Lubbock-LV-7
Lubbock-LV-8
Lubbock-LV-9
Lubbock-LV-12
Lubbock-LV-13
Lubbock-LV-14
Standard selenite enrichment
Salmonella Volume enriched
detected mL
200
+ 200
+ 200
+ 100
100
+ 25
Double enrichment3
Salmonella Volume enriched
detected mL
+ 100
+ 100
+ 10
+ 1
+ 10
+ 1
+ 0.1
+ 1
+ 0.1
+ 0.01
+ 0.1
0.01
1
+ 0.1
a Kaper et al. (1977)
97
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(4) Mycobacterium—Mycobacteria were assayed quantitatively by a
procedure which almost totally suppresses sewage saprophytes while
permitting recovery of most Mycobacteria. The sample was treated for 20 to
30 minutes with 500 ppm of benzalkonium chloride (Zephiran®), diluted and
plated onto the surface of previously prepared plates of Middlebrook 7H11
agar plus OADC enrichment modified by the addition-of 3 ug/mL of
amphotericin B. Plates were incubated at 37°C in a C02 atmosphere and
examined over a period of one month for the appearance of typical colonies
of Mycobacteria. Suspect colonies were identified by examination of
stained (Ziehl-Neel sen) smears for acid-fast bacilli. Additionally, all
nonchromogens were subcultured onto Lowenstein-Jensen tubed medium and
subsequently tested for niacin production, a distinguishing characteristic
of _M. tuberculosis.
If the density of Mycobacteria was low, a concentration
procedure was employed to improve detection sensitivity. A 50-mL volume of
Zephiran®-treated samples was centrifuged at approximately 5,000 x g for 20
minutes. The supernatant fluid was discarded, the pellet resuspended in
1.0 mL of phosphate-buffered saline, and this volume plated as described
above.
(5) Kl ebsiel 1 a—Appropriate aliquots of wastewater were
dilution-plated in triplicate to eosin-methylene blue (EMB) agar and
incubated at 35°C. Mucoid colonies were counted and tested for an oxidase-
negative reaction. Suspect Klebsiella isolates were identified by typical
biochemical reactions in TSI and MIO medial
(6) Yersinia enterocol itica--As the detection of this organism
was inconsistent during baseline monitoring using either enrichment or
direct plate procedures, comparative testing of alternative methods was
completed as described below.
Lubbock wastewater (trickling filter composite) was used
unseeded and seeded with approximately 1 x 104 cfu/mL of Y_. enterocolitica
ATCC 23715. The different variables tested included the following:
1) Plating media
a) Salmonella-Shigella agar (SS)
b) MacConkey agar (Mac)
c) Cellobiose arginine lysine agar (CAL)
2) Cold enrichment media
a) 0.067 M phosphate-buffered saline, pH 7.6 (PBS)
b) PBS with 1% mannitol, pH 7.3 (PBS-Man)
c) 0.85% NaCl with 25 ug/mL potassium tellurite (NS-PT)
98
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3) Sampling periods
a) Direct
b) 3 days
c) 7 days
d) 14 days
e) 21 days
4) Treatment of inocula
a) Untreated
b) Potassium hydroxide treatment (KOH-NaCl).
Portions (150 ml) of the unseeded and seeded wastewater were
filtered through separate 1-g diatomaceous earth plugs. One third of each
plug was placed into the respective enrichment medium. The enrichment
media were incubated in a refrigerator at 4°C. The seeded and unseeded
wastewaters were sampled prior to filtration and enrichment, immediately
after filtration and placement into the enrichment media (i.e., "zero
time"), and after cold enrichment for 3, 7, 14 and 21 days. In each case,
inocula for the plating media were untreated and treated by mixing 20 uL of
sample with 0.1 mL of 0.5% KOH in 0.5% NaCl just prior to plating. The
plates were streaked by the four quadrant plating method and incubated at
25°C for 48 hours. Characteristic colonies were identified using the
API 20E® system.
Results of the comparisons of procedures of recovery of Y_.
enterocolitica from the seeded and unseeded samples are shown in Tables
4.25 and 4.26, respectively. A semi quantitative index of the numbers of
this organism present was obtained by reporting the highest quadrant in
which the organisms were isolated as discrete colonies. It is apparent
that these results that Y_. enterocolitica could readily be isolated from
both the seeded and unseeded wastewater samples.
The cold enrichment medium (NS-PT) previously employed
(Sonnenwirth, 1974) proved to be markedly inhibitory to the organism in
both seeded and unseeded samples; however, both PBS and PBS-Man yielded _Y_.
enterocolitica at the different sampling periods, particularly when the
inocula were treated with KOH-NaCl . Y_. enterocolitica was recovered from
each of the plating media. However, the greatest percentage of isolates
picked that proved to be Y_. enterocolitica by the API 20E® were from CAL.
Colonies of the organism were very distinctive on CAL in contrast to Mac
and SS agars.
Based on these results, Y_. enterocolitica was detected by
the following enrichment procedure beginning with samples collected on
March 23-24, 1981. A measured amount of wastewater was filtered through a
1-g diatomaceous earth plug which was subsequently dispersed in PBS
99
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TABLE 4.25. COMPARISON OF PROCEDURES FOR RECOVERY OF YERSINIA
ENTEROCOLITICA--UNSEEDED SAMPLES
Recovery of
Y. enterocol
quadrant at plating
Enrichment
None
0.067 M PBS
0.067 M PBS with
1% mannitol
0.85% NaCl with
potassium
tellurite
(25 pg/mL)
Medium
CAL
CAL
MAC
MAC
SS
SS
CAL
CAL
MAC
MAC
SS
SS
CAL
CAL
MAC
MAC
SS
SS
CAL
CAL
MAC
MAC
SS
SS
Treatment
KOH-NaCl
KOH-NaCl
KOH-NaCl
KOH-NaCl
KOH-NaCl
KOH-NaCl
KOH-NaCl
KOH-NaCl
KOH-NaCl
KOH-NaCl
KOH-NaCl
KOH-NaCl
Di rect
from
sample
Oa
2
0
0
0
1
Zero
0
2
0
2
0
0
0
2
0
2
1
0
0
0
1
0
0
0
3
days
0
3
0
2
0
0
0
2
0
2
0
0
2
0
0
0
0
0
7
days
3
2
0
3
0
2
0
2
0
3
0
2
0
0
0
0
0
0
itica
time
14
days
0
2
0
3
0
0
0
3
0
3
0
2
0
0
0
0
0
0
from
21
days
0
2
0
3
0
1
0
3
0
3
0
3
0
0
0
0
0
0
a 0 = none detected
100
-------
TABLE 4.26. COMPARISON OF PROCEDURES FOR RECOVERY OF YERSINIA
ENTEROCOLITICA-SEEDED SAMPLES
Recovery of
Y. enterocol
quadrant at plating
Enrichment
None
-
0.067 M PBS
0.067 M PBS with
1% mannitol
0.85% Nad with
potassium
tellurite
(25 yg/mL)
Medium
CAL
CAL
MAC
MAC
SS
SS
CAL
CAL
MAC
MAC
SS
SS
CAL
CAL
MAC
MAC
SS
SS
CAL
CAL
MAC
MAC
SS
SS
Treatment
KOH-NaCl
KOH-NaCl
KOH-NaCl
KOH-NaCl
KOH-NaCl
KOH-NaCl
KOH-NaCl
KOH-NaCl
KOH-NaCl
KOH-NaCl
KOH-NaCl
KOH-NaCl
Di rect
from
sample Zero
Oa
0
0
0
0
0
4
3
3
3
2
1
3
3
0
2
3
2
3
0
0
3
0
0
3
days
4
3
0
2
0
1
3
2
3
2
4
1
0
0
0
0
0
0
7
days
0
2
4
3
3
2
3
3
4
2
0
1
0
0
0
0
0
0
itica
time
14
days
3
2
0
3
0
3
0
4
0
4
4
3
0
0
0
0
0
0
from
21
days
0
3
0
2
0
2
0
3
0
0
0
2
0
0
0
0
0
0
a 0 = none detected
101
-------
(50 mL). A volume was removed for plating at this time and after three
days of incubation at 4°C. Plating volumes were treated with KOH-NaCl and
plated onto CAL agar. Typical colonies were isolated after 48 hours of
incubation at room temperature (22 to 25°C) and identified using API 20E®
and oxidase tests.
(7) Clostridium perfringens--An MPN procedure was used to
enumerate both vegetative and sporulated Clostridia. Prior to analysis, a
portion of the wastewater was heated at 80°C for 30 minutes. Both this
heat-shocked and the untreated sample were diluted appropriately in
phosphate-buffered saline and inoculated into three tubes of differential
reinforced Clostridia medium (DRCM) at each dilution. Following incubation
at 35°C for 72 hours, a loopful of sample from each DRCM tube was
transferred to litmus milk and subsequently examined for typical stormy
fermentation to confirm the presence of £. perfringens. Organism densities
were computed from the MPN tables in Standard Methods (1975).
An alternate membrane filtration procedure for the
enumeration of C_. perfringens as described by Bisson and Cabelli (1979) was
evaluated in parallel with the MPN procedure described above. A volume of
wastewater was filtered through a 0.45-u membrane filter (Gelman GC-6)
which was placed onto mCP agar containing cycloserine and polymyxin B
sulfate as inhibitory agents. Plates were incubated anaerobically in a BBL
Gas Pak® system at 45°C for 18 to 24 hours. Sucrose positive, cell obiose
negative (yellow colored) colonies were counted and tested for positive
reactions for acid phosphatase and gelatinase. Further confirmation
involved subculture to litmus milk with stormy fermentation followed by
testing for lactose, mannose and sucrose (with gas production) fermentation
and nonfermentation of cellobiose, mannitol and salicin. Additionally,
Gram-positive rods were visualized from litmus milk cultures.
Results of parallel testing are presented in Table 4.27. The
multiple tube technique detected a higher level of vegetative C_.
perfringens (nonheated sample) than the membrane filtration method in all
of the samples analyzed. The membrane filtration method detected a higher
level of sporulative C_. perfringens (heated-treated sample) on two of four
samples. This result could be attributed to the milder heat treatment
process used in the membrane filtration method. Perhaps more importantly,
the confirmation of £. perfringens by visualization of Gram-positive,
nonmotile rods was nearly equivalent for both procedures.
Due to the nature of the membrane filtration technique, it
may be desirable to use this procedure where larger volumes of samples are
to be processed. It should be remembered, however, that baseline
environmental data collected to date by the multiple tube technique cannot
be interchanged or extrapolated should an alternate procedure be used.
102
-------
TABLE 4.27. PARALLEL TESTING OF CLOSTRIDRIUM PERFRINGENS ASSAYS:
COMPARISON OF MULTIPLE TUBE INOCULATION AND
MEMBRANE FILTRATION TECHNIQUES
Sample
Lubbock 4
Wilson 4
Lubbock 4
Wilson 4
Lubbock 5
Wilson 5
Lubbock 5
Wilson 5
Lubbock 6
Wilson 6
Lubbock 6
Wilson 6
Heat
treatment3
+A
+A
-A
-A
+A
+A
-A
-A
+A
+A
-A
-A
Clostridrium
Multiple tube
(MPN/100 ml)
7.5 x 104
4.3 x 10*
2.1 x 106
7.5 x 104
1.1 x 105
2.4 x 104
2.8 x 105
4.6 x 106
1.5 x 104
2.1 x 104
1.1 x 106
1.1 x 105
perfrigens enumerated by
Membrane filtration
(cfu/100 ml)
3.5 x 104
5.0 x 103
5.0 x 104
1.5 x 104
no growth*5
no growth
no growth
no growth
5.9 x 104
6.9 x 104
6.0 x 104
7.6 x 104
Sample heated at 80°C for 30 minutes on multiple tube procedure and 65°C
for 15 minutes on the membrane filtration procedure (A = heat).
Increased volumes of sample tested were also negative for isolated
colonies of C. perfringens.
103
-------
(8) Campylobacter fetus ssp. jejum'--Beginm'ng with samples
collected in July 19.8k,-an assay to allow the detection of £. fetus ssp.
jejuni in wastewater was included in the microbiological screen. Aliquots
of wastewater were spread onto the surface of Campy-BAP agar plates
supplied by San Antonio Biological Company. This medium consisted of
brucella agar base with 5% sheep erythrocytes and vancomycin (10 mg/L),
trimethoprim (5 mg/L), polymixin B (2500 I.U./L), amphotericin B (2 mg/L),
and cephalothin (15 mg/L). Plates were incubated in a microaerophilic
environment (Campy-Pakll®) for 48 hours at 37°C. Suspect colonies were
subcultured to 5% sheep blood agar, incubated as before, and nonhemolytic
reactions typical of £. fetus ssp. jejuni were noted. Further tests for
this organism included catalase production, oxidase production, growth in
1% glycine, lack of growth in 3.5% NaCl , sensitivity to nalidixic acid (30
yg disk) and darting mobility as observed microscopically in wet mounts.
(9) Candida a1bicans--Testing for this organism was initiated as
part of wastewater screens in July 1981. Appropriate dilutions of
wastewater were spread onto Sabouraud dextrose agar (SDA) supplemented with
200 ug/nt chloramphenicol. Plates were incubated at 37°C for 48 hours.
Suspect colonies were subcultured onto SDA prior to confirmatory testing
which consisted of positive germ tube formation in bovine serum, positive
chlamydospore production on corn meal-Tween 80® agar, and assimilation of
sucrose as the sole carbon source.
(10) Gram-negative enteric bacteria—Both oxidase-negative and
oxidase-positive enteric bacteria including all members of the family
Enterobacteriaceae were sought using the screening procedures diagrammed in
Figure 4.17. Wastewater samples were diluted appropriately in sterile
phosphate-buffered saline and spread-plated over three plates per dilution
on MacConkey agar. After incubation at 35°C for 24 hours, all colonies
were counted and isolated at a dilution yielding a total of approximately
100 colonies over three plates. Discrete colonies were streaked onto
quadrants of heart infusion agar plates to allow growth and confirmation of
purity.
Subsequent identification involved oxidase testing and the
use of API 20E® identification strips. The API 20E® system consists of a
preset battery of 20 microtubes which allows the performance of 22
biochemical tests for the identification of 49 species/subspecies of
Enterobacteriaceae and 38 group/species of other Gram-negative bacteria.
c. Bacteriophages—Coliphages indigenous to wastewater were assayed
as plaque-forming units (pfu) using Escherichia col i K13 as the host
organism. Tests in this laboratory have shown strain K13 to yield the
highest coliphage titers when compared to other E. coli hosts. Appropriate
104
-------
Wastewater
Direct Plating on Selective
Medium, e.g.
- MacConkey Agar
Counting, Subculture
Oxidase Test
API
Identification by Profile Index
Figure 4.17. Isolation of Gram-negative enteric bacteria
from wastewater
105
-------
volumes (0.1, 0.5, or 1.0 ml) of the wastewater and 0.5 ml of overnight
culture of host cells were added to 3.5 ml of liquefied tryptose-phosphate
soft agar and poured while warm (45°C) onto 100-mm petri dishes prepared
with 10 ml of solidified tryptose-phosphate agar base layer. When firm,
the plates were inverted and incubated at 35°C for approximately 18 hours
prior to counting. For each sample, a minimum of five plates was used.
d. Human enteric viruses—During 1980, two concentration techniques
were used in parallel for the recovery of human enteric viruses from
wastewater samples. Both bentonite adsorption and organic flocculation
were used to concentrate indigenous viruses from the five effluent samples.
This approach was deemed necessary due to the nature of the wastewater
entering the Lubbock treatment plant, i.e., both industrial and domestic
wastes.
Positive viral recoveries were made consistently from the
bentonite concentrates, while parallel assays of the organic flocculation
concentrates were less successful due to toxicity and contamination. The
standard bentonite concentration procedure has performed adequately on both
Lubbock and Wilson wastewater effluents. Viral concentration efficiencies
based on the recovery of poliovirus 1 (Chat) have been consistent with a
mean of 67 _+ 27% for Lubbock wastewater (13 samples) and 55 _+ 13% for
Wilson effluent (13 samples). Concentrated volumes have been suitable for
both plaque and tube culture assay.
In addition, the bentonite adsorption technique has isolated a
wide spectrum of enteroviruses as shown in Table 4.28. It should be noted,
however, that this concentration technique is not expected to recover
either reoviruses or adenoviruses.
TABLE 4.28. VIRAL TYPES RECOVERED FROM WASTEWATER BY
THE BENTONITE ADSORPTION PROCEDURE3
Cell line Viruses isolated
HeLa Poliovirus 1, 2, 3
Coxsackievirus Al, A7, A9, A10, A16
Coxsackievirus B3, B4, B5
Echovirus 1, 3, 6, 7, 11, 21, 25
BGM Poliovirus 1, 2, 3
Coxsackievirus B2, B3, B4, B5
Echovirus 11, 25
RD Poliovirus 2, 3
Coxsackievirus Bl
Echovirus 6. 7. 11. 19. 22. 24. 30. 33
a Isolated from Rilling Road, Lubbock, and Wilson
samples; identified by a microneutralization
technique using Lim Benyesh-Melnick typing pools.
106
-------
Based on these observations, the bentonite adsorption procedure
as described below has been used as the sole viral concentration technique
for wastewater effluents.
For detection of human enteric viruses, a maximum of 4 L of
treated wastewater was concentrated in the laboratory using a standard
bentonite adsorption technique (Moore et al., 1979). Briefly, wastewater
was placed in a vessel of convenient size and 100 mg/L of expanded
bentonite added along with sufficient CaCl2 to bring the wastewater to
approximately 0.01 M. The pH of the sample was adjusted to 6.0 with HC1 ,
and it was mixed for 30 minutes. After mixing, the virus-solids-bentonite
complex was sedimented by low speed centrifugation. Tryptose-phosphate
broth (TPB) was added to the pellet to facilitate viral elution at a ratio
of 10 to 15 ml of TPB per liter of sample concentrated. Elution was
accomplished by sonicating the TPB-solids-virus suspension for 5 minutes in
an ice bath. The suspension was separated by centrifugation (8,000 x g),
and a portion of the supernatant fluid containing the eluted virions was
assayed. The remaining sample was held at -76°C.
Indigenous enteric viruses were enumerated by plaque assay on
selected cell monolayers. Testing conducted as part of the wastewater
pathogen screens during 1980 led to the selection of HeLa and RD cell lines
for viral recovery from environmental samples. Data presented in Tables
4.28 and 4.29 substantiate the choice of these cells in a complementary
assay system. In this laboratory HeLa cells recover the greatest variety
of enteric viruses. During baseline monitoring, the RD cell line showed a
preferential recovery of echoviruses, even in the presence of polioviruses
and Coxsackieviruses as evidenced by results from Lubbock-1 and Lubbock-2
samples (see Table 4.29). Additional testing has shown that echoviruses
can be isolated as plaques on the RD cell line. To further enhance the
recovery of a broad spectrum of enteroviruses, a portion of each
concentrated volume was neutralized for all three poliovirus serotypes
prior to the assay.
Beginning in January 1981, the assay matrix shown in Table 4.30
was used.
TABLE 4.30. ENTEROVIRUS ASSAY MATRIX FOR
WASTEWATER SAMPLES
Cell line/assay system
HeLa
HeLa + polio antisera
RD + polio antisera
Number of 100 mm
Undiluted
10
10
10
plates/ dilution
10-1
10
0
5
107
-------
TABLE 4.29.
VIRAL ISOLATES RECOVERED FROM THE SAME WASTEWATER SAMPLES
BY VARIOUS ASSAY PROCEDURES
Sample
Type
assay
Cell
line
Viruses isolated3
Lubbock-1
Lubbock-2
Wilson-1
Plaque
Tube
Tube
Plaque
Tube
Tube
Plaque
Tube
Tube
HeLa Poliovirus 1, 2, 3
Coxsackievirus Al, A7, A9, A16
Coxsackievirus B3, B4, B5
Echovirus 1, 3, 6, 11, 21, 25
BGM Coxsackievirus B2, B3, B4, B5
Echovirus 11, 20, 24
RD Poliovirus 1
Coxsackievirus Bl
Echovirus 6, 15, 24, 25, 29, 33
HeLa Poliovirus 2, 3
Coxsackievirus B2, B3, B5
BGM Coxsackievirus B2, B3, B5
RD Echovirus 11, 15, 19, 30
HeLa Poliovirus 1, 3
Coxsackievirus A10
Echovirus 25
BGM Poliovirus 1
Coxsackievirus B2, B5
Echovirus 25
RD Poliovirus 3
Coxsackievirus Bl
Echovirus 24
Identified by a microneutralization technique using Lim Benyesh-Melnick
typing pools.
108
-------
At the time of inoculation each series of ten (or five) plates were
assigned a number (1 through 5 or 109 as appropriate). A random ranking of
numbers was created for each assay system by lottery draw. The numbers
were recorded on the assay sheet in the order in which they were pulled.
After the appropriate incubation period, pfu were counted on those plates
yielding countable plaques. Plaques were picked for confirmation and
storage from plates at the dilution which allowed the best separation of
pfu and reflected the viral level to be reported. Selection of pfu from
plates followed the previously recorded order. Thus, if the ranked order
of RD plates (undiluted sample) was 3, 1, 89 2, etc., all plaques on plate
3 were picked followed by plates 1, 8, etc., until the desired maximum
number of pfu were acquired. If one plate was unacceptable due to overlap
of pfu or contamination, the next listed plate was used. The following
guidelines were followed in picking plaques for confirmation and future
identification: 25 pfu from the unaltered HeLa assay and 15 pfu from each
assay of polio-neutralized sample on HeLa and RD cells. In those cases
when fewer than the specified number of viral plaques were evident, all pfu
were picked. All pfu were confirmed by passage in the homologous cell
line, logged, and frozen at -76°C until identification.
Poliovirus neutralization was done using commercially available
rabbit antisera (M.A. Bioproducts). During 1981 the commercial supply of
specific poliovirus antisera was discontinued. Subsequently, lypholized
monkey or equine sera were obtained from the National Institutes of Health
for use in the poliovirus neutralization assays. Each lot of antisera was
used at a level which had previously demonstrated at least a 2.5 logjo
plaque reduction of homologous laboratory strains of poliovirus. Sample
and antisera against polio 1,2, and 3 were mixed, incubated at 37°C for 30
minutes, and plated.
The generalized procedure for plaque assay consisted of
inoculating confluent cell monolayers grown in 100-mm plates with 1.0 ml of
sample. After a 60-minute infection period, monolayers were overlaid with
an agar-based Eagle's minimal essential media containing bovine serum and
antibiotics. Infected plates were held at 37°C in a 5% C02 humidified
incubator. Two to three days post-infection, a second overlay containing
30 ug/mL of neutral red was placed on each plate. Plates were read on each
succeeding day and scored for plaques through five to seven days.
Possible viral isolates were picked from areas exhibiting
characteristic cytopathic effect (CPE) 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. Agar
overlaying the entire plaque was asceptically collected using a
microspatula. The sample was placed in 0.5 ml of medium 199 containing
antibiotics and held at -76°C until confirmation.
109
-------
Confirmation of potential viral isolates was performed in
homologous tube culture systems. Culture tubes were grown out to 50 to 75%
confluence and inoculated with 0.2 ml of sample. After 48 hours of
incubation at 37°C, tubes were observed daily for evidence of CPE. When
characteristic CPE was observed, the sample was removed and frozen at
-76°C. After seven days, all samples not showing CPE were harvested and
blind-passaged. Those isolates that demonstrate CPE after a second passage
were reported as viruses (pfu).
Viral isolates were identified using the Lim Benyesh-Melnick
pools for typing enteroviruses (Pools A-H and J-P) in a microneutralization
procedure.
e. Physical-chemical analysis—Total suspended solids (TSS), total
volatile suspended solids (TVSS), and total organic carbon (TOO were
analyzed following procedures outlined in Standard Methods (1975) which are
accepted by the USEPA.
Routine wastewater samples—Routine waste water samples were intended
to allow a determination of potential exposure of the study population when
the wastewater is used in irrigation. Samples were cooled to 4°C in wet
ice and shipped to UTSA at that temperature for analysis.
The routine wastewater samples were analyzed for total and fecal
col i forms, coliphage, fecal streptococci, Mycobacteria, enteric viruses,
TSS, TVSS, and TOC. Analytical procedures were those described above under
"Microbiological Screens."
Enterovirus identification samples—Composite samples were collected
from the Lubbock treatment plant trickling filter effluent or from flow in
the pipeline at the irrigation site (when available) and from the Wilson
Imhoff tank effluent. Samples were cooled to 4°C and shippped to UTSA.
The enterovirus identification samples were analyzed for human enteric
viruses, fecal coliform, TSS, TVSS, and TOC following the procedures
described above under "Microbiological Screens." Plaques were picked,
confirmed and frozen at -76°C for future identification. Within the limits
of the assay systems employed, the analysis of these samples allowed the
determination of enterovirus types present in the sprayed wastewater and
circulating within the Wilson population.
Limited bacterial screen samples—Composite samples of Lubbock
trickling filter effluent (or when available pipeline flow) were collected
and shipped to UTSA as part of the enterovirus identification samples
described above. In addition to physical-chemical analyses, the following
potential microbiological pathogens were sought using procedures described
110
-------
under "Microbiological Screens": Salmonel1 a , Shi gel 1 a , Yersi nia ,
Staphylococcus aureus, and Klebsiella-like organisms. On March 23, 1981,
both Campylobacter fetus spp. jejuni and Candida albicans were added to
this list of pathogenic organisms following methods described above.
Beginning June 29, 1982, fluorescent Pseudomonas sp. was substituted for _S.
aureus. As part of an effort to characterize Wilson wastewater, the same
limited bacterial evaluation screen covering these seven organisms was
initiated on Imhoff tank effluent beginning in July 1981. The occurrence
of selected organisms with human pathogenic potential in wastewater
destined for irrigation can thus be documented.
Legionella samples—Vlastewater from the Lubbock sewage treatment plant
was piped to three reservoirs located on the Hancock site and used for
spray irrigation either directly or from these reservoirs. A total of nine
separate wastewater samples were received by the University of Illinois
during 1982. Five of these samples (one trickling filter effluent sample
from March; three pipeline effluent samples from February, March and June;
and one reservoir sample from June) were processed and inoculated into
guinea pigs. Two samples (pipeline effluent and reservoir samples from
July) were examined by direct fluorescent antibody (DFA) techniques for
Legionella antigen. The two remaining samples (both reservoir samples from
August) have not been tested.
Complete testing for Legionella-group agents involved tenfold
concentration of wastewater samples by centrifugation. Aliquots of the
sample were then examined by DFA using available conjugates and diluted
(serial tenfold) for total bacterial counts using standard methods. The
purpose of this latter step was to avoid "overloading" guinea pigs with
more than 106 to 10^ non-Legionella and it was anticipated that samples
would be diluted to this level. However, this concentration was generally
found in the tenfold concentrated or unconcentrated samples, making further
dilution unnecessary. Guinea pigs were inoculated intraperitoneally with
1.0 ml of samples. Samples seeded with a standard amount of virulent _L_.
pneumophila 1 were included as controls. Guinea pigs were observed daily
and rectal temperatures recorded. Animals having a fever for two
consecutive days were euthanized. A fever was defined as a 0.5°C increase
in rectal temperature above prei nocul ation values. Since animals
inoculated with this type of material would be expected to develop fevers
unrelated to Legionella infection after inoculation, fever three days post-
inoculation was taken as the start of fever indicating Legionella
infection. All animals were euthanized on the seventh day post-inoculation
and were autopsied within hours of euthanization or dying. Sterile
techniques were used to collect peritoneal exudates and spleens. Samples
of these fluids or tissues were examined by DFA for Legionella and were
inoculated onto a variety of nonselective and semi selective agar media.
Ill
-------
Potential Legionella colonies were passed on charcoal-yeast extract (CYE)
agar. Second passage material was inoculated onto trypticase soy agar
(ISA) plates. CYE colonies failing to grow on ISA were considered possible
evidence of Legionella.
A number of attempts were made to isolate Legionella directly from
wastewater samples. These included inoculation of samples onto plates of
the semiselective medium BMPAa (Edelstein, 1981) which contains
cefamandole, polymyxin B, anisomycin, an organic buffer, and
a-ketoglutarate and pretreatment of samples with an acid buffer (pH 2.2) as
described by Bopp and associates (1981) followed by inoculation onto BMPAa.
Aerosol Samples--
The composite samples of sprayed wastewater taken during the
microorganism aerosol runs were analyzed for the same microorganism groups
and water quality measurements as the routine wastewater samples. The
aerosol sampler fluids from the microorganism aerosol runs and background
runs and the aerosol and wastewater samples from the quality assurance runs
were assayed for fecal coliforms, coliphage, fecal streptococci, and
Mycobacteria or Clostridium perfringens. Assays for human enteric viruses
were conducted on the wastewater and aerosol samples from the enterovirus
runs. Procedures for the indicator bacteria, Mycobacteria, C_. perfringens,
coliphages and human enteric viruses are described in "Microbiological
Screens."
The aerosol concentration procedure for human enteric viruses
described by Moore et al. (1979) was developed to be performed at a field
site. Due to the relative proximity of the Wilson site and the reduced
interval between sample collection and arrival at the laboratory, organic
flocculation was evaluated as an alternate concentration procedure. It was
considered probable that this procedure might provide higher viral
recoveries.
Three enteric viruses were used in the procedure development and
comparison testing: poliovirus 1, coxsackie B3 virus and echovirus 6.
These viruses were differentiated by using two cell lines and monospecific
antiserum in the following combinations. To determine poliovirus 1 titers
the sample was neutralized for coxsackie B3 virus and assayed on HeLa
cells. Coxsackie B3 virus and echovirus 6 were assayed from samples
treated with poliovirus 1 antisera and titered on HeLa and RD cells,
respectively (echovirus 6 will not plaque on HeLa cells; likewise coxsackie
B3 virus will not plaque on RD cells). This assay scheme allowed all three
viruses to be detected in one sample.
Typically, organic flocculation is performed by adding organics (beef
extract) to a sample. These organics are precipitated out of solution when
the pH is lowered to approximately 3.5. Virions are entrapped in the
112
-------
organic floe and removed by centrifugation. The amount of organics present
in a solution frequently dictates viral recovery rates; therefore,
experiments were performed to determine the optimal amount of beef extract
that should be added to the sampler fluid [brain heart infusion (BHI) +
0.1% Tween
Poliovirus 1, coxsackie B3 virus and echovirus 6 were added to three
liters of BHI + 0.1+ Tween 80® to give a final concentration of
approximately 10 to 100 pfu/mL and mixed for 15 minutes. Ten milliliters
of the sample were removed to establish actual input titers and the
remaining sample was aliquoted into 500-mL test volumes. Beef extract was
added, resulting in final concentrations of 0%, 1%, 2% and 3%. The pH of
each aliquot was adjusted to 3.5 by the dropwise addition of IN HC1. The
samples then were mixed for 30 minutes and centrifuged for 10 minutes at
8000 x g. After the supernatant was decanted, each pellet was resuspended
in 10 mL of 0.15 M NaHP04 (pH 9.0), and subsequently the pH was adjusted to
7.0. The final volume was measured and the sample assayed as previously
described. For comparative testing, a 500-mL aliquot of seeded sampler
fluid was concentrated by two-phase separation as described by Moore et al.
(1979).
Results shown in Table 4.31 demonstrate that the addition of 2% beef
extract provided optimal recovery when compared to the other beef extract
concentrations evaluated.
TABLE 4.31. CONCENTRATION EFFICIENCY OF ORGANIC
FLOCCULATION AND TWO-PHASE SEPARATION
Concentration% Polio la% CB3a% Echo 6b
procedure recovered recovered recovered
Organic flocculation
Q% beef extract
1% beef extract
2% beef extract
3% beef extract
Two-phase separation
a Results are an average
b Results are an average
33
41
55
33
50
of four experiments.
of two experiments.
53
61
77
62
61
60
79
84
81
43
Organic flocculation using 2% beef extract also consistently outperformed
two-phase separation, especially in the recovery of echovirus 6.
Therefore, the following protocol was adopted for the detection of viruses
in aerosols.
113
-------
The total volume of BHI + 0.1% Tween 80® from an aerosol run was
measured and 100 mL of the sample removed for routine organism
determinations. The amount of beef extract added to the sample was
calculated on the basis of total volume minus 100 ml. The beef extract was
added to a final concentration of 2% and mixed until the beef extract went
into solution. The pH of the sample was then lowered to 3.5 with IN HC1 .
After 30 minutes of mixing the organic floe was recovered by centrifugation
at 8000 x g for 10 minutes. The pellet was resuspended in 140 ml of 0.15 M
NaHOP4 (pH 9.0). The pH of the final eluate was adjusted to 7 and
subsequently split into two equal portions, one to be assayed on1 HeLa cells
and the other on RD cells. Prior to being assayed, the sample was treated
with chloroform to reduce bacterial and fungal contamination.
i
Plaque assay conditions and viral confirmation and identification
utilized the protocols described under "Microbiological Screens."
Fly Samples--
An effort was made to isolate enteric bacteria and viruses from
houseflies trapped at the farmhouses and at the effluent ponds. The
insects were processed as outlined in Figure 4.18. The clinical
bacteriology and virology procedures described previously were followed.
114
-------
Houseflies
I
C02, Packaged; Shipped by Air
- Add 10 ml diluent per 1 gm flies
- Homogenize in tissue grinder
Bacterial
Analysis
Streak plate through
Clinical Bacterial
Isolation scheme
(see Figure 4, Feces)
1
Viral
Analysis
Centrifuge at 8,000 x g
for 10 min, recover
supernatant fluid
Inoculate through
Clinical Viral Isolation
Scheme (see Figure 6)
Figure 4.18. Analyses of insect vectors
115
-------
DATA MANAGEMENT
-------
DATA MANAGEMENT
The activities in processing the data gathered in the LHES are
summarized below. Basically they consist of generating sample labels,
developing and revising data report forms, monitoring and processing data,
establishing and utilizing a data verification system, updating and
correcting the data base, and generating data summary tables, graphs and
computer files.
Several different types of data were collected in this study. These
include information obtained from the household health diary, scheduled
fecal specimens, illness specimens, electron microscopy, activity diary,
tuberculin test, household and participant interview, polio immunization,
and blood samples. A health data processing status report was developed to
monitor the receipt of this data and its subsequent processing. A current
report is contained in Tables 4.32 and 4.33.
The letter codes listed in Tables 4.32 and 4.33 indicate the status of
each data category. The code L indicates that sample labels were
generated, S denotes that the sample has been stored, A designates that the
data activity has been conducted, R indicates that the data have been
received for processing, C and K denote that the data were coded and
keypunched, P designates that the data were entered in preliminary form on
the data base, V indicates the data are verified, and D denotes that data
processing has been completed.
Sample Labels
A sample identification system based on a coded label was used to
preserve the integrity of the collected data. A computer-generated label
is affixed to each sample's container (e.g., wastewater, aerosol, blood
serum, fecal specimen, throat swab), each sample aliquot, and each source
record (e.g., medical history, health diary). An alphanumeric code on the
label specifies the participant ID number, sample medium (e.g., blood,
feces, wastewater), sampling period, and type of sample analysis so the
sample is uniquely identified. The key elements of the code also are
interpreted on the label in order to facilitate sample processing. The
sample code is reported to data processing along with the analytical result
and is keypunched and placed on the data base with the data. The sample
code functions as the index key for the data base. Typically, 11 different
labels are generated for collecting and archiving blood samples, 13 labeled
are produced for gathering and processing routine fecal specimens, 2 types
of labels are used in collecting the health diary data and 1 sample label
is generated for gathering activity diary data.
117
-------
TABLE 4.32. LHES HEALTH DATA PROCESSING STATUS REPORT
(March 15, 1983) (excluding serology)
Data Household
collection Start health
period date diary
Oil
012
013
014
015
016
017
018
019
020
108
109
110
111
112
113
114
115
116
117
118
119
201
202
203
204
205
206
207
208
209
210
211
212
213
214
215
216
217
218
219
220
221
222
223
224
225
226
1980
May 18
Jun 1
Jun 15
Jun 29
Jun 13
Jul 27
Aug 10
Aug 24
Sep 7
Sep 21
1981
Apr 5
Apr 19
May 3
May 17
May 31
Jun 14
Jun 28
Jul 12
Jul 26
Aug 9
Aug 23
Sep 6
1982
Jan 3
Jan 17
Jan 31
Feb 14
Feb 28
Mar 14
Mar 28
Apr 1 1
Apr 25
May 9
May 23
Jun 6
Jun 20
Jul 4
Jul 18
Aug 1
Aug 15
Aug 29
Sep 12
Sep 26
Oct 10
Oct 24
Nov 7
Nov 21
Dec 5
Dec 19
ARKP
ARKP
ARKP
ARKP
ARKP
ARKP
ARKP
LARKP
LARKP
LARKP
LARKP
LARKP
LARKP
LARKP
LARKP
LARCK
LARCK
LARCK
LARKP
LARKP
LARKP
LARKP
LARKP
LARKP
LARKP
LARKP
LARKP
LARKP
LARKP
LARKP
LARKP
LARKP
LARKP
LARKP
LARKP
LARKP
LARKP
LARKP
LARKP
LARKP
LARKP
LARKP
LARKP
Schedu led
fecal
specimens
ARCKVD
ARCKVD
ARCKVD
LARKVD
LARKVD
LARKVD
LARDVD
LARKVD
LARKVD
LARKVD
LARKVD
LARKVD
LARKVD
LARKVD
LARK
Household/ Polio
Illness Electron Activity TB participant Itmnunl-
speclmens microscopy diary test Interview zatlon
ARKP
ARKP
ARKP
ARKP
ARKP
ARKP
ARKP
ARKP
ARKP
ARKP
ARKP
ARKP
ARKP
ARKP
ARKP
ARKP
ARKP
ARKP
A
A
A
A
ARKP
AR
AR
AR
AR
AAR
AR
AR
A
AR
AR
AR
S
ARK
S
LARCKVD
S
ARCKVD
S
S LARC
S
A
A
A
A
A
A
continued.,
118
-------
TABLE 4.32. (CONT'D)
Data
col lection
period
301
302
303
304
305
306
307
308
309
310
311
312
313
314
315
316
317
318
319
320
321
322
323
324
325
326
Household Scheduled Household/ Polio
Start
date
1983
Jan 2
Jan 16
Jan 30
Feb 13
Feb 27
Mar 13
Mar 27
Apr 10
Apr 24
May 8
May 22
Jun 5
Jun 19
Jul 3
Jul 17
Jul 31
Aug 14
Aug 28
Sep 11
Sep 25
Oct 9
Oct 23
Nov 6
Nov 20
Dec 4
Dec 18
health fecal Illness Electron Activity TB participant Immunl-
dlary specimens specimens microscopy diary test Interview zatlon
LARKP A
LA A
LA LA A A
LA A
LA A
L
L
L L
L
Status Codes
L - labels generated
S - samples stored
A - activity conducted
R - received by data manager
C - coded by data processing group
K - keypunched
P - preliminary on data base
V - verified
D - data processing completed
119
-------
TABLE 4.33. LHES SEROLOGY DATA PROCESSING STATUS REPORT
(March 15, 1983)
Agent
Serum col lection
Hepatitis A
Polio 1
2
3
Coxsackle A9
B5
Echo 1
5
9
11
17
20
25
Adenovirus 7
Reovirus 1
2
3
Rotavirus
Norwalk
Leg i one 1 la
Inf luenza
012
Jun 80
LA
R
RK
RK
RK
RK
RK
RK
RK
RK
Serum collection period /col lection date
025 113 201 212 225
Dec 80 Jun 81 Jan 82 Jun 82 Dec 82
LA LA LA LA LA
R R R
RK RK RK RK
RK RK RK RK
RK RK RK RK
RK
RK RK
RK RK
RK RK
RK RK
Status Codes
L - labels generated
A - activity conducted
R - received by data manager
C - coded by data processing group
K - keypunched
P - preliminary on data base
V - verified
D - data processing completed
120
-------
Reporting Forms
An efficient reporting system has been developed. It includes special
data reporting forms that have been devised for use with the household
interviews, participant interviews, fecal, health diary, and serology data.
These forms allow for the transmission of the collected data in suitable
formats for direct keypunching. Each form, which can be found in Appendix
J, contains the sample identification number, participant's name, sample
period (where applicable), and type of sample analysis. Each category of
data is entered onto the data form as soon as the appropriate field or
laboratory analysis for a sampling period are complete. These forms are
sent directly to the data manager for keypunching and processing onto the
data base.
Data Processing
The data flowchart given in Figure 4.19 illustrates the steps involved
in processing the LHES data. Initially, when data or updates are received,
any necessary coding or corrections are made after the data are reviewed by
the processing staff. The completed data reporting forms then are sent to
be keypunched. Any noted errors detected in keypunching or in verifying
the keypunched data are corrected and the data are entered onto the
computer data base.
At this stage in the process computerized range and logic checks based
on individual variables are run in order to verify the accuracy of the
data. If a data record fails either of these checks, it is rejected, the
errors are corrected, and the data are reentered onto the data base.
Following these checks additional quality control programs are run to check
on intervariable consistency. Again, noted errors are corrected and the
revised data are reprocessed.
After all computer checks are complete, data listings and summary
reports are prepared and sent to the specified individuals who completed
the data reporting forms. These personnel, in turn, verify further the
accuracy of the data and relate any errors to the data processing staff.
The data then are corrected and updated for the final time. New computer
listings and summary tables are generated and sent to all data users for
use in their analysis and interpretation of the data.
All data are processed using the Scientific Information Retrieval
(SIR) data base management system. This system allows for easy data
storage and retrieval and provides a variety of means for inputting,
modifying, deleting and controlling the contents of the data file. It also
enables data users to interface with other computer programs in order to
perform statistical analysis on the data. Both versions 1 and 2 of SIR
have been used during the course of this study.
121
-------
RECEIVE DATA
(ADDS OR UPDATES)
COD IN6
NECESSARY?
KEYPUNCH
DATA
ERRORS
NOTED BY
\ KEYPUNCH? /
I
CORRECT ERRORS
ADD DATA TO
DATABASE'
ANY DATA
REJECTED?
CORRECT
REJECTED DATA
RUN ADDITIONAL
QC PROGRAMS2
CORRECT ERRORS
GENERATE
-LISTINGS OF NEW DATA
-REPORTS INCLUDING NEW DATA
MAIL LISTINGS
AND REPORTS3
NOTE 1: At this stage, any range
or logic checks that are built Into
the schema are run. If a record
falls either a range or logic
check, the record Is rejected.
NOTE 2: Additional QC programs are
programs that could not be built
Into the schema definition.
NOTE 3: Any correct I on Indicated
by Ul or UiSA should be coded on
the appropriate coding form and
submitted to data processing.
DATA CUSTODY
University of Texas at
San Antonio
' Fecal data
University of Illinois
• Activity diaries
' Health diaries
' Serology
Southwest Research Institute
- Data processing
* Keypunch
" Data base
* Report generation
Currently, coded data forms are not returned to the sender. Both Ul and UTSA are keeping
copies of submitted data forms.
Once processed, data forms are kept In ACCO-type binders.
Figure 4.19. Data flowchart for LHES
122
-------
Data Verification
Both computerized and manual verification systems are used to ensure
the accuracy of the LHES data. Initially, a manual review is made of the
received data reporting forms to be certain that all vital information,
such as participant ID or the data collection period, is entered onto the
coding sheets. Next after the initial keypunching of the data onto
computer cards, an independent verification is made of the keypunched data
by a separate keypunch operator. This two-step process in keypunching
eliminates almost all of the possible errors in transcription of data from
the reporting form to the data base. .
Software was developed for crosschecking IDs on all data records and
for ensuring that the data for a participant had the same status code as
that listed on the participant data file. These checks are made when the
data initially is entered onto the data base. Also included at this step
are computer checks on the type and range of all variables to be certain
that no observations are given an invalid code or have an incorrect format.
A simple manual data verification procedure follows the above computer
checks. Data lists or specified summary tables are compiled from the
validated keypunched data and returned to the data sender for final
verification. This process is the primary verification system used for the
fecal specimen data. ;
A computerized verification system has been developed for the
household health diary data due to its detail and complexity. A list of
diary variable edit checks was provided by the UI staff and implemented on
the SIR data base system. These checks assure intervariable consistency
and logic and allow for joint interaction between the UI personnel and the
SwRI processing staff in correcting data errors or inaccuracies. A similar
computerized system also is being developed for verification of the
serology data.
Data Summaries
Routine data processing services are provided on a continuing basis.
Primary among these is the updating and revising of the data base and the
generation of data summary tables and computer files. Participant and
donor status changes frequently during the study and this necessitates
monthly updates to the data base to ensure data accuracy. Major status
changes include moving a participant's data records from active to inactive
or adding a participant to the active list.
Summary tables are generated on a regular basis for certain data
categories. This includes summarizing the bacteriology data for the
scheduled fecal specimens and providing illness incidence and prevalence
123
-------
summaries for the health diary data. Computer files of the health,
exposure, and demographic data also are generated for usage in the
statistical analyses.
New variable schema listings are provided to all data users at the
annual meetings along with summary reports needed for presentations at
these sessions. Between annual meetings, a variety of summary table and
crosstabulations are generated to aid the data users in understanding and
interpreting their collected information.
124
-------
QUALITY ASSURANCE
-------
QUALITY ASSURANCE
Health Match
Field representatives have been contacted by phone on a weekly basis
for a summary of health diary information. UI staff then attempted to
contact all households which field representatives reported as no-contacts.
The field representatives submitted the written health diary summaries to
UI on a biweekly basis. These completed diaries are reviewed and coded for
data entry. In order to achieve consistency in classification of illness
information, all illnesses are coded according to a standardized list of
illnesses and conditions. Telephone reports and written diaries were
compared for discrepancies, and whenever possible, any discrepancies were
resolved. Computer-generated illness information will be compared to
original health diaries for further verification of information.
In order to assure that blood specimens are properly labeled at time
of collection, either the health watch manager or a field representative
places the name labels on the tubes. This avoids the problem of the
phlebotomist misidentifying similarly named study participants.
Aerosol Measurejnent _Pr_e_cj_sj_oji
Inspection of the microorganism aerosol density data shows
considerable variation, even between paired samplers. This measurement
variation may result from differences in many factors, including aerosol
density fluctuation, sampler operating procedures, undetected sampler
contamination, shipping difficulties (e.g., temperature above 4°C),
analytical laboratory techniques, and random error.
Two quality assurance aerosol runs were conducted to investigate the
amount and source of imprecision of the aerosol density measurements for
each microorganism group. Nine samplers were operated 3 meters apart in a
line at the same distance from a nozzle line so all samplers were
theoretically sampling the same aerosol density. The 100 ml of sampler
collection fluid is normally split at the laboratory into four 25-mL
portions for the four microorganism assays. On the quality assurance runs,
each sample was split in the field into 25-mL portions labeled for specific
analyses. Three of the four portions were labeled for assay for the same
microorganism group. Hence, portion variation, which reflected shipping
and laboratory-related variation, could be compared to measurement
variation to deduce the magnitude of sampling-related variation relative to
shipping/laboratory variation.
The data from the quality assurance runs are presented in Table 4.34.
The microorganism density in air determined from portions from the same
125
-------
TABLE 4.34. SAMPLED MICROORGANISM DENSITIES ON THE QUALITY ASSURANCE AEROSOL RUNS
ro
cr>
Quality
assurance
run
number
Qla»b
(75 m from
nozzle 1 ine)
Q2
(50 m from
nozzle line)
Sampler
alignment
(from left
to right)
(Wastewater cone. ,
no./mL)
123
201
210
211
217
219
223
226
227
(Wastewater cone. ,
no./mL)
210
219
226
106
227
123
211
223
217
Microorganism concentration in air
Fecal
col i forms
(cfu/m3)
(51,000)
160,170,160
250
640
TNTC,TNTC,TNTC
TNTC
(50,000)
87,90,80
52
_ ._.--- „..--,
430
180,170,170
200
Fecal
streptococci
(cfu/m3)
(4,800)
120,330,330
260
270
280,210,280
390
(3,600)
70,78,76
120
270
53,60,50
38
Mycobacteria
(cfu/m3)
(20,000)
3.5
2.6,5.3,5.3
4.4
4.2
8.2,5.3,4.0
(25,000)
X).60C
<0.15,<0.15,0.15
0.30
X).90C
2.0,0.52,0.67
Coliphaje
(pfu/m3)
(1,100)
6.7,8.0,5.9
11
12
i
8.3,10.4,8.3'
16
(720d)
4.6,d3.8 d9.6d
4.0d
6.0d
7.0,d6.6 d7.2d
10°
TNTC - too numerous to count
a Conducted during a dust storm.
b Portions received at laboratory at elevated temperature (9°C).
c A large number of colonies with indistinguishable morphology were present. Since only
representative colonies were examined for acid fastness, reported data are minimal values.
d Possible laboratory contamination due to phage aerosolization.
-------
sampler often exhibited less variation than the measurements from different
samplers, but there were exceptions.
The precision of a sample of n determinations can be measured by the
coefficient of variation, which is the ratio of the unbiased sample
standard deviation to the sample mean:
CV = an s/x
where x - sample mean = zx/n
S - sample standard deviation = [z(x-x)2/(n-l)]l/2
an - bias correction factor = [(n-l)/2]1/2r[(n-l)/2]/r(n/2)
The bias correction factor an adjusts for the bias in the sample standard
deviation s as an estimator of the population standard deviation a. The
values of an approach 1.0 as n increases: 02=1.253, 03=1.128, 04=1.086,
and 05=1.064.
To investigate the consistency of measurement variation over the
entire range of aerosol densities sampled in the field, measurement
coefficients of variation were determined for all situations in which
several samplers were theoretically sampling the same true density of
microorganisms in air. These situations were the paired samplers at three
locations on each microorganism run and the samplers assigned the same
microorganism assay on a quality assurance run. Coefficients of variation
were calculated when microorganisms were detected in at least one of the
sampler assays, assuming assays in which no microorganisms were recovered
had a value of half the detection limit. The coefficients of variation for
microorganism run pairs in the same density range were averaged to yield a
more stable estimate of the measurement variation.
The average measurement coefficients of variation throughout the
density range sampled are presented for each microorganism group in Table
4.35. Because the standard deviation calculated from a small sample is
very imprecise, the average coefficients of variation are quite variable
over a microorganism's density range. However, there is no consistent
pattern in the magnitude of the coefficient of variation with increasing
aerosol density. Hence, average measurement coefficients of variation were
determined over all sample sets, with the values 0.43 for coliphage, 0.46
for fecal streptococci, 0.67 for fecal coliforms, 0.72 for Clostridium
perfringens, and 0.81 for mycobacteria. Hence, the precision standard
deviation of the aerosol density measurements ranged from 43% of the
measured value for coliphage to 81% of the measured value for fecal
streptococci.
An investigation of the relative magnitude of the various sources of
the measurement variation was conducted based on the quality assurance
127
-------
TABLE 4.35. CONSISTENCY OF AEROSOL MEASUREMENT PRECISION OVER DENSITY RANGE
Microorganism group/
sample set
Fecal coliforms (cfu)
Usual detection limit
M1-M20: 6 pairs with C<1
M1-M20: 8 pairs with 110
Q2: 5 samplers
Ql: 3 samplers
AVERAGE OVER ALL SETS
Fecal streptococci (cfu)
Usual detection limit
M1-M20: 14 pairs with C"<1
M1-M20: 10 pairs with 150
Q2: 5 samplers
Ql: 5 samplers
AVERAGE OVER ALL SETS
Mycobacteria (cfu)
Usual detection limit
M1,M3-M16: 11 pairs with C<1
Q2: 5 samplers
M1.M3-M16: 6 pairs with Q>1
Ql: 5 samplers
AVERAGE OVER ALL SETS
Clostridium perfringens (cfu)
Usual detection limit
Sporulated: M17-M20: 3 pairs
Vegetative: M2.M17-M20: 7 pairs
AVERAGE OVER BOTH SETS
Coliphage (pfu)
Usual detection limit
M1-M20: 13 pairs with C<1
M1-M20: 9 pairs with 15
AVERAGE OVER ALL SETS
Mean
aerosol
density
(no. /FIT)
0.1
0.3
3.3
21
190
350
0.3-350
0.1
0.3
3.7
27
75
110
290
10.3-290
i
I 0.1
0.3
0.9
2.9
4.5
0.3-4.5
0.3
0.8
1.6
0.1
0.3
3.4
6.7
11.0
11.1
0.3-11
Average coefficient
of variation
for replicate
measurements
0.70
0.60
0.37
0.84
0.82
0.67
0.71
0.52
0.21
0.20
0.90
0.21
0.46
0.75
1.26
1.02
0.20
0.81
0.69
0.74
0.72
0.56
0.20
0.37
0.33
0.71
0.43
128
-------
runs. Portion coefficients of variation were determined for each run,
where the values from different samplers were averaged. The assay result
reported by the laboratory is an average, i.e., the total number of
colonies or plaques counted in spreading aliquots of the sample or its
serial dilutions over several plates (usually 3 plates for fecal coliforms
and fecal streptococci and 25 plates for coliphage). To estimate
laboratory sources of variation, an average aliquot coefficient of
variation was calculated for all assays on each quality assurance run using
the aliquot standard error s/Yrf in place of the standard deviation to
obtain a variability measure comparable to the measurement and portion
coefficients of variation. Since variances of independent variables are
additive, the variation attributable to field sources was estimated by
subtracting the variance for shipping and laboratory sources from the
measurement variance. Similarly the variation attributable to shipping
sources was estimated from the portion and aliquot coefficients of
variation.
Each of these coefficients of variation are presented in Table 4.36.
While the aliquot variation estimates are quite stable, the variation
attributed to other sources was highly variable due to the limited amount
of quality assurance data. Although a much broader range of aerosol
densities were sampled in comparison with the Pleasanton study (Johnson
et al., 1979), the average measurement coefficients of variation were
similar for fecal coliforms, mycobacteria, and Clostridium perfringens.
However, the LHES data for fecal streptococci and coliphage only exhibited
about 60% as much measurement variation as in the Pleasanton study.
Laboratory Analysis
Serology (hepatitis A)--
Quality assurance for the determination of antibody to hepatitis A
virus was that built into the HAVAB® test system (see above). This
involved the use of both positive and negative controls provided by the
test system manufacturer and determining that only repeatably reactive
specimens (minimally two tests conducted on separate days) were considered
to be positive for anti-HAV by the HAVAB® test. As a further control
measure, each analytical series of 100 tests included two or three sera
from participants whose HAVAB® reactivity had been established previously.
In addition an internal quality assurance (QA) program was conducted
during May and June 1981. One test series was comprised of eight coded
sera which had been tested previously for hepatitis reactivity. These
unknowns were analyzed in tests conducted on different days. Results from
this internal QA program are presented in Table 4.37 and show analytical
agreement for all eight samples. In addition an external blind QA program
was created by sera shipped to UTSA under a three-digit code by Dr.
129
-------
TABLE 4.36. ESTIMATED MAGNITUDE OF SOURCES OF PRECISION VARIATION
co
o
Average coefficient of variation (s/xj
Microorganism group/
quality assurance run
Fecal coliforms (cfu)
Q2
Ql
Fecal streptococci (cfu)
Q2
Ql
Mycobacteria (cfu)
Q2
Ql
Coliphage (cfu)
Q2
Ql
a Determined by subtraction
b Subtraction gives negative
Mean
density
in air
(no./m3)
190
350
110
290
0.88
4.5
6.7
11.0
Measurement Portion
variation
(all
sources)
0.84
0.82
0.90
0.21
1.26
0.20
0.37
0.33
variation
(shipping and
lab sources)
0.053
0.040
0.085
0.35
1.40
0.41
0.32
0.16
Al iquot
variation
(lab
sources)
0.1
0.06
0.08
0.08
0.4
0.17
0.16
Presumed
shipping
sources3
_b
-
0.04
0.3
1.3
0.3
0
Presumed
field
sources3
0.8
0.8
0.9
-
-
-
0.2
0.3
of variances.
variance;
presumably 1
ittle variation
due to this
source.
-------
TABLE 4.37. QUALITY ASSURANCE TESTING OF UNKNOWN SERA USING THE
HAVAB® COMPETITIVE BINDING ASSAY (Abbott Labs)
QA
number
1
2
3
4
5
6
7*
8*
ID
number
403010
409120
221020
531021
228110
215120
223020
225111
Previous HAVAB® Reactivity QA
June December reactivity
_
_
+ +
+ +
_
_
'
- -
* December 1980 sera tested; all other sera taken from June 1980.
131
-------
Northrop's laboratory. At the time of HAVAB® testing, it was assumed that
the samples represented new participants' sera. After testing had been
completed, a list was provided by Dr. Northrop showing that of the 28 sera
tested, 26 represented duplicate samples. Results of these HAVAB tests are
presented in Table 4.38. Once again, excellent reproducibil ity was noted
for the qualitative HAVAB® test.
Virus Serology--
The objective of measuring the antibody titer for nearly 30 different
infectious agents in the sera of the study population is to determine the
incidence of new infections caused by these selected agents. Following an
infection by any one of these specific viruses, neutralizing (protective)
antibody develops in the normal individual whether there is an associated
illness or not. The serological documentation that a new infection
occurred is based on demonstrating a four-fold increase in antibody titer
in serum obtained after infection compared to the titer in serum collected
at an earlier time from the same individual. In fully susceptible people,
antibody to a given agent is not detectable, and after infection, the titer
rises minimally from <2 to 4, or it may rise remarkably from <2 to 1024 or
more.
In the event that the incidence rate of new infections is more than
expected, it would be important to determine whether reinfections also
occur. Reinfections can be determined if there is a four-fold rise in
antibody titer to a given agent where the antibody level in the
prereinfection serum is low but measurable, i.e., 4, 8, 16, etc. These low
level antibodies suggest that a person had the same infection sometime in
the past but this protection to the agent is low and a limited, usually
subclinical, infection can occur.
The search for new infections and reinfections will be based on
comparing the antibody titers of each blood sample obtained from every
individual throughout the study period. It can also be analyzed by
comparing the geometric mean titers (GMT) for the entire study population
or subpopulation at different time intervals throughout the study period.
In order to accurately interpret rates of new infections or changes in
GMT, it is essential that there be knowledge of the reproducibil ity of the
serological results. This is particularly important when hundreds of sera
will be tested at one time using one virus type and then another group of
sera are run months later using the same virus type. The reproducibility
of the test is, essential in comparing these titers either individually or
as a group (GMT).
The following information outlines the laboratory procedures to be
followed for quality assurance, specifically reproducibility of the testing
format.
132
-------
TABLE 4.38. HAVAB RESULTS FOR REPLICATE SERA SHIPPED UNDER
THREE-DIGIT CODE BY NORTHROP1S LABORATORY
ID
number
114010
5330109
219141b
130020
219120b
402130a
130110
110010
219130b
402020b
217020b
130010
554010
a Previously
b Previously
Serum HAVAB®
code results
576 +,+
587 +,+
577
588 +,-
578 +,+
589 +,+
598 +,+
579 +,+
585 +,+
580 +,+
600 +,+
575
581
582
601
583
592 +,-
584 +,+
591 +,+
586 +,+
596 +,+
590 +,+
595 +,+
594 +,-
602
597 +,+
598 +^+
collected sera negative.
collected serum positive.
CPM Interpretation
ratio (hep A antibody)
0.12, 0.11 +
0.10, 0.11 +
1.8
0.80, 2.1
0.13, 0.13 +
0.16, 0.11 +
0.16, 0.11 +
0.13, 0.10 +
0,10, 0.14 +
0.14, 0.11 +
0.13, 0.13 +
2.1
1.1
1.3
1.4
2.2
0.74, 1.6
0.17, 0.15 +
0.15, 0.13 +
0.18, 0.14 +
0.20, 0.17 +
0.14, 0.13 +
0.16, 0.15 +
0.59, 1.8
1.9
0.79, 0.82 +
0.91, 0.47 +
133
-------
Quality control methods--
a. Confirmation of poliovirus antibody titers by a reference
laboratory--
After a group of sera have been tested in the laboratory and
titers for poliovirus types 1 to 3 are recorded, sera with no, intermediate
and high titers of antibody are selected and sent to a reference laboratory
performing neutralizing antibody tests. The titers for these sera from the
two respective laboratories are compared for consistency. This is a
suboptimal control. Different laboratories use viruses, tissue culture
cells and protocols that are not identical. Therefore, neither agreement
nor disagreement in the titers for the same serum is acceptable as
confirmation or failure to confirm the titration results.
b. Internal quality control in the testing laboratory--
To determine the variation in titers of one serum assayed
repeatedly at the same time for one virus type, the following procedure has
been and will be used. Reference sera known to have no, intermediate or
high antibody levels will be titrated approximately six times on one day
using one preparation of virus, one suspension of cells and performed by
one technician. By simple geometric analysis this range of variability in
titers of the six titrations will be determined under these defined
conditions. From this the range of variation expected for different titers
recorded on one day can be calculated and used in interpreting the
reproducibility of the titers for one day.
Another quality control procedure to be used now and in the
future is to determine variation in titers of one serum assayed at
different times for each virus type. Each time an assay is performed,
which may be at 3- to 6-month intervals in this study, a reference sera
will be run. During and at the completion of all the testing, the
variability in the titers of these sera for each virus type can be
determined. Knowing the range variation in the titers of sera run at
different times can be calculated and used to interpret the results
obtained at different times in the same laboratory.
c. Consultants used to develop.quality control methods--
Extensive discussions were held with Dr. L. Hatch, Enterovirus
Laboratory Section, Dr. J. La Monte, Laboratory Liscensure Program, and Dr.
W. Taylor, Laboratory Assurance Program of CDC, to develop the quality
control format presented above.
Dr. La Monte emphasized the need for the low and high titer
control sera for the positive control assays. Dr. Hatch discussed the
technical aspects of the neutralization test and the availability of
reference sera and Dr. Taylor commented on the discrepancies in laboratory
results reported by different laboratories performing the same tests on the
same sera.
134
-------
Protocols Used for Quality Control of Different Virus Groups--
a. Poliovirus serology quality control--
Because it has been determined, not unexpectedly, that
polioviruses are present in the Lubbock and Wilson wastewater, the LHES
assumed the responsibility to identify those study participants who are
susceptible to one or more of the three types of polioviruses. Antibody
titrations by serum neutralization testing have been done for participants
providing preexposure blood samples. According to numerous reports any
detectable level of antibody greater than a titer of 2 is considered
minimal for protection against disease. In this study individuals with a
titer of <2, 2, or 4 were recommended to be immunized. For quality
control, procedure a above has been or will be done and procedure b above
will be conducted by another state department of health laboratory.
b. Quality control for coxsackie-, echo-, and adenoviruses--
Only quality control method b described above will be used for
coxsackie-, echo- and adenoviruses because no one laboratory is available
to conduct control a for the specific viruses being used in this study and
because confirmation is not guaranteed as discussed in a and c above.
Sera obtained in Period 201 from the LHES and LLTP staff was
selected when a sufficient volume was available for use as positive and
negative control sera. These sera are titrated as described in b initially
and in each separate run. Titer variations of control sera determined for
one day and on different days are reported as quality control data.
c. Confirmatory titrations of seroconversions--
Whenever a four-fold rise in antibody titer for a given virus
type is observed in test sera collected at different times during the
project, these sera will be retested for confirmation of that rise. Paired
sera will be taken before and after the presumed time of seroconversion and
will be titrated concurrently in one assay under the same conditions. In
addition, a number of paired sera equal to those having rises but having no
change in titers in two tests will be receded by the Illinois Department of
Public Health (IDPH) staff before retitration. This "blind" reading will
serve as a further control for the reproducibility of seroconversions.
These tests, with all of the positive and negative control sera included,
will constitute a confirmed seroconversion.
d. Supervision of serology testing--
All of the serological testing described here will be performed
by University of Illinois personnel under the supervision of the director
of the Virology Section, IDPH. Virus stocks, tissue culture cells
reference sera, and reagents will be provided by IDPH or secured from
American Type Culture Collection (ATCC) and the work performed in IDPH and
UI laboratories jointly.
135
-------
Clinical Bacteriology--
Quality control in the Clinical Bacteriology Laboratory involved a
program of internal monitoring, seeded unknowns, and replicate, split
clinical specimens. Internal monitoring included testing each new batch of
culture media, testing reagents and stains, and quality control of
biochemical tests. In addition, the plating and enrichment media and
biochemical test media were assigned expiration dates that prevent use
beyond the point where consistent results can be obtained. Periodic
seeded, unknown specimens ensured the proficiency of the laboratory in
correctly identifying organisms and determining the levels of organisms in
the specimens.
Selected specimens were split and coded as unknowns for clinical
analysis in April and May 1981. A listing of coded split samples
(generated during preanalysis sample handling) was forwarded to the
laboratory supervisor on a weekly basis. Results of this QA testing for
clinical bacteriology are presented in Table 4.39 and indicated a very
successful program. Of the 22 split samples, total agreement on both
isolate identification and quantitation was recorded on 15 specimens (68%).
In all remaining samples, the variance between results of known and QA
tests involved a difference of a single quadrant level of microorganism
detection. For example, in handling specimen 559131 (period 108) as a
split sample, Escherichia coli was reported as moderate and heavy,
respectively, while Enterobacter cloacae was detected as no more than 10
colonies on one sample. These results indicated excellent reproducibil ity
in the clinical bacteriology laboratory.
Results of additional quality assurance unknowns performed in November
1982 are shown in Table 4.40. The unknown samples were given to the
technician as seeded autoclaved fecal specimens in buffered glycerol saline
(from routine fecal specimens that had been preserved by freezing for use
in quality assurance unknowns). Each of the four unknowns were correctly
identified both with respect to identity of organisms and the level of
seeding.
The concentration of organisms represented by different levels of
growth on MacConkey agar plates streaked by the four quadrant method is
suggested by the results of Table 4.41. Each of the values represents
laboratory reports on "blind" (unknown) samples seeded with known
concentrations of three organisms. The unknown samples were given to the
technician as buffered glycerol saline suspensions (E_. col i) or seeded
autoclaved fecal specimens in buffered glycerol saline (!£. pneumom'ae and
£. aeruginosa).
A program of surveillance procedures for selected laboratory equipment
also is being used in the clinical laboratory. This includes a time
schedule (e.g., each time or use for pH meters, daily for incubators) and
136
-------
TABLE 4.39. QUALITY ASSURANCE, CLINICAL BACTERIOLOGY
ID number
Period 108
557131
559131
321111
434141
211121
533131
324121
123111
123021
310111
426131
227121
539131
E.
E.
E.
E.
K.
E.
S.
C.
C.
E.
K.
H.
E.
C.
S.
K.
K.
E.
E.
S.
K.
E.
E.
K.
E.
C.
E.
E.
K.
Fl
E.
S.
Reported results
coli3
coli (M)
coli (M)
coli (M)
oxytoca (L)
coli (H)
aureus (L)
freundii H2S+ (L)
freundii H2$~ (VL)
coli (H)
oxytoca (L)
alvei (L)
cloacae (L)
albicans (L)
aureus (H)
pneumoniae (VL)
pneumoniae3
sakazakii3
coli (H)
aureus (L)
pneumoniae (VL)
cloacae (VL)
coli (M)
pneumoniae (VL)
coli (M)
albicans (VL)
coli (M)
coli (M)
oxytoca (VL)
. pseudomonas (VL)
coli (H)
aureus QJ
E.
C.
E.
E.
E.
E.
K.
E.
S.
C.
K.
E.
K.
H.
E.
C.
S.
K.
K.
E.
E.
S.
K.
E.
E.
C.
E.
E.
E.
C.
E.
E.
E.
S.
QA results Agreement
coli3 +
albicans (VL)
coli (H) +
cloacae (VL)
coli (H) +
coli (M) ++
oxytoca (L)
coli (H) +
aureus (L)
freundii (L)
pneumoniae (VL)
coli (H) ++
oxytoca (L)
alvei (L)
cloacae (L)
albicans (L)
aureus (H) ++
pneumoniae (VL)
pneumoniae3
sakazakii3
coli (H) ++
aureus (L)
pneumoniae (VL)
cloacae (VL)
coli (M)
albicans (VL)
coli (M) ++
albicans (VL)
coli (M) ++
albicans (VL)
coli (M) ++
coli (M) +
coli (H) ++
aureus (L)
continued.
137
-------
TABLE 4.39. (CONT'D)
ID number
122021
408121
E.
K.
-C.
E.
Fl
Reported results
coli (M)
oxytoca (VL)
freundii (VL)
coli (M)
. pseudomonas (VL)
QA results
E. coli (M)
K. oxytoca (VL)
C. freundii (VL)
C. albicans (VL)
E. coli (M)
Fl . pseudomonas (VL)
Agreement
+
++
Period 110
559121
559131
426131
404111
122111
533121
E.
S.
E.
K.
S.
E.
K.
E.
E.
C.
E.
K.
C.
coli (M)
aureus (L)
coli (M)
pneumoniae (VL)
epidermidis (VL)
coli (M)
oxytoca (L)
coli (H)
coli (H) ,
albicans (M) I
coli (H)
pneumoniae (L) i
albicans (VL)
E. coli (M) +
S. aureus (L)
E. cloacae (VL)
E. coli (M) ++
K. pneumoniae (VL)
S. epidermidis (VL)
E. coli (M) -H-
K. oxytoca (L)
E. coli (H) ++
E. coli (H) ++
C. albicans (M)
E. coli (H) ++ .
K. pneumoniae (L)
C. albicans (VL)
a Isolated by enrichment procedures, therefore nonquantitative.
138
-------
TABLE 4.40. CLINICAL BACTERIOLOGY QUALITY ASSURANCE UNKNOWNS
Specimen Identification reported Level Correct identification Level
1
Klebsiella pneumoniae H
Shi gel la flexneri H
Yersenia enterocolitica H
Enterobacter cloacae H
Salmonella species H
Serratia marcescens H
Staphylococcus aureus H
Klebsiella pneumoniae H
Shi gel la flexneri H
Yersenia enterocolitica H
Candida albicans H
Escherichia coli H
Proteus vulgaris H
Klebsiella pneumoniae H
Shigella flexneri H
Yersenia enterocolitica H
Enterobacter cloacae H
Salmonella typhimurium H
Serratia marcescens H
Staphylococcus aureus H
Klebsiella pneumoniae H
Shigella flexneri H
Yersenia enterocolitica H
Candida albicans H
Escherichia coli H
Proteus vulgaris H
139
-------
TABLE 4.41. QUANTITATION OF GROWTH BY THE FOUR QUADRANT METHOD
Organism
E. coli
K. pneumoniae
P. aeruginosa
Seeded organism
concentration
9 x lOVmL
9 x 102/ml_
9 x 103/mL
9 x lOVmL
4.5 x 106/mL
9 x 106/mL
4.5 x 107/mL
9 x 107/mL
0
33/mL
3.3 x 103/mL
3.3 x 105/mL
3.3 x 106/mL
7/mL
700/mL
7.0 x 104/mL
7.0 x 106/mL
7.0 x 107/mL
Level of quantitation from
clinical lab report
NG
L
L
L
M
M
M
M
NG
VL
NG
L
M
NG
NG
L
M
H
VL
L
L
L
L
L
M
H
NG
NG
NG
M
H
NG
NG
L
M
H
NG - negative
VL - very light
L - light
M - moderate
H - heavy
140
-------
set tolerance limits for incubators, refrigerators, freezers, water baths,
and pH meters.
Clinical Virology--
As described above, split samples coded as unknowns were also screened
for viruses in parallel with routine clinical specimens. Unfortunately, no
viruses were recovered from any of the 35 fecal samples received during
April and May 1981. Therefore, the split-sample approach did not yield
definitive data concerning laboratory precision for clinical virology.
A similar split-sample program was initiated in August 1981 in an
effort to test the reproducibility of viral isolation in tube cultures from
clinical specimens. Detailed results of this testing are presented in
Table 4.42. Of the 33 participant samples used in this program, only two
specimens yielded virus as part of the routine analysis while five isolates
were recorded in QA testing. Noteably, both samples found to be positive
in routine testing were also positive in QA testing, although in one
instance the isolation was made in different cell lines. These results
also highlighted the low likelihood of recovering viruses from routine
specimens when assay volumes were limited by tube culture inoculation.
Subsequently, assay procedures were modified as described in a previous
section to increase the amount of sample inoculated into susceptible cell
monolayers.
In addition, a specific quality assurance program was followed for
viral identification. On a quarterly schedule, three "unknown" animal
viruses (from laboratory stocks) were handled for identification using the
serological protocols described under "Laboratory AnalysiS--C1 inical
virology." An acceptable performance required the recovery of each unknown
virus in at least one cell line and the correct identification of each
isolate.
Electron Microscopy--
Photographs of each positive specimen are taken for documentation of
visual identification. The electron micrographs are evaluated against
micrographs published in peer reviewed journals with regard to size and
distinctive morphological characteristics. Positive specimen material is
maintained at -70°C for future reference. Poliovirus is used as the
reference standard for size determination. All examinations are performed
on the same JEOL 100CX electron microscope. The microscope is maintained
under a service contract and undergoes periodic maintenance and performance
checks by qualified personnel.
As 1982 included the first stool specimens collected after irrigation
was initiated, a procedure to eliminate possible bias during the EM
141
-------
TABLE 4.42. VIRAL QUALITY ASSURANCE TESTING
- '••' C.i IT'J !:
Period
116
117
and
118
Sample
1
2
3
4
5
6
7
8
9
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
18
16
17
19
20
21
22
23
25
26
27
28
29
Participant
227125
22712b
12211
12202
10413
55912
55913
32412
23112
32411
53912
53911b
53911b
20211
53313
22512
40311
40312
56211
56202
45114
53312
45113
40312b
40312b
40311b
40311b
40216
12202
12211
55715
32412
53911
55911
22512
40214
: 43613
Routine Anaiysisa QA Analysis3
HeLa
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
1/2
0/2
1/2
0/2
0/2
0/2
': 0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
RD
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
HeLa
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
2/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
RD
0/2
2/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
1/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
0/2
1/2
0/2
0/2
1/2
0/2
0/2
0/2
0/2
0/2
0/2
a Number of tubes showing viral cpe/total number of tubes inoculated for
each cell line listed. Other cells used with negative results were BGM
and primary RhMK.
b Replicate QA sample.
142
-------
examination of these specimens was implemented. All post-irrigation fecal
specimens, along with an equal number of preirrigation specimens, were
coded before examination. The identity of individual specimens under
examination remains unknown to the microscopist until all coded specimens
have been examined.
Environmental Samples—
In addition to the equipment and media performance testing described
above for "Clinical Bacteriology," the internal quality assurance program
for analysis of environmental samples involved two approaches. Wastewater
samples seeded with several laboratory strains of enteric bacteria were
analyzed for the quantitative recovery of the unknowns. Likewise, selected
known viruses were recovered and identified as described above for
"Clinical Virology."
In addition, a series of split analyses for enterovirus concentration
and assay on HeLa cells and for indicator bacteria enumeration by membrane
filtration were incorporated into the enterovirus identification and/or
routine wastewater analyses conducted monthly during April and May 1981.
Results are summarized in Table 4.43. Both bacterial and viral analyses
were within an acceptable reproducibility range. Such split sample
analyses will continue semiannually, unless future experience requires
additional QA documentation.
External QA data has been generated by compiling data for indicator
bacteria in Lubbock wastewater reported by both the LCCIWR laboratory and
the UTSA laboratory. Composite samples were collected by either SwRI or
LCCIWR personnel, split and shipped as part of routine monitoring described
previously. Results presented in Table 4.44 show that for those analyses
completed on or before March 10, 1981, values obtained by the UTSA
laboratory generally were lower by 10 to 70% than comparable results
reported by LCCIWR. While transit time might be evoked as an explanation
of bacterial inactivation, samples collected subsequently show an opposite
trend. Overall, however, the agreement between laboratories can be
considered good for microbiological parameters.
Data Management
A sample identification system based on a coded label is used to
preserve the integrity of the sample data. A computer-generated label is
affixed to each sample's container (e.g., wastewater, aerosol, blood serum,
fecal specimen, throat swab), each sample aliquot, and each source record
(e.g., medical history, health diary). An alphanumeric code on the label
specifies the participant ID number, sample medium (e.g., blood, feces,
wastewater), sampling period, type of sample analysis, etc., so the sample
is uniquely identified. The key elements of the code are also printed in
143
-------
TABLE 4.43. QUALITY ASSURANCE, REPLICATE ENVIRONMENTAL ANALYSES
Analysis
Bacteriology3
Fecal coliform
Total coliform
Fecal streptococci
Vi rol ogy b
HeLa (unaltered)
Source
Wilson LV-9
Lubbock LV-9
Wilson LV-10
Lubbock LV-9
Lubbock LV-9
Lubbock LV-9
Wilson LV-10
Sample 1
4.2 x 106/100 mL
8.8 x 106/100 mL
6.9 x 107/100 mL
1.5 x 107/100 mL
4.2 x 105/100 mL
1.1 x 102 pfu/L
1.6 x 102 pfu/L
Sample 2
3.9 x 106/100 mL
8.4 x 106/100 mL
6.2 x 107/100 mL
1.6 x 107/100 mL
4.8 x 105/100 mL
1.2 x 102 pfu/L
1.7 x 102 pfu/L
Mean
4.1 x 106
8.6 x 106
6.6 x 107
1.6 x 10?
SI
4.5 x lOf
r:
r~
1.2 x 102
1.7 x 102
a Membrane filtration.
b Values not corrected for concentration efficiency.
-------
TABLE 4.44. COMPARISON OF BACTERIAL INDICATOR VALUES REPORTED
BY SEPARATE LABORATORIES
Sample
date
Total coliform
(cfu/100 ml)
LCCIWR
UTSA
Fecal coliform
(cfu/100 ml)
LCCIWR
UTSA
6-4-80
7-23-80
11-14-80
1-20-81
2-17-81
3-10-81
3-24-81
4-21-81
5-5-81
4.1 x 107
4.8 x 107
3.0 x 107
1.0 x 107
1.4 x 107
2.5 x 107
1.8 x 107
3.9 x 107
2.9 x 107
3.5 x 107
4.8 x 107
1.4 x 107
6.0 x 106
4.1 x 106
1.2 x 107
1.6 x 107
5.2 x 107
Not done
Not done
2.5 x 107
1.0 x 107
1.5 x 106
1.1 x 107
4.0 x 106
4.0 x 106
4.8 x 106
5.9 x 106
8.7 x 106
7.2 x 106
8.8 x 106
1.5 x 106
3.4 x 106
1.6 x 106
8.3 x 106
5.9 x 106
8.6 x 106
145
-------
English on the label to facilitate sample processing. The sample code is
reported to data management along witJvJhe analytical result and is
keypunched and placed on.the data base with the result. The sample code
functions as the index key for the data base. Data processing errors are
minimized by.judicious inspection and editing of participant-furnished
data, inspection of field- and laboratory-reported data, key verification
of keypunched data, and reliance on automated data processing accompanied
by checks on the coherence of the data.
Archiving of Clinical Specimens
A portion of all clinical specimens (blood, feces, and throat swabs)
taken in the health watch were preserved and frozen at -76°C. A cross-
referenced catalog system allows ready access to specific samples should
retroactive studies be undertaken. Master lists of blood donors and
clinical specimen donors (based on samples received at UTSA) were updated
each period, reflecting each individual's cumulative participation in the
health watch program.
Archived aliquots of sera collected during June 1980, December 1980,
and June 1981 are in storage at UT-Austin. All sera collected after this
time are held in storage at UI;
Based on decisions reached at the third annual meeting held in
December 1982, routine clinical samples collected in 1980 and 1981 were
discarded with selected exceptions. All samples which had been found to
contain viruses either by recovery of an infectious agent or by electron
microscopic examination were retained as archived specimens. Current
inventory includes these routine specimens from baseline collections, all
illness specimens, and all routine specimens collected in 1982 and 1983.
146
-------
DATA ANALYSIS
-------
DATA ANALYSIS
The general objective of the health effects study is to detect any
possible adverse effects on human health resulting from the use of
wastewater for irrigation of crops in the Lubbock Land Treatment Project.
This will be achieved through the accomplishment of three specific
objectives:
1) maintain surveillance of the health status of the study
population;
2) describe the pattern of infections in the study population;
3) determine if the incidence of infections to agents found (or
presumed) to be prevalent in the wastewater is associated with
exposure to sprinkler irrigation of wastewater.
This section includes a discussion of the second and third specific
objectives as well as the procedures that will be used in evaluating the
expected health risks.
Describe Pattern of Infections
Incidence data from the serology survey, clinical specimens and health
diaries will be used to describe the patterns of infection in space and
time. Distribution of illness and infections in the study population will
be formed by age, race, sex, income, location of dwelling, outdoor activity
and medical history. Such distributions will be used to describe the
natural history of infections in the study population and show their
relation in space and time to irrigation activities, community-wide health
trends, weather, seasonal activities (such as farm work, school, holidays)
and other health-related events.
Individual and family units (as the unit of infection and route of
transmission) will be examined and procedures for exploring the route of
transmission via family units will be designed.
Association of Infection with Exposure
The effect of interest on the human population is any increase in the
incidence of infections due to the wastewater pathogens. Statistical
procedures for detecting such increases are given below for the four types
of observations: serological screening, analysis of fecal specimens,
tuberculin skin tests, and health diaries. Some of the statistical
procedures utilize a comparison of incidence rates of infections between
groups of individuals exposed to wastewater and those not so exposed. The
methods for estimating exposure and assigning individuals to exposure
147
-------
groups will be explained. The criteria for selection of agents to be
tested in the serological and fecal specimens will be discussed.
Considerations for interpretation of evidence from all phases of the study
are also explored. A flow diagram showing the order of these procedures is
given in Figure 4.20.
Exposure Estimation--
l An exposure index /is calculated for each participant during each;
period of irrigation to estimate the participant's exposure to the;
wastewater aerosol, assuming exposure primarily occurs through the aerosol
inhalation route. For a given participant and irrigation period, the
exposure index is computed from activity diary data and historical wind
data as
4 .
Exposure Index (El) = S(PnTn + E ^-1-,-)
where h - household location
i=l - blue map area (Hancock farm)
i=2 - orange map area (surrounding Hancock farm)
i=3 - white map area (remainder of study area)
i=4 - outside map area
Tn - weighted average of hours that the participant is at home
during the applicable weeks of the activity diaries
T-j - weighted average of hours that the participant is in region i
(i=l,4) excluding hours at home during the applicable weeks
of activity diaries (T^ + zT-j = 168)
Pn -. predicted relative aerosol concentration at the participant's
home
P-j - average predicted relative aerosol concentration in exposure
region i (i=l,4) calculated as geometric mean of all Pn
values in the region (£4=0)
S - proportion of days during the irrigation period that the
participant is reported to be in the study area (from
the health diary)
As the product of estimated relative microorganism concentration in the air
of a given ^region and time spent in that region accumulated over all
regions in the study area, the exposure index provides a crude estimate of
inhaled dose.
The predicted relative aerosol concentration of microorganisms at a
given distance d from the edge of the nearest irrigation rig on the Hancock
farm is estimated as
148
-------
ASSOCIATION OF INFECTION
USING EXPOSURE GROUPS
(2x2 TABLE)
EXPOSURE
ESTIMATION
ASSOCIATION OF INFECTION
USING INDIVIDUAL EXPOSURE,
AGE, HOURS IN LUBBOCK, ETC.
(LOGISTIC REGRESSION)
I
EPIDEMIOLOGICAL INTERPRETATION USING WASTEWATER EVIDENCE, MEDICAL
HISTORY, TRANSMISSION OF INFECTIONS THROUGH FAMILIES,
AND ANY OTHER POTENTIAL SOURCES OF EXPOSURE
PEAK EXPOSURE ESTIMATE
COVERING RANGE OF
HIGH EXPOSURE GROUP
Figure 4.20. Flow diagram for specific objective 3, association of infection with exposure
-------
p _ D exd/u = D e(-0.005 sec-d/^.O m/sec) _ D e-0.001 d
where d - distance in meters from edge of nearest irrigation
rig on Hancock farm
A=-0.005 sec"* - median decay rate of aerosolized wastewater micro-
organisms determined in Pleasanton study (Camann,
1980)
u - average wind speed = 5.0 m/sec for Lubbock
(1969-73)
Dd - normalized aerosol concentration at point d
resulting from diffusion, based on 1969-73 wind
patterns for Lubbock for the months of the
irrigation period
The normalized aerosol concentration D
-------
still lower exposure (left white) was chosen utilizing a Dj diffusion
isopleth modified slightly for landmarks recognizable by participants and
for microorganism die-off. The map used with activity diaries collected
during the school year (in March, April, and December) was based on a Dj
isopleth from the historical February-April wind data since that was the
primary period of irrigation during these months. The summer activity
diary map used a D^ isopleth from the historical July-August wind data.
Exposure index estimates will be computed for each of four irrigation
periods:
Winter/Spring 1982: Feb 16-May 4, 1982
Summer 1982: Jul 23-Sep 16, 1982
Winter/Spring 1983: Feb-Apr 1983
Summer 1983: Jul-Aug 1983
A weighted average of the time reports from the applicable activity diaries
will be employed to estimate Tn, T]_, T2, and T3 for each irrigation period.
Activity diaries were collected for six weeks (cf. Figure 4.21): M2
(March 21-27, 1982), A2 (April 20-26, 1982), U2 (August 1-7, 1982), D2
(November 28-December 4, 1982), A3 (April 10-16, 1983) and J3 (July 10-16,
1983). Full weight will be given to concurrent activity diaries, half-
weight to diaries from the same season of the other year, and quarter
weight to other diaries during the same school year. The weighted averages
are:
Winter/Spring 1982: T = (2TM2 + 2TA2 + T/\3)/5
Summer 1982: T = (2TU2 + TJ3)/3
Winter/Spring 1983: T = (4TA3 + 2TM2 + 2TA2 + TD2)/9
Summer 1983: T = (2TJ3 + Tu2)/3
Cases in which the T^ and TI time reports from the lesser weighted activity
diaries differ substantially from the concurrent activity diaries will be
evaluated to determine whether all the data are applicable to the exposure
estimate. Missing activity diaries will be excluded in calculating the
weighted average. The participant's T value from the most similar season
will be substituted if none of the activity diaries in the weighted average
were provided. If a participant provides none of the six activity diaries,
his T values will be estimated as the maternal values (for preschool
children) or as the average reported by responding participants in the same
geographic (rural or Wilson) and age/gender (0-5, 6-18, 19-65 males, 19-65
females, or over age 65) group.
Exposure groups of participants can be defined for each irrigation
period by stratifying the participants according to their exposure index
151
-------
en
ro
£ o
ID
I
_
O
o:
cc
ct
til
UJ i:
> CE
UJ O_
-I V)
Heovy-
Modecale —
Light-
Very Light-
LUES MONITORING
Activity Diary
1982
J|F M|A
AA
M2 A2
U2
S|O|N|O
A
D2
1983
M|A
A
A:
M|J
J|A
A
J3
s|o
N|D
Figure 4.21. Relation of activity diary collection weeks to periods of wastewater irrigation
-------
value El. To perform the 2x2 table statistical analysis, all participants
must be placed in either a "high exposure" or a "low exposure" group for
each irrigation period. The El cutpoint to be used as the boundary between
these two exposure groups will be the El value equivalent to spending an
average of 24 hours per week on the Hancock farm during the irrigation
period, i.e., EIcutp0-jnt = (PI)-(24 hours). To investigate a dose-response
gradient during an irrigation period, incidence rates and relative risk
should be determined for at least three exposure groups. The exposure
structure of the study population lends itself to three such groups,
defined by the following El cutpoints: 1) the relatively small group
residing or working on or adjacent to the Hancock farm with El >^ (PI) "(48
hours), 2) a large group having intermediate exposure that includes the
Uilson residents with (Pi)(12 hours) < El < (Pi)(48 hours), and 3) another
smaller rural group living farther from the Hancock farm with El _< (Pi)(12
hours).
Identification of Infection Episodes--
When an infection episode is identified from the health watch that may
have occurred during or after a period of irrigation, the infection episode
will be investigated by statistical methods to determine its association
with wastewater exposure. An infection episode is defined operationally
within the LHES as the observation in the study population of a number of
similar infection events (either serologically or in serial clinical
specimens) within a restricted interval of time. Episodes will be
statistically analyzed for association with wastewater exposure when the
infectious agent(s) was(were) found (or can be presumed) to be present in
the wastewater that was sprayed during that period.
To express these ideas more precisely, denote by \2 the number of
infections in the high exposure group of size i\2 due to a given agent and
let Xi be the number of infections due to the same agent in the low
exposure group. A "high" rate of infections will be said to occur when a
sufficient number of infections (Xj + fy 2. bo) are detected in the entire
monitored study population. The number b0 is chosen so if all these
infections had occurred in the high exposure group and none in the low
exposure group, the appropriate statistical test would reject the null
hypothesis of no association between exposure and infection. The critical
number b0 of infections in the study population sufficient to constitute an
infection episode are given in Table 4.45 for realistic values of nj and
r\2> The selected level, a, and the ratio (r=n2/ni) will influence the
definition of a "sufficient" number of infections. The ratio (n2/n^) will
be previously determined by assignment of individuals to low exposure (n^)
and high exposure (n2) groups. The level a=0.05 will be used to define an
infection episode.
153
-------
TABLE 4.45. NUMBER OF CASES (b0) REQUIRED3 FOR REJECTION OF ?i=P2 IN
FAVOR OF ?i
-------
Statistical Approach--
Previous studies of the effect of wastewater and associated aerosols
upon the health of such diverse groups as sewer and sewage treatment
workers (Clark et al., 1980; Sekla et al., 1980), agricultural workers
(Shuval and Fattal, 1980), school children (Camann et al., 1980), and
suburban residents (Johnson et al., 1980; Fannin et al., 1980; and Northrop
et al., 1980) suggest that any health effects seen in this study are likely
to be rather subtle. To ensure that the analysis is sensitive enough to
detect such effects, care has been taken to employ statistical tests for
which both the level and power can be calculated. In most instances this
leads to the use of rather simple tests of the main hypotheses. More
elaborate and sophisticated analyses often involve tests whose power is
unknown or known only approximately. These are presented as exploratory
techniques to be employed only after the primary test with controlled error
probabilities has been conducted.
This objective will be addressed through statistical analysis of a set
of selected infection episodes. The study design permits observation of
infections due to many agents over four distinct periods of irrigation
(winter-spring 1982, summer 1982, winter-spring 1983, and summer 1983).
The agents of observable infections (via serology, skin test, clinical
bacteriology, and clinical virology) which might serve as dependent
variables are listed in Table 4.1. The quantity (Q) or presence (+/-) of
many of these agents are being measured in the wastewaters from Lubbock and
Wilson on either a regular (R) or infrequent (I) basis, as shown in Table
4.3. However, some agents including Hepatitis A virus, rotavirus, and
Norwalk virus will not be measured in wastewater.
Detecting an association between irrigation with wastewater and
adverse changes in human health will be approached by statistical analyses
of four types of data; each of these is discussed below.
Serology—
The serological analysis consists of measurement of the concentration
(titer) of antibodies generated by the body in response to infection by
specific pathogens. Thus, if the titer of a given antibody in an
individual's blood is low at one time and higher at a later time, it is
presumed that an "infection" has occurred during the intervening interval
for that individual. Comparison of incidence rates of such infections
between groups of individuals exposed to relatively high concentrations of
wastewater aerosols and those less exposed will allow an evaluation of
serologic effects of the differential exposure.
A "seroconversion" for a given individual will be said to occur if the
antibody titer for a certain pathogen undergoes a fourfold increase from
one time of measurement to another. Thus, if the titer at any follow-up
determination equals or exceeds four times the prior titer for that
155
-------
individual, he will have "converted" with respect to that pathogen. If
that prior titer is b.elow the lowest dilution titer, any increase to one
titer above the lowest dilution (effectively, a fourfold rise or more) will
be considered a seroconversion.
Thus, we choose as a response variable to measure the health effect of
direct and indirect exposure of a group of individuals to wastewater, the
number of seroconversions as measured by comparing a titer with its
follow-up titer with respect to each of a specific set of pathogens. After
each follow-up titer, each susceptible individual within the group could be
classified as "converted" or "did not convert" for each pathogen. The
proportion converting would be an incidence rate and an appropriate
probability model would be the binominal with parameter P, the true
probability of conversion within the group during the period of
observation.
To determine the effect of wastewater exposure with regard to a given
pathogen or group of pathogens, the observed conversion rate within an
exposed group must be compared to the observed conversion rate within a
control group of individuals who have had little or no exposure. It is
important that the two groups be comparable in every pertinent respect
except wastewater exposure. Ideally, this comparability would be achieved
by random assignment to treatment groups, but since this is not feasible in
this study, we can check for comparability by examining variables such as
age, gender, socioeconomic status (income a-nd education), medical history,
and occupation. If significant imbalance i!s found, adjustment can possibly
be made by post-stratification or covariate1 analysis.
The serologic investigation for each pathogen can be summarized by the
following 2x2 contingency table
Converted
No Yes
Exposure
Low
High
"1
n2
wherein the row totals nj and n2 are fixed. The two rows represent the
outcomes of two binomial experiments, with probabilities of converting
being PI and P2, respectively. An appropriate statistic for testing the
null hypothesis P2 = PI against the alternative P2 > PI is
x^ = E (observed-expected)2/expected
156
-------
or when expected values are small, Fisher's exact test may be used. The
one-sided alternative is appropriate since ?2 < PI suggests that people
exposed to wastewater have fewer seroconversions than those not exposed, a
seemingly remote and uninteresting possibility. Furthermore, the test of
P2 = P! against ?2 > PI is more powerful than the test against the two-
sided alternative, given the same level and sample size. If the level, a,
and power, 1-p, of the test are specified and if the true probabilities of
seroconversion are PI and ?2 anc' ^ r> tne ratio of the sample sizes in the
susceptible low exposure and high exposure groups is given, the method of
Fleiss, Tytun and Ury (1980) can be used to calculate the required sample
sizes. Note that the characteristics of the analysis depend not only upon
the total sample size, but upon the ratio, r = n2/ni, of the samples from
each of the two groups to be compared. Table 4.46 shows the relationship
of nj, r\2 and r for several total sample sizes. The most favorable
situation for analysis occurs when r = 1, i.e., when nj = r\2 = n/2. The
results for several choices of a, e, P^, ?2, and r are displayed in Table
4.47.
Choice of appropriate values for a and 0 is of necessity subjective.
If the hypothesis ?2 = PI is rejected when it is in fact true, we would
conclude that exposure to wastewater in the manner of this study has
undesirable health effects and thus would be led to recommend more
extensive treatment or more guarded use of wastewater. This would
unnecessarily increase the expense or decrease the usefulness of irrigation
with wastewater. If, on the other hand, ?2 > PI and we fail to reject the
hypothesis that ?2 - PI> we would conclude that wastewater has no
significant health effect (as measured by incidence of seroconversion),
thus leading to possibly hazardous use of wastewater.
Of course, decisions with such far-reaching implications will not be
based solely on the outcome of this study, but its result can become an
important bit of evidence. In view of these considerations, choosing a = 3
seems appropriate. Table 4.47 displays several choices of testing
procedures available to use for which 0=3. If, for example, the
incidence rate of seroconversions in the low exposure group is PI = 0.01,
there are n^ = n2 = 214 individuals in each group, and a = 3 = 0.10, we can
expect to detect a seroconversion rate of ?2 = 0.06 (or greater) in the
high exposure group with probability 1-6 = 0.9 (power). Sample sizes for
given values of P]_, ?2» «> B, and r likely to be used in this study are
displayed in Table 4.48.
Note that if the cell expectations are small, the x^ approximation to
the distribution of the test statistic may be poor, and the corresponding
entries in Table 4.47 may be in error. It is usual in such instances to
use Fisher's exact test to test the hypothesis that PI = ?2 versus Pj < ?2>
but this test is conditional on having observed the total number of
157
-------
TABLE 4.46. SUBGROUP SAMPLE SIZES (nlt n2) FOR SELECTED VALUES
OF TOTAL.SAMPLE SIZE__(n). AND_IHE RATIO r = r\2/r\i
n
25
50
100
150
200
250
300
350
400
r =
"1
13
25
50
75
100
125
150
175
200
1
n2
12
25
50
75
100
125
150
175
200
r =
nl
17
33
67
100
133
167
200
233
267
0.5
"2
8
17
33
50
67
83
100
117
133
r =
nl
21
42
83
125
167
208
250
292
333
0.2
"2
4
8
17
25
33
42
50
58
67
r =
nl
23
45
91
136
182
227
273
318
364
0.1
"2
2
5
9
14
18
23
27
32
36
158
-------
TABLE 4.47. SAMPLE SIZE REQUIRED FOR TESTING P? = Pi VERSUS
P2 > PI IN TWO BINOMIAL POPULATIONS (ENTRIES ARE THE TOTAL
NUMBER REQUIRED FOR BOTH GROUPS)
o =
0 =
r =
a =
0 =
r* ~
a =
0 =
r =
a =
$ =
r =
a =
0 =
r =
a =
0 =
r =
0.10
0.10
1
0.10
0.10
0.5
0.10
0.10
0.2
0.10
0.10
0.1
0.15
0.15
1
0.15
0.15
0.5
Pi
0.0010
0.0100
0.0200
0.0300
0.0500
0.1000
0.1500
0.0010
0.0100
0.0200
0.0300
0.0500
0.1000
0.1500
0.0010
0.0100
0.0200
0.0300
0.0500
0.1000
0.1500
0.0010
0.0100
0.0200
0.0300
0.0500
0.1000
0.1500
0.0010
0.0100
0.0200
0.0300
0.0500
0.1000
0.1500
0.0010
0.0100
0.0200
0.0300
0.05
338
428
526
620
804
1266
1594
426
527
636
742
947
1416
1826
752
912
1086
1254
1579
2323
2971
1315
1584
1875
2158
2704
3949
5032
246
304
368
432
552
828
1068
306
372
444
513
0.07
236
282
330
378
470
680
862
298
348
402
456
557
790
992
526
606
691
775
937
1304
1622
920
1054
1198
1339
1608
2222
2752
172
202
234
264
326
462
582
215
248
283
318
P2 - P!
0.10
162
182
206
228
272
370
456
204
227
252
278
325
435
528
359
396
437
475
551
722
869
627
690
758
823
949
1234
1477
118
132
146
162
190
256
312
147
162
179
195
0.15
104
112
122
132
150
192
226
131
141
151
162
181
226
264
228
230
245
262
278
308
378
402
427
455
482
532
648
744
76
82
88
94
106
134
156
94
101
108
114
0.20
76
80
84
90
100
122
140
94
99
105
111
120
114
163
166
174
182
191
206
241
270
289
303
317
331
356
415
462
54
58
62
64
72
86
98
69
72
75
79
...continued
159
-------
TABLE 4.47 (CONT'D)
a = 0.15
0 = 0.15
r = 0.2
a = 0.15
6 = 0.15
r = 0.1
Pi
0.0500
0.1000
0.1500
0.0010
0.0100
0.0200
0.0300
0.0500
0.1000
0.1500
0.0010
0.0100
0.0200
0.0300
0.0500
0.1000
0.1500
0.05
648
954
1222
535
641
755
866
1079
1566
1990
934
1111
1302
1488
1845
2660
3368
0.07
384
537
669
376
428
485
540
646
886
1093
655
743
837
930
1107
1508
1856
P2 - PI
0.10
227
298
360
257
281
307
334
383
496
592
448
488
532
576
659
845
1005
0.15
128
157
182
164
175
186
197
217
263
300
287
305
322
340
374
449
512
0.20
86
101
114
119
125
130
136
146
169
188
208
217
226
234
252
289
321
P! - probability of event in population 1 ,
?2 - probability of event in population 2 :(
a - probability of Type I error (level) i
3 - probability of Type II error (1-p = power)
r = (size of sample from population 2)/(size of sample from population 1)
NOTE: These calculations are based upon the chi-squared approximation to
the distribution of Pearson's goodness-of-fit statistic. In tables
with small expected values (<5), these approximations may be in
error. In such cases, use of Fisher's exact test is appropriate.
160
-------
TABLE 4.48. SAMPLE SIZE REQUIRED FOR TESTING
VERSUS ?i
-------
seroconversions in the two groups. A procedure suggested by Fears et al.
(1977) shows how to ^calculate exact unconditional level and power so the
hypothesis tested is the same as in the usual x^ test for homogeneity,
i.e., the unconditional test that PI = ?2-
Once the major test has been accomplished wherein a and p are
carefully controlled, the way is open to use other exploratory techniques.
One possibility is to fit a multiple logistic model to the data whereby the
probability of "seroconversion" is calculated for each individual as a
function of certain explanatory variables such as age, race, gender,
socioeconomic status, and exposure (Truett, Cornfield, and Kannel, 1967).
This model is of the form
P (seroconversion) = [1 + exp(-X'j5)]-l
where X_' = (xj, X2>>>X|() is the vector of predictor or explanatory
variables mentioned above and §_' = (BQ, e^...^) is a vector of
coefficients whose magnitudes are related to the importance of the
associated variable in predicting the outcome. Thus, for example, if
exposure is an important predictor of seroconversion we would expect the
coefficient to be large, but if age is unimportant, its coefficient is
likely to be small. The distribution of the e's can be approximated so
that meaningful judgments of relative size can be made.
The maximum likelihood estimates of |jj_ can be calculated using the
Walker-Duncan procedure (1967) or if we wi'sh to consider not only presence
or absence of seroconversion, but the times until seroconversion, the Cox
regression solution for g_ would be useful (Cox, 1972).
In the discussion above, it was assumed that seroconversion was as
likely to occur in an individual who had an elevated titer at baseline as
in one who had very low titer at baseline. Some concern has been expressed
as to whether individuals with elevated titers have the same level of
susceptibility to reinfection as those with low titers. For some agents,
the lowest titer at which an individual is susceptible to reinfection is
not known. When baseline data indicate a significant difference between
the rates of new infection and reinfection, it may be advisable to study
seroconversion rates using one or two titers as cutpoints for
susceptibility.
An extension of the 2x2 contingency table is proposed which will
include two levels of exposure and three levels of susceptibility (see
Table 4.49). The effects of exposure and susceptibility on seroconversion
rates will then be tested using either weighted least squares regression
analysis or the Mantel-Haenszel chi-square test. Logistic regression will
also be performed as originally proposed to assess the association of
seroconversion with wastewater exposure and other explanatory factors
(including baseline titer level).
162
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TABLE 4.49. SEROLOGY DATA TABLE FOR TWO LEVELS OF EXPOSURE
AND THREE LEVELS OF SUSCEPTIBILITY
Exposure
Susceptibility
No seroconversion
Sereconversion
Low
High
High
Medium
Low
High
Medium
Low
Fecal Specimens—
In this part of the study, fecal specimens will be obtained at
consecutive four-week intervals over seasons that span wastewater exposure.
The presence (or level) of each of a prespecified group of microorganisms
will be determined for each sample. It is presumed that increased
concentration of these organisms in the environment will tend to be
reflected in increased prevalence in fecal samples obtained from
individuals in the high exposure group as compared to the low exposure
group.
The issues and procedures discussed with regard to the serological
analysis are also pertinent to the analysis of clinical specimens. The
fecal specimens are to be analyzed for evidence of infection from many
agent groups.
The statistical analysis of the clinical (fecal) specimen data follows
the same pattern as that of the serology data. For each participant, a
fecal specimen will be obtained just prior to the period of irrigation with
wastewater and again four weeks later. Isolation of an agent in the
specimen or an increase from a low level in the previous specimen to a
heavy level will constitute an event. That individual will be said to have
experienced an infection by that organism. As described earlier, each
individual can be assigned to either a high exposure or low exposure group.
The outcomes can then be recorded in a 2x2 contingency table:
Exposure
Low
High
Infection
No Yes
nl
n2
163
-------
The hypothesis to be tested is that the incidence rate of infection is
the same in the low and high groups, and the alternative of interest is
that the rate in the high group exceeds that of the low group. An
appropriate test statistic is
x^ = ^(observed - expected)^/expected
and the critical region is to be one-sided in accord with the stated
alternative hypothesis.
It is anticipated that about 80 paired samples will be obtained during
each irrigation season. Reference to Table 4.47 shows that for fairly rare
organisms (P^ _< 0.01) and relatively balanced groups (r >^ 0.5), increase in
incidence rates of 0.20 would be detected with probability 0.85 or greater.
In addition to the two collection periods just discussed, a third
specimen will be obtained from each individual eight weeks after irrigation
is begun, i.e., four weeks after collection of the second specimen. Since
significant exposure for a given individual may occur somewhat after the
beginning of irrigation and since several days may be required for
transmission and incubation of a given type of organism, the third specimen
will furnish an opportunity to detect infections that develop after the
second collection. The additional data would be incorporated into the
statistical analysis by redefining an infection as an event occurring at
either of the two follow-up examinations. By broadening the definition of
a positive response in this way, additional sensitivity to delayed
responses can achieved. Further, each participant is at risk of
experiencing an infection at each follow-up collection and only one
statistical test with controlled level and power is conducted for each
agent or group of similar agents at each irrigation season. If a
significant difference is found in this manner, the incidence rates from
baseline to four weeks and from four weeks to eight weeks can be examined
to determine the period in which most of the infections occurred.
An alternative analysis could be based upon the formation of a three-
dimensional contingency table with these data. Let Xjj|< denote the
presence (=1) or absence (=0) of the i^ organisms at the j*" measurement
on the kth individual and let the cell probabilities be denoted by
Then a log-linear model for these probabilities is
log Pjjk = M + oj + 3j + Yk + «Bij + QYik + Bvjk + eijk
where y - overall mean
aj - effect of itn organism, 1=1, 2, ...9
164
-------
Bj - effect of jtn time of measurement, j=l, 2, 3, 4
Y|< - effect of ktn individual
e - error.
From this point onward the analysis parallels that of the analysis of
variance for repeated measures experiments (Koch et al., 1977).
Three hypotheses are to be tested at prespecified levels
H2: ctj =0
H3: 6j = 0
Rejection of HI implies the rejection of H2 and H3 with the conclusion that
the incidence of some of the organisms changes with time while the
incidence of the others either does not change or changes in some other
manner. Inspection of tables of means and interactions would show the
location and nature of the interactions. If HI is not rejected, then H£
and H3 can be tested separately. If H2 is rejected we would conclude that
the incidence rate varies among organisms (an expected result) and if H3 is
rejected we would conclude that incidence rate varies with time. At this
point, the location of the changes could be determined by contrasts or by a
multiple comparison procedure.
Tuberculin Test--
The skin test for TB shows by the individual's reaction whether his
body has generated antibodies to the tubercle bacillus or to "atypical"
Mycobacteria. Presence of a large induration at a given time and absence
of a such an induration at a later time is taken to be evidence of a TB or
"atypical" infection during the intervening period in the manner of the
serological analysis. Presence of tubercle bacilli in the aerosol spray
would presumably be reflected by increased incidence of "primary
infections." If a positive reaction is taken to be the pertinent response,
the experimental design and analysis described for seroconversion will be
applicable. With this design, the experimental units would be individuals
without positive reactions as shown by the baseline test before exposure to
wastewater. These "susceptible" individuals would be divided into two
exposure groups and the analysis would consist of comparing the incidence
rates of conversion between these two groups.
The experiment may be summarized by the following table
165
-------
Response to
TB Test
Negative Positive
Exposure
Low
High
nl
n2
wherein the row totals are fixed. The rows contain the outcome of two
independent binomial experiments, with the probability of converting in the
high exposure group being P2 and in the low exposure group, P^.
Testing procedures, level and power considerations, and sample size
calculations from this point onward are the same as for the serology
experiment.
Data for estimating the conversion rate as defined in this experiment
for low exposure population (i.e., P^) are not yet available, but it seems
likely that it is rather small. Several examples showing the sample sizes
required for detecting differences of 0.05 and 0.10 in various situations
have been selected from Table 4.47 and are presented in Table 4.48. These
examples emphasize the need for obtaining the maximum possible number of
valid observations from the study in order to maintain useful standards of
level (a), power (1-p) and sensitivity (P2-Pi) for the statistical
analysis.
Health Diaries--
Since the health diaries are more subjective and more likely to be
biased or incomplete than the other types of data, this information will be
interpreted mainly as supplementary or supportive of the other analyses.
Analysis of the diary data should consider the following points:
1) Comparison of "operational" illness rates with baseline illness
rates will be confounded with secular community-wide cycles or
trends. This is particularly true of communicable infections
such as upper respiratory infections.
2) Data from different observation periods are correlated since the
same individuals are reporting their illness events.
3) Illness events within families are likely to be correlated.
4) Reporting within families is likely to be correlated.
166
-------
Response variables can be chosen and probability models formulated which
take into account some, but likely not all, of these factors.
To avoid confounding the community-wide trends, comparisons should be
made between "exposed" and "not exposed" groups simultaneously as was done
for serology, fecal, and skin tests.
The results of several comparisons should be interpreted in light of
the multiple test phenomenon, i.e., that the overall probability of finding
a false positive result increases with the number of tests performed.
These remarks apply mainly to the formal calculations and comparison of
illness rates. Tables and graphs showing the distribution of these events
in time and space and showing their relation to other health and
environmental data will also be helpful.
Some illnesses tend to be shared by individuals within families. If
this is judged to be an important complicating feature, the experimental
unit could be considered to be the family as represented by a chosen
individual (or individuals). This would avoid the unwanted correlations at
the expense of effective sample size.
Interpretation of the Statistical Results
To evaluate the possible relationship of sprinkler land application of
wastewater and infectious disease in the nearby population, the LHES is
investigating all observed episodes of infection. For each identified
episode of infection, a statistical assessment will be made by the
procedures described above of the association of the infections with
wastewater exposure. In the statistical analysis of each infection
episode, the null hypothesis is that the incidence of infection is the same
in the high exposure group and in the low exposure group (but not
necessarily the same for different infection episodes). The probability
levels (i.e., p values) of the statistical analyses conducted for the
infection episode will be reported. There will be one p value for the
initial 2x2 analysis and another p value for the exposure term in the
logistic regression analysis.
Influential factors which cannot be controlled in the statistical
analysis and pertinent data from other sources will also be considered and
objectively evaluated as supportive evidence for the findings derived from
the infection episode. These pertinent data include: 1) wastewater
evidence of the agent's presence, prevalence, and transmission that varies
in quality depending upon the agent (cf. Table 4.50), 2) strength of the
dose-response relationship (i.e., relative risk) among three exposure
groups, 3) timing of the infections relative to the period of irrigation,
4) related sources of health data on the infection episode (illness
specimens, health diaries, serology, fecal specimens, etc.), and 5)
167
-------
TABLE 4.50. CRITERIA FOR JUDGING QUALITY OF WASTEWATER EVIDENCE FOR EACH MICROORGANISM
Category of quality
Quality of evidence of agent in sprayed wastewater
1. Excel lent
2. Good
3. Fair
4. Presumptive
Source measurement
(Hancock wastewater)
Frequency
Specificity
Transmission measurement
(Wilson wastewaterc)
Frequent9
Serotype
Frequent
Species/genus
Occasional'5
Genus/species
None
oo
Frequency
Specificity
Agent monitored in
clinical specimens
(suitable as a dependent
variable in the statis-
tical analysis)
Frequent
Serotype
Specific coxsackievirus
Specific echovirus
Frequent
Species/genus
Salmonellae
Shigellae
Yers i n i a enteroco1111cae
Campylobacter fetus6
Fluorescent Pseudomonas6
Klebslella6
Mycobacteria (atypical)^
Candida a Ibicans6
None
Leg I one I la pneumophila
Staphylococcus aureus6
Proteus/C i trobacter6
Aeromonas/Serrat i ae
None
Hepatitis A vlrusT
Adenovirus
Reovlrus
Rotavirus*
Norwalk virus
Virus-like particles"
a Frequent: at least one measurement every four weeks.
b Occasional: about one measurement per irrigation season.
c Feces of rural donors may be substituted when the dependent variable is serologlc.
d Infections determined from serologic or fecal Isolate data.
e Infections determined from fecal iso I ate/1 eve I data.
f Infections determined from serologic data.
g Infections determined from skin test data.
h Infections determined by electron microscopy of fecal specimens.
-------
consistency of the pattern of infection in this episode with the observed
pattern for similar infectious agents during the same or other irrigation
periods. An attempt may be made to confirm and characterize the infection
episode (e.g., time of occurrence) within the context of the other
available health information: other participant data (i.e., illness
specimens, health diaries, routine fecal specimens and/or serology),
transmission through families, staff data, and local medical records. The
timing of the infection episode will be considered relative to the agent's
presence, abundance, and temporal pattern in the sprayed and Wilson
wastewaters and to the schedule of irrigation. The wastewater evidence
will be compared with the evidence regarding such other sources of
introduction as drinking water and time spent in Lubbock. The strength of
the association will be determined from the relative risk for the high and
middle of three exposure groups compared to the low exposure group.
Finally, the separate findings from each observed episode of infection
will be considered together to draw conclusions regarding wastewater
exposure and the incidence of infection. The relative quality and
reliability of the data upon which each finding was based will be utilized
to rank the findings. Consistency in the pattern of evidence across
several infection episodes would probably be needed to indicate a
relationship between wastewater irrigation and infectious disease.
169
-------
5. RESULTS AND
DISCUSSION
-------
HEALTH DATA
-------
SECTION 5
RESULTS AND DISCUSSION
HEALTH DATA
Description of the Study Population
This section reports information derived from interviews with members
of the 151 households initially participating in the health effects study.
The questionnaire used in the interviews was designed at the University of
Illinois School of Public Health and administered at the respondent's home
by trained personnel. A more detailed description of the interview
procedure is given in Section 4, Methods and Materials.
Information is presented concerning the demographic characteristics of
the households and individual members (Table 5.1), dwellings (Table 5.2),
crops and livestock (Table 5.3), exposure to wastewater (Table 5.4), and
health history of individuals (Table 5.5). This information was obtained
at the beginning of the study in May 1980.
Most of the items are answers to questions asked of the respondents
during the interview. These questions are cited verbatim with the
tabulated responses. In other instances the data were obtained from
observations by the interviewer or from other sources. In every case, the
source is indicated. A copy of the questionnaire can be found in
Appendix A.
There are a few omissions and inconsistencies in the data. Many of
these have been resolved by obtaining additional information. Efforts will
be made to resolve as many of these problems as possible before the final
report is made. The heading NR is used as an abbreviation for "not
recorded."
Health Watch Sampling
Table 5.6 lists the number of samples which were collected for the
various health watch activities during the first three years of the study.
At least one blood sample has been obtained from 95% of the current
population with 65% providing all of the requested blood samples. Fecal
specimens were received from 43% of the participants during 1982 with 21%
171
-------
TABLE 5.1. INITIAL INTERVIEW: DEMOGRAPHIC CHARACTERISTICS OF HOUSEHOLDS AND INDIVIDUALS
ro
Household Size
Question 4a: Including yourself, how many people live In this household?
Number In
household
Households
Percent
Individuals
Percent
1
33
22
33
8
2
53
35
106
24
3
24
16
72
17
4
15
10
60
14
5
11
7
55
13
6
8
5
48
11
7
2
1
14
3
8
2
1
16
4
9
3
2
27
6
Total
151
100
431
100
Mean number in household: 2.9.
Income
Question 32a: Considering all of the Income from employment, from net farm
income and from all other sources, please tell me which category best
describes your total household Income before taxes In 1979.
Income
($K)
Number
Percent
<5
19
12
5-
7.9
16
11
8-
9.9
12
8
10-
14.9
19
12
19.9
19
12
29.9
19
12
>30
27
18
Don't
know
10
7
NR
10
7
Total
151
99
Estimated Income
Question 32b*: Can you tell me if it was less than $10,000 or more than
$10,000?
Estimated
Income
Less than
$10,000
More~Ehan
$10,000
Don't
know
Total
Number
10
* Question 32b was asked of persons In NR (not recorded) category of
Question 32a.
Education
Question 30: (For the respondent and spouse)--What is the highest grade of
school which (you/ _) (have/has) completed?
Total
Grade
13-16
17+
Number
Percent
20
5
142
35
58
14
129
32
53
13
408
100
Gender
Question 5b: Gender of household members.
Gender
Male
"Female
"NTT
Number
Percent
212
49
217
50
2
1
Total
431
100
Age
Question 6: In what year (were you/was
by subtracting year from 1980.) .
Age
Number
Percent
9
55
13
10-
18
94
22
29
57
13
39
55
13
49
42
10
59
52
12
69
36
8
79
25
6
80+
10
2
NR
4
1
Total
431
100
Numbers Employed
Question 27: (For each household member born before
1962)--Are you (is _) currently working on any
part-time or full-time"~job?
WorkTng Yes
Number
Percent
171
61
~NQ-
104
37
ZEE.'
7
2
282
100
Unemployed
Question 28: (For each household member born before
1962 and not currently work1ng)--Are you (Is )
(category)?
Category *"
1.
2.
3.
4.
5.
6.
7.
Usually employed; just
temporarily out of work
Retired
Homemaker
Disabled or handicapped
Not usually employed
Student
Other
TOTAL
Number
14
42
49
6
1
2
0
104*
Percent
4
40
47
6
1
2
0
100
* Could be as many as lll--see Question 27.
Occupation
Question 29: (For those born before 1962 and either
employed, usually but not currently employed or
ret1red*)--Mhat (Is/was) (your/ 'si main
) born? (Age calculated
occupation or job title?
Occupation
1
2
3
4
5
6
7
8
9
10
11
1?
13
14
. Professional or technical
. Manager or administrator
. Sales workers
. Clerical
. Craftsman
. Operative
. Transport equipment operator
. Laborer (nonfarra)
. Fanner
. Farm laborer
. Service worker
. Private household worker
. Unemployed or homemaker
. Retired-occupation unknown
NR
TOTAL
Number
19
12
2
24
10
9
1
5
66
15
31
1
47
17
23
282
Percent
7
4
1
9
4
3
0
2
23
5
11
0
17
6
8
100
Apparently the question was asked of everyone born
before 1962.
-------
TABLE 5.2. INITIAL INTERVIEW: DWELLINGS
CO
Dwelling Type
Question 37: (Observed by interviewer)--Does the
respondent live in a single family dwelling? duplex?
apartment house (3 to 4 units)? apartment house (5 or
more units)?
Type of
dwel 1 i ng
Number
Percent
Single
family
141
93
Apartment (5 or
more units)
1
1
NR
9
6
Total
151
100
Fara Dwellings
Question 38: (Observed by -1 nterviewer)--Is the
household located on a farm?
Nonfarm
Farm
"NT
Total
Number
Percent
76
50
• 66
44
9
6
151
100
Drinking Water Source
Question 2: Do you obtain your drinking water from a
private well or a public water supply?
Water
source
Number
Percent
Private
well
75
50
Public
supply
68
45
NR
8
5
Total
151
100
Sewage Disposal
Question 3: Do you dispose of sewage through a septic
tank or cesspool or (the) city sewage system?
Sewage
disposal
septic tank
or cesspool
City sewage
system
NR
Total
Number
Percent
76
50
67
45
151
100
Air Conditioning
Question la: Do you have air conditioning (AC) in your
home?
AIT
TeT
Total
Number
Percent
125
83
17
11
9
6
151
100
Air Conditioning Type
Question Ib: Do you have central air conditioning,
window or wall units or both?
AC Type
Central
W1ndow
Both
Total
Number
Percent
76
61
48
38
1 125
1 100
Air Conditioning Use
Question Ic: During the summer, do you have the air
conditioning on all or most of the time, some of the
time every day, only when It is very hot or never?
AC use
All or
most
Some
When hot Never Total
Number
Percent
28
22
36
29
60
48
125
100
-------
TABLE 5.3. INITIAL INTERVIEW: CROPS AND LIVESTOCK
Crop Types
Question 11: What crops are you producing on your farm
this year? Please tell me each crop which you are
growing and the amount of acreage~devoted to It (cotton,
wheat, other).
Crop
No. of farms
Percent
No. of acres
Percent
"Cotton
56
74
16236
88
"Wat
5
7
105
1
•QTher
26
34
2155
11
76*
18496**
100
*
**
More than one crop Is grown on some farms.
Not all acreage Is cropland.
Irrigation
Question 13a:
Irrigation
Do you currently irrigate your farm land?
Yes
HcT
TotaT
Number
Percent
60
79
8
11
76
100
Irrigation Water Source
Question 13b:
other)?
Source
What Is the source of that water (well,
HeTI OCTer
ToTaT
Number
Percent
60
100
0
0
60
100
Livestock Types
Question 12: What types of livestock are you raising on your farm this
year? Please tell each type of livestock and the numbers of animals
(cattle, hogs, sheep, fowl, horses, other).
Livestock
No. of farms
Percent
No. of animals
Percent
Cattle
13
17
340**
20
Hogs
8
11
886 1
54
Sheep
3
4
175
11
Fowl
12
16
246
15
Horses
1
1
2
0
Other
1
1
1
0
Total
76*
1650
100
* Not all farms had livestock; some had more than one kind.
** One farm had 100 head of cattle.
t One farm had 700 hogs.
Acres Fanred
Question 14: Approximately how many acres of land do you farm, Including
pasture, fallow ground and grazing land?
Reponses: 64 Maximum acres: 1,800
Total acres: 31,529 Minimum acres: 2
Average acres: 492 Median acres: 320
-------
TABLE 5.4. INITIAL INTERVIEW: EXPOSURE TO WASTEWATER
-o
en
Location of Dwellings
Households
Number
Percent
Location of
TndTvTduTI s
Number
Percent
Rural
80
53
Individuals
Rural
216
50
~" WfTsofr"
71
47
Wilson
215
50
Total
151
100
" TofaT"
431
100
Fara Work Within Study Area
Question 15a,b: Asked only if household not located on
a farm (see Question 38 and Table 3.3). Do you or does
anyone in your household every work on a farm within the
outlined area (see map, Figure )? Who Is that?
WoYk~oh farm
within area
Yes
No
NR
Total
Number
Percent
* AddTTfona
50
177
nformation needed to complete this table.
Weeks Per Year of Farm Work
Question 15c: How many weeks per year (do you/does
) work on a farm?
Weeks
11
12-
25
39 52 NR Total
Number
Percent
14 13
10
*Additional information needed to complete l*rs table.
Days Per Week of Faro Work
Question 15d: How many days per week (do you/does
) work on a farm when (you/ )
work(s)?
Days per
week
Number
Percent
2
1
4
3
5
30
6
11
7 NR Total
3 * *
Location of Job or School
Question 76: Looking at this map (Figure
) works or goes to school.
Location ZonTT*Zone"?*
Lubbock
), please show me where (you/
Otfier NRToTaT
Number
Percent
40
17
113
48
40
17
39
" 17
1
1
233
100
Zone l--within 500 meters or on Hancock farm.
Zone 2--within study area but outside Zone 1.
Hours Per Week Outside Study Area
Question 8: Approximately how many hours per week (do you/does
spend outside the outlined area shown on this
Hours
each week
Number
Percent
0
72
17
1-
8
134
31
9-
16
70
16
24
28
7
25-
36
28
7
37-
48
37
9
map (see Figure )?
49-
60
19
4
61-
72
6
1
73-
120
9
2
NR
28
6
Total
431
100
Average hours per week: 17
Hours Per Day Out of Doors
Question 9a,b: During the nonwinter months, how many hours per day (do you/
does ) generally spend out of doors, within the outl1ned area (see
map, Figure ) on weekdays? weekends?
Hours per day
Number (weekdays)
Number (weekends)
0-1
52
51
2-4
104
121
5-7
103
132
8+
111
98
NR
23
29
Total
393
431
Hours Per Week of Fara Work
) spend doing
farm work out of
Hours per week
Number
Percent
doors?
0
67
16
1-9
40
9
10-19
6
1
20-39
22
5
40+
52
12
NR
244
57
Total
431
100
Seasons of Faro Work
Question 15e: During which seasons (do you/does
) generally work on a farm?
Season*
Spring Summer
Total
Number 18
* Not mutually exclusive.
46
22
49
-------
TABLE 5.4 (CONT'D)
o>
Visits Per Month to Lubbock
Question 16a: Approximately how many times per mcHith^ (do you/does
) travel to Lubbock? ~~
Work Outside of Hone
Question 7a: Do you (does
to school outside your home or farm?
) have a job or go
Visits per month 0
Number 17
Percent 4
T-T~~ 6-icr n-rs i6-2~o"
233 67 22
54 15 5
Ti«e Per Visit to Lubbock
Question 16b: Approximately how much time (do
Lubbock each visit?
Hours per visit 0-5
Number 303
Percent 70
Hours Per Month In Lubbock
Calculation of hours per month
6-10
72
17
44
10
you/does
(number of visits
11+
9
2
x number
"21+ MR TotaV
20 28 431
5 7 100
) spend in
NR Total
47 431
11 100
of hours for each
*
Outside job
or school
Number
Percent
Bottled and
Question 17b
ever drink
)
Drinking
water
Number
Percent
Yes
233
54
No
168
39
Tap Water
,c: Do you or does anyone In
bottled water regularly?
ever drink water from the tap?
Bottle Bottle
only tap
11 31
2 7
or Tap
only
365
85
NR
30
7
Total
431
100
your household
(Do you/does
NR
24
6
Total
431
100
individual).
Hours per
month
0-
5
6-
15
16-
25
45
46-
95
145 <146 NR Total
Number
Percent
71
16
122
28
83
19
46
11
34
8
11
3
34
8
31
7
431
100
-------
TABLE 5.5. INITIAL INTERVIEW: HEALTH HISTORY
Respiratory Conditions
Question 18a,b,c,d: Have you or anyone in this household ever seen a doctor
for any of these respiratory illnesses or conditions? Who is that? Which
Illness or condition (do you/does _ _ ) have? How old (were you/was
) when the (condition) appea'red?
Condition
Allergies
Chronic bronchitis
Emphysema
Asthma
Tumor or cancer of
the lung
Tumor or cancer of
mouth or throat
Other
F-5"-
28
3
0
11
0
0
0
~6~IT"
16
1
0
2
0
0
0
T2--TT
2
0
0
1
0
0
0
TsV
9
1
0
3
0
0
0
~J1~5"0~
5
3
2
2
0
0
1
-?IT-
6
1
4
0
0
0
1
~m
5
1
0
0
0
0
0
Total
71
10
6
19
0
0
2
Cardiovascular Conditions
Question 19,a,b,c,d: Have you or anyone In this household ever seen a
doctor for any of these heart conditions? Who is that? Which type of heart
condition (do you/does ) have? How old (were you/was )
when the (condition) first occurred?
Condition
High blood
pressure
Stroke
Heart attack
Angina
Other
0-5
0 00 O 0
6-11
0
0
0
0
0
12-17
0
0
0
0
0
Age
18-30
5
0
0
0
0
31-50
21
1
2
0
1
51+
31
1
3
1
3
NR
1
0
0
0
0
Total
58
1
5
1
4
Gastrointestinal Conditions
Question 20a,b,c,d: Have you or anyone In this household ever seen a doctor
for any of these stomach or abdominal conditions? Who Is that? Which of
these conditions (do you/does ^ ) have? How old (were you/was
) when the (condition) first appeared?
Condition
Tumor or cancer of
stomach
intestine
colon
esophagus
Peptic or duodenal
ulcer
Ulcerative colitis
Diverticulitis
Gall bladder
problems
Other
V-TS~
0
0
0
0
1
0
0
0
1
6-11
0
0
0
0
1
1
0
0
0
r<*-ir
0
0
0
0
i
0
0
0
0
Age
-JttO
1
0
0
0
9
0
0
2
3
31-50
0
0
0
1
9
1
2
11
6
51 +
0
0
0
0
4
1
6
6
2
~w
0
0
0
0
0
0
0
i
0
Total
1
0
0
1
25
3
8
20
12
Other Conditions
Question 21a,b,c,d: Have you or anyone In this household ever seen a doctor
for any of these types of conditions? Who is that? Which of these
conditions (do you/does ) have? How old (were you/was )
when the (condition) first appeared?
Condi tion
Skin cancer
Leukemia
Hodgkin's disease
Other cancers
Arthritis
Diabetes
Anemia
Immunologlcal
disorder
Rheumatic fever
Serum hepatltis-B
Infectious hep-A
Infectious
mononucleosis
Other chronic cond.
TPi>
0
0
0
0
1
0
1
0
0
0
1
0
5
6-11
0
0
0
0
1
1
0
0
3
1
5
0
3
12-17
0
0
0
0
1
0
0
0
0
0
2
1
1
Age
18-30
3
0
0
1
7
1
1
0
0
0
1
0
3
31-50
7
0
0
2
15
0
0
0
0
1
2
1
4
51 +
7
0
0
2
31
5
1
0
0
0
0
1
7
NR
0
0
0
0
5
0
2
0
1
0
0
0
2
Total
17
0
0
5
60
7
5
0
4
2
11
3
25
Blood Transfusions
Question 23a,b: Have
transfusion?
Blood transfusion
Number
Percent
you or has anyone In this household ever had
Yes No Don't know NR
39 372 2 18
9 86 1 4
a blood
Total
431
100
Howdialysls
Question 24a,b: Have you or anyone in this household ever been on a kidney
machine or hemodlalysis?
Yes
Hemodlalysis
~fio~
TotaT
Number
Percent
2
1
411
95
18
4
431
100
Tuberculosis
Question 25a,b: Have you or anyone in this household ever been in close
contact with a person who had TB? Who Is that?
Contact with TB
Yes
No
NR
Total
Number
Percent
12
3
401
93
18
4
431
100
Sinking
Question 26a,b: Do you or does anyone In this household smoke cigarettes
regularly? Who Is that?
SmoVe YesHo N~R~~Total
Number
Percent
60
14
353
82
18
4
431
100
-------
TABLE 5.6. SAMPLES COLLECTED FOR HEALTH WATCH ACTIVITIES
Data
collection
period
1980
001
002
003
004
005
006
007
008
009
010
on
012
013
014
015
016
017
018
019
020
021
022
023
024
025
026
1981
101
102
103
104
105
106
107
108
109
110
111
112
113
114
Blood Fecal Health Skin Activity Illness
specimens specimens diaries tests diaries specimens
318 265
342
22 366
365
34 351
343
50 332 3
337
363 33
24 392
385
11 381
358
45 346
287 365 187
30 359
178
-------
TABLE 5.6. (CONT'D)
Data
collection Blood
period specimens
1981 (Continued)
115
116
117
118
119
120
121
122
123
124
125
126
1982
201 330
202
203
204
205
206
207
208
209
210
211
212 310
213
214
215
216
217
218
219
220
221
222a
223
224
225 268
226
Fecal
specimens
11
43
108
128
127
123
119
125
Health Skin
diaries tests
340 '
324
313
319
348
344
332
339
338
326
335
345
349
355
348
350
355
349
342
329
336
338
340
348
342
174
175
175
180 245
Activity Illness
diaries specimens
11
194
7
156
2
4
5
1
6
261 6
1
3
15
15
8
4
5
332 15
7
a Onset of sentinel families program.
179
-------
of the population providing all six requested specimens. At least one
activity diary was obtained from 90% of the households in 1982. Tables 5.7
through 5.9 summarize compliance information by zone for blood samples,
fecal specimens, and activity records.
Health Diary Data
Tables 5.10 through 5.13 summarize the illness information that has
been obtained from the health diaries. Table 5.10 lists the incidence
rates for all self-reported illnesses (excluding trauma and surgery) by
data collection period and year of collection. Table 5.11 compares the
incidence rates for specific types of illness in Zone 1 to the rates
observed in the lower exposure sampling zones. Prevalence information is
presented in Tables 5.12 and 5.13.
Illness Specimens
Examination of the results from the throat swab illness specimens
collected over the period from January to December 1982 revealed some
anomalies (Table 5.14). All throat swabs are examined for Group A
Streptococci by the fluorescent antibody technique and by isolation and
identification of 6-hemolytic colonies on sheep blood agar. A MacConkey
agar plate also is inoculated to detect unusual levels of enteric
organisms. Table 5.15 (Youmans et al ., 1980) summarizes types of
microorganisms found in the normal human oropharynx. Various members of
the Enterobacteriaceae or Pseudomonas occasionally are found in small
numbers from oropharyngeal swabs of healthy humans. However, heavy
colonization of the upper respiratory tract by these organisms, as
discussed in the section that follows, is a situation that occurs under
unusual circumstances. As can be seen in Table 5.14, levels of enteric
Gram-negative organisms similar to those observed with the fecal specimens
were first observed in July. All six throat swabs in July and four of the
five in August that yielded unusual levels of the enteric flora were from
four different members of the same family (210). The Achromobacter
xylosoxidans (H) in August occurred in a different household (403). Five
of the six unusual isolations in September occurred in new individuals
(four in household 557 and one in 509). The two unusual isolations in
October occurred in new individuals (in Households 447 and 533).
In an attempt to characterize this phenomenon, 23 throat swabs were
obtained from three groups of well participants in mid-September: Hancock
farm residents and workers, Wilson residents (Zone 4), and distant rural
residents (Zone 5). Table 5.16 shows that while the Hancock farm sample
had a higher recovery rate (3/7=43%), enteric Gram-negative bacteria were
also recovered from the well participants in Wilson (1/8) and Zone 5 (2/8).
180
-------
TABLE 5.7. LHES BLOOD DONOR STATUS FOR PARTICIPANTS CURRENTLY IN STUDY
(December 1, 1982)
Zone
Number of
participants
Number given
all 5 samples
Number given
1-5 samples
Number given
0 samples
1
2
3
4
5
6
50
85
27
84
92
9
36 (72%) 50
46
14
55
54%
52%
65%
82
26
76
74 (80%) 89
2 (22%) 5
(100%)
(96%)
96%
(90%)
(97%)
(56%)
0
3
1
8
3
4
(4%)
(4%)
(10%)
(3%)
(44%)
TOTAL
347
227 (65%)
328 (95%)
19 (5%)
181
-------
TABLE 5.8. SUMMARY OF FECAL DONOR INFORMATION FOR PARTICIPANTS DURING 1982
(December 1, 1982)
Households
Total Number of
Zone possible donors
1
2
3
4
5
6
TOTAL
22
29
9
32
31
4
127
18 (82?)
20 (69%)
7 (78$)
24 (75$)
23 (74$)
2 (50$)
94 (74$)
Al 1 participants
Total Number of
possible donors
50
85
27
84
92
9
347
24 (48$)
37 (44$)
10 (37$)
38 (45$)
38 (41$)
3 (33$)
150 (43$)
Number given
all 6 samples
19 (38$)
16 (19$)
4 (15$)
13 (15$)
19 (21$)
1. (11$)
72 (21$)
Adults
Total Number of
possible donors
37
53
21
53
61
6
231
20 (54$)
20 (38$)
7 (33$)
24 (45$)
26 (43$)
2 (33$)
99 (43$)
Chi Idren
Number given
al 1 6 samples
16 (43$)
8 (15$)
2 (10$)
7 (13$)
13 (21$)
1 (17$)
47 (20$)
Total Number of
possible donors
13
32
6
31
31
3
116
4 (31$)
17 (53$)
3 (50$)
14 (45$)
12 (39$)
1 (33$)
51 (44$)
Number given
all 6 samples
3 (23$)
8 (25$)
2 (33$)
6 (19$)
6 (19$)
0
25 (22$)
00
PO
-------
TABLE 5.9. ACTIVITY DIARY COMPLIANCE FOR CURRENT POPULATION
(December 1, 1982)
Households
Zone
1
2
3
4
5
6
TOTAL
Total
possible
22
29
9
32
31
4
127
Number given
1-3 diaries
20 (91$)
28 (97$)
8 (89$)
27 (84$)
29 (94$)
2 (50$)
114 (90$)
Number given
all 3 diaries
11 (50$)
8 (28$)
4 (44$)
13 (41$)
15 (48$)
1 (25$)
52 (41$)
Number given
0 diaries
2 (9$)
1 (3$)
1 (11$)
5 (16$)
2 (6$)
2 (50$)
13 (10$)
Total
pos s i b 1 e
50
85
27
84
92
9
347
Participants
Number given
1-3 diaries
48 (96$)
83 (98$)
23 (85$)
64 (76$)
85 (92$)
7 (78$)
310 (89$)
Number given
al 1 3 diaries
25 (50$)
20 (24$)
9 (33$)
19 (23$)
24 (26$)
3 (33$)
100 (29$)
Number given
0 diaries
2 (4$)
2 (2$)
4 (15$)
20 (24$)
7 (8$)
2 (22$)
37 (11$)
i— •
00
co
-------
TABLE 5.10. COMPARISON OF TOTAL ACUTE ILLNESS INCIDENCE RATES
FOR FIRST THREE YEARS OF STUDY
(Number of New Illnesses/1000 Persondays of Observation)
Data collection
period
01
02
03
04
05
06
07
08
09
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
1980
(001-026)
7.10
6.09
5.28
4.88
5.24
4.95
3.82
1981
(101-126)
9.50
6.18
8.75
4.62
4.23
3.17
3.81
2.96
0.86
4.41
4.52
1982
(201-226)
10.71
9.22
8.24
10.99
5.98
10.88
9.73
10.62
10.39
7.66
4.36
2.70
3.66
7.14
9.07
6.42
7.77
9.43
14.04
12.99
10.90
10.80
11.11
17.89
8.07
184
-------
TABLE 5.11. INCIDENCE OF SELF-REPORTED ACUTE ILLNESSES IN STUDY POPULATION
(Number of New Illnesses/1000 Persondays of Observation)
Data
col lection
period
1980
014
015
016
017
018
019
020
1981
108
109
11V
12
13
14
15
17
•- 18
oo ,982
201
202
203
285
206
207
208
209
210
211
212
213
i\A5
216
217
218
219
220
222*
223
224
225
Total
Zone 1
11.90
4.29
3.11
5.95
0
8.31
4.87
11.20
4.04
ifcfl
3.17
1.58
0
0
7.76
3.32
9.52
Ills
11*80
13.22
1.56
7.46
1.49
7.60
2' 87
it: 12
1.68
7.02
5.01
11.94
10.51
\'M
7.61
18.78
8.98
1 Iness
Zones 2-6
6.43
6.38
5.60
4.71
5.87
4.45
3.66
9.25
6.52
8.94
3.69
4.40
3.40
3.45
3. 12
1.00
4.99
11.17
10.07
8.03
"$'.92
10*74
9.11
12.08
10.87
8.66
3.79
2.63
3.80
2:?8
7.21
7.89
10.14
14.39
13.40
11: ?i
12*,41
17.55
7.74
Respiratory
Zone 1
5.10
1.43
1.55
2.98
0
1.66
3.25
4.20
1.35
g.98
0
0
3.14
1.79
0
0
7.76
3.32
4.76
fcS
13*22
1*56
1.49
1.49
4.56
2*87
i79
1.68
3.51
0
5.97
4.50
$.56
6.09
7.82
5.99
1 1 Iness
Zones 2-6
2.38
1.14
2.24
2.59
1.64
1.98
2.19
6.31
3.04
0'.21
0*73
0.68
8.69
.24
0.33
2.10
8.74
5.52
5.27
Ml
1:13
7.05
4.94
3.13
1.35
Q\'.zi
3:?9
2*77
3.42
3.20
10.10
6.95
l:?i
7l33
13.46
3.32
Gl
Zone 1
1.70
0
0
0
0
1.66
0
4.20
0
1:8
1.59
0
1.57
0
0
0
8
3.17
8
a*37
0
2.99
0
0
o'53
f:2g
0*
3.51
3.34
2.99
1.50
a*29
0
3.13
1.50
I 1 Iness
Zones 2-6
0.71
1.82
0.22
0.94
2.82
1.24
0.24
0.84
1.96
Hi
1*71
0.45
1.38
1*68
0
1.05
U68
1.51
I'.ll
2*30
2*77
2.96
3.61
1.35
8:7769
i:I2
2*77
2.63
4.27
2.78
4.47
4.02
6.29
2.26
3.51
2.21
Other
Zone 1
1.70
1.43
0
1.49
0
1.66
1.62
2.80
1.35
3*f?
0*
0
1.57
0
0
0
8
0
8
1.69
0
0
0
0
3.04
8
l.*6370
0
0
0
0
0
8
1.52
4.69
0
acute
Zones 2-6
1.19
1.14
0.90
0.47
0.23
0.49
0.24
0.63
0.43
8:8
0.49
0.23
0.23
0.48
0.33
0.53
1.44
0.50
1.21
0.25
1.34
0.26
0.75
1.24
0.48
0.27
O-2,6!
1:8
0.28
0.79
1.33
0.26
1.49
Olil
1.69
0.59
1.11
Beginning of sentinel families program.
-------
TABLE 5.12. COMPARISON OF TOTAL ACUTE ILLNESS PREVALENCE RATES
FOR FIRST THREE YEARS OF STUDY
(Number of Persondays of Illness/1000 Persondays of Observation)
Data collection
period
01
02
03
04
05
06
07
08
09
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
1980
(001-026)
13.16
19.84
16.24
18.11
23.66
24.74
18.65
1981
(101-126)
43.68
31.07
32.91
12.24
12.49
9.90
20.68
15.86
1.73
18.58
24.01
1982
(201-226)
139.62
34.79
42.70
51.56
32.03
61.13
64.77
49.61
73.34
37.46
23.88
13.26
20.44
31.35
54.86
23.31
56.70
35.20
96.09
60.05
47.96
55.26
79.84
98.38
38.35
186
-------
TABLE 5.13. PREVALANCE OF SELF-REPORTED ACUTE ILLNESSES IN STUDY POPULATION
(Number of Persondays of Illness/1000 Persondays of Observation)
oo
Data
col lection
period
1980
014
015
016
017
018
019
020
1981
108
109
110
111
Hi
114
115
117
118
119
1982
201
202
203
204
205
206
207
208
209
210
211
212
213
214
215
216
217
218
219
220
221
222*
223
224
225
Total
Zone 1
30.61
25.71
10.87
11.90
0
13.29
38.96
37.82
12.13
17.86
57.45
8:3?
26.69
0
0
0
19.23
41.93
14.95
44.44
68.03
22.22
38.79
69.02
6.23
17.91
19.32
41.03
10.74
31.56
31.59
73.48
15.08
21.05
20.03
41.79
51.05
13.16
31.91
63.93
84.51
20.96
II Iness
Zones 2-6
10.71
18.90
17.02
19.09
26.55
26.45
15.60
44.56
34.13
35.12
5.53
'i:li
\9\80
17.99
2.00
21.01
24.77
154.89
37.65
42.42
49.23
33.56
64.68
64.01
56.62
82.51
40.39
20.82
13.69
18.48
31.31
51.78
24.67
67.04
37.63
105.28
61.54
53.28
64.04
85.73
103.57
44.78
Respiratory
Zone 1
13.61
0
7.76
11.90
0
6.64
16.23
22.41
4.04
0
0
8
10.99
0
0
0
19.23
41.93
14.95
30.16
68.03
22.22
18.55
69.02
6.23
5.97
19.32
16.72
6.13
31.56
0
22.36
15.08
8.77
0
25.37
33.03
0
31.91
53.27
42.25
13.47
1 1 Iness
Zones 2-6
5.71
5.69
8.28
10.14
11.75
10.87
9.02
31.32
18.48
21.16
0.69
0:§6
4!37
1.68
0.33
7.88
7.43
97.84
22.30
32.63
32.58
25.91
34.35
31.75
38.00
32.11
20.92
8.11
0
8.86
12.83
16.12
10.53
43.38
14.68
87.35
36.23
37.20
35.45
59.22
88.94
23.22
61
Zone 1
5.10
0
0
0
0
3.32
0
7.00
0
2.98
12.42
6.35
4.71
0
0
0
0
0
0
9.52
0
0
6.75
0
0
5.97
0
0
4.60
0
11.51
4.79
0
12.28
13.36
4.48
6.01
13.16
0
0
18.78
4.49
1 1 Iness
Zones 2-6
2.38
4.33
0.22
2.59
9.16
5.68
0.49
3.57
8.04
4.58
1.61
6.84
2.94
9.44
6.95
0
3.15
9.91
0
4.56
4.77
9.41
1.97
6.71
2.60
7.55
8.65
12.74
9.46
1.84
1.01
10.78
14.27
5.82
10.52
9.34
13.13
15.14
8.54
25.73
8.46
12.29
11.06
Other
Zone 1
5.10
5.71
0
0
0
0
22.73
8.40
5.39
14.88
26.40
0
0
10.99
0
0
0
0
0
0
0
0
0
5.06
0
0
0
0
24.32
0
0
16.48
19.17
0
0
0
0
0
0
0
10.65
10.95
0
acute
Zones 2-6
0.95
3.42
4.70
1.89
0.47
2.47
3.41
3.36
1.30
3.05
2.77
1.95
0.91
1.84
3.36
1.67
5.51
3.85
0
5.70
3.01
5.07
0.74
8.86
0.78
3.77
8.89
1.92
1.89
1.32
4.56
6.16
15.06
1.11
4.21
6.94
3.03
9.18
7.29
2.29
13.54
2.34
4.42
Beginning of sentinel families program.
-------
TABLE 5.14. SUMMARY OF CLINICAL BACTERIOLOGY RESULTS FOR
ILLNESS SPECIMEN THROAT SWABS
1982
month
January
February
March
Apri 1
May
June
July
August
September
October
November
December
Percent
Normal flora
100 (6)
100 (5)
100 (6)
100 (1)
-
86 (6)
30 (3)
50 (5)
58 (18)
79 (11)
75 (9)
87 (13)
(number) positive
Group A strep
0 (0)
0 (0)
0 (0)
0 (0)
-
14 (1 low level by FA)
10 (1 low level by FA)
0 (0)
23 (4 low levels
by FA, 3 high levels
plate and FA)
7 (1 low level by FA)
25 (3)
13 (2)
Other
0 (0)
0 (0)
0 (0)
0 M
_
0 (0)
60 (6J)
50 (5b)
19 (6C)
14 (2d)
0 JO)
0 (0)
a E. coli (M), E. cloacae (M), K. oxytoca (M); E. col i (H), E. cloacae
(H); E. coli (H), E. cloacae (M); two with E. coli (M), E. cloacae (M);
E. cloacae (M)
b K. oxytoca (M); E. coli (M), E. cloacae (M), K. oxytoca (M); K. oxytoca
(M), Pseudomonas sp. (M); Achromobacter xylosoxidans (H); E. cloacae (M)
c E. cloacae (H); Gr. A Strep (H), K. oxytoca (H), Pseudomonas sp. (H); K.
pneumoniae (H); Gr. A. Strep (H), CDC Gr. V E-2 (H); E. cloacae (H); S.
liquefaciens (M)
d E. cloacae (H); E. cloacae (M)
188
-------
TABLE 5.15. MICROORGANISMS FOUND IN THE OROPHARYNX
Range of prevalence
Microorganisms _ .
Staphylococcus aureus 35-40
Staphylococcus epidermidis 30-70
Aerobic corynebacteria (diphtheroids) 50-90
Streptococcus pyogenes (Group A) 0-9
Streptococcus pneumoniae 0-50
Alpha- and nonhemolytic streptococci 25-99
Branhamella catarrhalis 10-97
Neisseria meningitidis 0-15
Haemophilus influenzae 5-20
Haemophilus parainfluenzae 20-35
Gram-negative bacteria, e.g.
Klebsiella pneumoniae _ Uncommon
Youmans et al . , 1980
189
-------
TABLE 5.16. SUMMARY OF CLINICAL BACTERIOLOGY RESULTS FOR 23 REQUESTED
THROAT SWABS FROM WELL PARTICIPANTS: SEPTEMBER 1982
Group of well
participants
Normal Positive for enteric
flora Gram-negative bacteria
7 Hancock farm residents
and workers
8 Wilson (Zone 4)
residents
8 distant rural (Zone 5)
residents
4 3 (43%)
- C. diversus-levinea (H),
E. aerogenes (H)
- E. coli (M)
- E. cloacae (H),
E. agglomerans (M)
7 1 (13%)
- E. agglomerans (M)
6 2 (25%)
- E. cloacae (H)
- Acinetobacter calcoaceticus
var. anitratus (H), K.
oxytoca (H)
190
-------
Hence, the phenomenon of moderate and heavy levels of enteric Gram-negative
bacteria in the upper respiratory tract appears to have been prevalent
throughout the study area, in both ill and well throats.
Clinical Bacteriology
Data-
Routine fecal specimens from health watch participants in the second
year of monitoring were analyzed using procedures summarized in Figures
4.13 and 4.14 (Methods Section). In all cases, the organisms isolated are
reported as a function of the level of growth (very light to heavy)
observed on primary plating media. Results from 106 specimens collected in
the last preirrigation period (Period 201) are shown in Table 5.17.
Results from 378 fecal specimens collected from participants during all
periods prior to any irrigation are shown for comparison in Table 5.18.
Likewise, summary data from 607 fecal specimens collected during all post-
irrigation periods through 219 are given in Table 5.19.
Three indices of an infection event, as determined by the
bacteriological analyses of fecal specimens, were utilized. An infection
event was defined by any one of the three following criteria:
1) isolation of a major enteric bacterial pathogen (i.e., specific
procedures were designed to attempt the isolation and
identification of any Salmonella species, Shi gel la species,
Campylobacter fetus subsp. jejuni, or Yersim'a enterocolitica);
2) marked elevation (to heavy level) of a possible significant
organism (i.e., API Group I, Candida albicans, Chromobacterium,
Citrobacter, Klebsiella, Morganella, Proteus, Providencia,
Serratia, and Staphylococcus aureus);
3) isolation at moderate or heavy levels of selected organisms
uncommon in feces but prevalent in effluent (i.e., Aeromonas
hydrophila and the fluorescent Pseudomonas group: _P. aeruginosa,
P_. fluorescens, and P_. putida).
An infection event is not equated with disease. Infection is used in
the broader sense of entrance and multiplication of the organism in the
body. Disease is indicated by detectable alterations in normal tissue
functions (i.e., the overt clinical manifestations of illness). The
distinction between the two terms is important. Generally, infection is an
essential prerequisite to production of microbial diseases. Important
exceptions would be diseases associated with ingestion of preformed
microbial toxins (e.g., staphylococcal food intoxication, botulism in
adults). However, even overt microbial pathogens (bacterial, fungal, or
191
-------
TABLE 5.17.
ro
ORGANISMS ISOLATED FROM FECAL SPECIMENS IN SAMPLING PERIOD 201
(106 Specimens)3
Quantitation of growth^ [percent (number) positive]
Organism
Candida albicans
Citrobacter freundii
Citrobacter spp.
Enterobacter aerogenes
Enterobacter agglomerans
Enterobacter cloacae
Enterobacter spp.
Escherichia coli
Fluorescent Pseudomonas gr.
Klebsiella oxytoca
Klebsiella pneumoniae
Proteus mirabilis
Proteus rettgeri
Proteus vulgaris
Staphylococcus aureus
a 106 of 107 routine fecal
b quantisation of growth on
Heavy
_
-
-
0.9(1)
-
-
38.7(41)
-
-
0.9(1)
-
-
-
specimens received
primary culture plates
Moderate
3.8(4)
-
-
-
-
2.8(3)
-
38.7(41)
0.9(1)
-
4.7(5)
0.9(1)
0.9(1)
-
3.8(4)
Light
4.7(5)
1.9(2)
-
0.9(1)
-
2.8(3)
-
15.1(16)
0.9(1)
2.8(3)
2.8(3)
-
-
0.9(1)
5.7(6)
Very light
7.5(8)
3.8(4)
0.9(1)
1.9(2)
0.9(1)
9.4(10)
0.9(1)
6.6(7)
8.5(9)
3.8(4)
4.7(5)
1.9(2)
-
0.9(1)
2.8(3)
Total
16.0(17)
5.7(6)
0.9(1)
3.8(4)
0.9(1)
15.6(16)
0.9(1)
99.1(105)
10.4(11)
6.6(7)
13.2(14)
2.8(3)
0.9(1)
1.9(2)
12.3(13)
Heavy -
Moderate -
Light -
Very light -
growth on three or all quadrants
growth on first two quadrants
growth on first quadrant
one to ten colonies on plate
-------
TABLE 5.18. ORGANISMS ISOLATED FROM FECAL SPECIMENS DURING ALL PREIRRIGATION PERIODS
(378 Specimens)3
10
CO
Quantitation of growthb [percent (number) positive]
Organism
Aeromonas hydrophila
Candida albicansc
Citrobacter di versus
Citrobacter freundii
Citrobacter spp.
Enterobacter aerogenes
Enterobacter agglomerans
Enterobacter cloacae
Enterobacter sakazakii
Enterobacter spp.
Escherichia coli
Hafnia alvei
Klebsiella oxytoca
Klebsiella pneumoniae
Klebsiella spp.
Morganella morganii
Proteus mirabilis
Proteus rettgeri
Proteus vulgaris
Providencia alcalifaciens
Fluorescent Pseudomonas gr.
Pseudomonas spp.
Serratia liquefaciens
Serratia odorifera
Staphylococcus aureus
Staphylococcus epidermidis
Yersinia enterocolitica
a from Sampling Periods 015,
b quantisation of growth on i
Heavy
-
-
0.5(2)
-
0.3(1)
-
0.3(1)
-
-
39.7(150)
-
-
0.8(3)
-
-
-
-
-
-
-
-
-
-
0.3(1)
-
3
017, 019, 108, 110, 112
Drimary culture plates
Moderate
1.9(6)
-
1.6(6)
-
0.3(1)
-
2.6(10)
0.3(1)
-
42.1(159)
-
0.5(2)
3.4(13)
-
-
0.3(1)
0.5(2)
-
0.3(1)
0.3(1)
-
-
-
2.9(11)
-
positive by
, 114, 117,
Light
0.3(1)
6.2(20)
-
2.9(11)
-
0.8(3)
-
4.0(15)
0.3(1)
-
13.0(49)
0.8(3)
4.0(15)
7.1(27)
0.5(2)
0.5(2)
0.3(1)
-
0.3(1)
0.3(1)
1.3 5
0.3(1)
0.3(1)
0.5(2)
18.0(68)
0.3(1)
enrichment
118 and 201
Very light
0.3(1)
11.9(38)
0.5(2)
4.5(17)
0.5(2)
1-1(4)
0.3(1)
5.8(22)
0.8(3)
0.3(1)
3.4(13)
-
2.9(11)
8.7(33)
-
0.5(2)
0.5(2)
-
0.3(1)
0.5(2)
4.5(17)
-
-
-
6.6(25)
0.8(3)
onl^y
Total
0.5(2)
20.0(64)
0.5(2)
9.5(36)
0.5(2)
2.4(9)
0.3(1)
12.7(48)
1.3(5)
0.3(1)
98.1(371)
0.8(3)
7.4(28)
20.1(76)
0.5(2)
1-1(4)
1.1(4)
0.5(2)
0.5(2)
1-1(4)
6.1(23)
0.3(1)
0.3(1)
0.5(2)
27.8(105)
1-1(4)
0.8(3)
Heavy - growth on three or all quadrants Light - growth on first quadrant
Moderate - growth on first two quadrants Very light - one to ten colonies on plate
based on 320 specimens (procedures for isolation of C. albicans began in Sampling Period 019)
-------
TABLE 5.19. ORGANISMS ISOLATED FROM FECAL SPECIMENS DURING ALL POST-IRRIGATION PERIODS IN 1982
(607 Specimens)3
10
Quantitation of growthb [percent (number) positive]
Organism
API Group I
Aeromonas hydrophila
Candida albicans
Chromobacterium
Citrobacter amalonaticus
Citrobacter di versus-! evinea
Citrobacter freundii
Citrobacter spp.
Enterobacter aerogenes
Enterobacter agglomerans
Enterobacter cloacae
Enterobacter sakazakii
Escherichia coli
Hafnia alvei
Klebsiella oxytoca
Klebsiella pneumoniae
Morganella morganii
Proteus mirabilis
Proteus vulgaris
Providencia alcalifaciens
Fluorescent Pseudomonas gr.
Pseudomonas aeruginosa
Pseudomonas spp.
Salmonella spp.
Serratia font i col a
Serratia marcescens
Serratia odorifera
Staphylococcus aureus
a from Sampling Periods 205,
b quantitation of growth on
Heavy
0.2(1)
_
0.2(1)
-
-
-
-
-
0.7(4)
0.2(1)
2.0(12)
-
36.6(222)
0.2(1)
0.2(1)
4.9(30)
'-
-
-
-
0.2(1)
-
-
0.2(1)
0.2(1)
-
-
207, 212, 216 and 219
Drimary culture plates
Moderate
—
_
0.5(3)
0.2(1)
0.8(5)
1.2(7)
-
1.0(6)
-
3.5(21)
0.2(1)
45.8(278)
0.2(1)
2.1(13)
8.1(49)
-
0.5(3)
-
0.2(1)
1.2(7)
-
-
-
-
-
-
1.8J11)
Li ght
—
0.2(1)
2.6(16)
0.5(3)
0.2(1)
0.2(1)
1.3(8)
0.2(1)
1.8(11)
0.3(2)
4.3(26)
0.8(5)
13.3(81)
0.2(1)
3.5(21)
9.1(55)
0.3 2
0.3(2
0.2(1
-
1.8(11)
0.7(4J
0.2(1)
-
-
0.3(2)
0.2(1)
6.6(40)
•Very light
0.2(1)
0.2(1)
10.7(65)
-
-
-
0.8(5)
-
0.2(1)
-
1.8(11)
-
2.5(15)
0.2(1)
0.8(5)
3.1(19)
0.5(3)
-
-
-
0.5(3)
-
-
-
-
-
-
7.2(44)
Total
0.3(2)
0.3 2)
14.0(85)
0.7(4J
0.2(1)
1.0(6)
3.3(20)
0.2(1)
3.6(22)
0.5(3)
11.5(70)
1.0(6)
98.2(596)
.0.7(4)
6.6(40)
25.2(153)
0.8(5)
0.8(5)
0.2(1)
0.2(1)
3.5(21)
0.7(4)
0.2(1)
0.2(1)
0.2 1
0.3(2)
0.2(1)
15.7(95)
Heavy - growth on three or all quadrants
Moderate - growth on first two quadrants
Light - growth on first quadrant
Very light - one to ten colonies on plate
-------
viral) may produce inapparent or subclinical infections. Likewise, an
immune response can occur without overt clinical disease.
The organisms of Category I, overt enteric bacterial pathogens, are of
major clinical significance because they often are associated with disease
and even inapparent or subclinical infections may provide a source of
infection and disease to others. Organisms in Categories 2 and 3 may be
associated with enteric disease if isolated in large numbers from stools.
Overgrowth in the intestine by some of the organisms may be associated with
a compromised state or with intensive use of antibiotics. The latter
possibility can be recognized by flagging specimens of individuals who have
recently been or who are on antimicrobial therapy. If an infection event,
as defined by Criterion 2 or 3, occurs in such individuals, the organism
will be tested for susceptibility to antimicrobials in an attempt to rule
out overgrowth of a resistant type as opposed to a new infection event.
The clinical significance of organisms in Categories 2 and 3 is
questionable and should be treated as such. However, increased numbers and
frequency of isolations of such organisms previously have been associated
with environmental exposure. In this study they provide an additional, as
well as sensitive and rapid, indicator of possible exposure to wastewater
associated with irrigation operations.
The summary data for all preirrigation (Table 5.18) and post-
irrigation (Table 5.19) periods are compared in Table 5.20 for the three
suggested indices of infection events„ The major differences are the
increased frequency of K^. pneumoniae (Category 2) and the Fluorescent
Pseudomonas group (Category 3) in the post-irrigation periods. K_,
pneumoniae was present at the heavy level in 0.8% of specimens Tn
preirrigation periods compared to 4.9% of specimens for all post-irrigation
periods. Interestingly, the percentages of heavy level K^ pneumoniae in
preirrigation Period 201 was 0.9%, in post-irrigation Periods 205 and 207
0.8% and 0.9%, respectively, but jumped to 7.9% in Period 212, 7.5% in
Period 216, and 7.2% in Period 219.
Patterns of Infection--
The definition for a new infection event for organisms in the three
categories is as follows. A "new infection" event for organisms in the
first category would require isolation at any level or by enrichment from
an individual whose previous specimen was negative for the respective
pathogen. For organisms in the second category, isolation would have to be
at the heavy level and the previous specimen negative to light. For
organisms in the third category, isolation at a moderate or heavy level and
a previous negative to light isolation would be required.
Investigation of the ]<. pneumoniae isolations in Category 2 for "new
infections" (Tables 5.21 and 5.22) suggests a possible infection episode
195
-------
TABLE 5.20.
COMPARISON OF CLINICAL BACTERIOLOGICAL ANALYSES OF FECAL SPECIMENS
BETWEEN PREIRRIGATION AND POST-IRRIGATION
Criterion
Prei rrigationa
Post-irrigation"
Isolation of major bacterial
enteric pathogen:
Campylobacter fetus
Salmonella spp.
Shigella spp.
Yersinia enterocolitica
Y. enterocolitica from 3
specimens (0.8%) by enrichment
only (3 different individuals,
two in Period 112 and one in
114)
Salmonella spp. (Group Cl) from 3
specimens (0.5%) by direct
plating (two different specimens
from same person in Periods 212
and 213 and one from his son in
214)
10
CTl
2. Marked elevation (to heavy Citrobacter freundii 0.5%
level) of a possibly sig- Klebsiella pneumoniae 0.8%
nificant organism Staphylococcus aureus 0.3%
3. Isolation at moderate or
heavy level of selected
organisms uncommon in feces
but prevalent in effluent:
Aeromonas hydrophila
Fluorescent Pseudomonas gr.c
Fluorescent
0.3% (1 M)
2
3
(1)
Pseudomonas gr.
API Group I 0.2%
Candida albicans 0.2%
Klebsiella oxytoca 0.2%
Klebsiella pneumoniae 4.9%
Serratia fonticola 0.2%
Fluorescent Pseudomonas gr.
1.3% (1 H, 7 M)
i)
(l)
(30)
(1)
a 378 specimens from Periods 015, 017, 019, 108, 110, 112, 114, 117, 118 and 201
b 609 specimens (including 2 illness specimens) from Periods 205, 207, 212, 213, 214, 216, and 219
c P. aeruginosa, P. fluorescens, and P. putida
-------
TABLE 5.21. POSSIBLE EPISODE OF BACTERIAL INFECTION IN JUNE 1982
DETERMINED FROM SCHEDULED FECAL SPECIMENS
Scheduled fecal collection period
Donor Age 201 205 207 212
jm in 1982 Jan 4-8 Mar 1-5 Mar 29-Apr 2 Jun 7-11
New Klebsiella pneumoniae Infections (H) In 212
21611 <2 H
21915 8 - - - H
23111 14 NS NS H
40411 8 NS VL - H
53101 74 NS - - H
53311 13 NS - NS H
60111 (HF) <1 NS L H
Other Klebsiella pneumoniae Infections (H) in 212
23113
40702 (ND)
42801
50701
6
36
74
73
NS
NS
NS
NS
NS
NS
M
NS
NS
NS
H
NS
H
H*
H
H
NS no specimen provided
organism not isolated
HF resides on or frequents the Hancock farm
* illness specimen in Period 213
ND nondonor
Quantisation of growth on primary culture plates;
H heavy—growth on three or all quadrants
M moderate—growth on first two quadrants
L light—growth on first quadrant
VL very light—one to ten colonies on plate
197
-------
TABLE 5.22. POSSIBLE EPISODE OF BACTERIAL INFECTION IN AUGUST AND SEPTEMBER
1982 DETERMINED FROM SCHEDULED FECAL SPECIMENS
Scheduled fecal collection period
Donor Age 212 216 219
ID in 1982 Jun 7-11 Aug 9-13 Sep 13-17
New Klebsiella pneumoniae (or K. oxytoca) Infections
H H
H
H H
H M
H H
H
H -)
H
M - H
M - H
H - H
L VL H
M H L
NS H E
NS H H
Fluorescent Pseudomonas group Infections
205 207
22102
31502
32413
41302
44502
45311
(54502
22511
22512
41501
50701
54202
Other Klebsiella
12202
40402
40403
67
67
<2
57
68
10
54
17
14
74
73
67
pneumoniae infections
29
28
59
10801
11402
11902
41601
45201
73 - M
62
55
67
Elderly
NS
-
_
VL
NS
M
L
M
-
L
_
M
_
M
M
NS no specimen provided
organism not isolated
Quantitation of growth on primary culture plates;
H heavy—growth on three or all quadrants
M moderate—growth on first two quadrants
L light—growth on first quadrant
VL very light—one to ten colonies on plate
E positive by enrichment only
198
-------
beginning in Period 212. Seven j<. pneumoniae "new infections" (defined as
discussed previously) were noted in Period 212 (Table 5.21). An additional
three "new infections" may have occurred, but previous specimens were not
available. As shown in Table 5.229 seven "new infections" occurred in
Period 216 and five in Period 219,, Interestingly, 75% (15/20) of the K_.
pneumoniae "new and other infections" in these periods occurred in
individuals 54 or older or less than 2 years of age. All of the five
fluorescent Pseudomonas group infections occurred in individuals 55 or
older.
Interpretation of Fecal and Illness Specimen Bacterial Data--
As indicated by Table 5.15, the oropharynx of healthy humans is not
commonly assumed to be an environment favoring growth or persistence of
Gram-negative enteric flora. Johanson et al . (1969) examined the
prevalence (in terms of presence or absence only) to Gram-negative bacilli
in the oropharyngeal flora of five groups of adult subjects: nonhospital-
associated normal subjects, hospital-associated normal subjects, physically
normal hospitalized patients, moderately ill hospitalized patients, and
moribund patients. Only 2% of normal subjects, whether nonhospital
associated or hospital associated, yielded throat cultures containing Gram-
negative bacilli. Essentially the same level of positive cultures (0 to
2%) was observed with patients on the psychiatry service. In contrast, the
levels of positive cultures in a single culture survey of moderately ill
and moribund patients was 16% and 57% increasing to 22% and 63%,
respectively, in a multiculture survey. Administration of antibiotics had
no significant effect on the prevalence of Gram-negative bacilli in the
oropharyngeal flora of the physically ill patients.
Ramirez-Rhonda et al. (1980) observed in a Puerto Rican hospital that
Gram-negative organisms (presence/absence only) were found in the
oropharynx of 14% of "normal" adult outpatients. Colonization of the
oropharynx of hospital staff with Gram-negative organisms ranged from 12 to
18% in the absence of illness but increased to 38 to 60% in individuals
with upper respiratory illness (URI), presumably of viral origin. j£.
pneumoniae was the most frequent isolate, followed by JE. coli and
Enterobacter spp. The total numbers of all gram-negative bacilli per
milliliter of oropharyngeal fluid also was determined. Among hospital
staff with URI (151 subjects), the levels of these organisms in positive
individuals were <10 cfu (9%), 10 to 100 cfu (54%), 100 to 300 cfu (38%),
and >300 cfu (1%). The remarkable aspect of the results of illness
specimen throat swabs of some LHES participants during July to October
(Table 5.14) is not the mere presence of Gram-negative enterics, but the
unusually high levels of the organisms. Gram-negative enteric bacilli have
been observed occasionally before and after these dates at the VL or L
level, but not at the M or H levels. From preliminary studies, it would
199
-------
appear that isolation at the M or H level would require >105 to 10? cfu/mL
of the organisms (cf. Table 4.40). Such numbers would be inconsistent with
all but the 1% of subjects with URI in the study of Ramirez-Rhonda et al.
(1980) who may have had comparable levels of organisms (i.e., the >300
cfu/mL group).
Additional factors other than severity of illness in a hospital
environment and URI in normal subjects may influence pharyngeal carriage of
enteric Gram-negative organisms. The rates of carriage of Gram-negative
enterics increases dramatically with duration of hospitalization (e.g.,
Johanson et al., 1972; Haverkorn and Michel, 1980), although many patients
become colonized on the first day of admission. Different sites of
colonization by enteric Gram-negative organisms also have been compared.
Hart and Gibson (1982) examined 260 hospital patients who were involved in
an outbreak of carriage and infection due to gentamicin-resi stant
enterobacteria during a two-year period beginning in January 1979.
Klebsiella spp. were the most frequently isolated organisms followed by £.
coli, Citrobacter, and Enterobacter spp. Moribund patients were found to
more likely demonstrate carriage than less severely ill patients. The
intestinal tract was the most frequent site (of seven examined) of
gentamicin-resistant Klebsiella, as had been observed previously for less
resistant Klebsiella. Positive isolations from the intestine were followed
in decreasing frequency from the vagina, groin, mouth, umbilicus, axillae,
and nose. The studies of Phil pot and MacDonald (1980) suggested that
pharyngeal carriage rates of enteric Gram-negative bacilli may differ
substantially between different groups of normal individuals and challenged
the common assumption that a high rate of carriage of the organisms
exclusively is associated with hospitalization or debility. The prevalence
of all enteric Gram-negative bacilli (presence/absence) recovered from
throat swabs of healthy Australian adults (31 subjects), Malaysian adults
(25 subjects), and Malaysian children (25 subjects) were 9%, 36% and 4%,
respectively. The prevalence of the organisms (28% and 12%, respectively)
in Malaysian adults (25 subjects) and children (25 subjects) with sore
throats was not markedly different from that observed for the healthy
counterparts. It is interesting and perhaps relevant to the data of Table
5.14 that the investigators noted that "in each case the numbers of these
bacteria detected were not great." They suggested that the higher carriage
rate in Malaysian as opposed to Australian adults might be due to "food
preferences or other social habits."
The role of antibiotics as a predisposing factor for colonization of
the oropharynx by Gram-negative bacilli is a subject of some controversy.
For example, in the extensive studies of Johanson et al . (1969, 1972)
increased prevalence of the organisms in the oropharynx of most patients
was not correlated with antibiotic administration. In contrast, Haverkorn
and Michel (1979) noted isolation rates of Klebsiella from the throat and
200
-------
feces were significantly higher in patients who received antibiotic
treatment in the week before bacteriological examination than in patients
who did not. The major effect of antibiotics would be to reduce
susceptible components of the indigenous flora that normally play a role in
prevention of colonization of other organisms through "bacterial
interference." For example, pharyngeal colonization with
-------
(Tables 5.21 and 5.22) have been suggested at meetings of investigators in
the LHES or in personal conversations with the investigators. These
include:
1) antibiotic selection,
2) ingestion of organisms on garden vegetables,
3) exposure to Gram-negative bacteria associated with heavily
contaminated cotton,
4) wastewater irrigation operations,
5) fecal contamination of drinking water.
Antibiotic selection is an unlikely cause of the unusual isolations
from the fecal specimens (Table 5.22) since the isolations were observed
with routine, not illness, specimens. The throat swabs (Table 5.14) were
illness specimens (use of antibiotics uncertain). However, a number of
requested nonillness specimens (throat swabs) in a follow-up study (Table
5.16) also yielded increased oropharyngeal colonization. Ingestion of
contaminated garden vegetables also is an unlikely cause, even though
Wright et al. (1976) reported that salads may be heavily contaminated
with Gram-negative bacilli. Wright et al. (1976) studied the flora of
foods served to patients in a hospital and recovered enteric bacteria and
Pseudomonas aeruginosa from vegetable salads. The organism most
frequently isolated was Enterobacter agglomerans (85% of samples, 1Q2 to
106 cfu/g). Other organisms isolated frequently and at mostly high
counts were Enterobacter cloacae (48%) and Klebsiella (46%). The studies
of Casewell and Phillips (1978) and Cooke et al. (1980) challenge some of
the interpretations of Wright et al. (1976). Casewell and Phillips
(1978) noted that food prepared for intensive care patients was
frequently contaminated with Klebsiella but noted that the hospital
kitchen was the main source of contamination. Likewise Cooke et al.
(1980) examined hospital food for the presence of Klebsiella. Salads and
cold meat were the most frequently contaminated foods. However,
Klebsiella al so was widely distributed in the hospital kitchen
environment which was considered, at least in part, to be the source of
the organisms found in the food. It should also be noted that
Enterobacter agglomerans, the most frequent isolate from salads in the
study of Wright et al. (1976), was not associated with the ill throat
swabs of LHES participants (Table 5.14) and has been isolated only rarely
from fecal specimens (Tables 5.18 and 5.19) but was occasionally isolated
from the well throat swabs (Table 5.16).
Exposure to Gram-negative bacteria associated with heavily
contaminated cotton also is an unlikely cause of the unusual isolations
202
-------
of Tables 5.14, 5.21, and 5.22. Morey et al. (1983) recently reported
that seed cotton and cotton plants collected from Lubbock, Texas were
heavily contaminated with Gram-negative bacteria. The organisms were not
identified, but the investigators noted that Enterobacter agglomerans was
the predominant species in other similar studies. £. agglomerans is a
relatively recent designation for a group of organisms which include the
former Herbicola-Lathyri bacteria which were included in the plant
associated genus Erwinia. The nature of the flora of the contaminated
cotton and the lack of relationship to the isolations of Tables 5.14,
5.21 and 5.22 make contaminated cotton an unlikely source. It should
also be noted that while K^. pneumoniae is widely distributed in the
environment, strains isolated from humans and animals may be routinely
different in properties. Bagley and Seidler (1977) noted that 85%
(49/58) of J^. pneumoniae of human and bovine origin were fecal col iform
(FC) positive whereas 16% (19/120) of environmental strains were FC
positive. Strains of K_. pneumoniae that are FC positive have been
recently shown to have other unique properties (Edmondson et al., 1980).
The fact that the unusual isolations of Tables 5.14, 5.21, and 5.22
occurred over a defined period also tends to argue against possibilities
1 through 3, but not 4 and 5. The strongest argument against wastewater
being the source of organisms is the widespread distribution of the
unusual isolations over the study area (i.e., in low and high exposure
groups of participants). This observation would support possibility 5.
Fecal contamination of drinking water as a consequence of contamination
of the city of Wilson and individual wells is a distinct possibility,
particularly since the unusual isolations occurred following a period of
unusually heavy rains in the Lubbock area.
Clinical Virology
A summary of viral isolations made during the scheduled fecal
collection periods is illustrated in Table 5.23 as a function of sampling
month. The baseline results from 1980 and 1981 are consistent with other
published data of viral isolations from children where the greatest
number of enterovirus isolations were reported during the summer and
early fall (Honig et al ., 1956). Total virus recoveries from stool
specimens ranged from none to 32% positive. If poliovirus isolations are
presumed to reflect immunizations, the number of positive recoveries
attributed to naturally acquired nonpoliovirus infections was maximally
17 to 18% in July and August 1980.
A more detailed listing of viruses recovered from clinical
participants during sampling in 1980 and 1981 is presented in Table 5.24.
Positive isolations were made from 29 individuals during baseline
monitoring, all of whom were children. Of these, multiple viral
203
-------
TABLE 5.23. VIRAL ISOLATES RECOVERED FROM SCHEDULED FECAL SPECIMENS
1980 — Basel
Number of samples analyzed
Number positive for virus
Percent positive for virus
Number of samples yielding
designated viral type
Adenovlrus
Echovirus
Pol iovlrus
Coxsackievirus
Unidentified
a Samples from Periods 117 and
ro
o
JuT
22
7
32
0
0
3
3
1
118
Aug
36
8
22
0
2
2
3
1
(does not
ine
Sep
50
7
14
0
1
2
2
2
include
1981— Basel ine
Apr
24
0
0
0
0
0
0
0
dupl
May
11
0
0
0
0
0
0
0
i cates
Jun
45
4
9
1
0
3
0
0
Jul
30
5
17
2
1
1
0
1
from individuals
Aug
43a
6
14
0
1
1
0
4
during
Jan
4-8
107
11
10.3
8
3
0
0
0
Per i od
1982 — Operational
Mar
1-5
127
11
8.7
4
1
5
0
1
118).
Mar 29-
Apr 2
127
13
10.2
3
2
5
0
3
Jun
7-11
124
7
5.6
4
0
1
0
2
/\ug
9-13
121
5
4.1
1
0
0
1
3
Sep
13-17
126
11
8.7
1
4
1
2
3
-------
TABLE 5.24. VIRAL ISOLATES RECOVERED FROM INDIVIDUALS DURING BASELINE MONITORING
(July 1980 to September 1981)
ID
number
21111
43414
22712
42711
30612
40812
53912
53913
53911
32412
12311
20211
21916
21915
ro 55914
S 55913
10414
55715
55714
40411
32112
32111
21012
21011
53313
43511
40216
45112
12211
Period 015 Period 017
Coxsackle B-3
unidentified
-
polio 1
-
unidentified*
echo 11»
polio 3 polio 1
-
Coxsackie B-5
-
Coxsackle B-2
Coxsackle B-3 Coxsackle B-3
Coxsackle B-3
•
-
polio 3
echo 24
-
po 1 i o 3
Viral isolates from fecal specimens
Period 019 Period 108 Period 110 Period 112 Period 114 Period 117 Period 118 Period 119
_
-
unidentified polio 1
_
polio 1
unidentified
unidentified
echo 11
_
adeno -
adeno
polio 1
Coxsackie B-5
Coxsackle B-5
- - polio 3
adeno
_
- - echo 5 unidentified
- polio 3
_
_ _
polio 3
unidentified
-
- - unidentified
echo 24
unidentified
polio 1
- - -
- No viral isolate recovered from fecal specimen
* Illness convalescent specimen
(Blank) No specimen obtained.
-------
isolations were made from seven children. The most commonly recovered
enterovirus during the two baseline years of the study were poliovirus
(12 isolates) with Coxsackie B viruses (8 isolates) a close second.
Clinical virology results for fecal specimens from the six scheduled
collection weeks in 1982 (Periods 201.through 219) are also shown in
Table 5.23. The percentage of positive isolates range from a high of
10.3% in January to a low of 4.1% in August. These results cannot be
considered significantly different from those observed during the
baseline years, because most of the fecal donors in 1982 were adults (cf.
Table 5.8).
The use of a fluorescent staining procedure permitted the
identification of 21 isolates as adeno-like viruses from the 1982
specimens with the highest number of isolates recovered during January
sampling. This number represents 36% of the total isolates during the
year. Polioviruses had the next highest isolation rate (21%) followed by
echovirus types (17%) and Coxsackie viruses (5%). Twelve isolates have
yet to be identified.
Fecal specimens from the first year of full irrigation were separated
into two groups, corresponding to the preplanting irrigation (Periods 201
to 207) and summer irrigation (Periods 212 to 219) in an effort to
determine any obvious correlation between irrigation and viral
infections. A fecal collection, i.e. 201 and 212, was taken prior to the
start of each irrigation period. Isolates from positive samples
collected during these pre-irrigation periods were considered endemic to
the population and not related to irrigation events. Viral recoveries
from the study population during 1982 are shown in Table 5.25. A new
viral infection is defined as the isolation of a virus (either a specific
type or unidentified) from the second of a pair of consecutive fecal
specimens which was not isolated from the first specimen. These new
infections are indicated by boxes in Table 5.25. Table 5.26 presents a
summary of all new viral infections (events) during the periods of
irrigation.
A total of 15 infection events could be assigned to the preplanting
irrigation period by comparing periods 201 to 205 and 205 to 207, based
on available viral identifications. Of these, 80% were incurred by rural
inhabitants, while 20% were observed in Wilson. Poliovirus infections
predominated causing 40% of the infections, followed by adenovirus
infections (20%) and echovirus 17 (13%). Four of the isolates have not
been identified.
The viral isolations for Periods 212 through 219 were used to
determine new viral infections during the summer irrigation of 1982.
Tables 5.25 and 5.26 show that of 13 events observed during these
206
-------
TABLE 5.25. VIRAL RECOVERIES AND NEW VIRAL INFECTIONS FROM SCHEDULED FECAL SPECIMENS IN 1982
ID
201
(Jan 4-8)
Fecal Collection Period In 1982
205
(Mar 1-5)
207 212
(Mar 29-Apr 2) (Jun 7-11)
216
(Aug 9-13)
219
(Sep 15-17)
10201
10414
10901
10913
11402
11902
12501
12602
13211b
13212b
20502
20713
21012
21112
21301
21611
21915
21916
22712
23112
23614
23615
32202
32411
32412
40312
41302
41601
42801
45113
45312
45314
50501
53901
53911
53912
54502
55913
60101
60111
+
(adeno)
(adeno)
(adeno)
(adeno)
(echo 5)
(adeno)
(adeno)
(echo II)
(adeno)
(adeno)
(echo 5)
(polto 3) |
Ho 3)
(po
(adeno)
(adeno)
(adeno)
| (polio 1)|
(echo 17)
(adeno)
(pol
Ton
(polto 1)1
(adeno)
(po
Ho 3)
(echo 17)
(adeno)
(poll
(polfo 3)I
(echo 27)
(TS CB 4)
(echo 31)
(adeno)
(adeno)
(CB 5)
(adeno)
(adeno)
(adeno)
[ (CB*4)
(CB 5)
(polio 2)
(echo 30)
(echo 31)
(II I-adeno)
a positive throat swab.
b recipient of oral vaccine In mid-February.
ne» viral Infection
(Blank) No fecal specimen obtained
- No viral Isolate recovered from fecal specimen
+ Viral Isolate recovered from fecal specimen
207
-------
TABLE 5.26. SUMMARY OF NEW VIRAL INFECTIONS (EVENTS) IN SCHEDULED FECAL SPECIMENS DURING IRRIGATION PERIODS IN 1982
Between Periods
201 and 205
(Jan-Mar)
Between Per i ods
205 and 207
(Mar-Apr)
Between Periods
212 and 216
(Jun-Aug)
Between Periods
216 and 219
(Aug-Sep)
Locat i on
Rural
(Zones 1,3,5,6)
Wi Ison
(Zones 2,4)
Total events
Number of
events Agent
1 Po 1 i o 1
1 Po 1 i o 3
1 Echo 17
1 Adeno
4
Number of
events
2
1
1
2
3
1
1
11
Agent
Po 1 i o 1
Po 1 i o 3
Echo 17
Adeno
Unidentified
Po 1 1 o 1
Unidentified
Number of
events Agent
1 Unidentified
1 Coxsackle B4
1 Unidentified
3
Number of
events
1
1
2
1
2
1
2
10
Agent
Echo 27
Echo 30
Echo 31
Po 1 i o 2
Coxsacki e
Adeno
B5
Unidentified
ro
o
oo
-------
periods, 77% occurred in Period 219. Unlike the spring data, these
infection events were more evenly distributed between Wilson (62%) and
the rural population (38%). Echoviruses represented 31% of the isolated
infectious agents, Coxsackieviruses 23%, polioviruses 8%, and
adenoviruses 8%.
Electron Microscopy of Fecal Specimens
As of February 1983, 261 stool specimens had been received at HERL-
Cincinnati for EM examination. Results of this examination and specimen
status are indicated in Table 5.27. One hundred and twenty specimens
have been examined. These include: 41 from 1980, 52 from 1981, 25 from
1982 and 2 from 1983. All illness specimens have been examined.
Picorna/parvovirus-like particles have been detected in only one specimen
received in September 1980.
Coronavirus-1ike particles were detected in the stools of five
individuals. None of the stools were illness specimens. The particles
were observed in all subsequent specimens from two of these individuals.
Norwalk-like particles were detected in one acute illness specimen
(211121, May 1982). This specimen, a related specimen (211111), and four
pairs of sera were sent to Dr. N. R. Blacklow's laboratory at the
University of Massachusetts for examination by RIA. Both stools were
negative for Norwalk antigen and no seroconversions to Norwalk were
detected.
Calicivirus-like particles were detected in a second illness specimen
from 211121 (December 1982). Astrovirus-like particles were detected in
an November 1982 illness specimen from 202111. Stools received in
January 1983 from 202111 and 211121 were negative for virus-like
particles as were all other illness specimens.
The remaining stools (primarily post-spray irrigation) are being
examined under blind conditions with a similar number of prespray
irrigation stools as controls.
Tuberculin Test Data
The results of the tuberculin skin test are shown in Table 5.28. Only
two new positive reactions were found in the population in 1982.
Serologic Data
Serum Neutralization Serology—
A summary of the serum neutralization serology for study participants
is presented in Table 5.29. The post-irrigation seroconversion rate was
209
-------
TABLE 5.27. EM ANALYSIS OF FECAL SPECIMENS
Date
Shipment received
1 7-29-80
Period 015
2 8-26-80
Period 017
3 9-23-80
Period 019
4 4-29-81
Period 108
5 6-23-81
Period 110
Period 112
Specimen
ID
104141
122111
123111
207131
207141
210111
211111
227111
306121
306131
503111
503121
514111
202111
210111
213111
219161
231131
407131
427111
436131
539111
539131
539131
104131
202111
210121
215141
219151
227111
231111
303111
321121
404121
408121
409141
436141
503111
514121
539121
123021
227121
310111
408121
426131
434141
533131
557131
559131
122111
404111
426131
533131
559131
559141
104141
122111
321121
Particles
detected
_
-
-
CV
-
-
-
-
-
-
-
-
-
-
-
-
CV
-
-
-
-
-
-
-
-
-
-
CV
-
-
-
-
_
-
PC/PV
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
CV
-
-
-
Date Specimen
Sh 1 pment rece I ved 1 D
Period 112 324111
(cont'd) 402141
403111
436131
436141
510111
557141
6 7-29-81 211111
Period 114 225121
321111
403111
404121
434131
533121
557151
559111
559121
801131
801141
7 9-9-81 104131
Period 116 231121
324121
559141
Period 118 123111
202111
225111
227121
402151
402161
403121
427111
539111
539121
539131
8 2-24-82 119010
Period 201 119020
122111
202021
202111
207111
219151
219161
221020
225111
303021
321111
322020
324 1 1 1
324121
411010
414010
504010
505010
510121
523010
526010
535010
53901.0
539111
Particles
detected
_
_
_
_
_
_
-
_
-
-
-
-
-
-
-
CV
-
-
-
-
-
CV
-
.
.
-
-
-
-
-
-
—
-
ND
ND
ND
ND
ND
ND
ND
CV
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
continued.
210
-------
TABLE 5.27 (CONT'D)
Shipment
9
10
11
12
Date
rece 1 ved
Period 201
(Cont'd)
3-30-82
Period 205
5-5-82
Period 207
5-25-82
Period 210
6-29-82
Period 212
Specimen
ID
545020
559141
103012
108012
207141
223022
225111
231111
236022
301022
324121
410012
415012
416012
418012
428012
447022
451141
523012
531012
533131
539012
559131
106012
118012
125012
129012
207141
211121
216111
219151
223022
231121
235022
301011
402141
403111
404111
408121
520010
539121
540012
211111
211121
107022
121012
126022
129012
202111
216111
219151
219161
221022
223022
231131
236010
236110
236141
Particles
detected
ND
CV
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
-
ND
ND
ND
ND
ND
ND
ND
ND
ND
-
ND
ND
ND
ND
ND
ND
ND
ND
ND
-
ND
ND
ND
-
NK
ND
ND
ND
ND
ND
ND
ND
CV
ND
ND
ND
ND
ND
ND
Date Specimen
Shipment received ID
Period 212 236151
(Cont'd) 324111
404040
411021
427020
428012
432012
438012
451141
506022
507012
532012
540010
540020
540110
540121
540121
540131
553012
557141
557151
559020
559131
561012
13 9-1-82 107022
Period 216 122121
211111
216111
219151
219161
221022
223022
234012
236022
236151
315022
324121
402141
402151
404020
404121
428012
432012
453020
453131
453141
505012
507012
532012
540121
601111
14 10-20-82 107022
Period 219 122121
126022
202111
207112
210111
219161
234012
236141
Particles
defected
ND
ND
_
ND
-
ND
ND
ND
ND
ND
ND
ND
-
ND
-
-
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
CV
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
CV
ND
ND
ND
-
_
ND
ND
ND
ND
ND
ND
CV
ND
ND
continued.
211
-------
TABLE 5.27 (CONT'D)
Date Specimen
Shipment received ID
Period 219 324020
(Cont'd) 324121
324131
403121
403121
404040
415012
416012
428012
432012
453020
453131
505012
Particles
detected
ND
ND
ND
-
_
ND
ND
ND
ND
ND
ND
ND
ND
Date
Shipment received
Period 219
(Cont'd)
•
Period 220
15 12-21-82
Period 223
Period 225
16 1-12-83
Period 301
Specimen
ID
526012
532012
545022
555012
601111
403121
202111
211121
202111
211121
Particles
detected
ND
ND
ND
ND
ND
-
AS
CL
-
-
ND - not done, samples not yet processed
- negative sample
AS - astrovlrus-lIke particles
CL - ca11clvirus-like particles
CV - coronavirus-1 Ike particles
NK - Norwalk-like particles
PC/PV - picornavirus/parvovIrus-1 Ike particles
212
-------
TABLE 5.28. TUBERCULOSIS SKIN TEST RESULTS
yize of induration
Testing date
0 mm
1-5 mm
6-9 mm
>10 mm
Previous
reactor
June 1980 265
December 1980 33
June 1981 172
December 1982 240
2
0
8
1
6
1
3
0
18
1
5
1
4
1
1
3
213
-------
TABLE 5.29. SUMMARY OF SERUM NEUTRALIZATION SEROLOGY FOR LHES STUDY PARTICIPANTS DURING 1982
Microorganism/ Data
col lection period
ADENO 7
Per i od
Per i od
COXSACKIE
Per i od
Period
ECHO 1/8
Period
Period
ECHO 5
Period
Period
ECHO 9
Period
Period
ECHO 11
Period
Period
201
212
B5
201
212
201
212
201
212
201
212
201
212
Titer Distribution
<10 (%)
73
70
63
52
81
87
72
65
55
63
86
58
= 10 (.%)
16
21
14
18
10
8
12
14
12
17
9
18
>10 (%)
11
10
23
31
5
5
16
19
33
21
4
25
Number
of sera
312
309
312
309
312
309
312
309
312
309
312
309
Geometric Baseline Post- 1 rrl gat ion Baseline Post-lrr igatlon
mean seroconvers ion seroconverslon decrease decrease
titer ratea rate3 rateb rateb
1.71 0.77 0 0
6.64
6.84
2.78 3.87 1.28 0
8.24
10.09
1.44 000
5.93
5.78
1.50 6.93 0.43 0.77
7.59
7.76
2.78 0 0.85 0.77
11.09
8.31
0.85 29.28 0.43 0
5.61
9.10
a Number of fourfold Increases per 100 people per year. All reported Increases will be reverifled using paired sera. Baseline
period is defined as the period from June 1980 to January 1982. Post-1rrlgat Ion is defined as the period from January 1982 to
June 1982.
b Number of fourfold decreases per 100 people per year.
-------
elevated for Coxsackie B5, Echo 5 and Echo 11. However, a breakdown of
household seroconversions by zone (Table 5.30) suggests that the
seroconversions which were observed appear to be evenly distributed
throughout the sampling zones. In addition to the agents listed,
baseline sera were tested for antibody to Coxsackie B3 and Coxsackie A9.
These two agents were not used in testing post-irrigation bloods when it
was determined that more than 60% of the population had antibody to these
two agents.
Reoviruses--
A preliminary screen of 41 paired sera (Periods 012 and 201) from
study participants and 46 sera (Period 201) from staff was performed.
Based on Period 201 titers, 76%, 68% and 34% of the 41 participants had
antibody levels of 1:8 or greater to reovirus types 1, 2 and 3,
respectively. Significant (fourfold) increases in antibody levels to
types 1 and 2 were detected in four and three participants, respectively.
These test results were based on endpoints defined as any indication of
hemagglutination-inhibition. In order to improve reproducibil ity, this
definition has been modified as described in the Serologic Methods
section.
Hepatitis A--
Since the beginning of the health watch program, a total of 427
participants have been tested for IgG antibody to hepatitis A virus in
sera from June 1980 through June 1982. An overview of the distribution
of HAVAB® positive individuals whose age was available (396 total) is
shown in Table 5.31. Only 7% of the participants age 10 or under showed
a prior exposure to this viral agent, while all participants over the age
of 70 were positive. To date, only three conversions from negative to HA
VAB® positive have been observed, as shown in Table 5.32.
This distribution of pre-existing antibody to infectious hepatitis is
consistent with other published results. In a study reported by Szmuness
and associates (1976), 45% of an adult population (n = 947) in New York
City was hepatitis A positive using an immune adherence hemagglutination
test. Antibody was detected in a larger proportion of lower class
participants (72 to 80%) than in the middle and upper classes (18 to
30%). Study results also showed hepatitis A antibody prevalence was
closely related to age. In middle class whites and blacks, the rate was
two to four times higher in those 50 or more years old than in the 18 to
19 year olds. Further in samples of healthy children from the same area,
the rate of hepatitis A antibody detection varied between 10 and 20%. A
more recent report by Snydman and co-workers (1981) noted that of 73
people tested as part of a control group, 32% had IgG antibody to
hepatitis A virus as detected by solid-phase radioimmunoassay.
Following the completion of Period 212 HAVAB® testing, all sera
(except archived aliquots) from Periods 012, 025, 112, 201 and 212 were
215
-------
TABLE 5.30. HOUSEHOLD SEROCONVERSION RATE BY ZONE
DURING POST-IRRIGATION PERIOD
Zone
1
2
3
4
5
6
Overall
Echo 5
2%
2%
8%
3%
1%
0%
3%
Echo 11
12%
4%
12%
16%
7%
0%
10%
Coxsackie B!T
4%
5%
8%
8%
2%
20%
6%
216
-------
TABLE 5.31. DISTRIBUTION OF HEPATITIS A ANTIBODY (IgG)
AS DETERMINED BY AN RIA TEST
(June 1980-June 1982)
Age in 1980 (years)
<10 11-20 21-30' 31-40 41-50 51-60 61-70 >70
Male
Total tested3 30 39 19 26 21 21 14 14
Number positive 29 5 10 9 14 12 14
Percent positive 7 23 26 38 43 67 86 100
Femal e
Total tested15 28 47 27
Number positive 297
Percent jDOsitive 7 19 26
30
9
30
20
13
65
25
18
72
21
20
95
14
14
100
a excludes 20 participants of unknown age
b excludes 11 participants of unknown age
TABLE 5.32. CONVERSION FROM NEGATIVE TO HAVAB
(1980-1982)
POSITIVE
ID
012
025
112
201
212
438012
516010
557151
217
-------
shipped to the University of Illinois to replenish their dwindling
stocks. Only archived sera from Periods 012, 025 and 112 remain in
storage at the University of Texas at Austin.
218
-------
ENVIRONMENTAL DATA
-------
ENVIRONMENTAL DATA
Microorganism Levels in Wastewater
24-Hour Composite Samples--
During the two baseline years of this health effects study, composite
wastewater samples were collected at both the Lubbock Southeast Trickling
Filter plant and the Wilson Imhoff tank. Beginning with the delivery of
treated Lubbock sewage to the Hancock farm site in February 1982, pipeline
effluent and reservoir water were analyzed for a variety of microbiological
and selected physical-chemical parameters. Results of these analyses are
presented in Tables 5.33, 5.34 and 5.35 for Lubbock wastewater (and
subsequently pipeline effluent), Hancock farm reservoir water and Wilson
wastewater, respectively. Time series of key microbiological and physical
parameters are graphed in Figures 5.1 and 5.2.
Lubbock wastewater effluent may be classified as relatively strong
based on both microbial and chemical analyses. A review of data presented
in the figures and Table 5.33 shows that fecal coliform levels in effluent
sampled at the Lubbock treatment plant routinely exceeded 10^ cfu/mL, while
total organic carbon (TOO values ranged from 40 mg/L to over 200 mg/L.
During the first two years of the study, total enterovirus levels as
measured on HeLa cell monolayers ranged from 0.045 pfu/mL to over 1.0
pfu/mL in the summer of 1980. Typically, enterovirus levels were evaluated
in summer and early fall.
The first pipeline effluent was sampled at the Hancock farm in
February 1982 and represented a highly atypical sample microbiologically.
Once a daily wastewater flow to the Hancock site was established, the
microbial and physical profile of the wastewater delivered to the
irrigation site was not dissimilar from the wastewater previously
characterized at the treatment plant. In 24-hour composite samples,
maximal viral levels of about 0.1 pfu/mL were observed during spring
monitoring, while levels approaching 0.5 pfu/mL were detected during the
summer 1982 irrigation season.
Similar data for Hancock reservoir water collected from June to
September 1982 are shown in Table 5.34. A comparison of both indicator
bacteria and virus levels shows that, in general, organism concentrations
in reservoir water were two logio units lower than comparable pipeline
effluent. Likewise, the diversity of bacteria recovered during microbial
screens was much greater in the Lubbock effluent (Table 5.36) than in
reservoir water (Table 5.37). Of the five samples of reservoir water
concentrate which were successfully assayed, enteroviruses were detected in
four samples with a maximal level of about 0.06 pfu/mL.
Microorganism concentrations in Wilson wastewater are profiled in
Table 5.35 and Figure 5.1. Greater variability in organism levels in these
219
-------
TABLE 5.33. MICROORGANISM CONCENTRATIONS IN LUBBOCK WASTEWATER
ro
PO
o
Samp 1 inq date
24-Hour composite
samples analyzed
Bacteria (cfu/mL)
Standard plate count
Total col i forms
Fecal col i forms
Feca 1 streptococc i
Mycobacteria sp.
Clostridium perfringens3
- vegetat i ve
- sporulated
Staphylococcus aureus
Sa Imonel la sp.
Shigel la sp.
Yerslnia enterocol itica
Campy lobacter fetus ssp. jejuni
Candida albicans
Fluorescent Pseudomonas sp.
Klebsiel la sp.
Viruses (pfu/mL)
Bacterlophage
Enterovi ruses
HeLa, 5 day (uncorrected)
HeLa, polio-neutralized
RD, polio-neutralized
Poliovlrus concentration
efficiency (?)
Physical Analyses (mg/L)
Total organic carbon
Total suspended solids
Total volatile suspended solids
PH
Jun
3-4
3,600,000
350,000
87,000
4,700
1,200
7,500
930
<30
< 0.004
< 0.004
1 0, 000
<33, 000
1,400
0.78
38
83
96
65
6.5
I960
Jul
28-29
5,700,000
380, 000
72, 000
2,000
170,000
1 1 0, 000
430
<3
>0.002
<0. 002
<0.004
6,200
130,000
3,200
1.2
42
40
78
52
6.6
Nov
3-4
3,400,000
140,000
88,000
5,100
1,100
2,400
930
<3
<0.002
<0.002
<0.004
3,100
53, 000
2,600
0.73
39
215
135
7.2
Jan
19-20
60, 000
15,000
880
0.096
97
115
184
130
7.0
1981
Feb Mar
16-17 9-10
110,000 120,000
34,000 16,000
0.054 0.059
78 26
133 141
151 234
120 178
7.3 7.0
Mar
23-24
160,000
83, 000
<10
>0.01b
>0.01
100
66
<0.3
130,000
-
0.046
105
91
89
74
7.1
continued...
-------
TABLE 5.33. (CONT'D)
ro
ro
Sampl Ing date
24-Hour composite
samples analyzed
Bacteria (cfu/mL)
Standard plate count
Total col I forms
Fecal conforms
Fecal streptococci
Mycobacterla sp.
Clostridium perfrlngens3
- vegetat I ve
- sporulated
Staphy lococcus aureus
Salmonel la sp.
Shigel la sp.
Yersinla enteroco 1 1 t 1 ca
Campy 1 obacter fetus ssp. jejuni
Candida albicans
Fluorescent Pseudomonas sp.
Klebsiella sp.
Viruses (pfu/mL)
Bacterlophage
Enterovl ruses
HeLa, 5 day (uncorrected)
HeLa, polio-neutralized
RD, po 1 1 o-neutra 1 1 zed
Po 1 1 ov i r us con centrat I on
efficiency ($)
Physical Analyses (mg/L)
Total organic carbon
Total suspended solids
Total volatile suspended solids
PH
Apr
20-21
9,600,000
520,000
59, 000
6,900
400,000
1 1 0, 000
460
3
>1
>0.008
<0.005
<3
<3
220, 000
230, 000
1,600
0.057
0.018
0.008
69
127
200
147
7.5
May
4-5
86, 000
<3
>10
>1
>0. 005
<3
<3
2,600
0.11
0.006
0.033
95
104
115
92
7.6
May
18-19
360, 000
1 1 0, 000
1,100
<3
>100
<0.01
<0.01
200, 000
0.1
0.065
0.15
79
47
47
44
6.5
1981
Jun
29-30
120,000
50, 000
8,700
<3
>10
<0.01
<0.01
>20(f
<3
30, 000
0.085
0.055
0.1
77
100
51
36
7.6
Jul
20-21
3,000,000
380, 000
1 00, 000
2,400
14,000
230
210
<10
>10
<0. 007
<0. 007
<10
<10
23, 000
66, 000
2, 1 00
0.065
0.02
0.93
85
100
43
33
7.2
Aug
17-18
91,000
<3
>10
<0.008
<0.008
<0.01
<3
50, 000
0.045
0.053
0.42
34
79
68
49
6.4
Nov
17-18
60, 000
<3
>10
<0.01
<0.01
<3
<3
130,000
0.055
0.0013
0.13
80
100
118
87
7.3
continued..
-------
TABLE 5.33. (CONT'D)
ro
ro
ro
24-Hour composite
samples analyzed
Bacteria (cfu/mL)
Standard plate count
Total coliforms
Fecal coliforms
Feca 1 streptococc i
Mycobacteria sp.
Clostridium perfringens3
- vegetative
- s populated
Staphy lococcus aureus
Sa Imonel la sp.
Sh igel la sp.
Yersinla enterocol Itica
Campy lobacter fetus ssp. jejuni
Candida albicans
Fluorescent Pseudomonas sp.
Klebsiella sp.
Viruses (pfu/mL)
Bacterlophage
Enterovl ruses
HeLa, 5 day (uncorrected)
HeLa , po 1 i o-neutra 1 i zed
RD, polio-neutralized
Pollovirus concentration
efficiency (?)
Physical Analyses (mg/L)
Total organic carbon
Total suspended solids
Total volatile suspended solids
pH
Feb
15-16d
11 , 000
11 , 000
130,000
900
0.037
<0. 003
<0. 003
227
136
111
96
7.1
Feb
16d,e
15,000
240
39
120
1 , 000
210
28
<3
<0.04
<0. 01
<0.01
<0.01
<3
30
180
750
0.033
<0. 005
<0.002
103
143
90
8.8
Mar
1-2
56, 000
1,000
28, 000
2.5
>0.04
<0.01
<0.01
<3
<3
50, 000
1,000
0.07
0.034
<0.002
50
98
150
113
7.1
Samp 1 ing date
1982
Mar Mar
8-9e 15-16
75,000 79,000
5,900 3,500
53, 000 30, 000
<3
<0.01
40
<3
66,000
1,800 780
0.11 0.11
0.022 0.017
86 f
116 95
178 92
153 82
7.1 7.4
Mar
22-23
57,000
81,000
7,900
13,000
<3
100
<0.01
<0.01
<3
<3
260, 000
50, 000
1,500
0.063
0.004
0.010
63
151
269
170
7.3
Mar
29-30
50, 000
5,000
1 0, 000
69
0.012
0.002
0.034
125
205
165
7.1
continued..,
-------
TABLE 5.33. (CONT'D)
PO
ro
CO
Samp 1 Ing date
24-Hour composite
samples analyzed
Bacteria (cfu/mL)
Standard plate count
Total col (forms
Fecal collforms
Fecal streptococci
Mycobacterla sp.
Clostrldium perfrlngens3
- vegetative
- sporulated
Staphylococcus aureus
Sa Imonel la sp.
Shlgel la sp.
Yerslnla enterocol itlca
Campy lobacter fetus ssp. jejunl
Candida alblcans
Fluorescent Pseudomonas sp.
Klebslel la sp.
Viruses (pfu/mL)
Bacterlophage
Enterovl ruses
HeLa, 5 day (uncorrected)
HeLa , po 1 1 o-neutra 1 1 zed
RD, po 1 1 o-neutra 1 1 zed
Po 1 1 ov 1 r us concentrat 1 on
efficiency (?)
Physical Analyses (mg/L)
Total organic carbon
Total suspended solids
Total volatile suspended solids
pH
Apr
5-6
84, 000
2,800
20,000
<3
0.01
1.0
<0.01
100
10
40
130,000
830
0.042
0.016
0.010
54
71
118
96
7.6
Apr
26-279
9,100
1,800
8,500
220
0.028
0.008
0.004
69
98
98
79
7.5
1982
Jun
14-15
66,000
1,000
13,000
840
0.026
0.026
< 0.002
64
72
77
66
7.2
Jun
29-30
68, 000
4,200
43, 000
>1.0
<0.01
<0.01
<10
<10U
9,000"
1 00, 000
840
0.49
0.39
0.056
59
lit
84
7.3
Jul
26-27
1,300,000
120,000
58, 000
2,300
13,000
750
9
<3
>0. 1
<0.01
<0.01
10
<3
6,000
5,000
"l,100
0.060
0.030
0.007
69
140
106
7.5
continued.
-------
TABLE 5.33. (CONT'D)
ro
PO
Samp 1 inq date
24-Hour composite
samples analyzed
Bacteria (cfu/mL)
Standard plate count
Total col 1 forms
Fecal col I forms
Feca 1 streptococc 1
Mycobacteria sp.
Clostridlum perfrlngens8
- vegetat 1 ve
- sporulated
Staphy lococcus aureus
Sa Imonel la sp.
Shlgel la sp.
Yerslnla enterocol Itlca
Campy lobacter fetus ssp. jejuni
Candida albicans
Fluorescent Pseudomonas sp.
Klebsiel la sp.
Viruses (pfu/mL)
Bacteriophage
Enterovi ruses
HeLa, 5 day (uncorrected)
HeLa, polio-neutralized
RD, po 1 1 o-neutra 1 1 zed
Po 1 1 ov 1 rus concentrat I on
efficiency (%)
Physical Analyses (mg/L)
Total organic carbon
Total suspended solids
Total volatile suspended solids
PH
Aug
9-10
35, 000
2,500
>1
<0.01
<0.01
<3
<3
730
26, 000
0.087
0.074
156
67
105
74
7.6
1982
Aug
30-316
200
30
760
<0.01
<0.01
<0.01
<3
<3
30, 000
300
30
i
I
0.018
47
52
51
39
7.3
Sep
13-14
65, 000
3, 500
1,400
>0.01
<0.01
<0.01
>10
<300
2,000
40, 000
~
0.022
0.008
42
58
50
42
7.8
a Most probable number (MPN)/mL.
b A new procedure was used for detection of Salmonella spp. (Kaper et al., App. Environ. Microblol., 33:829-35, 1977) beginning
in March 1981.
c Value calculated from representative colonies Identified as C. fetus ssp. jejuni, actual number may be higher.
d On February 16, 1982 the sample source was changed from the trickling filter to the pipeline; the first set of data on
February 16 was sampled from the trickling filter while the second set was collected from the pipeline.
e Chi or(nation of wastewater at treatment plant.
f Lost.
g Chi or I nation in Lubbock of a portion of the sampled wastewater.
h Beginning with samples collected on June 29-30, 1982 fluorescent Pseudomonas sp. was substituted for Staphylococcus aureus as
part of the limited bacterial screen.
i HeLa cells used for the assay were contaminated; results could not be obtained.
-------
TABLE 5.34. MICROORGANISM CONCENTRATIONS IN HANCOCK RESERVOIR
ro
no
en
24-Hour composite
samples analyzed
Bacteria (cfu/mL)
Standard plate count
Total coliforms
Fecal col I forms
Feca 1 streptococc 1
Mycobacteria sp.
Clostridium perfringens3
- vegetative
- sporulated
Staphy lococcus aureus
Salmonel la sp.
Shlgel la sp.
Yersinla enteroco 1 1 t 1 ca
Campy lobacter fetus ssp. jejuni
Candida albicans
Fluorescent Pseudomonas sp.
Klebslel la sp.
Viruses (pfu/mL)
Bacter iophage
Enteroviruses
HeLa, 5 day (uncorrected)
HeLa, polio-neutralized
RD, polio-neutralized
Poliovirus concentration
efficiency ($)
Physical Analyses (mg/L)
Total organic carbon
Total suspended solids
Total volatile suspended solids
pH
Jun
14-15
520
20
4,000
14
0.002
0.005
<0.002
81
33
218
50
7.6
Jun
29-30
60
3
200
<0.01
<0.01
<0.01
<10
<10u
23 Ob
10
19
0.014
0.056
<0.017
21
67
28
7.9
Samp 1
Jul
26-27
36,000
500
190
3
<10
430
4
<3
<0.01
<0.01
<0.01
<3
<3
13
30
0.9
<0. 002
<0.002
0.004
14
21
20
8.0
ing date
1982
Aug
9-10
390
6.6
<0.01
<0.01
<0.01
<3
<3
16
130
0.002
0.004
0.004
87
27
24
21
8.1
Aug
30-31
10
0.3
1,000
<0.01
<0.01
<0.01
<3
<3
2,000
<50
0.8
c
c
<0.002
61
23
24
19
7.9
Sep
13-14
350
10
550
<0.01
<0.01
<0.01
<10
<10
250
1,000
<0.002
0.002
27
28
44
34
8.4
a Most probable number (MPN)/mL.
b Beginning with samples collected on June 29-30, Fluorescent Pseudomonas sp. was substituted for Staphylococcus aureus as part
of the limited bacterial screen.
c HeLa cells used for the assay were contaminated; results could not be obtained.
-------
TABLE 5.35. MICROORGANISM CONCENTRATIONS IN WILSON WASTEWATER
ro
ro
24-Hour composite
samples analyzed
Bacteria (cfu/mL)
Standard plate count
Tota 1 co 1 1 forms
Feca 1 co 1 1 forms
Feca 1 streptococc 1
Mycobacteria sp.
Clostrldlum perfrlngens3
- vegetative
- sporulated
Staphy lococcus aureus
Salmonel la sp.
Shlgel la sp.
Yersinia enteroco 1 1 t i ca
Campy lobacter fetus ssp. jejunl
Candida albicans
Fluorescent Pseudomonas sp.
Klebslel la sp.
Viruses (pfu/mL)
Bacterlophage
Enterovl ruses
HeLa, 5 day (uncorrected)
HeLa, polio-neutralized
RO, po 1 1 o-neutra 1 1 zed
Po 1 1 ov 1 rus concentrat i on
efficiency (.%)
Physical Analyses (mg/L)
Total organic carbon
Total suspended sol Ids
Total volatile suspended solids
PH
Jun
3-4
1,600,000
270, 000
1 00, 000
6,800
1,400
1 1 , 000
1,500
33
<0.004
<0.004
< 0.002
8,300
1 00, 000
410
0.047
56
87
68
39
6.5
1980
Jut
28-29
3,300,000
160,000
30,000
2,300
1,900
24,000
240
<3.3
<0.002
<0.002
<0.004
1,500
70,000
3,300
15
47
64
45
29
6.6
Samp 1
Jan
19-20
390,000 52,
64,000 15,
3,100
<0. 0009
55
90
64
54
7.0
inq date
Feb
16-17
000
000
0.22
69
159
97
77
7.3
1981
Mar
9-10
98, 000
44, 000
0.002
46
96
73
58
7.0
Mar Apr
23-24 20-21
98, 000
19,000 80,000
-
0. 001 0. 003
<0.001
0.002
76
87 200
70 151
51 89
7.2 7.7
continued..,
-------
TABLE 5.35. (CONT'D)
Samp I ing date
24-Hour composite
samples analyzed
May
4-5
May
18-19
Jun
1-2
1981
Jun
15-16
Jun
29-30
Jul
20-21
Aug
17-18
ro
Bacteria (cfu/mL)
Standard plate count
Total col if or ins
Fecal coliforms
FecaI streptococcI
Mycobacterla sp.
Clostrldlum perfrlngens3
- vegetative
- sporulated
Staphylococcus aureus
Salmonella sp.
Shi gel la sp.
Yerslnla enterocolItlca
Campy Iobacter fetus ssp. jejunl
Candida alb!cans
Fluorescent Pseudomonas sp.
Klebsfella sp.
Viruses (pfu/mL)
Bacterlophage
Enterov!ruses
41,000
66,000
110,000 110,000 36,000 54,000 53,000
H
>0.01d
<0.007
<0. 007
<3
<0.01
<0.008
<0.008
<0.01
<3
56,000
20,000
HeLa, 5 day (uncorrected)
He La, polio-neutralized
RD, po 1 1 o-neutra 1 1 zed
Po 1 1 ov 1 rus concentrat 1 on
efficiency (?)
Physical Analyses (mg/L)
Total organic carbon
Total suspended solids
Total volatile suspended solids
PH
0.025
<0. 001
1.5
32
92
75
60
7.8
0.17
0.004
0.14
c
108
80
59
6.5
0.078
0. 001 5
b
74
57
44
36
6.4
<0. 001
<0.014
0.075
55
56
30
26
6.5
0.99
0.008
0.058
42
97
26
22
7.6
0.006
0.002
0.053
53
101
57
42
7.3
0.013
0.001
1.5
63
80
30
23
6.9
continued.
-------
TABLE 5.35. (CONT'D)
ro
oo
Sampl inq date
24-Hour composite
samples analyzed
1981
Sep
14-15
Nov
17-18
Feb
15-16
Mar
1-2
1982
Mar
8-9
Mar
22-23
Apr
5-6
Bacteria (cfu/mL)
Standard plate count
Total col I forms
Fecal conforms 8,700 44,000
Fecal streptococci
Mycobacterla sp.
Clostridlum perfrlngens9
- vegetative
- sporulated
Staphylococcus aureus <3
Salmonella sp. >0.006 >1
Shlgellasp. <0.006 <0.005
Yerslnla enterocolitlca <0.005
Campy Iobacter fetus ssp. jejuni <3 <3
Candida albicans <3 <3
Fluorescent Pseudomonas sp.
Klebslella sp. 7,500 130,000
Viruses (pfu/mL)
Bacteriophage
Enteroviruses
17,000
10,000
<0.01
<0.01
<0.01
<3
<3
50,000
130,000
140,000
81,000
110, 000
<3
<0.01
<0.01
<0.01
<3
<3
100,000
<3
<0.01
<0.01
<0.01
<3
<3
1,000
HeLa, 5 day (uncorrected)
HeLa, polio-neutralized
RD, polio-neutralized
Pollovirus concentration
efficiency (?)
Physical Analyses (mg/L)
Tota 1 organ I c carbon
Total suspended solids
Total volatile suspended solids
PH
0.001
<0.001
1.0
50
75
57
7.4
0.06
<0.002
0.15
96
72
60
50
7.4
<0. 0007
<0. 001
<0. 003
233
102
82
73
7.5
<0. 0008
e
87
92
98
74
7.2
0.12
0.012
74
103
82
76
7.2
0.11
<0.002
86
87
70
67
7.3
1.5
0.085
b
77
89
72
59
7.7
continued...
-------
TABLE 5.35. (CONT'D)
ro
ro
Samp 1 inq date
24-Hour composite
samples analyzed
Apr
19-20
May
3-4
May
17-18
1982
Jun
14-15
Jun
29-30
Jul
19-20
Bacteria (cfu/mL)
Standard plate count
Total col I forms
Fecal col I forms
FecaI streptococc i
Mycobacteria sp.
Clostrldium perfringens9
- vegetative
- sporuIated
Staphylococcus aureus
Salmons I la sp.
Shi gel la sp.
Yerslnla enterocolitica
Campy Iobacter fetus ssp. jejuni
Candida a Ibicans
Fluorescent Pseudomonas sp.
Klebsiella sp.
Viruses (pfu/mL)
Bacteriophage
Enteroviruses
HeLa, 5 day (uncorrected)
HeLa, polio-neutralized
RD, poIi o-neutra11zed
Poliovirus concentration
efficiency (?)
Physical Analyses (mg/L)
270,000 37,000
140,000 150,000
0.27
0.003
0.045
58
0.70
0.008
0.008
72
0.0076
<0.0002
72
< 0.002
<0.002
<0.002
61
85,000 120,000
>0.01
<0.01
<0.01
11,000f
16,000
>0.01
<0.01
<3
<3
9,300
35,000
0.034
0.036
0.036
0.44
0.004
0.004
Total organic carbon
Total suspended solids
Total volatile suspended solids
pH
92
74
65
7.6
68
89
69
7.5
81
60
50
7.5
75
67
56
7.2
69
70
61
7.0
76
44
41
7.5
continued.
-------
TABLE 5.35. (CONT'D)
ro
CO
o
Samp 1 ing date
24-Hour composite
samples analyzed
Aug
9-10
Aug
30-31
1982
Sep
13-14
Sep
27-28
Oct
11-12
Bacteria (cfu/mL)
Standard plate count
Total conforms
Fecal conforms
FecaI streptococcI
Mycobacteria sp.
Clostridlum perfringens3
- vegetative
- sporulated
Staphylococcus aureus
Sa Intone I la sp.
Shigella sp.
Yerslnla enterocolitica
Yerslnia intermedia
Campy Iobacter fetus ssp. jejunl
Candida albicans
Fluorescent Pseudomonas sp.
Klebsiella sp.
Viruses (pfu/mL)
Bacterlophage
Enterovlruses
130,000
120,000 81,000
18,000 51,000
<0.01
<0.01
<0.01
<0.01
>0.01
<0.01
<0.01
<3
<3
9,700
36,000
<3
<3
11,000
26,000
<300
9,500
30, 000
>0.1
<0.1
<0.01
<0.1
30, 000
350, 000
<0.01
<0.01
<0.01
>1,000
750
40, 000
HeLa, 5 day (uncorrected)
HeLa, polio-neutralized
RO, polio-neutralized
Po 1 1 ov I rus concentrat i on
efficiency (%)
Physical Analyses (mg/L)
Total organic carbon
Total suspended solids
Total volatile suspended solids
pH
a Most probable number (MPN)/mL.
b Toxic concentrate.
c Sample lost — tube broken during handling.
d A new procedure was used for detection of Salmonella spp
in July 1981.
e Not done; no pfu were recovered on HeLa mono layers.
0.058
0.012
0.007
83
59
49
7.5
. (Kaper et al.
f Beginning with samples collected on June 29-30, Fluorescent Psuedomonas
of the limited bacterial screen.
g HeLa cells used for the assay were contaminated; results
g 0.61
g 0.85
0.016
47 33
93 81
66 54
55 48
7.3 7.5
0.043
0.045
81
123
70
7.5
, Appl. Environ. Microbiol., 33:829-35,
sp. was substituted
for Staphy lococcus
0.008
<0. 002
92
89
27
25
7.6
1977) beginning
aureus as part
could not be obtained.
-------
LUBBOCK
HANCOCK RESERVOIR
WILSON
3 5
O
O
O
O
UJ
O
O
PO
oo
— TRICKLING FILTER
-PIPELINE
J J
I960
N JFMAMJJA
1981
FMAM JJAS
1982
SAMPLING MONTH
^EXTENDED DETENTION IN PIPELINE
5E 4
O
U.
J *
(9
O
CC
O
U.
O
(J
J
4
O
Ul
O
O
MJJ AS
1982
JJ
1980
JFMAMJJAS
1981
FMAMJJASO
1982
SAMPLING MONTH
SAMPLING MONTH
tu
vt
3
K
O
CC.
O
O
TRICKLING FILTER
PIPELINE
q
10
in
tL
>
O
ac
z
UJ
o
o
KJ
•t
•»r
(0
Ul
CO
o
IT
U
t-
UJ
o
O
J J
1980
N FMAMJJA
1382
SAMPLING MONTH
N JFMAMJ JA
1981
MJ JAS
1982
SAMPLING MONTH
SAMPLING MONTH
*HaLo CORRECTED FOR CONCENTRATION EFFICIENCY
Figure 5.1. Time series of fecal coliform and corrected enterovirus densities in Lubbock
pipeline, Hancock reservoir, and Wilson wastewater.
-------
LUBBOCK
250
J 200
5 150-
OC
O
100
50
- 250r
- 200-
(\J ti
ro o
rO OT
O
Ul
O
z
Ul
Q.
(0
ISO
100
50
JJ JFMAMJJA N FMAMJJAS
1980 1981 1982
SAMPLING MONTH
JJ N JFMAMJJA N FMAMJJAS
I98O 1981 [98J
SAMPLING MONTH
-TRICKLING FILTER
-PIPELINE
-TRICKLING FILTER
-PIPELINE
O
O
M
o
Ul
O
If.
Ul
£L
CO
CO
I
2SO
2OO
ISO
100
SO
HANCOCK RESERVOIR
WILSON
3
9
**
Z
o
OD
5
O
u
X
o
g
_J
<
o
250
200
ISO
100
90
O
-
-
-
-
V
1 1 1 1 1
1982
SAMPLING MONTH
MJJAS
1982
SAMPLING MONTH
^ 250
~ 200
O
o
3j .50
o
ce
o
o
100
SO
J
o
o
111
o
1.
Ul
a.
CO
3
U>
I
250-
200
ISO
100-
SO
TOTAL VOLATILE
SOLIDS (%)
C» -4 5
O 01 O
i, IOO
TRICKLING FILTER J
—-PIPELINE g
yVL^IrV i *°
~-— x /! ' 3
•/ > 25
JJ N JFMAMJJA N F MA M J J A S -1
1980 1981 1981 J*
SAMPLING MONTH £ 0
!f
1 1 1 1 1
MJJAS
1982
lOOr
75
50
JJ JFMAMJJA N FMAMJJASO
I98O 1981 1962
SAMPLING MONTH
JJ -JFMAMJJAS N FMAMJJASO
1980 1981 1982
SAMPLING MONTH
j j
1980
JFMAMJJAS N FMAMJJASO
1981 1982
SAMPLING MONTH
SAMPLING MONTH
Figure 5.2. Time series of physical analyses of Lubbock pipeline, Hancock
reservoir, and Wilson wastewater.
-------
TABLE 5.36. BACTERIAL SCREEN3—LUBBOCK, TEXAS
Sampl Ing date
Organisms (10^ cfu/mL)
Enterobacter 1 aceae
API— Group ld
Citrobacter amalonaticus
Cltrobacter dl versus
C i trobacter f reund 1 1
Enterobacter aerogenes
Enterobacter agglomerans
Enterobacter cloacae
Enterobacter sakazaki I
Escherichla col i
Escherichia coll alkalescens
Klebslel la oxytoca
Klebslel la ozaenae
Klebslel la pneumonlae
Morgana 1 la morgan 1 1
Prov 1 dene 1 a a 1 ca 1 1 f ac 1 ens
Provldencia rettgeri
Serratia liquefaclens
Serratia marcescens
Serratia rubldaea
Vibrio fluvialis
Yerslnla enterocol Itica
Yerslnla krlstensenl 1
Non-En terobacter i aceae
Achromobacter spp.
Achromobacter xylosoxldans
Aclnetobacter calcoaceticus
var. Iwoffi
Aeromonas hydrophila
A leal i genes sp.
API— Group ld
CDC Group 1 1 K-2
CDC Group V E-2
Chromobacterium sp.
Eikenella corrodens
F 1 avobacter 1 urn odoratum
Fluorescent Pseudomonas gp.
Pasteurel la multoclda
Pseudomonas cepacla
Pseudomonas fluorescens
Pseudomonas mat tophi Ma
Pseudomonas put Ida
Pseudomonas putrefaclens
Pseudomonas stutzerl
Pseudomonas sp., other
Vibrio alglnolytlcus
Jun
3-4
_
-
-
15
5
16
20
5
20
-
7
5
5
-
-
-
-
5
3
-
10
-
-
5
-
93
5
-
-
-
5
-
3
-
10
10
15
5
30
60
to
25
-
1980
Jul
28-29
_
-
-
-
10
10
30
-
20
-
-
-
-
-
-
-
-
-
-
-
-
-
-
20
-
560
10
-
-
-
-
-
-
-
-
10
10
-
-
20
-
140
-
1981
Nov
3-4
_
-
-
10
10
20
-
-
30
-
10
-
10
-
-
-
-
-
-
-
-
-
-
-
-
590
-
-
-
-
-
10
-
-
-
-
-
-
30
10
-
-
-
Apr
20-21
_
-
-
10
10
-
20
-
20
50
20
-
-
-
10
-
-
-
10
-
-
-
-
-
-
510
10
-
10
-
-
-
-
-
-
10
10
-
20
20
-
-
-
Jul
20-21
_
-
-
5
-
-
15
-
25
-
5
-
15
-
-
15
-
-
-
-
-
-
-
-
-
210
-
10
-
-
-
-
-
5
-
10
-
-
10
-
-
10
5
15-16
_
0.05
0.05
0.43
-
0.13
-
-
0.4
-
-
0.025
0.025
-
-
-
-
-
-
-
0.025
-
-
-
1.3
-
-
-
-
-
-
-
-
0.13
0.025
-
-
-
-
-
0.025
-
1982
Mar
22-23
1
-
-
4
-
-
2
-
4
-
4
-
2
1
-
-
-
-
-
-
-
-
3
5
-
8
1
-
-
-
-
-
1
-
-
-
1
-
3
-
-
8
-
Jul
26-27
_
-
-
6.6
3
10
16
-
10
-
3
-
6.6
-
3
-
3
-
-
56
-
-
-
10
3
150
10
3
-
10
6.6
-
-
-
-
20
-
-
-
-
-
-
-
a Highest levels observed on either MacConkey agar or brilliant green agar and Identified by
API 20E® biochemical tests.
b On February 15, 1982 the sample source was changed from the trickling filter on the
pipe!ine.
c Chior I nation of wastewater effluent at treatment plant.
d A group of organisms which to date have been described by CDC and have been designated
temporarily by API as API Group I.
233
-------
TABLE 5.37. BACTERIAL SCREEN3—HANCOCK RESERVOIR
Sampling date
Jul 26-27,
Organisms (103 cfu/nl) 1982
Enterobacterlaceae
Enterobacter cloacae 0.4
Klebsiella oxytoca 0.1
Klebsiella ozaenae 0.1
Non-Enterobacteri aceae
Achromobacter xylosoxidans 0.9
Acinotobacter calcoaceticus 0.2
var. Iwoffi
Aeromonas hydrophila 4.3
Alcall genes sp. 0.5
CDC Group V E-2 0.1
Pseudomonas sp. 0.5
Pseudomonas cepacia 0.1
Pseudomonas maltophilia 0.3
a Highest levels observed on either MacConkey agar or
brilliant green agar and identified by API 20E®
biochemical tests.
234
-------
samples has been observed primarily as a result of the smaller collection
system in the city of Wilson. Fecal coliform densities have ranged from
1()3 to 10^ cfu/mL over the three-year monitoring period. Total enterovirus
levels as enumerated on HeLa cell monolayers have varied from no virus
detected to in excess of 1.0 pfu/mL. . A relatively broad diversity of
bacteria were recovered during screens in 1980 (Table 5.38).
Of particular interest in the 1982 monitoring period were the
unusually high levels of polioviruses persisting in the Wilson wastewater
from March to May. Viruses did not appear in the Wilson sewage until three
weeks after the first Lubbock wastewater (also containing predominantly
polioviruses) was collected at the Hancock farm. Noteably, polioviruses 2
and 3 comprised most of the identified isolates from both sources.
Specific viral identifications of environmental isolates are provided
in Tables 5.39 and 5.40 (Lubbock effluent) and 5.41 and 5.42 (Wilson
effluent). In addition to the expected recovery of all three poliovirus
types, all five Coxsackie B viral serotypes have been recovered during the
course of this study as well as a diverse number of echoviruses. As
indicated by the differential assay results, polioviruses were prevalent in
pipeline effluent during the spring of 1982 with Type 2 predominating
(Table 5.40). Coxsackievirus B5 and selected echoviruses were recovered
also. During the months of June and July 19829 Coxsackievirus B5 was
prevalent in water irrigated from the pipeline. Similar observations were
made in Wilson wastewater with poliovirus 2 comprising a significant number
of the 1982 spring isolates (Table 5.42) and Coxsackievirus B5 appearing at
elevated levels in both June and September 1982 samples.
In reviewing the occurrence of specific viral types during baseline
monitoring it is interesting to note that as in 19829 polioviruses 2 and 3
were predominant in Lubbock wastewater in April 1981, while Coxsackievirus
B5 represented a large proportion of the isolates recovered in June and
July 1981. Insufficient information exists to allow similar evaluations to
be made concerning the seasonal occurrence of specific enteroviruses within
the Wilson community.
Search for Legionella Isolates from 24-Hour Composite Samples-
Table 5.43 lists a summary of results from UI efforts to isolate
Legionella from wastewater samples. Isolates of these agents have not been
recovered from any of the seven samples processed, although antigens from a
variety of serogroups and species have repeatedly been demonstrated in
wastewater samples and in tissues of guinea pigs inoculated with those
samples. Most isolates of potential Legionella group agents grew readily
on TSA or blood agar. One isolate, not growing on TSA in the UI
laboratory, was forwarded to the Illinois Department of Public Health
Bacteriology Laboratory where it grew on a number of media, including TSA,
suggesting the isolate was not Legionella.
235
-------
TABLE 5.38. BACTERIAL SCREENa--WILSON, TEXAS
Organism
Sampling Date
Jun 3-4. 1980 Jul 28-29, 1980
ENTEROBACTERIACEAE (103 cfu/mL)
Citrobacter diversus
Citrobacter freundii
Citrobacter sp., other
Enterobacter agglomerans
Enterobacter cloacae
Enterobacter sakazakii
Escherichia coli
Hafnia alvei
Klebsiella oxytoca
Klebsiella ozaenae
Klebsiella pneumoniae
Serratia liquefaciens
Serratia rubidaea
Yersinia enterocolitica
NON-ENTEROBACTERIACEAE (103 Cfu/mL)
Achromobacter sp.
Achromobacter xylosoxidans
Aeromonas hydrophila
Alcaligenes sp.
CDC Group II K-2
Eikenella corrodens
Morgenella morgani
Pasteurella multocida
cepacia
fluorescens
putida
putrefaciens
sp.t other
Pseudomonas
Pseudomonas
Pseudomonas
Pseudomonas
Pseudomonas
5
30
5
20
30
5
40
5
55
5
5
10
5
5
150
5
5
20
5
5
15
15
15
25
45
10
30
30
90
10
20
120
20
10
50
50
Highest levels observed on either MacConkey agar or brilliant green agar
and identified by API 20E® biochemical tests.
236
-------
TABLE 5.39. VIRUSES ISOLATED FROM LUBBOCK EFFLUENT DURING BASELINE YEARS3
ro
GO
Assay
HeLa (unaltered concentrate)
Concentration (pfu/L)
Virus type
Polio 1
Polio 2
Po 1 1 o 3
Coxsackie A1
Coxsackle A7
Coxsackie A16
Coxsackle B1
Coxsackie B3
Coxsackie B4
Coxsackie B5
Echo 1
Echo 3
Echo 6
Echo 11
Echo 14
Echo 21
Echo 24
Echo 25
Echo 30
Unidentified
TOTAL SAMPLED
HeLa (polio-neutralized)
Concentration (pfu/L)
Virus Type
Polio 2
Coxsackie B3
Coxsackie B5
Echo 14
Unidentified
TOTAL SAMPLED
RD (polio-neutralized)
Concentration (pfu/L)
Virus type
Coxsackie A16
Coxsackle B4
Echo 5
Echo 7
Echo 1 1
Echo 12
Echo 13
Echo 15
Echo 19
Echo 20
Echo 24
Echo 27
Echo 31
Unidentified
TOTAL SAMPLED
a Plaque forming units on cell
b Labeling error precluded sep
1980
Jun 3-4 Jul 28-29 Nov 3-4
780 1,200 730
2
20 14 16
3
19 24
1
1
1
4
2
1
1
21 2
81 18 20
300
19
19
mono layers.
arating neutralized/unaltered viruses.
Samp 1 1 ng Date
Apr 20-21
57
1
16
7
1
25
18
4
2
6
8
2
2
4
198
Jun 15-16
u.
100b
1
1
3
4
1
25
1
1
1
4
42
^
150
1
1
3
1
5
11
1
Jul 20-21
65
6
11
4
21
20
11
11
93
1
6
4
1
3
1
2
18
Aug 17-18
45
3
4
4
1
1
13
5.3
1
1
1
1
4
420
5
2
1
1
1
1
1
1
16
-------
TABLE 5.40. VIRUSES ISOLATED FROM LUBBOCK PIPELINE EFFLUENT DURING 1982
no
CO
00
Samp 1 inq Date
1982
Assay
HeLa (unaltered concentrate)
Concentration (pfu/L)
Virus type
Polio 1
Polio 2
Polio 3
Coxsackle B2
Coxsackie B4
Coxsackle B5
Echo 11
Unidentified
TOTAL SAMPLED
HeLa (polio-neutralized)
Concentration (pfu/L)
Virus Type
Po 1 1 o 3
Coxsackle B5
Echo 1
Echo 31
Unidentified
TOTAL SAMPLED
RO (po no-neutralized)13
Concentration (pfu/L)
Virus type
Coxsackle A16
Coxsackle A19
Coxsackie B5
Echo 12
Echo 15
Unidentified
TOTAL SAMPLED
a Ch 1 or 1 nation of wastewater effluent
b Identification of plaques selected
Mar
8-9a
110
3
6
2
1
6
18
22
1
1
1
6
9
Incomplete
at treatment
from RD monolc
Mar
22-23
63
1
4
3
1
1
10
4.0
Incomplete
10
Incomplete
plant.
Apr
5-6
17
8
1
9
3.9
1
1
2
44
2
1
1
10
14
lyers is Incomplete due to
Apr
19-20
42
3
6
2
7
2
20
16
5
1
6
10
1 ncomp 1 ete
problems in the
Jun
29-30
490
23
23
390
11
11
56
1
1
4
7 .
13
maintenance
Jut
26-27
60
1
2
5
1
9
30
6
6
6.6
3
3
of this
Sep
13-14
22
1
2
2
1
3
2
11
8.0
4
-
4
1 ncomp lete
ce 1 1 lines! nee
September 1982.
-------
TABLE 5.41. VIRUSES ISOLATED FROM WILSON EFFLUENT DURING BASELINE YEARS3
Assay
Samp I Ing date
1980
1981
Jim 3-4 Jul 28-29 Jim 15-16 Aug 17-18
HeLa (unaltered concentrate)
Concentration (pfu/L)
Virus type
Polio 1
Polio 2
Polio 3
Coxsackle A10
Coxsackle B3
Echo 2
Echo 25
Unidentified
TOTAL SAMPLED
HeLa (polio-neutralized)
Concentration (pfu/L)
Virus type
RO (polio-neutralized)
47
2
16
1
1
5
25
15,000
<1
12
15
13
12
<1.4 1.0
Incomplete
Concentration (pfu/L)
Virus type
Polio 2
Coxsackie A9
Echo 5
Echo 27
Echo 31
Unidentified
TOTAL SAMPLED
75
1
5
1
1
1
9
1,500
1
6
7
a Plaque forming units on cell mono layers.
239
-------
TABLE 5.42. VIRUSES ISOLATED FROM WILSON EFFLUENT DURING 1982
Assay
Mar
8-9
Samp I Ing date
Apr
5-6
1982
Jun
29-30
Aug
9-10
Sep
13-14
HeLa (unaltered concentrate)
Concentration (pfu/L)
Virus type
Polio 1
Polio 2
Polio 3
Coxsackie B5
Echo 11
Echo 24
Unidentified
TOTAL SAMPLED
120
1
10
8
1500
1
23
19
25
34
1
1
10
12
58
8
3
6
3
1
2
1
24
610
1
20
4_
25
HeLa (polio-neutralized)
Concentration (pfu/L)
Virus type
PoIi o 2
Coxsackie 84
Coxsackie B5
Echo 11
Unidentified
TOTAL SAMPLED
RO (polio-neutralized)3
<2
85
4
1
5
36
2
10
13
12
850
14
14
Concentration (pfu/L)
Virus type
Echo 13
Unidentified
TOTAL SAMPLED
12 b
Incomplete
36
3
__3
6
6.6
1
1
1 ncomp 1 ete
a Analysis of samples using RD cells Is Incomplete due to problems In maintaining this cell
line since September 1982.
b Toxic sample.
240
-------
TABLE 5.43. SPECIES OF LEGIONELLA DETECTED IN WASTEWATER SAMPLES BY DIRECT FLUORESCENT ANTIBODY
STAINING OF THE ORIGINAL SAMPLES OR TISSUES FROM GUINEA PIGS INOCULATED WITH THOSE SAMPLES
L.
L. pneumophila L. L. L. L. longbeacheae
Sample 123456 bozemannii dumoffii gormanii micdadei 1 2
February 16, 1982
Pipeline effluent + - - + NA NA - - - NA NA NA
March 22-23, 1982
Trickling filter + - - + NA NA - + + NA NA NA
Pipeline effluent + - - - NA NA - + + NA NA NA
June 29-30, 1982
Pipeline effluent + - - + NA NA + + - NA NA NA
Reservoir + - - + NA NA - + - NA NA NA
July 26-27, 1982a
ro
£ Pipeline effluent __ + + + + + + + + + +
Reservoir -- + + -- - + + - + • -
NA - Conjugates not available.
a Examination of wastewater sample only.
-------
The UI experience in isolation attempts of Legionella from aqueous
samples appears not to be unusual; others have also been unable to recover
viable Legionellae from DFA positive samples. Factors influencing the
inability to recover Legionella include the susceptibility of experimental
animals to Legionella infection, viability and virulence of Legionella
present in wastewater samples, and the levels of both Legionella-group and
non-Legionella agents present in those samples.
The inability to consistently recover L_. pneumophila from guinea pigs
inoculated with up to 10^ cfu of yolk-sac passed stock cultures suggests
that there may be differences in susceptibility or that extremely high
doses of Legionella are needed to infect some animals. The difference in
lethal doses of Legionella pneumophila in egg-passed and agar-passed
cultures, reported by McDade and Shepard (1979) suggests facultative
differences in virulence factors and it is possible that the Legionella
observed in Lubbock wastewater samples are relatively avirulent. It is
also possible that these agents are nonviable, since isolates were not
recovered from samples inoculated onto artificial media. The low levels of
Legionella and high levels of non-Legionella present in the samples have
undoubtedly influenced the results. Isolation work using guinea pigs
involves a trade-off between concentrating samples sufficiently to obtain
infectious doses of Legionella consistently and diluting samples to
nonlethal levels of other agents.
30-Minute Composite Samples--
Composite wastewater samples generally of 30 minutes duration were
collected in 1982 during each microorganism, virus and quality assurance
aerosol run and assayed for the microorganisms monitored in the aerosol,
for enteroviruses, and for selected physical-chemical parameters. Results
of these analyses are presented for pipeline wastewater during the
preplanting irrigation (Table 5.44) and during the summer crop irrigation
(Table 5.45) and for reservoir wastewater during the crop irrigation (Table
5.46).
The 30-minute composite wastewater samples had similar values of all
monitored parameters to those observed in the 24-hour composite samples for
the same wastewater source. Thus, the aerosol sampling data should be
representative of the microorganism levels in air generated by the
irrigation system in 1982. Because aerosol sampling was conducted daily
during some weeks, the 30-minute composite samples provide an indication of
daily variability. The enterovirus level (5-day assay on HeLa cells) in
the pipeline water was markedly elevated during the 2-day period when Virus
Run V3 was conducted (August 3-4, 1982). The polio-neutralized enterovirus
assays indicate that over 90% of the enteroviruses in the pipeline
wastewater on these days were polioviruses. Differential assays also
242
-------
TABLE 5.44. WASTEWATER SAMPLES COLLECTED DURING 1982 AEROSOL MONITORING (30 MINUTE COMPOSITES)
WASTEWATER FROM PIPELINE DURING PREPLANTING IRRIGATION
ro
-P»
co
Sampling date/aerosol run
Parameter
Bacteria (cfu/mL)
Fecal conforms
Fecal streptococci
Clostridium perfringens3
Vegetative
Sporulated
Mycobacterla sp.
Viruses (pfu/mL)
Bacterlophage
Enterovi ruses (uncorrected)
HeLa, 5 day
HeLa , po 1 1 o-neutra 1 1 zed
RD, po 1 1 o-neutra 1 1 zed
Pollovlrus concentration
efficiency (?)
Physical Analyses (mg/L)
Total organic carbon
Total suspended solids
Total volatile suspended
solids
Sample conditions
pH
Temperature (°C)
Feb 22
Ml
1 00, 000
4,400
16,000
1,200
0.054
0.015
0.051
57
135
147
121
6.9
2
Feb 23
M2
1,000,000
7,200
360
360
1,500
0.093
0.024
0.012
49
161
182
152
6.8
3
Feb 24
M3
1 1 0, 000
6,300
45, 000
1,400
0.047
Incomplete
Incomp lete
76
168
217
176
7.0
8
Mar 15
01
51,000
4,800
13,000
1,100
0.067
0.0084
0.034
60
92
87
74
7.2
9
Mar 16
VI
81,000
4,500
20, 000
840
0.16
0.035
0.030
Lost
100
101
90
7.4
9
Mar 17
M4
39,000
1,900
29,000
530
0.11
0.022
0.067
64
158
245
207
7.0
4
Mar 18
M5
57,000
5,800
13,000
1,100
0.12
0.047
Incomplete
49
128
92
90
7.1
2
Mar 19
M6
68,000
16,000
15,000
940
0.028
0.0023
Incomplete
63
164
185
160
7.1
5
Most probable number (MPN)/mL
-------
TABLE 5.45. WASTEWATER SAMPLES COLLECTED DURING 1982 AEROSOL MONITORING (30 MINUTE COMPOSITES)
WASTEWATER FROM PIPELINE DURING SUMMER CROP IRRIGATION
Parameter
Bacteria (cfu/tnL)
Fecal col 1 forms
Feca 1 streptococc 1
Jul 7
M7a
44, 000
4,200
Jul 8
MS
31,000
3,200
Samp 1 ing
Jul 13
Q2
50,000
3,600
date/aerosol run
Jul 14 Jul 15
Mil M12
13,000 76,000
4,600 5,600
Aug 2
V2
180,000
2,000
Aug 3
M14
37,000
4,900
CI ostr i d i ufn perfringens
Vegetat i ve
Sporulated
Mycobacteria sp.
Viruses (pfu/mL)
100,000
550, 000
250
11,000
10, 000
4,000
5,300
Bacterlophage
Enterovi ruses (uncorrected)
HeLa, 5 day
HeLa , po 1 i o-neutra 1 1 zed
RD, polio-neutralized
Po 1 1 ov i rus concentrat i on
efficiency (%)
Physical Analyses (mg/L)
Total organic carbon
Total suspended solids
Total volatile suspended
solids
Sample conditions
PH
Temperature (°C)
1,700
0.54
0.47
0.17
64
128
307
213
7.0
1
930
0.51
0.55
0.26
57
92
213
161
7.3
10
720
0.14
0.067
0.020
39
76
82
66
7.4
3
16
0.013
0.002
0.016
50
51
67
54
7.6
3
1,100
0.078
0.097
0.018
49
80
170
119
7.6
5
880
0.10
0.10
0.004
80
52
79
62
7.4
2
1,900
1.5
0.10
0.011
Incomplete
71
86
68
7.5
2
continued...
-------
TABLE 5.45. (CONT'D)
no
en
Sampling date/aerosol run
Parameter
Bacteria (cfu/mL)
Fecal col i forms
Feca 1 streptococc 1
Clostrldium perf rlngens*5
Vegetative
Sporulated
Mycobacterla sp.
Aug 4
V3
5,600
2,600
2,300
Aug 5
M15
30, 000
2,700
6,000
Aug 23
M17C
16,000
300
460
200
Aug 24
V4C
93
Lost
<5.0
5.0
Aug 25
M18
29, 000
830
360
190
Aug 27
M200
360
10
93
230
Viruses (pfu/mL)
Bacteriophage
Enterovl ruses (uncorrected)
HeLa, 5 day
HeLa, polio-neutralized
RD, polio-neutralized
Poliovirus concentration
efficiency (?)
Physical Analyses (mg/L)
Total organic carbon
Total suspended solids
Total volatile suspended
solids
Sample conditions
pH
Temperature (°C)
1,600
2.2
0.060
0.020
Incomplete
66
93
75
7.5
3
1,200
0.21
0.080
0.022
94
65
69
57
7.8
2
820
0.39
0.34
0.34
Incomplete
58
62
49
7.2
2
150
0.066
0.051
0.008
85
61
58
47
7.3
3
2,100
0.10
0.11
0.28
Incomplete
46
48
36
7.4
3
140
0.044
0.13
0.28
Incomplete
-
63
49
41
7.3
3
a Presumed pipeline source based on microbial parameters.
b Most probable number (MPN)/mL.
c Chlorinated.
-------
rvs
•p»
en
TABLE 5.46. WASTEWATER SAMPLES COLLECTED DURING 1982 AEROSOL MONITORING (30 MINUTE COMPOSITE)
WASTEWATER FROM RESERVOIR DURING SLIMMER CROP IRRIGATION
Parameter
Bacteria (cfu/mL)
Fecal collforms
Fecal streptococci
Jul 9
M9
230
30
Sampl
Jul 11
M10
40
13
ing date/aerosol run
Jul 16 Aug 6
M13 M16
1,100 450
53 3.0
Aug 26
M19
750
3.0
Clostridium perfringens3
Vegetative
Sporulated
Mycobacteria sp.
Viruses (pfu/mL)
Bacterlophage
Enteroviruses (uncorrected)
430
100
1.2
0.40
230
15
10
2.4
3.0
5.3
HeLa, 5 day
HeLa, polio-neutralized
RD, polio-neutralized
Poliovirus concentration
efficiency (?)
Physical Analyses (mg/L)
Total organic carbon
Total suspended solids
Total volatile suspended
solids
Sample conditions
pH
Temperature (°C)
0.034
0.002
<0.002
61
19
26
26
8.2
5
0.002
< 0.002
<0. 002
71
16
27
24
8.0
1
0.004
0.013
0.002
52
16
21
19
7.8
8
0.12
0.008
0.002
108
43
35
35
8.5
2
8.7
0.006
< 0.002
Incomplete
-
17
12
12
7.9
2
a Most probable number (MPN)/mL.
-------
indicate that the predominant enteroviruses in the sprayed pipeline
wastewater were polioviruses during the preplanting irrigation and
nonpoliovirus during the summer irrigation (except August 3-5), consistent
with the 24-hour composite results. As expected, chlorination at the
Lubbock treatment plant reduced bacterial,indicator levels in the sprayed
pipeline wastewater but had no apparent effect on enteric virus levels.
Microorganism Levels in Air
Aerosol sampling data from the dye, particle size, background,
microorganism, and virus runs follow. Data from the quality assurance runs
were presented in the Aerosol Measurement Precision section of Methods and
Materials.
Aerosolization Efficiency--
Four dye runs were conducted to provide estimates of the
aerosol ization efficiency (i.e., the fraction of wastewater aerosolized
during application) of the center pivot sprinkler system at the Hancock
farm. During injection of the Rhodamine dye, wastewater grab samples were
collected at 1-minute intervals and assayed to determine the source
strength of the dye. The dye concentrations in wastewater are presented in
Table 5.47. Sampling was conducted only for minutes when dye was visible
in the sprayed wastewater. The dye concentrations sampled in air are
presented in Table 5.48. The lowest dye concentrations in air exceeded the
method detection limit of 0.2 x 10~° yg/m^ by a factor of 2. These data
will be input into a diffusion model to calculate the aerosolization
efficiency during each of the four dye runs.
Aerosol Viable Particle Size--
The distribution of sizes of all the viable particles able to
reproduce on standard plate count agar was determined upwind and at three
downwind distances from the irrigation nozzle line using six-stage Andersen
samplers. The relationship of the particle size ranges sampled by the six
stages to usual site of deposition when inhaled into the human respiratory
system is depicted in Figure 5.3. The data from the five particle size
runs are presented in Table 5.49. Fungal spores and aggregate organisms
frequently yielded plates which could not be counted and were reported as
TNTC (too numerous to count). To permit interpretation, the TNTC values
were inferred either as large densities or as probable fungal
contamination, based on the values of the corresponding stage from the
paired sampler and of adjoining stages. The upwind data exhibit little
consistency with regard to viable particle size. For most runs, the
density of large viable particles in air decreased with increasing downwind
distance from the irrigation nozzle line. The density of small viable
particles sometimes increased with downwind distance. These patterns are
247
-------
TABLE 5.47. SOURCE STRENGTH OF RHODAMINE IN WASTEWATER DURING DYE RUNS
Dye
Run
Dl
D2
D3
D4
Rhodamine concentration in wastewater
Min 0 Min 1
96 126
53 94
112 119
95 111
TABLE 5.48.
Min 2 Min 3 Min 4
183
93
118
109
RHODAMINE
95
91
108
112
AEROSOL
10
91
110
115
Min 5
12
91
102
113
CONCENTRATION
Rhodamine concentration
Dye
run
Dl
D2
D3
D4
Tower
3
5
6
4
6
4
5
3
Near
(Dist)
(31
(40
(25
(25
(25
(25
(40
(40
pairs
L
m) 22
m)
1.1
m) 80
m)
m)
m)
m)
m)
1.9
2.3
3.7
3.7
2.5
sample, mg/L
Min 6
25
87
99
105
DURING
Min /
88
6.7
' Min
9.0
8
DYE RUNS
in air, 10~6
yg/m3
Far pairs
4
0
0
7
9
0
6
2
R
.5
.89
.46
•5
.7
.47
.3
.4
(Dist)
(81 m)
(115 m)
(75 m)
(75 m.)
(75 m)
(75 m)
(80 m)
C80 m)
L
0.
1.
0.
2.
0.
1.
1.
1.
38
1
67
3
71
9
3
0
R
1.
0.
0.
1.
0.
0.
2.
1.
5
96
87
3
50
79
4
8
248
-------
STAGE 1
7 microns & above
STAGE 2
4.7-7
pharynx
STAGE 3
3.3-4.7
STAGE 4
2.1-3.3
secondary
bronchi
STAGE 5
1.1-2.1
STAGE 6
O.65—1.1
terminal
bronchi
alveoli
trachea A primary
bronchi
Figure 5.3. Particle sizes of the Andersen sampler stages are
designed to simulate deposition in the human respiratory system
249
-------
TABLE 5.49. SAMPLED STANDARD PLATE COUNT IN AIR BY PARTICLE SIZE
PO
in
o
Run no.
Run date
Run time
PI
2-23-82
1609-1619
P2b
3-16-82
1539-1549
P3
7-8-82
1510-1518
P4
7-14-82
1519-1527
Andersen Range of
sampler particle
stage sizes (y)
1
2
3
4
5
6
1
2
3
4
5
6
1
2
3
4
5
6
1
2
3
4
5
6
>7.0
4.7-7.0
3.3-4.7
2.1-3.3
1.1-2.1
0.65-1.1
>7.0
4.7-7.0
3.3-4.7
2.1-3.3
1.1-2.1
0.65-1.1
>7.0
4.7-7.0
3.3-4.7
2.1-3.3
1.1-2.1
0.65-1.1
>7.0
4.7-7.0
3.3-4.7
2.1-3.3
1.1-2.1
0.65-1.1
Standard plate
count concentration in air by particle size, cfu/m^
Upwind
L
81
47
68
72
60
TNTC
260
60
22
78
95
22
16
27
38
38
11
32
110
5
5
<1
5
16
R
170
65
86
65
60
TNTC
94
64
64
47
210
43
370
330
400
180
290
37
_c
<1
37
>540
37
11
L
36
260
130
240
300
180
82
33
1700
1200
1300
390
190
29
20
1080
1200
340
120
95
5
35
1200
660
TNTC
290
46
10
Downwind
R
m
240
190
540
140
140
100
m
2300
2500
1500
650
100
20
m
TNTC
1300
650
87
130
<1
m
520
550
550
150
45
110
of irrigation nozzle line
L
61
200
140
280
TNTCa
220
70
58
210
210
960
130
43
TNTC
45
TNTC
380
240
68
15
10
60
410
390
300
83
15
29
R
m
280
150
70
280
250
90
m
1700
540
1200
920
180
78
m
1500
590
200
87
110
<1
m
390
690
370
150
31
CS
L
75
290
110
240
170
TNTC
74
83
290
920
450
190
120 .
TNTC
70
350
180
59
74
53
48
85
280
64
160
43
37
64
R
m
140
150
180
170
170
TNTC
m
110
140
180
120
TNTC
TNTC
m
528
169
77
67
56
10
m
640
180
360
72
82
5
continued..,
-------
TABLE 5.49 (CONT'D)
ro
en
Run no.
Run date
Run time
P5
8-25-82
1730-1738
Andersen Range of
sampler particle
stage sizes (y)
1
2
3
4
5
6
>7.0
4.7-7.0
3.3-4.7
2.1-3.3
1.1-2.1
0.65-1.1
Standard plate
count concentration in air by particle size, cfu/m3
Upwi nd
L
1000
160
TNTC
150
100
59
R
410
540
11
150
11
160
L
35
TNTC
TNTC
1420
880
310
26
Downwind
R
m
TNTC
TNTC
TNTC
490
150
5
of irrigation nozzle line
L
60
600
400
640
190
130
180
R
m
880
1000
250
160
87
26
L
85 m
640
520
370
140
140
43
R
490
630
270
250
110
31
CS - fungal contamination
a TNTC - either too numerous to count (>2500 cfu/m3 for PI to P3; >1500 cfu/m3 for P4 and P5) or
fungal contamination
b Standard plate count of wastewater = 5.1 x 108 cfu/mL
c Sample lost.
-------
consistent with gravitational settling of heavy low-energy particles and
size reduction through drying or desiccation in the sprinkler aerosol.
Background Runs--
The outdoor air near but in an upwind direction from the homes of
eight participant households was monitored in summer before any irrigation
commenced to measure ambient microorganism levels in the vicinity of homes.
A ninth sampler was located downwind on the Wilson effluent pond to
determine if it was a source of aerosolized microorganisms.
Four background aerosol runs were conducted in nine locations in the
study area before sunrise on the mornings of August 5 through August 8,
1980. A detailed description of the methodology, sampler locations and
sampling conditions are contained in the Methods Section. All runs were
conducted at the same time of day, same season, and with the same wind
direction to minimize sources of variability.
The sampled densities of the standard plate count, fecal coliforms,
fecal streptococci, mycobacteria, and coliphage in the ambient air during
the four background runs are presented in Table 5.50. The Wilson effluent
pond does not appear to be an appreciable source of aerosolized
microorganisms. Geometric means calculated over the four runs are provided
in Table 5.51 to estimate ambient microorganism levels just upwind of
homes.
Fecal coliforms were only detected in 1 of the 30 air samples near
homes (at location F). Assuming there is a constant background level near
homes throughout the study area, this background level of fecal coliforms
is estimated as 0.01 cfu/m3. AS anticipated, no coliphage were detected in
the 80 air samples near homes, yielding a coliphage background level below
0.005 pfu/m3. Mycobacteria were detected in 9 of the 30 air samples near
homes for an estimated background level of 0.05 cfu/m3. Standard plate
count, monitored as a positive control, indicated that background bacterial
concentrations in the air near homes was about 450 cfu/m3.
Fecal streptococci were surprisingly prevalent in these background air
samples and were found in 27 of the 30 air samples near homes, at
concentrations ranging from 0.1 cfu/m3 to 11 cfu/m3. Geometric mean air
concentration of fecal streptococci ranged from about 0.2 cfu/m3 at
locations D, E, G, and H to 2 cfu/m3 at location A. The Wilson sites (0.87
cfu/m3 geometric mean) appear to differ from the rural sites (0.32 cfu/m3
geometric mean), with locations A, C, and F having higher air levels of
fecal streptococci than the other locations.
The sources of the aerosolized fecal streptococci and mycobacteria are
unknown. The prevalence and wide distribution of fecal streptococci
densities in air between about 0.1 cfu/m3 and 1 cfu/m^ suggests a normal
252
-------
TABLE 5.50. MICROORGANISM DENSITIES IN AIR ON BACKGROUND AIR RUNS
Back-
ground
run Wilson Wilson Wilson
no. A B C
Standard Plate Count (cfu/m3)
Bl - 1150 260
B2 530 680 CS
B3 1050 CS 500
B4 CS 430 630
Fecal Coli forms (cfu/m3)
Bl - <0.4 <0.1
B2 <0.1 <0.1 <0.4
B3 <0.1 <0.2 <0.1
B4 <0.1 <0.1 <0.2
Fecal Streptococci (cfu/m3)
Bl - 0.5 8.0
B2 0.9 0.3 2.1
B3 0.7 <0.1 0.3
B4 11 0.6 0.3
Mycobacteria (cfu/m3)
Bl - <0.2 0.1
B2 <0.1 0.1 <0.3
B3 <0.1 <0.1 0.1
B4 <0.1 <0.1 <0.1
Coliphage (pfu/m3)
Bl - <0.4 <0.1
B2 <0.2 <0.1 <0.4
B3 <0.1 <0.2 <0.1
B4 <0.1 <0.1 <0.2
Samp
ler location
Ef f 1 uent
pond
D
1900
430
370
73
<0.
<0.
<0.
<0.
<0.
0.
0.
0.
<0.
<0.
<0.
3.
<0.
<0.
<0.
<0.
2
1
3
1
1
3
2
3
1
1
2
4a
2
1
3
2
Rural
• E
2800
1220
280
65
<0.
<0.
<0.
<0.
<0.
0.
0.
0.
<0.
0.
<0.
<0.
<0.
<0.
<0.
<0.
3
2
1
1
2
2
2
3
2
1
1
1
3
2
1
1
Rural
F
CS
990
1030
130
<0
<0
0
<0
1
1
2
1
<0
<0
<0
<0
<0
<0
<0
<0
.1
.3
.3
.1
.1
.3
.3
.5
.1
.2
.1
.1
.2
.4
.1
.1
Rural
G
390
CS
-
60
<0.
<0.
-
<0.
0.
0.
-
0.
0.
<0.
-
0.
<0.
<0.
-
<0.
1
2
1
1
3
2
1
1
1
1
2
1
Rural
H
190
3500
200
CS
<0.2
<0.2
<0.1
<0.2
<0.1
0.3
<0.1
0.8
0.1
<0.1
<0.1
<0.1
<0.2
<0.2
<0.1
<0.2
Rural
I
CS
450
260
500
<0.
<0.
<0.
<0.
0.
0.
2.
0.
0.
<0.
0.
<0.
<0.
<0.
<0.
<0.
4
1
4
1
3
1
4
2
3
1
5
1
4
1
4
1
- - No sample collected.
CS - Contaminated sampler (presumptive).
a Cows grazing approximately 300 to 500 m upwind from sampling site.
253
-------
TABLE 5.51. GEOMETRIC MEAN AIR LEVELS SAMPLED ON BACKGROUND RUNS
ro
in
-P.
Background microorganism concentration in air
Sampler location
(near participant home)
Wilson-A
Wilson-B
Wilson-C
Wilson Effluent Pond-D
Rural (Hancock) -E
Rural (NE)-F
Rural (SE)-G
Rural (SW)-H
Rural (NW)-I
Wilson (Geometric) Mean
Rural (Geometric) Mean
Estimated Area Background
(A- I, excluding D, Geometric
Mean)
Standard
plate count
(cfu/m3)
750
700
430
390
500
510
150
510
390
610
380
450
Fecal
col i forms
(cfu/m3)
<0.03
<0.04
<0.04
<0.04
<0.04
0.09
<0.04
<0.04
<0.04
<0.01
0.02
0.01
Fecal
streptococci
(cfu/m3)
2
0.3
1.1
0.2
0.2
1.5
0.2
0.2
0.3
0.87
0.32
0.47
Mycobacteria
(cfu/m3)
<0.03
0.04
0.07
0.4
0.04
<0.03
0.07
0.04
0.2
0.04
0.06
0.05
Coliphage
(pfu/m3)
<0.04
<0.04
<0.04
<0.04
<0.04
<0.04
<0.04
<0.04
<0.04
<0.012
<0.008
<0.005
Note: < x cfu/m3 implies none detected in any samples at this location.
-------
background of this order of magnitude throughout the study area. Further,
there is no known feed lot or similar operation south or southeast of the
Wilson area which might produce the observed effect. High air levels of
fecal streptococci were observed consistently at locations A and F and
occasionally at C. Twelve of the fecal streptococci colonies from the
first air sample at location C (8 cfu/m3)'were characterized: four were
classified as j^. durans, which may be of human origin, and eight were
categorized as _S. bovis or _S. equinus, which are more likely of animal than
human origin. A plausible hypothesis is that the passage of air through
Wilson elevates the levels of aerosolized fecal streptococci of both human
and animal origin. The data at location F suggests there also are
comparable isolated local sources in some rural areas.
A high level of mycobacteria (3.4 cfu/m3) was observed on the fourth
air sample taken downwind of the Wilson effluent pond (location D); cows
were grazing approximately 300 to 500 m upwind during this sampling.
Representative mycobacteria colonies from this sample were speciated. All
isolates tested belonged to the "M^. avium complex," consisting of M_. avium
and M_. intracellulare, of Runyon group III. Traditionally, these species
are the major disease-associated strains of Runyon group III and hence are
classified as pathogens.
Microorganism Runs—
The densities of microorganisms in the air upwind and at four
distances downwind from the irrigation nozzle line were determined during
20 microorganism runs. The wastewater density and the sampled densities in
air of fecal coliforms, fecal streptococci, mycobacteria, Clostridium
perfringens, and coliphage are presented, respectively, in Tables 5.52
through 5.56. These data are segregated into three groups, based on the
source of wastewater and irrigation period: 1) preplanting irrigation with
pipeline wastewater direct from the Lubbock sewage treatment plant, 2)
summer crop irrigation with pipeline wastewater, and 3) summer crop
irrigation with wastewater stored in a reservoir.
These air data provide convincing evidence that sprinkler irrigation
of wastewater directly from the pipeline was a substantial source of each
of the monitored microorganism groups under most conditions of actual
operation of the irrigation system. The air densities within 100 meters
downwind of pipeline irrigation were markedly elevated above upwind levels,
ranging from two orders of magnitude elevation for mycobacteria to four or
more orders of magnitude elevation for fecal coliforms. Under some
conditions, particularly at night or with high wind speeds (>7 m/sec),
sprinkler irrigation of pipeline wastewater appeared to elevate the ambient
(upwind) density in air of fecal coliforms, fecal streptococci,
Clostridiuim perfringens, and coliphage to at least 400 meters downwind and
of mycobacteria to about 300 meters downwind.
255
-------
TABLE 5.52. SAMPLED FECAL COL I FORM DENSITIES ON THE MICROORGANISM AEROSOL RUNS
Fecal
col iform
Aeroso 1 concentrat I on
run i n wastewater
number (cfu/mL)
WASTEWATER
Ml
M2
M3
M4
M5
M6
WASTEWATER
M7b
M8
Mil
M12
M14
S M15
°^ M17c'd
M18d
M200
WASTEWATER
M9
M10
M13
M16
M19
FROM PIPELINE
1 00, 000
1 , 000, 000
1 1 0, 000
39,000
57,000
68,000
FROM PIPELINE
44,000
31,000
13,000
76, 000
37,000
30, 000
16,000
29, 000
360
Feca I co I i
Upwind of
irrigation
rig 20-39 m 40-59 m
DURING PREPLANTING IRRIGATION
<0.2 <0.2 >250
<0. 1 CS 150
<0.2 <0.4 190
<0. 1
<0. 1
<0.2
<0.1
<0.1
<0.1
DURING SUMMER CROP
<0.3
<0.2
<0.3
<0.3
CS
<0. 1
<0.2
<0.2
CS
<0.7
<0.3
<0.3
CS
<0. 1
<0. 1
<0.2
<0.2
<0.2
0.2a
120
IRRIGATION
140
900
form concentration in air
Ccfu/m3 of air)
Downwind of irrigation nozzle line
60-89 m
>250
110
330
133a
120
49
83
137
90-149 m
21 15
2.3 2.1
36 26
<0.3 <0. 1
CS
7.7 4.3
14 10
0.3 1.2
CS
37
<0.4
<0.2
0.5
0.4
150-249 m
3.2
<0. 1 <0. 1
40 13
<0 1 <0 1
16* 15*
CS 0.1
3.7 3.2
<0.3 <0.3
<0.3
70
0.2
<0.2
2.7
0.3
250-349 m
3.5
<0.4
0.1
<0. 1
<0. 1
3.5
3.8
<0.6
<0.3
<0. 1
CS
CS
27e
<0. 1
<0.3
350-409
<0.2 <0.
0.6 0.
<0.2 <0.
<0. 1 <0.
1.6 4.
0.6 4.
m
3
2
2
3
8
8
1
FROM RESERVOIR DURING SUMMER CROP IRRIGATION
230
40
1,100
450
750
<0.3
<0.3
CS
<0 1
<0.3
<0.3
<0.3
<0.4f
CS
<0.3 15
<3.3
CS
1.2
CS 5.7 0.3
CS
1.2
0.2
<0.4 <0.3
0. 1 0.3
CS
CS 0.3
0.5 <0.2
<0.3 <0.3
0.1 0.3
1.5
2.2 2.0
<0.3
<0.3
<0.4 <0.
3
CS - contaminated sample.
-------
TABLE 5.53. SAMPLED FECAL STREPTOCOCCUS DENSITIES ON THE MICROORGANISM AEROSOL RUNS
Fecal
streptococcus
Aerosol concentration
run in wastewater
number (cfu/mL)
ro
en
— i
WASTEWATER
Ml
M2
M3
M4
M5
M6
WASTEWATER
M7b
M8
Mil
M12
M14
M15
M17d'e
MIS®
M20d
WASTEWATER
M9
M10
M13
M16
M19
Upwind of
irrigation
riq
20-39
Fecal streptococcus concentration in air (cfu/m of air)
Downwind of irrigation nozzle line
m 40-59 m 60-89 m
FROM PIPELINE DURING PREPLANTING IRRIGATION
4,400 0.1 0.1 709 609
7,200 0.1 CS 65 65
6,300 <0. 1 0.3 69 110
1,900 <0. 1 <0. 1 70 600
5,800 <0. 1 <0. 1 95
16,000 <0.2 0.2 620 260
FROM PIPELINE DURING SUMMER CROP IRRIGATION
4,200 0.1 <0.7 140 120
3,200 0. 1C 2.7C 670 130
4,600 <0.3 <0.3
5,600 <0.3 CS
4,900 <0. 1 <0. 1
2,700
300
830
10
FROM RESERVOIR
30
13
53
3
3
1.3
<0.2
0.2
CS
1.
200 cfu/47 mm filter),
b Presumed pipeline source, based on microblal parameters.
c Possible contamination.
d Wastewater chlorinated at Lubbock treatment plant.
e Run conducted at night.
f Probable contamination, excluded from summary tables.
-------
TABLE 5.54 SAMPLED MYCOBACTERIA DENSITIES ON THE MICROORGANISM AEROSOL RUNS
ro
en
oo
Mycobacterla
Aerosol concentration
run In wastewater
number (cfu/mL)
WASTEWATER
Ml
M3
M4
M5
M6
WASTEWATER
M7a
M8
Mil
M12
M14
MJ5
WASTEWATER
M9
MfO
M13
M16
FROM PIPELINE
16,000
45,000
29, 000
13,000
15,000
FROM PIPELINE
1 00, 000
550,000
11,000
1 0, 000
5,300
6,000
Upwind of
Irrigation
Mycobacterla concentration in air (cfu/rrr of air)
rig 20-39 m 40-59 m
DURING PREPLANTING
IRRIGATION
1.3 1.3 7.0
<0. 1 <0.3 3.6
<0.1 <0. 1
CS CS
<0.1 <0. 1
DURING SUMMER CROP
<0. 1 <0.3
<0.1 <0.2
<0.1 <0.t
<0.2 <0.2
<0.1 0.7
CS <0. 1
FROM RESERVOIR DURING SUMMER CROP
430
100
230
10
<0.1 <0.2
<0.1 <0. 1
<0.2 <0.2
<0. 1 CS
7.0
20
IRRIGATION
0.3
1.4
IRRIGATION
<0.2
CS
0.2
Downwind
60-89 m
11
<0.4
7.9
9.0
32
0.2
<0.2
<0.2
CS
<0.2
of Irrigation
90-149 m
2.5 6.5
1.7 3.0
2.0 <0. 1
CS
2.0 3.7
0.3 0.3
0.2 <0. 1
0.2
0.5
0.7
5.0
<0.2 0.1
<0.1 <0.1
<0.2
CS 0.9
nozzle line
150-249 m
5.6
6.7 <0. 1
<0. 1 <0. 1
4.7 1.5
<0.2 <0. 1
<0.3 0.2
0.2 <0.2
<0.2
<0.2
<0.2
<0.2
<0.1 <0.1
<0.1 0.3
CS
<0. 1 <0.1
250-349 tn 350-409 m
4.1 CS
<0.2 <0.3 <0. 1 <0. 1
0.4 0.6 CS <0.2
<0.t 0.1 1.0 <0.2
<0.t 0.4 <0. 1 <0.3
<0. 1 <0. 1 <0.2 <0. 1
CS - contaminated sample.
-------
TABLE 5.55 SAMPLED CLOSTRIDIUM PERFRINGENS DENSITIES ON THE MICROORGANISM AEROSOL RUNS
Clostridium
perfringens
Aerosol concentration
ro
CJI
vo
run in
number
M2-Pipeline
Vegetative
M17-Pipelinea'b
Vegetative
Sporu lated
M18-Plpelineb
Vegetat i ve
Sporu lated
M19-Reservoir
Vegetat i ve
Sporu 1 ated
M20-Pipel lnea
Vegetat 1 ve
Sporu lated
-------
TABLE 5.56 SAMPLED COLIPHAGE DENSITIES ON THE MICROORGANISM AEROSOL RUNS
Col iphage
Col Iphage concentration In air (pfu/m of air)
Aerosol concentration Upwind of
run in wastewater irrigation
Downwind of Irrigation nozzle line
number (pfu/mL) rig 20-39 m 40-59 m 60-89 m 90-149 m 150-249 m
ro
cr>
o
WASTEWATER
Ml
M2
M3
M4
M5
M6
WASTEWATER
M7a
M8
Mil
M12
M14
M15
M17d'e
M18e
M20d
WASTEWATER
M9
M10
M13
M16
M19
FROM PIPELINE DURING PREPLANTING IRRIGATION
1,200 <0. <0. 1 38 50
1,500 <0. <0.3 4.0 8
1,400 <0. <0.3 5.7 11
530 <0. <0. 1 8.2 7.
1,100 <0. <0.1 6.
940 <0. <0. 1 23 12
FROM PIPELINE DURING SUMMER CROP IRRIGATION
1,700
-------
Tables 5.52, 5.53 and 5.56 also demonstrate that irrigation with
wastewater stored in Reservoir 1 was a source of aerosolized fecal
col i forms 9 fecal streptococci, and coliphage. These organisms were
sometimes transported 125 meters downwind and may occasionally have been
carried 200 meters from rigs irrigating with reservoir wastewater.
The aerosolized fecal col i forms exhibit more rapid die-off than the
other monitored microorganism groups. The aerosol data are consistent with
the hypothesis that a large proportion of the aerosolized colony forming
units of each microorganism are vulnerable and are rapidly inactivated,
while the remaining (hardy or protected) organisms survive without
detectable die-off out to the farthest distances sampled.
Virus Runs--
Four special virus runs were conducted to estimate enterovirus levels
in the air downwind from irrigation nozzles spraying pipeline wastewater.
The indigenous enterovirus levels ranged from 0.066 to 2.2 pfu/mL of
sprayed wastewater during these four runs, conducted on March 16 (Table
5.44) and August 2, 4 and 24 (Table 5.45). As shown in Table 5.57,
enteroviruses were recovered from the aerosol samples concentrate on every
virus run, at similar concentrations on the HeLa and RD cell lines. The
sampled enterovirus densities in wastewater and air are presented in Table
5.58 and compared to those obtained in 1977 in the two virus runs at the
Pleasanton, California wastewater irrigation system. The range of aerosol
densities of enteroviruses observed on three of the LHES virus runs (0.002
to 0.015 pfu/m^) at 46 to 60 meters downwind are comparable to those
observed at 63 meters downwind of the Pleasanton sprinkler line.
The identification of viral isolates recovered from the wastewater and
from the aerosol during the virus runs are presented in Table 5.59. The
specific viruses found in the aerosol sample were nearly always also
recovered from the wastewater being sprayed at the time, despite
differences in procedures used on the wastewater and aerosol samples.
Quantitative interpretation of Table 5.59 is difficult, because the
stability of various enteroviruses in the aerosol may differ.
During Virus Run V3 conducted on August 4, the enterovirus density was
elevated in the wastewater sample to 2.2 pfu/mL. However, the enterovirus
density was exceptionally elevated in the aerosol sample to a level (17
pfu/L) only one order of magnitude below those generally observed for the
indicator bacteria (cf. Tables 5.52 and 5.53). The degree of anomaly is
indicated in Table 5.58 by the ratio of aerosol to wastewater density of
7.4 for Run V3, compared to ratios ranging from 0.02 to 0.15 for the other
five virus runs. The majority of the aerosolized enteroviruses sampled on
Run V3 appear to have been poliovirus 1. A thorough evaluation of
laboratory procedures was conducted and indicated that laboratory handling
261
-------
TABLE 5.57. VIRUSES RECOVERED FROM AEROSOL SAMPLES DURING VIRUS RUNS3
Cell
line
Hela
RD
Virus
(Mar 16
pfu/mL
0.057
(2 pfu)
0.029
(1 pfu)
run vi
, 1982)
Total
expected
pfub
4
2
Virus
(Aug 2
pfu/mL
0.20
(3 pfu)
0.32
(9 pfu)
run V2
, 1982)
Total
expected
pfub
14
22
Virus
(Aug 4
pfu/nt
310
350
run Vi
, 1982)
Total
expected
pfub
22,000
25,000
virus
(Aug 24
pfu/mL
0.38
(5 pfu)
0.31
(9 pfu)
run V4
, 1982)
Total
expected
pfub
16
22
a Based on isolates confirmed as of November 19, 1982.
b 70 mL of concentrate from each aerosol run (VI: 3416 mL concentrated; V2:
2380 mL; V3: 2690 mL; V4: 2790 mL). Total number of plaques expected if
all 70 mL of concentrate were plated on a single cell line.
TABLE 5.58. SAMPLED ENTEROVIRUS DENSITIES ON VIRUS RUNS
Distance Enterovirus
Virus run
Date
density Re
from spray Cell in Wastewater in Air
line (m) line pfu/mL pfu/rr wa;
»tio of aerosol
density to
>tewater density
Lubbock Health Effects Study
VI
3-16-82
V2
8-2-82
V3
8-4-82
V4
8-24-82
PI easanton
V2-I
2-26-77
V2-II
4-9-77
60
46
44
49
Aerosol
63
63
HeLa 0.16
RD
HeLa 0.10
RD
HeLa 2.2
RD
HeLa 0.066a
RD
Monitoring Study
HeLa (5d) 0.036
HeLa (5d) 0.18b
0.0029
0.0015
0.011
0.018
16.2
18.3
0.010
0.013
0.0047
0.0070
0.018
0.11
7.4
0.15
0.13
0.039
a Pipeline water chlorinated at rate of 500 Ibs/day.
b Geometric mean of UTA and UTSA values.
262
-------
TABLE 5.59. IDENTIFICATION OF VIRAL ISOLATES RECOVERED DURING VIRUS RUNS
01
CO
Source of
isolates
Aerosol
Wastewater
Virus
Virus
Polio 2
Polio 3
TOTAL
Polio 2
Polio 3
Cox A9
Echo 5
Echo 11
Echo 13
Echo 17
Echo 19
Echo 20
Echo 21
Echo 25
Echo 27
on-going
TOTAL
run VI
Number of
isolates
2
1
T
4
4
3
1
1
1
1
2
1
2
2
1
8
3T
Virus
Virus
Polio 2
Cox B5
on-going
TOTAL
Polio 3
Cox A16
Cox B5
Echo 11
on-going
TOTAL
run V2
Number of
isolates
1
1
10
T7
1
1
24
1
7
"3T
Virus
Virus
Polio 1
Polio 2
Polio 3
Cox B5
Echo 11
on-going
TOTAL
Polio 1
Polio 2
Polio 3
Cox B5
Echo 11
Echo 12
Echo 24
Echo 25
on-going
TOTAL
run V3a
Number of
isolates
ia
18
22
1
1
11
^T
la
3
2
30
1
1
1
1
15
"57
Virus
Vi rus
Polio 1
Polio 2
Echo 13
on-going
TOTAL
Polio 1
Polio 2
Cox B2
Cox B5
Echo 24
Echo 25
Echo 33
on-going
TOTAL
run V4
Number of
isolates
8
2
1
3
PT
3
3
1
18
1
1
1
8
3F
The majority of the aerosol plaques (94%) were polio 1 based on neutralization with monovalent
antiserum. Only nonpolio 1 plaques were selected for identification using enterovirus pools.
-------
of aerosol-related samples had not compromised their integrity. Field
contamination of the Run V3 aerosol sample is not a plausible hypothesis
because the aerosol sample contained more plaque forming units than 10
liters of the wastewater and because there was no indication of any
irregularity in the field sampling. Hence, there is no laboratory or field
evidence of contamination to cast doubt on the validity of this anomalous
aerosol density.
Summary of Microorganism Data--
Table 5.60 summarizes the data from the microorganism and virus runs.
Estimated aerosol densities are determined as geometric means of all
sampled values in a distance range or as the detection limit for the pooled
air volume when all densities were below the detection limit. Caution must
be exercised in interpreting Table 5.60, since the estimated densities are
based on widely varying amounts of aerosol data and since environmental
conditions are not represented equivalently in the various distance
categories. Nevertheless, Table 5.60 does indicate the importance of
pipeline irrigation relative to reservoir irrigation as an aerosol source
of microorganisms.
Aerosol Exposure
The aerosol sampling data provide a basis for estimating the exposure
to the monitored aerosolized microorganisms received within 400 meters
downwind of Hancock farm irrigation relative to ambient background levels
in the study area. Ambient background densities of the monitored
microorganisms in the air just upwind of eight participant homes were
determined in the four background runs at dawn in early August 1980 prior
to irrigation or construction activities. Ambient background densities in
the fields were estimated from the upwind samplers from 18 of the 20
microorganism runs in 1982 in which there was no operating irrigation rig
or nearby human activity upwind of the upwind samplers. Aerosol densities
downwind of the irrigation nozzle line were determined for both pipeline
and reservoir sources of wastewater from the 20 microorganism runs and four
virus runs. The resulting estimated microorganism densities are presented
in Table 5.61.
Ambient background levels of the bacterial indicators, especially
fecal streptococci, are higher near homes than in the fields. Mycobacteria
and vegetative Clostridium perfringens were also present in the ambient
air, both with an average level in the fields of about 0.1 cfu/m^. AS
expected, coliphage was not found in the ambient air near homes or in
fields.
Pipeline irrigation was a substantial aerosol source of all the
monitored microorganisms. Reservoir irrigation was an aerosol source of
264
-------
TABLE 5.60. ESTIMATED DENSITIES SAMPLED ON MICROORGANISM AND VIRUS AEROSOL RUNS3
no
01
en
Microorganism concentration,
geometric mean*5
Aira (no/nr* air)
Microorganism
group Source-season
Fecal
coli forms
(cfu)
Fecal
streptococci
(cfu)
Mycobacteria
(cfu)
Clostridium
- Vegetative
- Sporulated
Coliphage
(pfu)
Pipeline-winter
Pipeline-summer
Reservoir- summer
Pipeline- winter
Pipeline- summer
Reservoir-summer
Pipeline-winter
Pipeline- summer
Reservoir- summer
perfringens (cfu)
Pipeline
Reservoir
Pipeline
Reservoir
Pipeline- winter
Pipeline- summer
Reservoi r- summer
Wastewater
(no./nt)
109,000
18,500
320
5,700
1,310
11
21,000
24,000
100
270
3
210
<1
1,060
630
2.5
Upwind
<0.01
<0.01
<0.03
0.08
0.2
0.04
0.2
0.07
<0.02
0.09
<0.2
<0.04
<0.2
<0.01
0.3
<0.01
Downwind of irrigation
25-89
180
200
2
140
200
0.4
8
0.4
0.08
9
<0.07
<0.07
11
7
0.03
90-149
6
2
0.2
38
5
0.2
2.1
0.6
0.1
2
<0.2
0.5
<0.2
4
1
0.06
150-249
3
2
0.6
23
5
0.2
0.9
0.08
0.06
2
1
2
0.7
0.07
nozzle line (m)
25U-349
4
0.8
<0.2
20
0.7
0.3
4
0.2
<0.05
1
.
0.4
0.9
0.1
0.06
35U-4U9
0.5
<0.2
0.6
0.2
0.1
<0.07
0.9
0.3
0.07
0.06
Enterovi ruses (pfu)
- HeLa cells
- RD cells
Pipeline
Pipeline
0.22
0.048
0.050
C —> 2C and C/2, where C is aerosol concentration.
-------
TABLE 5.61. ESTIMATED MICROORGANISM DENSITIES IN AIR DOWNWIND OF IRRIGATION
RELATIVE TO AMBIENT BACKGROUND LEVELS NEAR HOMES AND IN FIELDS
ro
Microorganism concentration
Microorganism group/
Wastewater source
Fecal coli forms (cfu)
Pipeline
Reservoir
Fecal streptococci (cfu)
Pipeline
Reservoir
Mycobacteria (cfu)
Pipeline
Reservoir
Clostrldium perfringens (cfu)
- Vegetative
Pipeline
Reservoir
- Sporulated
Pipeline
Reservoir
Coliphage (cfu)
Pipeline
Reservoir
Enterovi ruses6 (pfu)
Pipeline
a Geometric mean from aerosol
b From 20 microorganism runs.
c From background runs.
d From upwind samplers for 18
Ambient background
Homes0 Fieldsd
0.01 <0.006
0.5 0.07
0.05 0.1
0.08
<0.03
<0.005 <0.003
sampling.
microorganism runs with
Downwind
20-89 m
180
2
150
0.4
2.1
0.08
9
<0.07
<0.07
10
0.03
0.05
no upwind ri
in aira
(no./m3)
of irrigation line**
90-249
3
0.4
20
0.3
0.8
0.10
2
<0.2
0.8
<0.2
2
0.07
g in ope
m 250-409 m
0.8
<0.08
1
0.3
0.3
<0.03
1
0.3
0.13
0.06
(ration and no
nearby human activity.
From four virus runs.
-------
fecal coliforms, fecal streptococci, and coliphage. Table 5.61 summarizes
the estimated aerosol exposure with distance downwind from each source
relative to background levels.
Microorganism Levels on Flies
i
Fly collection for the baseline year was attempted on several
occasions, i.e., in August, September, and October 1980. After the first
attempt in August, collections were only made after flies were reported by
the local residents, since flies are not ordinarily seen except after rainyi
weather. On each occasion, traps were set for several days at the Wilson
effluent pond and at farmhouses on or adjacent to the Hancock farm. Flies
could be consistently collected by placing the traps adjacent to a piggery
located near the Wilson effluent pond. A single pooled sample of
houseflies was collected at the piggery on August 6 and 7, 1980. Viral
analysis of the flies yielded no positive isolates. However, a variety of
bacteria was recovered at densities ranging from very light to light (see
Table 5.62). Attempts to trap flies at four farmhouses (two with
livestock) during the same week yielded no flies.
Two samples of flies were trapped from October 15 to 17, 1980 at the
piggery near the Wilson effluent pond and from October 20 to 22, 1980 at
the barn of household 119 near the future west reservoir. No viruses were
detected in either sample. Bacterial profiles are compared with the
previous sample in Table 5.62. Staphylococcus aureus was present in
moderate numbers in both samples collected in October. Additionaly,
Proteus vulgaris (in moderate numbers) and Samonella arizonae were
recovered from the sample collected at the piggery.
Microorganism Levels in Drinking Water
Monitoring of drinking water samples for bacterial indicators and
selected pathogens commenced in October 1981 from 14 locations in the study
area: five on the Hancock farm, five within 400 m of the farm, two beyond
400 m and two from the city of Wilson. Eight additional wells, seven in
the low exposure area, were added in November 1982 to the locations
previously monitored. The 22 sampling locations are shown in Figure 5.4.
Water samples collected from these 22 locations were analyzed for total
coliforms, fecal coliforms, fecal streptococci and salmonella. Results
from the six sampling periods completed are shown in Table 5.63.
Activity Patterns
Activity diary data received during the 1982 preplanting irrigation
(March-April 1982) were used to describe the weekly activity patterns of
267
-------
TABLE 5.62. BACTERIAL ISOLATES FROM FLIES9
Piggery near Wilson effluent
pond, August 6-7, 1980
Piggery near Wilson effluent
pond, October 15-17, 1980
Barn near west reservoir,
October 20-22, 1980
PO
CTt
00
Escherichia coli (L)
Hafnia alvei (L)
Klebsiella pneumoniae (VL)
Proteus mirabilis (VL)
Providencia stuartii (VL)
Staphylococcus aureus (L)
Staphylococcus epldermldls (VL)
Escherichia coli H2S+ (L)
Fluorescent Pseudomonas gp (VL)
Hafnia alvei (VL)'
Klebsiella oxytoca (VL)
Proteus vulgaris (M)
Salmonella arizonae (VL)
Staphylococcus aureus (M)
Escherichia coli (L)
Fluorescent Pseudomonas gp (L)
Klebsiella oxytoca (VL)
Serratia marcesens (VL)
Staphylococcus aureus (M)
a Estimate of prevalence based on growth on primary culture plates (4 quadrants/plate):
M - moderate, growth on first two quadrants
L - light, growth on first quadrant
VL - very light, 1 to 10 colonies on the plate.
-------
531
0 Initial drinking water wells (October 81+) (13 wells)
* Initial treated drinking water sample (October 81+) (1 location)
O New drinking water wells (November/December 82+) (8 wells)
Figure 5.4. Drinking water sampling locations
269
-------
TABLE 5.63. ANALYSIS OF DRINKING WATER WELLS ON AND AROUND THE HANCOCK FARM
Total
col Iform
Household Dates (count/ 100 mL)
Feca 1
col Iform
(count/ 100 mL)
Fecal
streptococcus
(count/100 mL) Salmonella
N03-N
(mg/L)
On Hancock Far*
118
120
121
125
131
Within
109
114
116
122
126
320
10-14-81
1-6-82
2-15-82
6-22-82
11-4-82
12-14-82
11-5-81
1-5-82
2-16-82
6-16-82
11-4-82
12-14-82
10-15-81
1-4-82
2-15-82
6-16-82
11-3-82
12-14-82
10-15-81
1 -4-82
2-15-82
6-16-82
11-3-82
12-14-82
10-14-81
1-5-82
2-15-82
6-22-82
11-3-82
12-14-82
400 m of Hancock Farm
10-14-81
1-5-82
2-16-82
6-16-82
11-3-82
12-14-82
10-14-81
1 -6-82
2-16-82
6-22-82
1 1 -3-82
12-14-82
10-14-81
1-6-82
2-16-82
6-22-82
11-4-82
12-14-82
12-15-82
10-14-81
1-6-82
2-16-82
6-16-82
11-3-82
12-14-82
10-31-81
1-4-82
2-16-82
6-16-82
11-4-82
12-13-82
1-4-83
>2000
200
120
1300
47
0
570
6000
0
60
0
0
>2000
20
1
100
0
0
0
15
1700
1200
0
0
140
100
0
400
0
0
0
0
0
0
0
0
>2000
800
0
0
1
0
>2000
0
0
300
1
6
9
0
0
0
0
0
0
0
5
0
0
0
0
0
14
5
66
25
0
0
20
59
0
0
0
0
400
2
0
50
0
0
0
0
28
NR
0
0
30
0
0
3
0
0
0
0
0
0
0
0
0
20
0
0
0
0
20
0
0
30
0
6
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
9
0
0
0
0
0
330
0
0
49
0
0
3
1
1
0
0
9
1
0
0
3
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
4
53
27
1
0
0
0
0
0
0
0
0
0
0
0
0
0
Present
0
0
0
0
0
0
0
0
0
0
0
Present
Present
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
6.17
0.37
1.99
11.20
2.23
0.74
0.94
4.28
5.71
1.77
8.16
1.90
23.75
25.48
8.32
2.69
0.62
2.35
4.37
4.53
0.45
0.18
1.36
1.00
0.61
0.45
0.10
2.06
0.75
0.47
1.45
0.40
16.36
1.81
1.85
0.95
0.15
2.44
1.07
<0.01
1.45
0.26
3.96
1.70
1.30
9.47
l!l4
17.28
10.41
4.27
...connnuea
270
-------
TABLE 5.63. (Cont'd)
Household
City of Ml
298 (City
Well 1)
Dates
Ison
10-14-81
1-4-82
2-15-82
6-22-82
11-4-82
12-13-82
299 (Wilson 10-31-81
treated
water)
Beyond 400
103
315
399
504
531
540
545
546
555
1-4-82
2-16-82
6-22-82
11-3-82
12-13-82
• fro» Hancock
11-5-81
1-5-82
2-15-82
6-22-82
1 1 -3-82
12-13-82
11-4-82
12-15-82
10-14-81
1-4-82
2-16-82
6-16-82
11-4-82
12-13-82
12-15-82
11-4-82
12-13-82
12-15-82
12-15-82
12-15-82
12-15-82
Total
col Iform
(count/ 100 ml)
0
0
0
0
0
0
0
0
0
0
0
0
Farm
0
0
0
0
0
0
0
2
500
80
0
19000
21
100
190
0
1
0
0
2.8000
1100
Fecal
col Iform
(count/ 100 ml)
.
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
300
0
0
1000
2
3
0
0
0
0
0
160
3
Fecal
streptococcus
(count/100 ml)
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
160
0
0
60
3
0
82
0
4
0
0
27
16
Sa 1 mone 1 1 a
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Present
0
0
0
0
0
0
0
0
0
0
Present
0
N03-N
(mg/L)
• 5.42
1.55
7.15
9.04
<0.01
7.70
1.21
14.40
7.55
4.53
1.39
10.02
5.30
9.18
3.98
4.27
2.69
0.40
7.46
3.74
1.18
, 1.90
271
-------
the participants. Diary data were collected in two data collection
periods, 206 (March 21-27) and 208 (April 18-24).
During Period 206 diaries were received from 198 out of 384
individuals from 75 of the 135 'households. In Period 208 diaries were
received from 159 out of 375 persons from 68 of the 135 households. As
indicated in Table 5.64, this represents a response rate of 51.6% for
individuals in Period 206 and 42.4% for persons during Period 208.
Combined, 231 different participants from 93 households responded during
the two periods for an overall individual response rate of 61.6%.
The participants recorded the number of hours in the two given weeks
that they spent at six different locations (cf. Appendix D, Activity
Diaries). These included the home area, the blue area (i.e., the Hancock
farm), the orange area, the white area, the Lubbock area, and all other
areas. The data were recorded separately for each of the two collection
periods. During data processing the data were combined (into an average
when two diaries were completed by a given participant) in order to enlarge
the available sample size and provide an initial best estimate of exposure
during the 1982 preplanting irrigation, since later activity diary data is
not yet available.
Figure 5.5 indicates that approximately 8% of the respondents lived in
the blue area, 48% lived in the orange area, 41% lived in the white area
and 3% lived outside the study area. Table 5.65 illustrates the amount of
time each participant spent at home in a given week. Note that over 90% of
the respondents spent the majority of their time in the vicinity of their
homes so that home time dominated the exposure calculations.
Figure 5.6 indicates the amount of time that the participants spent in
Lubbock each week. Over 75% of these individuals visited Lubbock at some
time during the two data collection periods.
Exposure Estimates and Groups
The formula used for initial calculation of the exposure index for the
1982 preplanting irrigation period is given by
El = 0.13 T! + 0.025 T2 + 0.0014 T3 + 0 T4 + Ph-Tn
where T^ - weekly average of time in the blue area but not at home,
T2 - weekly average of time in the orange area but not at home,
T3 - weekly average of time in the white area but not at home,
272
-------
T4 - weekly time in any other area but not at home,
Tn - time at home,
Pn - relative concentration for the given home area.
Each of the Tn and T-j for i=l,2,3,4 was determined using the activity diary
information from data collection Periods 206 and 208 since these were the
only applicable periods available at the time of calculation.
The above exposure index is plotted as a function of frequency in
Figure 5.7. Over 60% of the participants would be placed in the low
exposure group for the 1982 preplanting irrigation period, having an
exposure index below the cutpoint of 3.12. This is equivalent to spending
24 or fewer hours per week on the Hancock farm (i.e., blue area). The
remaining 35 to 40% of the individuals would be placed in the high exposure
group since they had an exposure index exceeding 3.12 (i.e., equivalent to
more than 24 hours per week in the blue area). About 9% had an index
exceeding 11.4, equivalent to more than 50% of the time in the blue area.
Direct wastewater contact also was reported by some of the study
participants. This was accomplished through the usage of a query sheet
attached to the health diary. The information on direct contact for
Periods 205, 206 and 207 is summarized in Table 5.66. Note that 96% of the
participants did not report any direct contact with the Lubbock wastewater.
From later information, it is apparent that many Hancock farmers failed to
report minimal wastewater contact which they routinely experienced in their
work. Of the 4% reporting contact, the frequency of contact was evenly
spread across three contact categories: clothes/shoes, skin, and face.
273
-------
TABLE 5.64. ACTIVITY DIARY PARTICIPANTS
Data
collection
period
206
208
Combined
Households
Total %
75/135 55.6
68/135 50.4
93/135 68.9
Individuals
Total %
198/384 51.6
159/375 42.4
231/375 61.6
TABLE 5.65. PERCENT OF TIME SPENT AT HOME
Data
collection
period
206
208
Avg
Time
0
0.5
0
0.4
(hrs/wk)
0-40
3.0
3.8
2.6
spent at home
40-80
3.0
5.0
2.2
80-168
93.4
91.2
94.8
TABLE 5.66. FREQUENCY OF DIRECT WASTEWATER CONTACT
Data
collection
period
205
206
207
Combined
%
Type of contact
None
374
378
366
360
96.0
Cl othes/
shoes
1
4
3
5
1.3
Skin
3
2
4
5
1.3
Face
3
0
2
5
1.3
Total
381
384
375
375
100.0
274
-------
.PWieo 204_
.50
VJ.
2-
Ui ,
: -- :aq
.01
__BUJE ORAMSB -- WHITS - — OTHER-
LOCATIOM:
I PkHNo 208
-.50
.*"_.
U4_^
^z::
uj "-25
.OS
V52_
-BLUE-
WHITE «TH6R—
AVBWM
Ift
Jw
>.
o
jf|
a
o-
£
u.
UI •»
ot
Oft
U.A
• T|
•03
i r~
BLUE
WHITE 07W6J?—
Figure 5.5.. Location of home of activity diary respondents
275
-------
Pt«ie» 204
Ih.
^w
~» Ok
si
3
-------
HIGH
POSOR6
5 SOUP 1
31%—
*
10
9
X
F
C
j*»
j
5
f
I
«
10
1
1
w
f
J
E
5
5
i5
M
.
>P
(n=
io
98)
*P
,
1!
EXPOSURE INDEX
II
-
I
X
1
1
>(
f^
J
)
f
s
f
L
)
I
\
i
•
>
•
i
5
r
p
n
(i
01
•
1!
*
ZO
1]
>
6
^
EXPOSURE TNDEX
(
EXPOSURE
Figure 5.70 Distribution of preliminary exposure index for the 1982 preplanting
irrigation period
-------
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Goldman, D. A., J. Leclair and A. Macone. 1978. Bacterial Colonization of
Neonates Admitted to an Intensive Care Environment. J. Pediatr.
93:288-293.
Goldman, D. A. 1981. Bacterial Colonization and Infection in the Neonate.
Am. J. Med. 70:417-422.
281
-------
Hart, C. A. and M. F. Gibson. 1982. Comparative Epidemiology of
Gentamicin-Resistant Enterobacteria: Persistence of Carriage and
Infection. J. Clin. Pathol. 35:452-457.
Haverkorn, M. L. and M. F. Michel. 1979. Nosocomial Klebsiellas I.
Colonization of Hospitalized Patients. 0. Hyg., Camb. 82:177-193.
Honig, E. I., J. L. Melnick, P. Ipacson, R. Parr, I. T. Myers, and M.
Walton. 1956. An Epidemiological Study of Enteric Virus Infections.
Poliomyelitis, Coxsackie and Orphan (ECHO) Viruses Isolated from
Normal Children in Two Socioeconomic Groups. J. Exp. Med., 103:247-
262.
Johanson, W. G., Jr., A. K. Pierce, J. P. Sanford and G. D. Thomas. 1972.
Nosocomial Respiratory Infections with Gram-Negative Bacilli: The
Significance of Colonization of the Respiratory Tract. Ann. Int. Med.
77:701-706.
Johanson, W. G., A. K. Pierce and J. P. Sanford. 1969. Changing
Pharyngeal Bacterial Flora of Hospitalized Patients: Emergency of
Gram-Negative Bacilli. New Eng. J. Med. 281:1137-1140.
Johnson, D. E., D. E. Camann, K. T. Kimball, R. J. Prevost, and R. E.
Thomas. 1980. Health Effects from Wastewater Aerosols at a New
Activated Sludge Plant: John Egan Plant, Schaumburg, Illinois. In:
Wastewater Aerosols and Disease, H. Pahren and W. Jakubowski, eds.,
EPA-600/9-80-028, U.S. Environmental Protection Agency, Cincinnati,
Ohio.
Johnson, D. E., D. E. Camann, J. W. Register, R. E. Thomas, C. A. Sorber,
M. N. Guentzel , J. M. Taylor, and H. J. Harding. 1979. The
Evaluation of Microbiological Aerosols Associated with the Application
of Wastewater to Land: Pleasanton, California. Final Report to U.S.
Army Medical Research and Development Command and Environmental
Protection Agency, Cincinnati, Ohio. 482 pp.
Johnson, D. E., D. E. Camann, J. W. Register, R. J. Prevost, J. B. Tillery,
R. E. Thomas, J. M. Taylor, and J. M. Hosenfeld. 1978. Health
Implications of Sewage Treatment Facilities. EPA-600/1-78-032, U.S.
Environmental Protection Agency, Cincinnati, Ohio, 361 pp.
Kapikian, A. Z., S. M. Feinstone, R. H. Purcell, R. G. Wyatt, T. S.
Thornhill, A. R. Kalica, and R. M. Chanock. 1975. Detection and
Identification by Immune Electron Microscopy of Fastidious Agents
Associated with Respiratory Illness, Acute Nonbacterial
Gastroenteritis and Hepatitis A. In: Antiviral Mechanisms:
Perspectives in Virology, Vol. 9, Morris Pollard, ed., Academic Press.
p. 9-47.
282
-------
Koch, G. G., J. R. Landis, J. L. Freeman, D. H. Freeman, and R. G. Lehnen.
1977. A General Methodology for the Analysis of Experiments with
Repeated Measurement of Categorical Data. Biometrics 33:133.
Lennette, E. H., A. Balows, W. J. Hansler, Jr., and J. P. Truant, ed.
1980. Manual of Clinical Microbiology, 3rd edition. American Society
for Microbiology, Washington, D.C.
McDade, J. E. and C. C. Shepard. 1979. Virulent to Avirulent Conversion
of Legionnaires' Disease Bacterium (Legionella pneumophila)--Its
Effect on Isolation Techniques. J. Infect. Dis. 139:707-711.
Moore, B. E., B. P. Sagik, and C. A. Sorber. 1979. Procedure for the
Recovery of Airborne Human Enteric Viruses During Spray Irrigation of
Treated Wastewater. Appl. Environ. Microbiol. 38:688.
Morey, P., J. Fischer and R. Rylander. 1983. Gram-Negative Bacteria on
Cotton with Particular Reference to Climatic Conditions. Am. Ind.
Hyg. Assoc. J. 44:100-104.
Northrop, R., C. Becker, R. Cordell, M. Sulita, N. Altman, R. Anderson, and
J. Kusek. 1981. Health Effects of Sewage Aerosols: Additional
Serological Surveys and Search for Legionella pneumophila in Sewage.
EPA-600/S1-81-032, U.S. Environmental Protection Agency, Cincinnati,
Ohio.
Northrop, R., B. Carnow, R. Wadden, S. Rosenberg, A. Neal, L. Sheaff, J.
Holden, S. Meyer, and P. Sheff. 1980. Health Effects of Aerosols
Emitted from an Activated Sludge Plant. In: Wastewater Aerosols and
Disease, H. Pahren and W. Jakubowski, eds., EPA-600/9-80-028, U.S.
Environmental Protection Agency, Cincinnati, Ohio.
Philpot, C. R. and P. J. McDonald. 1980. Significance of Enteric Gram-
Negative Bacilli in the Throat. J. Hyg., Camb. 85:205-210.
Ramirez-Ronda, C. H., Z. Fuxench-Lopez and M. Hevarez. 1980. Increased
Pharyngeal Bacterial Colonization During Viral Illness. In: Current
Chemotherapy and Infectious Disease, Vol. II, J. D. Nelson and C.
Grassi, eds. American Society for Microbiology, Washington, D.C., pp.
1011-1013.
Rylander, R. and M. Lundholm. 1980. Responses to Wastewater Exposure with
Reference to Endotoxin. In: Wastewater Aerosols and Disease. H.
Pahren and W. Jakubowski, eds., EPA-600/9-80-028, U.S. Environmental
Protection Agency, Cincinnati, Ohio.
Schmidt, N. J. 1980. Enteroviruses and Reoviruses. In: Manual of
Clinical Immunology, 2nd edition, N. R. Rose and H. Friedman, eds.
American Society for Microbiology, Washington, D.C., pp. 672-677.
283
-------
Selden, R., S. Lee, W.L.L. Wang, J. V. Bennett and T. Eickhoff. 1971.
Nosocomial Klebsiella Infections: Intestinal Colonization as a
Reservoir. Ann. Int. Med. 74:657-664.
Sekla, L., D. Gemmill, J. Manfreda, M. Lysyk, W. Stackiw, C. Kay, C.
Hopper, L. Van Buckenhout, and G. Eibisch. 1908. Sewage Treatment
Workers and Their Environment: A Health Study. U.S. EPA Symposium on
Wastewater Aerosols and Disease, Cincinnati, Ohio, September 19-21,
1979.
Sheaffer and Roland, Inc. and Engineering Enterprises, Inc. 1980. Basis
of Design Report, Lubbock Land Treatment System Research and
Demonstration Project. EPA S806204010, March.
Shuval, H. I., and B. Fattal. 1980. Epidemiological Study of Wastewater
Irrigation in Kibbutzim in Israel. In: Wastewater Aerosols and
Disease, H. Pahren and W. Jakubowski, eds., EPA-600/9-80-028, U.S.
Environmental Protection Agency, Cincinnati, Ohio.
Snydman, D. R., J. L. Dienstag, B. Stedt, E. W. Bring, D. M. Ryan and K. A.
Fawaz. 1981. Use of IgM-Hepatitis A Antibody Testing—Investigating
a Common-Source, Food-Borne Outbreak. J. Am. Med. Assoc. 245(8):827-
830.
Sonnenwirth, A. C. 1974. Yersinia. E. H. Lennette, E. H. Spaulding, and
J. P. Truant, ed. American Society for Microbiology, Washington, D.C.
Standard Methods for the Examination of Water and Wastewater, 14th Edition.
1975. American Public Health Association, Washington, D.C.
Szmuness, W., J. L. Dienstag, R. H. Purcell, E. J. Harley, C. E. Stevens,
and D. Wong. 1976. Distribution of Antibody to Hepatitis A Antigen
in Urban Adult Populations. New Engl. J. Med. 295(14):755-759.
Texas Alamanac and State Industrial Guide 1980-1981. 1980. Fred Pass,
ed., A. H. Belo Corporation, Dallas, Texas.
Truett, J., J. Cornfield, and W. B. Kannel. 1967. A Multivariate Analysis
of the Risk of Coronary Heart Disease in Framingham. Journal of
Chronic Disease 20:511.
U.S. Environmental Protection Agency, U.S. Army Corps of Engineers, U.S.
Department of Agriculture. 1977. Process Design Manual for Land
Treatment of Municipal Wastewater. EPA 625/1-77-008.
Walker, S. and D. B. Duncan. 1967. Estimation of the Probability of an
Event as a Function of Several Independent Variables. Biometrika.
54:167.
284
-------
Wilkinson, H. W., D. D. Cruce, C. V. Broome. 1981. Validation of
Legionella pneumophila Indirect Immunofluorescence Assay with Epidemic
Sera. J7~Infect. Dis. 13:139-146.
Wright, C., S. D. Kominos and R. B. Yee. 1976. Enterobacteriaceae and
Pseudomonas aeruginosa Recovered from Vegetable Salads.App. Environ.
Microbiol. 31:453-454.
Youmans, G. P., P. Y. Paterson and H. M. Sommers. 1980. The Biologic and
Clinical Basis of Infectious Diseases, 2nd Edition. W. B. Saunders,
Philadelphia, Pennsylvania, p. 88.
285
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APPENDIXES
-------
LIST OF APPENDIXES
APPENDIX
A Personal Interview for Health Watch
B Personal Interview Update
C Informed and Parental Consent Forms
D Activity Diaries and Maps
E Procedure for Wastewater Sample Collection
Lubbock Southeast Water Reclamation Plant
F Procedure for Wastewater Sample Collection
Wilson Imhoff Tank Effluent
G Description of Litton Model M High Volume Aerosol Sampler
H Decontamination Procedure for Model M Samplers
I Collection Efficiency of Litton Model M Large Volume Samplers
J Data Reporting Forms
-------
APPENDIX A
PERSONAL INTERVIEW FOR HEALTH WATCH
-------
HH name
HH t
Phone t
HH size
University of Illinois
School of Public Health
Lubbock Land Treatment Project:
Personal Interview for Health Watch
(Time
Began
am
ASSURANCE OF CONFIDENTIALITY - All information that would permit identifica-
tion of individuals will be held in strict confidence, will be used only by
persons engaged in and for the purpose of the survey and will hot be disclosed
or released to others for any purpose. The results will be used only when
combined with those of many other people.
A-l
-------
First, I would like to ask you a few questions about your household.
1. a. Do you have air conditioning in your home?
Yes 1
No (Sfe-cp to 2. 2} 0
b. Do you have
central air conditioning or 1
window or wall units or 2
both 3
c. During the summer, do you have the air conditioning on:
all or most of the time 1
somt' of the time every day 2
only when it is very hot or 3
never 4
2. Do you obtain your drinking water from
a private well, or 1
public water supply 2
3. Do you dispose of sewage through
a septic tank or cespool or 1
city sewage system . 2
Now, I would like to ask you some questions about household members and
their activities.
4. a. Including yourself, how many people live in this household?
b. How many of these people are related to you?
Id theAe. a/ie. u.nn.&tcute.d houAikotd member (HM):
I will be asking you some questions about each of your family members.
I will be talking with unrelated household members separately.
A-3
-------
5. a. Beginning with yourself, please tell me the first name of each
person now living in the household who is related to you.
b. How is related to you?
(Recoid >Le.icutiont>k4.p and *ex)
6. In what year (were you/was ) born?
7. a. Do you (does ) have a job or go to school outside your home or
farm?
Yes 1
No (Sfex.p to Q. B) 0
b. Looking at the map, please show me where (you/ _ ) works
goes to school. (Indcco-te Zone)
or
Zone 1
Zone 2
Lubbock 3
Other area (excluding Lubbock) 4
8. Approximately how many hours per week (do you/does ) spend
outside the outlined area shown on this map? {Show map]
9. During the non-winter months, how many hours per day (do you/does )
generally spend out of doors, within the outlined area on: [Show map)
a. Weekdays
b. Weekends
less than 1 hour/day 1
more than 1; less than 4 hrs./day 2
more than 4; less than 8 hrs./day 3
more than 8 hrs./day 4
less than 1 hr./day 1
more than 1; less than 4 hrs./day 2
more than 4; less than 8 hrs./day 3
more than 8 hrs./day 4
Aife Qu.utA.onii 10 through 14 -c|j houAzhoid -it> ioc.cute.ct on a.
facuun. Skip to Q.. 15 -t|S kouAe.ko£d -C4 not loccutid on a. fiaA
10. How many hours per week (do you/does ) spend doing farm work
out of doors?
0 • 1
less than 10 hrs./week 2
more than 10; less than 20 hrs./week 3
more than 20; less than 40 hrs./week 4
more than 40 hrs./week 5
A-4
-------
We also need to find out a little bit about your farm, so we can more
accurately judge what types of farm work household members might be
doing.
11. What crops are you producing on your farm this year? Please tell
me each crop which you are growing, and the amount of acreage de-
voted to it. (Checfe OA many OA apply)
Crop Acreage
None None (000)
cotton
wheat
other
12. What types of livestock are you raising on your farm this year?
Please tell me each type of livestock and the number of animals.
(Check 04 many u apply]
Livestock Number
None None (000)
cattle
hogs
sheep
fowl
other
13. a. Do you currently irrigate your farm land?
Yes 1
No (Sfex.p *<> ^ 14) 0
b. What is the source of that water?
Well 1
Other (specify) 2
14. Approximately how many acres of land do you farm, including pastures,
fallow ground and grazing land?
Skip to fj. 16
A-5
-------
Aife Q.. 15 -i|5 houAehoid Lt, not located on a fagtun.
15. a. Do you or does anyone in your household ever work on a farm within
the outlined area? U/iOM) map)
Yes 1
NO (skip to Q.. u) o
) work on a farm?
_) work on a farm, when
) generally work on
Who is that?
How many weeks per year (do you/does
How many days per week (do you/does _
(you/ ) work(s)?
During which season(s) (do you/does
a farm? (Check at, many 06 apply)
a. Spring
b. Summer
c. Fall
d. Winter
16. a. Approximately how many times per month (do you/does ) travel
to Lubbock?
b. Approximately how much time (do you/does ) spend in Lubbock
on each visit?
17. a. Do you or does anyone in your household drink bottled water regularly?
b.
Who is that?
Do you/does
Yes 1
No (Skip to £. 18) 0
ever drink water from the tap?
Yes 1
No 0
A-6
-------
Now I would like to find out about any long term or chronic illnesses
or conditions which you or anyone in this household has ever had which
required consultation with a doctor.
18. a. Have you or anyone in this household ever seen a doctor for any of
these respiratory illnesses or conditions? (Show caAd A)
Yes 1
No (Skip to £. 19) 0
DK (S(u.p to Q.. 19)8
Who is that?
Fan. each, yu to Q.. l£a at>k:
Which illness or conditions (do you/does
(Check OA many at appty]
) have?
How old (were you/was
first appeared?
a. Allergies
b. Chronic bronchitis
c. Emphysema
d. Asthma
e. Tumor of cancer of the lung
f. Tumor of cancer of the mouth
or throat
g. Other (specify)
) when the
(Foi each
(mad condition]
ducted., A.e.coid age. on adja.ce.nt Line.}
What medications and/or treatments, if any, (are you/is
taking for (your/his/her) __^__^ ?
condition]
A-7
-------
19. a. Have you or has anyone in this household ever seen a doctor for any
of these heart conditions? (Show eoAd B) ,
Yes 1
No (Stop to 2. 20) 0
DK (Skip to Q,. 20) 8
b. Who is that?
Fo/i each yu to £. 19a tuk:
c. Which type of heart condition (do you/does ) have?
a. High blood pressure
b. Stroke
c. Heart attack
d. Angina
e. Other (specify)
d. How old (were you/was ) when the ^_^__^______ first
occurred? 1/iea.d condition)
e. What medications and/or treatments, if any, (are you/is )
taking for (your/her/his) _^ 1
(mod condition)
A-8
-------
20. a. Have you or has anyone in this household ever seen a doctor for
any of these stomach or abdominal conditions? (Sdou) caSid C]
Yes 1
No [Skip to Q.. 21) 0
DK (S(u.p to Q_. 21) 8
b. Who is that?
Fo/t each yu to Q. 20a. oife:
c. What of these conditions (do you/does have?
Tumor or cancer of the
a. Stomach
b. Intestine
c. Colon
d. Esophagus
e. Stomach (peptic) or intestinal
(duodenal) ulcer
f. Ulcer of the colon (ulcerative
colitis)
g. Diverticulosis
h. Gall bladder problems
. i. Other (specify)
d. How old (were you/was ) when the _^__^ _^ first
appeared? Ueod condi-tcon)
What medications and/or treatments, if any, (are you/is ) cur-
rently taking for (your/her/his) _^___^ ?
condition)
A-9
-------
21. a. Have you or has anyone in this household ever seen a doctor for
any of these other types of conditions? {Show aaJid V)
Yes
No (Sfe-tp to Q.
DK (S/U.p to Q.
Who is that?
1
23) 0
23] 8
Fan. za.c.h yu to Q_. 2/a aife:
Which of these conditions (do you/does ) have?
How old (were you/was
peared?
a. Skin cancer
b. Leukemia
c. Hodgkin's Disease
d. Other cancers
e. Arthritis
f. Diabetes
g. Anemia
h. Immunological disorder
i. Rheumatic fever
j. Serum hepatitis (Hepatitis B)
k. Infectious Hepatitis (Hepatitis A)
1. Infectious mononucleosis
m. Other chronic conditions
) when the
condition]
first ap-
What medications and/or treatments, if any, (are you/is
rently taking for the ?
c.ontLULion)
) cur-
22.
med4.c.a£Lon/t/iza.tme.ntA
A-10
-------
23. a. Have you or has anyone in this household ever had a blood trans-
fusion?
Yes 1
No (Skip -to <±. 24) 0
DK (Skip to 1. 24} 8
b. Who is that?
24. a. Have you or has anyone in this household ever been on a kidney machine
or hemodialysis?
Yes 1
No (Skip .to Q.. 25) 0
DK (Skip to Q.. 25) 8
b. Who is that?
25. a. Have you or has anyone in the household ever been in close contact
with (i.e. lived with or helped care for) a person who had TB (tuber-
culosis)?
Yes 1
No (Skip -to Q.. 26) 0
DK (Sfex.p to 0. 26) 8
b. Who is that?
26. a. Do you or does anyone in this household smoke cigarettes regularly?
Yes 1
No (Sfe^.p to Q.. 27) 0
DK (Sfe-tp to Q,. 27) 8
b. Who is that?
A-ll
-------
10
FOA. each HM boin beijoie 1962, n&k £. 27 thiu. 0. 29
27. Are you (is _ ) currently working at any part-time or full-time
job? (exclude hou^ew-^eAy]
Yes 1
No (Skip to 1. 29) 0
1^ HM ^4 not c.uMe.ntty wo.tkx.ng, oife:
28. Are you (is _ ): (Read ca£ego/U.e6)
Usually employed, but just out of work 1
temporarily
Retired 2
Homemaker (Sfe^p -to Q.. 30) 3
Disabled or handicapped [Slu.p to Q.. 30} 4
Not usually employed (Sfexp to Q.. 30) 5
Student (Sfe^.p to Q.. 30} 6
Other (Specify) (Sfu.p -to 2- 30) 7
29. a. What (is/was) your/ _ 's) main occupation or job title?
b. What kind of work (do/did) you/ _ ) do? That is, what (are/were)
(your/ _ 's) duties on the job?
(1^ occupoXum -a not "tfo/uneA" , cufe 4. 29c)
c. What (does/did) (your/ _ 's) employer manufacture or sell, or
what services does it provide?
Aife £. 30 onLy fan. n.uponde.nt, and -t|j
30. What is the highest grade of school which (you/ _ ) (have/has)
completed?
None 0
Elementary 12345678
High School 9 10 11 12
College 13 14 15 16
Some graduate or professional school 17
Graduate or professional degree 18
A-12
-------
11
31. Which household members contribute to the financial support of this
household?
32. a. Considering all of the income from employment, net farm income and
from all other sources, please tell me which category on this card
best describe your total household income before taxes in 1979?
{Show CAAd. E)
a. less than 5,000 1
b. 5,000 - 7,999 2
c. 8,000 - 9,999 3
d. 10,000 - 14,999 4
e. 15,000 - 19,999 5
f. 20,000 - 29,999 6
g. 30,000 and over 7
h. VK. (oife 32B) B
(oak 328) 9
b. Can you tell me if it was:
less than 10,000 or 1
more than 10,000 2
VK B
9
33. Now, in case the office finds I've missed something what would be
the best time to call you? a.m.
p.m.
************************************************************
A-13
-------
I
(Respondent)
Male...... . J
Female 2
19
(Age )
II
Uole 1
Female. ... 2
79 _
(Age )
III
Male 1
Female. . . .2
/9 '
(Age )
IV
itale 1
"emaJLe. 2
/9
(Age )
V
dale I
Female 2
19
(Age )
VI
\\aJLe. 1
Female 2
19 __
(Age )
34.
35.
Reco^xf Phone * on £t,oi*U:
-ta.ee 0(5 tiuponde.nt
2
3
Ilccan 4
5
37. Ooei the. lupondtnt Live, -in a:
Batlicng ^OA. 2 |$amc£x.e4 o-i
-------
APPENDIX B
PERSONAL INTERVIEW UPDATE
-------
«.
Card
^cols.
1"5
Uama 6 • ?. o
Phone | 21-27
I1H Size 26-29
Interviewer
University of. Illinois
School of Public Health
Lubbock Health Effects Study
Personal Interview I'pdate
of Interview
ASSURANCE OF CONFIDENTIALITY - All information that would permit identifi-
cation of individuals will be held in strict confidence, will be used only
by persons engaged in and for the purpose- of the survey and will not be
disclosed or released'to otheis for any purpose. The results will be used
only when combined with those of many other people.
B-l
-------
HOUSEHOLD INFORMATION
la.
Have you changed residences since you enrolled in the Health
Watch ?
Yes (Skip to Q. 2)
No
b. Have you made any of the following changes in your residence
since you enrolled in the Health Watch?
a. Installed air conditioning (Ask 2b-a)
b. Changed water supplies (Ask 3a)
c. Changed waste disposal (Ask 3b)
2a. Do you now have air conditioning in your home?
Yes 1
No (Skip to Q. 3) 2
b. Do you have central air conditioning or 1
window or wall units 2
or both 3
c. During the summer, do you have the air conditioning on:
All or most of the time
Some of the time everyday
Only when it is very hot
Never
Card Columns
30
31
3a. Do you now obtain your drinking water from
A private well, or
public water supply
b. Do you now dispose of sewage through:
A septic tank or cesspool
or city sewage system
End of Household
File
B-3
-------
PARTICIPANT INFORMATION
la Has anyone left your household permanently or temporarily since
you enrolled in the Health Watch?
Yes 1
No (Skip to Q. 2) 2
b. Who was that?
FOP each "yes" to Q. la-b, ask the following questions.
c. When did leave? (Record month, year)
d. Did leave permanently?
Yes (Sk.ip to Q. 2) 1
No 2
e. When did return? (Record month, year. If HM has not returned
record "NR" and ask If.)
f. When do you expect to return? (Record month, year. Record
"DK" if return not known.)
2a. Have you added any new members, including infants, to your household
z-7.:z yc'J enrolled in tl-.c. ";^ith ";tch?
Yes 1
No (Skip to .Q. 3) 2
*b. What is his or her name? (Record name in column at top of facing page.)
For each new household member, ask the following questions:
*c. How is related to you? (Record in column at top of facing page.)
*d. What is 's sex? (Record in column at top of facing page.)
*e. What is 's age? (Record in column at top of facing, page.)
f. When did enter your household? (Record; month, year.)
g. How long will be staying with you? (Record "permanently"
for infants and other permanent residents. Otherwise record length
of stay in weeks.)
B-4
-------
Now, I would like to ask you about any long-term or chronic
illnesses which you or anyone in your household may have develoued
sinc'e you enrolled in the Health Watch. If you are not sure whether
a household member developed a condition before or after enrolling
in the study, please tell me about it anyway and we can check that
later.
3a. Have you or has anyone in your household been newly diagnosed
as having any of these respiratory illnesses or conditions since
you enrolled in the study?
Read lic.t of conditions. Pause after each condition to allow
respondent to reply. For each "yes", ask "Who was that?" and
rec.ord condition in appropriate column.
a. Allergies
b. Chronic bronchitis
c. Emphysema
d. As thma
e. Tumor or cancer of the lung
f. Tumor or cancer of the mouth or throat
g. Other (specify)
Ask Zb. for each condition reported.
b. What medications and/or treatments, if any, (are you/is )
takinu for the ? (Record medications.)
(read condition)
'-!a. Have you or has anyone in your household been newly diagnosed
as having any of these cardiovascular conditions since you
'• enrolled in the study?
Read list of conditions. Pause after each condition to allow
respondent to reply. For each "yes", ask "who was that?" and
record condition in appropriate column.
a. High blood pressure
b. Stroke
c. Heart attack
d. Angina
e. Other (specify)
Ask 4b. for each condition reported.
b. What medications and/or treatments, if any, (are you/is )
taking for the ? (Record medications.)
(read condition)
B-5
-------
5a. Have you or has anyone in your household been newly diagnosed as
having any of these stomach or abdominal conditions since you
enrolled in the study?
Read list of conditions. Pause after each conditions to allow
respondent to reply. For each "yes", ask "Who is that? and
record condition in appropriate column.
Tumor or cancer of the:
a. Stomach
b. Intestine
c. Colon
d. Esophagus
e. Stomach (peptic) or intestinal (duodenal) ulcer
f. Ulcer of the colon (ulcerative colitis)
g. Diverticulosis
h. Gall bladder problems
i. Other (specify)
Ask Sb. for each condition reported.
b. What medications and/or treatments, if any, (are you/is )
taking for the ? (Record all medicationsT)
6a. Have you or has anyone in your household been newly diagnosed as
having any of these other types of conditions since you enrolled
in the study.?
Read list of conditions. Pause after each condition to allow
respondent to reply. For each "yes", ask "Who is that?" and
record condition in appropriate column.
a. Skin cancer
b. Leukemia
c. Hodgkin's Disease
d. Other cancers
e. Arthritis
f. Diabetes
g. Anemia
h. Immunological disorder
i. Rheumatic fever
j. Serum hepatitis (hepatitis B)
k. Infectious hepatitis (hepatitis A)
1. Infectious mononucleosis
m. Other chronic conditions (specify)
Ask 6b. for each condition reported.
b. What medications and/or treatments, if any, (are you/is )
taking for the ? (Record all medications.)
B-6
-------
7a. Have you or lias anyone in your household started working, stopped
working or changed jobs since you enrolled in the study?
Yes 1
No (Skip to Q. 8) 2
b. Who war, that?
Stopped working (Chip to Q. 8) 1
Started working 2
Changed .jobs 3
c. What is the name of the place where (you/ ) now work(s)? (Reocrd place)
d. What is (your/ 's) new job title? (Record job title)
Now, I would like to ask you about a couple of other types of health
conditions which are of interest to us. We want to know if you or
anyone in your household has ever seen a doctor for these conditions.
8a. Have you or has anyone in your household ever seen a doctor for a
goiter or other thyroid condition?
Yes 1
No (Skip to Q. 9) 2
DK (Skip to Q. 9) 8
b. Who is that?
c. Please tell me what the doctor called the thyroid condition, if
you know. (Record condition if known. Enter "DK" if not known.)
d. How old (were you/was ) when the thyroid condition first
occurred? (Record aas. )
e. Co you/does still have the thyroid condition?
Yes 1
No 2
f. What medications or treatments have yofl/fcas ___^ ever received
for the" thyroid condition? (Record all medications and treatments.)
g. Which of those medications or treatments, if any, (are you/
is ) currently taking for the thyroid condition?
(Record all current medications and treatments.)
B-7
-------
9a, Have you or has anyone in your household ever seen a doctor for
pneumonia?
Yes 1
No ('Skip to Q. 10., -if applicable) 2
DK (Skip to Q. 10,- if applicable ) 8
b. Who is that? (Record condition in appropriate column.)
c. HCw many times have you/has had pneumonia? (Record # times.)
d. How old (were you/was ) the last time that the pneumonia
occurred? (Record ageD
e. Were you/was ever hospitalized for pneumonia?
Yes 1
No 2
DK 8
f. Approximately how long did the pneumonia last the last time
that it occurred? (Record duration in weeks.)
This question is to be asked only for children 18 years of age or less.
Ask ayproyi'iate Questions for aye uj~ BUC.VI u'riild.
lOa. Where (did/does ) go to grammar school? (Record all schools
attended and location of school.)
b. Where (did/does ) to to junior high or middle school? (Record all
schools attended and location of school.)
c. Where (did/does ) go to high school? (Record all schools attended
and location of school.)
d. Did ever receive a polio immunization at school?
Yes 1
No (End of interview) 2
DK (End of interview) 8
e. Could you please tell me which school that was? (Record name and
location of school.)
B-8
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APPENDIX C
INFORMED AND PARENTAL CONSENT FORMS
-------
IMPORTANT INFORMATION
ABOUT POLIO AND INACTIVATED POLIO VACCINE
Please read this carefully
IP 10/1/80
WHAT IS POLIO? Polio is a virus disease that often
causes permanent crippling (paralysis). One person out
of every 10 who get polio disease dies from it. There
used to be thousands of cases and hundreds of deaths
from polio every year in the United States. Since polio
vaccine became available in the mid I950's. polio has
nearly been eliminated. In the last five years, fewer than
25 cases have been reported each year. It's hard to say
exactly what the risk is of getting polio at the present.
Even fpr someone who is not vaccinated, the risk is very
low. However, if we do not keep our children protected
by vaccination the risk of polio will go back up again.
INACTIVATED POLIO VACCINE (IPV): Immuniza-
tion with inactivated polio vaccine is effective in
preventing polio and has successfully controlled polio in
several countries. The vaccine is given by injection.
Several doses are needed to provide good protection.
Young children should get three doses in the first year of
life, each separated by 1 to 2 months, and another dose
6 to 12 months later, at about 18 months of age. A
booster dose is needed every 3 to 5 years, especially
when children enter school or when there is a high risk of
polio, for example, during an epidemic or when travel-
ing to a place where polio is common. The vaccine is ef-
fective in providing protection to over 90% of people
who receive it.
POSSIBLE SIDE EFFECTS FROM THE VACCINE:
Inactivated polio vaccine is not known to produce any
side effects.
PREGNANCY: Polio vaccine experts do not think inac-
tivated polio vaccine can cause special problems for
pregnant women on their unborn babies. However.
doctors usually avoid giving any drugs or vaccines to
pregnant women unless there is a specific need. Preg-
nant women should check with a doctor before taking in-
activated polio vaccine.
WARNING — SOME PERSONS SHOULD NOT
TAKE INACTIVATED POLIO VACCINE
WITHOUT CHECKING WITH A DOCTOR:
— Those who are sick right now with something more
serious than a cold.
— Those with allergies to antibiotics called neomydn
or streptomycin
— Pregnant women
NOTE ON ORAL POLIO VACCINE: Besides the inac-
tivated polio vaccine, there is also an oral polio vaccine
which is given by mouth and which after several doses
protects against polio for a long time, probably for life.
Many polio experts feel that the oral vaccine is more ef-
fective for preventing the spread of polio and for con-
trolling polio in the United States. However. It should
not be given to persons who have a low resistance to in-
fection or who live with persons with low resistance to
infections. It has been associated very rarely with
paralysis in persons who receive the vaccine or who are
in close contact with those recently vaccinated. Oral
polio vaccine is widely used in this country. It can be
given alone or in combination with IPV. If you would like
to know more about oral polio vaccine or combinations
of oral and inactivated vaccine, please ask us.
QUESTIONS: If you have any questions about polio or
polio vaccination, please ask us now or call your doctor
or health department before you sign this form.
REACTIONS: If the person who received the vaccine
gets sick and visits a doctor, hospital, or clinic in the 4
weeks after vaccination, please report it to:
IEXAS DEPARTMENT OF HEALTH
HORSING DIVISION 797-4331
PLEASE KEEP THIS PART OF THE INFORMATION SHEET FOR YOUR RECORDS
I have read tfte information on this torn about polio and the inactivated vaccine. I have had a chattel to ask questions which wen answered to my satisfaction. I believe I understand the benefits
and risks ot inactivated polio vaccine and request that it be given to me or to the person named below for whom I am authorized to make this request. ip | Q/ | /go
INFORMATION ON PERSON TO RECEIVE VACCINE
(Pleas* print first three Hnest
Name
Address
City
X
llastl (Nrstl Imiddlel Blrthdate
State
Signature of person to receive vaccine or person authorized to make the request
Age
County
Zip Code
Date
FOR CLINIC OSE
Clinic Ident.
Date Vaccinated
Manufacturer and Lot No.
Site of administration
r.-i
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INFORMACION IMPORTANTE ACERCA DE
LA POLIOMIELITIS Y LA VACUNA ANTIPOLIO ATENUADA
Favor de leer cuidadosamente
iOUE ES LA POLIOMIELITIS? La poliomielitls
(polio) es una enfermedad causada por un virus y que
muchas veces resulta en paralisis permanente. Muere
aproximadamente I de cada 10 personas que se
contagian de ella. Antes ocurian miles de casos de polio y
centenares de muertes causadas por esta enfermedad
todos los anos en los Estados Unidos. Desde que se hizo
disponible la vacuna antipolio a mediados de la decada
de los cincuentas. la poliomielitis ha sido cast totalmente
eliminada. En los ultimos 5 anos. se han reportado
menos de 25 casos en cada ano. Es diffcil senalar con
exactitud el riesgo actual de contagiarse de polio. Aun
para las personas no vacunadas. el riesgo es muy
reducido. Sin embargo, si no mantenemos la proteccibn
de nuestros hijos por medio de la vacunacibn regular, el
riesgo de contraer polio volvera a aumentar.
LA VACUNA ANTIPOLIO ATENOADA (IPV): La
inmunizaci6n por medio de la vacuna antipolio
atenuada sirve efectivamente para prevenir la
poliomielitis. y ha logrado controlar la enfermedad en
varios paises. La vacuna se administra en forma de
inyeccion. Se requieren varias dosis para lograr una
proteccibn satisfactoria. Los bebes deben recibir 3
dosis en su primer ano de vida. con una separacidn de I
o 2 meses entre cada dosis. y deben recibir otra dosis
entre 6 y 12 meses despues, a los 18 meses de edad
aproximadamente. Se requiere una dosis de refuerzo
cada 3 o 5 anos. particularmente cuando los nines
entren a la escuela o cuando haya un alto riesgo de
contraer polio, como por ejemplo durante una
epidemia. o durante viajes a lugares donde la
poliomielitis es una enfermedad comun. La vacuna
protege eficazmente a mas del 90% de las personas que
la reciben.
EFECTOS SECDNDARIOS DE LA VACUNA: Por lo
que se sepa. la vacuna antipolio atenuada no produce
efecto secundario algiino.
MUIERES EMBARAZAOAS: Los expertos en
vacunas antipolio no creen que la vacuna antipolio
atenuada cause problemas para mujeres embarazadas.
ni para sus ninos aun no nacidos. Sin embargo, los
medicos generalmente se abstienen de recetar drogas o
vacunas para mujeres embarazadas. a menos que haya
alguna necesidad especifica de ello. Las mujeres
IP 10/1/80
embarazadas deben consultar con un medico antes de
tomar la vacuna antipolio atenuada.
PRECADCION — ALGUNAS PERSONAS NO
DEBEN RECIBIR LA VACUNA ANTIPOLIO
ATENOADA SIN CONSULTAR PRIMERO CON
ON MEDICO:
— Las personas que sufren actualmente de
cualquiera enfermedad mi seria que un catarro.
— Las personas que padezcan alergias a los
antibibticos conocidos como Neomiclna y
Estreptomicina.
— Las mujeres embarazadas.
NOTA SOBRE LA VACDNA ANTIPOLIO DE
ADMINISTRACION ORAL: Ademas de la vacuna
antipolio atenuada. existe tambien una vacuna antipolio
de administracibn oral, que se toma por la boca. y que.
despueg de varias dosis. ofrece protecci6n contra la
poliomielitis por un tiempo largo, probablemente por
toda la vida. Algunos expertos creen que la vacuna oral
es mas eficaz para prevenir la propagacidn de polio y
para controlar esta enfermedad en los Estados Unidos.
Sin embargo, la vacuna oral no se debe administrar a
personas que tengan una baja resistencia a infecdones.
ni a las que vivan con otras personas que tengan una
baja resistencia a infecciones. En ciertas ocasiones
raras. esta vacuna ha sido asociada con la paralisis en
personas que han recibido la vacuna o que han estado
en contacto intimo con otras personas recien
vacunadas. La vacuna antipolio oral se usa ampliamente
en este pais. Puede ser administrada sola o junto con la
IPV (vacuna antipolio atenuada). Si usted desea saber
mas acerca de la vacuna antipolio oral, o acerca de las
combinaciones de vacuna atenuada y oral, por favor
consultenos.
PREGONTAS: Si tiene usted alguna pregunta acerca
de la poliomielitis o la vacunaci6n antipolio. por favor
hagala ahora mismo. a Name a su medico o su
Departamento de Salud antes de firmar esta forma.
REACCIONES: Si una persona que recibe la vacuna se
enferma y visita a un medico, algun hospital o alguna
clinica en las primeras 4 semanas despues de la
vacunaci6n. por favor rep6rtelo a:
FAVOR DE CUARDAR ESTA PARTE DE LA HO|A PARA SU INFOHMACION
Hi letdo la mbmaciin aue amtieneetta forma aarca de la poliomirlilis y la vacuna attnuada. He tenido la oportumdaddeliaaT fregutta. galas tuerm aiHeitadoi satatotoriamatU. Crto
Out enliendo loi baitticios y lof naam it la vacuaa anlifollo altnuada. g solicito que * me admmistre a mi on la tenom abaio meticimada. a tawrde auien Urao la auUridad de kaaraU
sol'uitud. IP 1011 /80
INFORMACION SOBRE LA PERSONA A QUE RECIBIR A LA VACUNA
IPor favor UM Inn de imprenta en la> primers) tres lineal)
Nombre (apellido)
Direccion
Ciudad
X
(primer) \segundo)
Estado
Firma de la persona qua recibira la vacuna o de la
persone autorizada para solicitarla.
Feche de Edad
nacimiento
Condado de reiidencia
Zip Code
Feche
PARA EL USO DE LA
CLINICA
Identidad de la clmica
Fecha de vacunacion
Fabricante V n'de lota
Lugsr de la inyeccidn
C-2
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ADULT* S CONSENT TOR PARTICIPATION IN A HEALTH
RESEARCH PROJECT
FORM Cft
I, , state that I am over twenty one(21)
(NAME OF PARTICIPANT)
years of age and wish to participate in an infectious disease study being
conducted by the School of Public Health at the University of Illinois under
the direction of Doctor Robert L. Northrop.
The purpose of the research is to ascertain the number and types of
infections and other illnesses I will have during the next three (3) years to
evaluate the Health effects, if any, of aerosols emitted from nearby
irrigation rigs spraying wastewater.
This project involves my allowing you to obtain from me six (6) blood
samples and taree (3) tuberculin tests in the next three (3) years.
I understand that there are no experimental -procedures to be performed
on me in this research and that there are no personal risks involved.
I acknowledge that I have been informed that this research is designed to
assist in maintaining or improving my personal health and will benefit me
personnally if causes for my infections are found.
I understand that in the event of physical injury resulting from this
research ther: is no compensation and/or payment for radical treatment from
The University of Illinois at the Medical Center for such injury except as
may be required of the University by law.
I acknowledge that Doctor Northrop, or his ispresentative, has fully
explained to rue the need for the research; has informed me that I may withdraw
from participation at any time and has offered tc answer any inquiries which I
may make concarning the procedures to be follower.
I freely and voluntarily consent bo my participation in this research
project.
(SIOJATURE OF VOLUNTEER)
(Witness to E
-------
MINOR'S CONSENT FOR PARTICIPATION
IN A HEALTH RESEARCH PPOJECT
FORM CM
I, , state that I am years of age
(UAME OF PARTICIPANT)
and wish to participate in a health watch program being conducted by the
School of Public Health at the University of Illinois under the direction
of Doctor tobert I... Northrop.
The purpose of the research is to ascertain the number and types of
infections I will have during the next three (3; years to evaluate the
health effects, i:: any/ of aerosols emitted fro in nearby irrigation rigs
spraying wastewatv;r.
This project involves my allowing you to obtain from me six (6) blood
samples and three (3) tuberculin tests in the next three (3) years.
I understand that there are no experimental procedures to be performed
on rae in this research and that there are no personal risks involved.
I acknowledge that I have been informed that this research is designed
to assist in maintaining or improving my personal health and will benefit me
personally if causes for my infections are found.
I understand that in the event of physical injury resulting from this
research there is no compensation and/or payment for medical treatment from
The University of Illinois at the Medical Center for such injury except as
may be required o: the University by law.
I acknowledge that Doctor Northrop or his representative has fully
explained to me tie need for the research, has informed me that I may
withdraw from participation at any time and has offered to answer any
inquiries which I may make concerning the procedures to be followed. I
freely and voluntirily consent to my participation in this research project.
(SIGNATURE OF MINOR)
Date
C-4
-------
PARENTAL CONSENT
FORM CM
We, parents or guardians of the above minor volunteer, agree to the
participation of che above minor in the research project set out above.
He have been informed of the need for the research, the benefits to be
derived from it, and the risks involved. He have been informed that the
research cannot bu conducted with adults only because of the nature of the
research.
We also understand that in the event of physical injury resulting from
this research the.re is no compensation and/or payment for medical treatment
from The Universi-y of Illinois at the Medical Center for such injury except
as may be require 1 of the University by law.
Being aware of the necessity for the participation of minors in this
research project and being informed that the procedures will also benefit
the above-named minor personally by reporting ta me/us, the parents or
guardians, and to his or her physician, test results which may assist in
diagnosis of an infectious illness the minor may have during this study, we
consent to the minor's participation.
(SIGNATUBE OP PARENTS OR GUARDIANS)
(SIGNATURE OF PARENTS OR GUARDIANS)
(WITNESS TO EXPLANATION)
(NOT TO SIGNATURE)
(DATE)
n-5
-------
APPENDIX D
ACTIVITY DIARIES AND MAPS
-------
.SV//CC/ cf IJniilif. Health
UNIVERSITY Om ILLINOIS A.T THEJ MH3DICA.L. CHJNTTH3R,,
.-In,-; (.,-.,;, .;;.', Tiicpi.uii: 99(>-6S:l(l
.>'ii;:lii'.;- At'iimr.: !'.<.:-. iiax rt.W.V • Unciiju., Illintiis 6'06'SYy
March 11, 1982
Dear Study Participant:
In order for us to get a better understanding of the relationship
between all of the environmental and health data which are being
collected, it is necessary for us to know how much time individuals
spend in various parts of the study area. Obviously, it would be
impossible for you to keep track of your whereabouts every day that
we are collecting health information, so we have developed an "activity
diary" which we would like study participants to keep for one week.
We hope that this week will be representative of people's normal
activities at this time of year.
We are asking that each member cf your household complete an
activity diary for the week of March' 21 - 27. Each person should
fill out the activity diary with his crc her name on it each day
for the one week period. (Mothers should fill cut the diary tor
young children.)
Included with the diary is a nap of the study ar-?a with
-------
The activity diaries should be returned to the University of
Illinois in the enclosed stamped, self-addressed envelope as soon
as they are completed.
We hope that filling out the activity diary will not be too
much of an inconvenience. The information which the diary will
provide is crucial to the health study, and we greatly appreciate
your efforts in completing it.
Sincerely yours,
Robert Northrop, Ph.D.
Associate Professor
Epidemiology-Biometry Program
RN/cb
D-2
-------
ACTIVITY DIARY
A. Basic Data
1. Name:
, First
2. Reporting week dates:
Last
to
B. Activity Information
Please record the number of hours per day which you spend within each area
(colunm) listed below. Use the reference maps to locate the areas for question
1; question 2 refers to specified locations familiar to you. This should be done
cnch day for one week. If you are out-of-town during the entire week, please
complete this diary the first week that you return home.
Don't forget to include sleeping hours when you record daily activities.
The number of hours for each day should total 24 hours.
Question 1: Kow many hours per day did you spend in the following areas?
Sunday
Monday
Tuesday
Wednesday
Thursday
Friday
Saturday
HOURS PER DAY
Blue Map
Area (Hancock
farm)
Orange
Map Area
White
Map Area
Outside
Map Area
Daily Total
(24 hrs.)
Question 2: In addition to the above, we would also like more detailed
information as to how many hours per day you spent in the following
specific locations.
HOURS PER DAY
In
Lubbock
At
Home
Sunday
Monday
Tuesday
Wednesday
Thursday
Friday
Saturday
D-3
-------
j
^ 3.0
! ...
UNION
^
D-4
-------
iS 1 I* 10 :
iy. LJL^ i
-1-
•••'•••••.••••••••a
,
r'-i — k'.isrsis! «*a s i — s-~\
'"
i iy""* i*. •„•'<»•* i ['•
D-5
-------
c-f Public Hiniih
MBUDIO.A.LI omNTTHR., CHICA.C»O
; Cede 312. Teifpiicne 996-66^0
.l-.liiras: P.O. 3ox i&9S • C.iiic.uro. Illinois fiO
July 27, 1982
Dear Study Participant:
1 believe you are familiar with the procedure for keeping the activity
diary, so 1 will not restate all the directions we have given to you pre-
viously. There are 3 important points about this diary:
1. Please keep the diary for the week of August 1st through 7th;
2. The completed activity diaries will be collected when fecal
specimens are collected during the week of August 9 through
August 13- The diaries can either be brought to the Wilson
Mercantile Building or arrangements can be made to pick up
these diaries at your home by calling Pearl Davidson (628-2961);
3. Please be sure to use the enclosed maps when you refer to times
spent in the colored areas. These maps are different from
previous activity diary maps.
If you have difficulty in keeping this diary, Parrie Graham will be
glad to answer your questions when she is at the Wilson Mercantile Build-
fng (628-2621) during the week of August 9 " 13-
This diary is particularly important to us since your activities may
be very different from previous times, particularly those of you who
would have been doing more farming than has been possible this year.
We really do appreciate your time in doing this task for us.
SlncereJ-y yours,
Robert Northrop, Ph.D.
Associate Professor
Epidemiology-Biometry Program
D-6
-------
COORD/MATE MAP
D-7
-------
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D-8
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APPENDIX E
PROCEDURE FOR WASTEWATER SAMPLE COLLECTION
LUBBOCK SOUTHEAST WATER RECLAMATION PLANT
-------
PROCEDURE FOR WASTEWATER SAMPLE COLLECTION
Operational Year - 1981
Trickling Filter Effluent - Lubbock Southeast Water Reclamation Plant
SwRI Project 01-6001
Purpose -
The purpose for collection of this sample is to determine relative
densities of a wide range of indigenous enteric bacteria and viruses
prevalent in the wastewater to be land applied at the Hancock
site. To accomplish this purpose a 24-hour flow-weighted composite
is derived by collecting three eight-hour time-weighted samples
from the Trickling Filter Plant (TFP) effluent followed by compositing
based on plant flow data for each eight-hour period.
Equipment Required -
Sample Collection -
ISCO Model 1580 Sampler with Nicad battery
109 ft. (3 m) of 3/8" O.D. x 1/4" I.D. Tygon tubing
Weighted stainer
3 clean 3-gallon polyethylene containers (for ISCO)
10 to 20 Ibs. cracked or cube ice (function of ambient conditions)
Sample Compositing -
5-gallon Nalgene (or requivalent) polypropylene carboy with
lid (sterile)
1-liter Nalgene (or equivalent) graduated cylinder (sterile)
1-liter Nalgene polypropylene bottles (sterile)
Sample Shipment
1 frozen Kool-Pac per six 1-liter sample bottles
1 insulated shipping container, labeled, with means of lid
attachment
1 counter-to-counter shipping ticket (Southwest or Braniff
Airlines)
Procedure
Preparation
1. Charge two Nicad batteries for 24 hours prior to sample
collection.
2. Check equipment for completeness including new Tygon
tubing with weighted strainer securely attached.
3. Place Kool-Pacs in freezer at least 24 hours prior to
sample compositing.
4. Sterilize equipment for compositing as appropriate.
E-1
-------
Sample Collection -
1. Locate sample adjacent to combined channel from the
secondary clarifiers of the TFP.
2. Place a 3-gallon container in the Sample Container Tub
with the false bottom open end up. Carefully add crushed
or cube ice to the tub without disturbing the position
of the container.
3. Replace the Pump and Controls Section and latch securely
making sure that the Stop Float Mechanism is free.
Attach the battery to the sampler and securely connect
the battery cable to the "12 VDC" socket on the side
of the control box. Attach the Tygon tubing to the
pump inlet, tape to secure, and lower weighted strainer
into the effluent channel. Tape tubing to side of sampler
to reduce strain on pump inlet connection.
4. Set the Control Panel as follows:
Mode Switch - Time
Time Interval Multiplier Control - 1.0
Suction Line Length Switch - 14 2/3' (1/4" I.D.)
Sample Rate Switch - 10 min.
Volume Selector Switch - 268 mL/sample (8' head)
Pump Switch - Auto
5. Turn Sample Rate Switch to the Manual Cycle position,
then return it to the 10 min. Time Inverval Position.
The pump should be automatically activated, first for
a brief period in the reverse mode to purge any liquid
in the line followed by a forward pumping action of
sufficient time to collect approximately 268 mL of sample.
This cycle is completed by a second reverse pumping
opertaion to again purge the sample line. If all functions
operate correctly in this test cycle, confirm the position
of all control switches, especially that the Pump Switch
is in the Auto Mode, then place and latch both the protective
lid over the Control Panel and the cover over the Pump
and Controls Section. Refer to the instruction manual
should problems be encountered.
6. Check the TFP Flow meter in the treatment plant office
for operation, and if necessary, mark the chart for
start of sample collection.
7. At the end of each 8-hour sampling period, turn the
Pump Switch to Off, remove the 3-gallon sample container,
label it, and place a clean sample container in the
tub. Turn the Pump Switch to the Auto position and
repeat Step 5. Renew the ice bath as required to maintain
the collected sample at 4°C. Store the collected sample
at 4°C until composited. At the conclusion of the 24-
hour sampling period, remove all equipment from the
sampling site.
E-2
-------
8. Prior to leaving the treatment plant obtain information
on TFP flows as follows:
(a) Remove the TFP flow chart recorder from the
instrument panel after disconnecting the multi-
lead sockets at the back.
(b) On a clean work table carefully unroll sufficient
chart paper from the take-up chart spool to
correspond to the 24-hour collection period.
Mark the 8-hour intervals, and using a transparent
straight edge, pencil a horizontal line through
a visually estimated average for each 8-hour
segment. Record average flows for each segment.
(c) Rewind the chart paper on the take-up spool
to the correct time, replace the recorder
in the instrument panel, and connect the multi-
lead sockets. Confirm that the recorder is
functional.
Sample Compositing
1. Based on total flow through the TFP during the 24-hour
composite period (z of the average flow for each 8-hour
sample segment), determine the fraction of total flow
for each segment.
2. Knowing the final volume of composite desired (18 L
max for 5-gal. jug), determine the amount of sample
needed for each segment based on the fraction of total
flow for that segment (final volume desired X fraction
of total flow).
3. Add appropriate amounts of each sample to the sterile
composite container using a sterile 1-L graduate. Cap
and shake to mix.
4. Apply sample labels to sterile, 1-L polypropylene bottles
and cover with a complete circle of clear protection
tape.
5. Transfer composite sample to 1-L bottles and cap tightly.
Sample Shipment
1. Samples should be shipped at 4°C. If samples are not
at this temperature and the shipping schedule permits,
place samples in a 4°C environment (refrigerator or
ice bath) prior to packing.
2. Pack a shipping container with the sample bottles.
Add a frozen Kool-Pac to the container insulating the
sample containers where necessary to prevent direct
contact between container and Kool Pac.
E-3
-------
3. Close container and strap securely. Check address label
for legibility.
4. Present shipping container with completed shipping ticket
at the passenger check-in counter or freight counter
of designated airline (Braniff or Southwest) at lease
45 minutes prior to scheduled departure. Shipment is
to be prepaid.
1/15/81
01-6001
J. Harding
bz:2H
-------
APPENDIX F
PROCEDURE FOR WASTEWATER SAMPLE COLLECTION
WILSON IMHOFF TANK EFFLUENT
-------
PROCEDURE FOR WASTEWATER SAMPLE COLLECTION
Operation Year - 1981
Wilson, Texas Imhoff Tank Effluent
SwRI Project 01-6001
Purpose -
The purpose for collection of this sample is to determine relative
densities of a wide range of indigenous enteric bacteria and viruses
prevalent in the wastewater from the Wilson community, the most
densely populated area adjacent to the Hancock Site. To accomplish
this purpose a 24-hour time-weighted composite sample is collected
by utilizing a self-contained automatic sampler.
Equipment Required -
Sample Collection -
• I SCO Model 1580 Sampler with Nicad battery
6 ft. (2m) of 3/8" O.D. x 1/4" I.D. Tygon tubing
Short length of pipe for tubing weight
1 clean 3-gallon polyethylene container for ISCO
10 to 20 Ibs cracked or cube ice (function of ambient conditions)
Sample Shipment -
1 frozen Kool-Pac per 6 (six) 1-liter sample bottles
1 insulated shipping container, labeled, with means of lid attachment
1 counter-to-counter shipping ticket (Southwest or Braniff Airlines)
Procedure -
Preparation -
1. Charge two Nicad batterys for 24 hours prior to sample collection.
If this sample is collected simultaneously with the Trickling
Filter Effluent from the Lubbock Southeast Reclamation Plant,
only one extra. Nicad battery needs to be charged.
2. Check equipment for completeness including new Tygon tubing
with weight attached to end.
3. Place Kool-Pacs in freezer at least 24 hours prior to sample
shipment.
Sample Collection -
1. Locate sampler adjacent to Imhoff tank effluent drain.
2. Place a 3-gallon container in the Sample Container Tub with
the false bottom open and up. Carefully add crushed or cube
ice to the tube without disturbing the position of the
container.
3. Replace the Pump and Controls Section and latch securely making
sure that the Stop Float Mechanism is free. Attach the battery
to the sampler and securely connect the battery cable to the
F-l
-------
-2-
"12 VDC" socket on the socket on the side of the control box.
Attach the Tygon tubing to the pump inlet, tape to secure, and
lower weighted end into the Imhoff tank drin. Tape tubing to
side of sampler to reduce strain on pump inlet connection.
4. Set the Control Panel as follows:
Mode Switch - Time
Time Internal Multiplier Control - 1.0
Suction Line Length Switch - 7 1/3' (1/4" I.D.)
Sample Rate Switch - 136 mL/sample (8' head)
Pump Switch - Auto
5. Turn Sample Rate Switch to the Manual Cycle position, then
return it to the 10 min. Time Internal position. The pump
should be automatically activated, first for a brief period
in the reverse mode to purge any liquid in the line followed
by a forward pumping action of sufficient time to collect
approximately 136 mL of sample. This cycle is completed by a
second reverse pumping operation to again purge the sample line.
If all functions operate correctly in this test cycle, confirm
the position of all control switches, especially that the Pump
Switch is in the Auto Mode, then place and latch both the
protective lid over the Control Panel and the cover over the
Pump and Controls Section. Refer to the instruction manual
should problems be encountered.
6. At the end of the 24-hour sampling period, remove the 3-gallon
sample container from the Sample Container Tub, cap and label it,
and remove all equipment from the sampling site.
Sample Shipment-
1. After thoroughly mixing the sample, fill the appropriate number
of 1-1 bottles and cap tightly.
2. Samples should be shipped at 4°C. If samples are not at this
temperature and the shipping schedule permits, place samples in
a 4°C environment (refrigerator or ice bath) prior to packing.
3. Pack a shipping container with the sample bottles. Add a
frozen Kool-Pac to the container insulating the sample
containers where necessary to prevent direct contact between
container and Kool-Pac.
4. Close container and strap securely. Check address label for
legibility.
5. Present shipping container with completed shipping ticket at
the passenger check-in counter or freight counter of designated
airline (Braniff or Southwest) at least 45 minutes prior to
scheduled departure. Shipment is to be prepaid.
2/02/81
01-6001
J. Harding
F-2
-------
APPENDIX G
DESCRIPTION OF LITTON MODEL M HIGH VOLUME AEROSOL SAMPLER
-------
APPENDIX G
DESCRIPTION OF LITTON MODEL M HIGH VOLUME AEROSOL SAMPLER
"The Model M Sampler is designed to continuously collect particulate
matter from a large volumetric flow rate of air (approximately 1000 liters/
minute) and deposite it into a small amount of liquid (flow rate of 2
mL/min). This effects a volumetric concentration factor on the order of 5
x 1Q5. Basically, the sampler is an electrostatic precipitator of a rather
unusual configuration. With reference to the schematic diagram, Figure
G-l, and an interior view, Figure G-2, aerosol is drawn into the unit
through a converging nozzle and passes through the center of the high-
voltage area. It then flows radially between this plate and a lower
rotating collection disc. An electric potential of 15,000 volts, which is
maintained across a 11/16-inch spacing between the plate and disc, creates
two effects: 1) A corona is emitted from a ring of 60 needles that is
located concentric to the air inlet. Particles, exposed to air ions
created from the corona, acquire an electrical charge. 2) The electric
field provides the driving force to precipitate charged particles onto the
lower disc.
"Liquid is pumped onto the center of the collection disc and, because
of the centrifugal force, forms a thin moving film over the entire disc
surface. Particles collected on the film are transported to a rotating
collection ring where the liquid is removed by the pickup. Subsequently,
the liquid drips into the collection funnel where it is pumped to a
receiver located outside the sampler.
"To accommodate a broad range of sampling situations, several variable
features are incorporated into the unit. These are:
Air Flow Rate 400 to 1200 liters/minute
Liquid Flow Rate 0 to 8 mL/minute
Disc Speed 0 to 45 rpm
High Voltage 0 to 20 kilovolts
"When the sampler is in operation, the air flow rate is read directly
from a calibrated meter on the front panel and is adjusted with a blower
control potentiometer (see Figure G-3). Both disc speed and pump flow rate
G-l
-------
CT5
I
NS
Corona Needles
High-Voltage Plate
Aerosol
Inlet
Liquid Inlet Tube
Collection Disc
Collection Ring
To Pump
and Receiver
Figure G-l.. Schematic Diagram of Large-Volume Air Sampler System
-------
Hinged Top
Strobelight
Ozone-Resistant
Casket Material
High-Voltage Plate
Ring Motor
Air Exhaust Fan
Strobelight
Circuitry
Fluid Supply Tank
Removable
Side Panel
Collection Disc
Speed Control
Air Inlet
Ceramic Insulator
Pickup Assembly
Collection Ring
Collection Disc
Disc Motor
Peristaltic
Pump Motor
Removable Side Panel
High-Voltage
Power Supply
Pump Speed Control
Electrical Connector
Figure G-2. Interior View of Large-Volume Air Sampler
6-3
-------
Air Flow
Rate Gauge~\.
Control
\
High-Voltage
Voltmeter
High-Voltage
Milliarnmeter
High-Voltage
Control
Potentiometer
High-Voltage
Circuit Breaker
and ON-OFF
Switch
Figure G-3.Instrument Panel of Model M Large-Volume Air Sampler
-------
are controlled by high and low range toggle switches, together with
potentiometers. Although no direct readouts are provided for these two
variables, calibrations are easily obtained so the arbitrary scales on the
potentiometers can be converted to actual speed or flow rates. The high-
voltage system is set with the aid of a potentiometer and is provided with
the meter to show voltage and current."!
To facilitate visual observation of the surface condition of the disc
in operation, the operator made observations through the windows with the
aid of a flashlight. The air flow rate was set at 1000 liters/minute.
1. Litton Model M Large-Volume Air Sampler: Instruction Manual, Report
3028. Minneapolis, Minnesota, 1966.
G-5
-------
APPENDIX H
DECONTAMINATION PROCEDURE FOR MODEL M SAMPLERS
-------
APPENDIX H
DECONTAMINATION PROCEDURE FOR LITTON MODEL M
SOLUTIONS:
1% Clorox
Buffers--KH2P04 (71 g/L) 50 mL i /, nT
/L DI
(115 g/L) 50 mL
Autoclave 50 mL of the buffer in 2-oz bottles.
Add 1 mL of 5% Clorox prior to use.
1% sodium thiosulfate
10 g NaThio/L DI H20
Sterile water
Autoclave 100 mL in 4-oz bottles prior to use.
PROCEDURE:
1. Calibrate air flow meter for 1000 1pm.
2. Disconnect electrical supply and remove side plate from unit.
3. Using Kimwipes dipped in 70% ethyl alcohol, wipe the inside top half
sides and all upper section parts.
4. Run disk (but not blower) and pump 1% Clorox solution through all
tubes. Hold Clorox solution in sampler tubing for a minimum of 30
minutes. The pump may be started periodically to move cleaning
solution through the tubing.
5. After decontamination with Clorox solution, flush the system with the
contents of a sodium thiosulfate bottle.
6. Rinse the system with the contents of a sterile water bottle. After
most of the liquid has been pumped out of the system, attach a
microfilter to the sampler inlet and run the blower until the disk is
dry.
7. Wipe the ends of the tubes with a Kimwipe saturated with 70% ethyl
alcohol. Place the ends of the tubes in a clean plastic bag and tape
shut. Seal the sampler inlet and exhaust ports with decontaminated
plastic caps.
01-6001-313
HJH 2/82
H-l
-------
APPENDIX I
COLLECTION EFFICIENCY OF LITTON MODEL M LARGE VOLUME SAMPLERS
-------
APPENDIX I
COLLECTION EFFICIENCY OF LITTON MODEL M LARGE VOLUME SAMPLERS
The Litton Model M large volume sampler (LVS), used to collect aerosol
data, is an electrostatic precipitator. During operation of an LVS, an
electrical potential of approximately 15,000 volts (15 kV) is maintained
across an 11/16-inch spacing between the plate and disk. This creates two
effects: 1) a corona is emitted from a ring of 60 needles thereby giving
the microorganism particles a charge and 2) the resultant electric field
attracts the charged particles to the collecting disk.
Collection efficiencies for electrostatic precipitators depend on the
operating high voltage producing the internal charging corona and electric
field. Sufficient voltage must be supplied to the corona source to charge
the particles suspended in air; the greater the voltage, the greater the
driving force to effect particle separation from air.
Electrostatic precipitators are usually operated at the highest
voltage possible without sparking (arcing). Sparking disrupts the
operation of the electrical equipment and lowers collection efficiency by
reducing the applied voltage, redispersing the collected particles, and
promoting current channeling (effectively reducing particle charging and
collection to localized areas).
Very high dust loadings increase the potential difference required for
the production of a corona and reduce the current due to the space charge
of the particles. This tends to reduce the average particle charge and
reduces collection efficiency. Compensation can be obtained by increasing
the potential difference when high dust loadings are involved.
The collection efficiency of an LVS is affected by many other factors
than simply the operating voltage and dust loading. The performance will
change according to intake air velocity, particle size distribution,
particle concentration in air, and environmental conditions (e.g., wind
gusts, wind speed, direction, and relative humidity).
Data obtained from field operation of the LVS are used in LHES to
calculate microbial concentrations in air as discussed in the second annual
LHES report (Calculation of Microorganism Density in Air section). The
resultant microbial concentrations assist the interpretation of the degree
1-1
-------
of aerosol exposure an individual would receive based upon the time of day
and distance from an operating rig whose source is either reservoir or
pipeline wastewater. Thus, it is important to correct all LVS sampling
data to a reference set of operating conditions to obtain internally
consistent data. For example, an LVS may measure 20 cfu/m3 of air with
operating conditions which result in a relative collection efficiency of
40% and its paired sampler may measure 40 cfu/m3 of air with different
operating conditions which have a relative collection efficiency of 80%.
If only the raw data were used to calculate microbial concentrations
without regards to operating conditions (i.e., collection efficiency), then
one would incorrectly conclude that the second sampler observed microbial
concentrations twice as great as the first sampler. If the reference set
of operating conditions had an effective collection efficiency of 100%,
then both samplers would be recorded as having measured 50 cfu/m3 of air.
To determine correction factors for operating conditions, rigorous
experimentation is required in a controlled environment. A few
environmental conditions can be reconstructed in the laboratory to evaluate
their effect on collection efficiency. However, certain factors such as
microbial concentrations, particle size, and wind gusts cannot be
evaluated. Thus, the calculated microbial concentrations will be subjected
to indeterminate errors; the magnitude of these errors cannot be estimated.
Some factors (e.g., operating voltage) are known to affect the collection
efficiency and since these can be evaluated, it is necessary to adjust the
raw data for these factors.
The Naval Biosciences Laboratory (NBL) in Oakland, California
conducted experiments on three separate occasions (1976, July 1980 and
October 1982) to develop a collection efficiency data base from which to
calculate correction factors. In all of the NBL studies, data were
obtained for relative collection efficiencies of LVS to all-glass impingers
(AGI) samplers in a controlled environment (an atomizer created a specified
amount of aerosol in an enclosed wind tunnel). In these studies the AGI
samplers had a high degree of precision (for November 1982 NBL data the
average s/x was 6.70%), but their accuracy was not evaluated. On the other
hand, the LVS performed with less precision, as is demonstrated by the
average s/x of 11.7% for operating voltages greater than 12 kV (precision
decreases for smaller operating voltages).
The experimental procedures employed by NBL to study the LVS
collection efficiencies are thoroughly documented in their three final
reports; a capsule summary of these reports follows.
Disinfecting procedures prior to a sampling period were identical for
all three NBL studies and SwRI field operations. Operating time for the
samplers varied in each study, but discrepancies among the reported results
should not be caused by this procedural change since the results are
1-2
-------
reported as relative collection efficiencies (relative to AGI samplers
operating simultaneously with the LVS in the same wind tunnel).
Bacillus subtilis var. Niger replaced Flavobacterium as the test
organism for the November 1982 NBL study. Bacillus subtilis var. Niger is
a hardy spore, but in spite of this, no problems of residual contamination
carryover were encountered.
Before the 1976 study, the samplers were completely overhauled;
defective and worn parts were either replaced or repaired. For the other
two NBL studies, the samplers were not overhauled; however, routine
preventative maintenance was continued. It is unknown whether the 1976
overhaul affected collection efficiencies differently than the routine
maintenance procedures.
The collection fluid (BHI) circulation rate varied among all three NBL
studies. In July 1980, NBL reported that no collection efficiency
differences were observed for a BHI rate greater than 8 nt/min; only data
obtained with BHI rates greater than this were used for correction factor
evaluations. The air intake sampling rates were approximately 1.0 nvVmin.
LVS operating voltages in the three NBL studies ranged from 8 to 18
kV. The 1976 study reported two LVS sampler responses at various operating
voltages (8 to 14 kV) were obtained by NBL for LVS samplers operated at the
highest voltage attainable without producing excessive arcing; these data
are reported as relative collection efficiencies at 15+ kV. The October
1982 data is reported on raw data sheets as relative collection
efficiencies at the actual LVS operating voltage.
During the effort to identify operating variables that influence the
LVS collection efficiency, NBL studied relative humidity and temperature
effects in the October 1982 study. According to NBL no strong effect of
relative humidity was observed for the range tested (relative humidities
from 51 to 81), and the rather narrow temperature range (unqualified) of
the tests showed no collection efficiency effects. These conclusions from
NBL are most likely incomplete for two reasons:
1) When relative humidity is plotted versus collection efficiency a
negative correlation between collection efficiency and relative
humidity for voltages greater than 12 kV is suggested (see Figure
I.I). This correlation is less apparent for operating voltages
of 12 kV. Insufficient data makes it impossible to evaluate the
effect of relative humidity at lower operating voltages.
2) NBL does not report the operating temperatures, but it seems
unlikely that the wide temperature range in the field (10 to
35°C) was adequately studied.
1-3
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Nevertheless, correction factors for temperature and relative humidity are
not applied to field data since insufficient data exist to formulate
accurate correction factors.
Only the October 1982 NBL report included data that could be used to
develop an air flow rate correction factor Cf. Each sampler will require a
unique Cf, however, NBL only reported one data value for each LVS. Since
neither reproducibility nor accuracy was demonstrated by NBL for Cf, it is
not recommended to use the "correction factors" to adjust field data; it is
presumed that exclusion of Cf will not result in severe deficiencies in the
final evaluation since none of these corrections changed the raw data by
more than 20%.
It would be possible to obtain sufficient data to derive a reasonable
correction factor by repeating these tests for each sampler set at the 1000
L/min mark. However, these data experiments are unwarranted, since during
field sampling wind gusts alter the sampling air flow rate making it
impossible to achieve the same laboratory precision in determining the air
flow rate.
In October 1982 NBL measured the voltage supplied to the corona source
at four different high voltage settings on nine different LVS samplers.
Calibration curves were drawn for each sampler by plotting the indicated
versus the measured voltage. Each calibration curve was a straight line
with a slope of approximately one but with various y-intercepts.
Repetition of the voltage measurements was not reported, so it is unknown
whether these results are reproducible. Consequently, the voltage
correction factor uses the recorded operating voltage as the independent
variable, not the actual measured voltage.
To determine whether each LVS should have an individual sampler
correction factor, the 1982 NBL data was analyzed at SwRI on a Cyber
170/171 with the SPSS package and the ANOVA subroutine. No consistent
differences were observed among samplers both at the 12 kV and at greater
than 12 kV (15+ kV). It appeared that the actual run numbers had greater
significance than individual samplers. The significance may be partly due
to relative humidity values; other operating variables (e.g., temperature)
may also contribute to the difference observed between runs.
In the 1976 NBL study, no effects from operating at voltages greater
than 12 kV were observed. However, in the October 1982 study, large
variations of collection efficiencies occur for LVS operating at voltages
greater than 12 kV. At this time there is no explanation for these
conflicting results.
The raw data from the October 1982 NBL study are plotted on a semilog
plot in Figure 1.2 (operating voltage versus relative collection
efficiencies of LVS to AGI samplers). From these data, four different
correction factor curves could be drawn.
1-5
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1-6
-------
In the field, several measurements were made with paired samplers.
These paired field samplers may help to identify the most valid correction
factor, i.e., the correction factor that minimizes the difference between
the reported microbial concentrations for all microorganisms for all paired
samplers.
The four possible correction factor curves are plotted in Figure 1.3.
Curve A represents no correction factor. Curve B is modeled after the 1976
data where data below 12 kV are corrected as an average between reported
values and above 12 kV no correction is made. The third method (Curve C)
was calculated from all averages at various voltages from the 1982 NBL
data. Curve D is a minimum correction factor.
The physical interpretation for Curve A is that an LVS sampler
operates similarly at all voltages. From the preceding discussion, it is
known that this is unrealistic.
Curve B assumes that once the operating voltage reaches 12 kV no
effect on collection efficiency is observed as long as operation occurs
below sparking. In addition, this correction factor has no minimum
asymptote for operating voltages below 12 kV.
The third correction factor (Curve C) demonstrates the same low
voltage correction as Curve B. High voltage operation distinguishes
between these two methods. In Curve C the NBL 1982 data is corrected to 12
kV. Since the data peaks at 14 kV, an inflection point is observed at 11.5
kV, a maximum at 14 kV, and then an asymptote at 14.5 kV. No minimum
asymptote exists. A physical interpretation could be the following: at
low voltages, the collection efficiency increases proportionately with the
operating voltage. At 11.5 kV all of the particles are charged. Greater
voltages affect a greater driving force for separating the charged
particles from the air. Above 14 kV visually undetectable sparking occurs
that reduces the effective voltage until it reaches an asymptote in which
the increased sparking is counteracted by the increased driving force from
the high voltage.
Curve D has a minimum asymptote that implies that under certain field
conditions, a low voltage will always be able to charge a few particles and
will be able to collect these. Moving from the asymptote, at higher
operating voltages, proportionately more particles are charged and
consequently collected. At 11.5 kV, an inflection point occurs that
implies that a different mechanism is responsible for greater collection
efficiencies. It is hypothesized that at 11.5 kV all particles are charged
but that the collecting electric field determines the percentage of
particles that are collected. Thus, an increase in operating voltage above
11.5 kV increases the electric field which in turn increases the collecting
1-7
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driving force. A maximum asymptote is then observed where an increase in
operating voltage increases the sparking phenomenon which reduces the
effective electric field. If operation had occurred while excessive
sparking occurred, it is predicted that Curve D would show a decrease in
collection efficiency beyond the maximum asymptote.
Differences in Curves C and D are a result of the calculational basis
of correction. Curve C was calculated from average efficiencies at various
operating voltages; Curve D was calculated from the highest efficiency
observed for operating voltages below 12 kV and lowest efficiencies
observed for voltages above 12 kV. The latter produces a conservative
correction that adjusts all data to the minimal degree expected. Thus, the
corrected data may be required to be adjusted further, but it will never be
overcorrected.
Curve D seems to be more realistic than Curves A and B because the
data suggest that some correction is required in both the high and low
voltage regions. It also seems to be more realistic than Curve C because
it will not result in overcorrections. This latter is an important
consideration since at low voltages (9 kV) an order of magnitude range was
observed in the experimental data (see Figure 1.2).
All of the field data are corrected using Curve D (minimum
corrections) and are presented in the aerosol data results section along
with a table of all of the operating field voltages. The correction
factors employed are in Table 4.15 of the Calculation of Microorganism
Density in Air section. With these data, the interested reader can develop
his own correction factor method and test it against the field data (paired
samples).
1-9
-------
APPENDIX J
DATA REPO.RTING FORMS
-------
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0 ID 11 1 ?. 13
(J_ (s
21 22 23 2-1 2S 26 27
Phone Number
_Q 3
28 29
Size of Household
1 A ? J. J
30 51 52 33 34
Air Typ l-'ro W;it Sew
_0 3 O 3.
33 56 37 58
III INI IM liil??!-:!.
M IS 16 17 IS l'i 20
o9 40 41
Cotton
CROPS
42 45 44" 45"
Wheat
LIVT.STOCK
47
Other
y,s "i
Catt
1-
60
Irr
(e
72
Inc
;>' sTi
le
%
oT
SRC
73
l-st
"fiT S2
Hogs
o _y
68 69
Acres
1
78"
iiace
S 5 '
O O
70 71
79"
Dwel
S4" SS SO S7" S8 SV> li~() OT' ()J 03" 0^1 i".
Sheep Fowl Horses Other
L
80
DWLOC
J-l
-------
HOUSEHOLD INTERVIEW UPDATE
1 1
Household Number
6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Name
21 22 23 24 25 26 27
Phone
.28 29
Size
30 31 32
Air Typ Fre
33
Wat
34"
Sew
J-2
-------
PARTICIPANT INTERVIEW
_.._ .-.
12345678
Card Form ID
No. No.
9 10 11 '12 13 14 IS 16 17 18 19 20 21 22
Last Name
23 24 25 26 27 28 29 30 31 32 33 34 3S~ 36 37 38"
First Name
O 1_ j2-3
39 40 41 42
Rel Sex Age
_L _L O.(f 3 3
43 44 45 46 47 SO
Job ZJob HR Job HOSWD
£f £" 1 _ _
51 52 S3 54 55
HOSWE HOSPM WFM WFARMWK
56 57 58 59 53"
WFARMDA SPRING SUMMER FALL WINTER
.
61 62
L-TRIPS
JO _
66 67
BOTLD TAPNTR
£i Q. 2?
63 64 65
L TINE
RESPIRATORY
D I
2_ copy cols.
1 2-8
Card No.
68 69 70 71 72 73 74 75 76 77 78 79 80 9 10
Con ' All Bro Emp Ast Cal Caz Oth
CARDIOVASCULAR
0
11 12 13 14 15 16 17 18 19 20 21
Con HBP Str Ilrt Ang Oth
GI
0
22 23 24 25 26 27 28 29 30 31 32 33 34 35 36* 37 38 39 40
Con Sto Int Col Eso Ulc Ulz Div Gal Oth
J-3
-------
PARTICIPANT INTERVIEW ( continued )
OTHER CONDITIONS
jQ __ ; __ __ __ __ __
41 42" 43 44 45 46 47 48 49 50 51 52 53 54 55
Con Skin Leuk HOD Oca Arth Dia Anemia
__ __ __ __ __ Q O O £ O
56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72
Imm RHE Hbv Hav Mon Oth Medic BldT Kdny TB Smoke
EMPLOYMENT 3_ Copy col
1 _ 1 05 _ix Tl"
73 74 75 76 77 78 79
Works Emp Contr Occup. Educ.
Stat
. PNEUMONIA
_ __ _ __
27 28 29 30 31 32 33
Pne Time .Age Hos DUR
34 35
Yr Birth
J-4
-------
PARTICIPANT INTERVIEW UPDATE
_____
12345673
Card Form ID
No. So.
9 10 11 12 13 14 15 16 17 18 19 20 21 22
23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38
First Name
3
39 40 41 42
Rel Sex Age
RESPIRATORY
icopy cola.
T 2-8
Card No.
68 69 70 71 72 73 74 75 76 77 78 79 80 9 10
Con All Bro Eap Aat Cal Caz Oth
CARDIOVASCULAR
11 12 13 14 15 16 17 18 19 20 21
Con HBP Str Hrt Ang Oth
GI
22 23 24
Con Sto
25 26
Int
27 28
Col
29 30
Eso
31 32
Die
33 34
tllz
35 36
Dlv
37 38
Cal
39
Oth
OTHER
40~ 4l~ 42 43 44 45 46 47 48 49~" 50 51 52~
Con Ski Leu Rod Oca Art Ola
5T~ 54~ 53" 35" 37" 33" F9~~6T~ 6T~6T~ oT~6"4~ 63" 6T" 57" 68"
Ane Itnm Rhe Hbv Hav Hon Oth Occ
PNEUMONIA
69 70 71 72 73 74 75
Pne Tim Age Hos Dur
3.2.
76 77
Yr Birth
J-5
-------
PERIOD
HEALTH DIARY REPORT
Hame:
id 5 1 0 1 1 1 pd / ^ ill 0. O O dur
T| y —S- —r —If ~T TV TT TT TT TT TT TT
con
dil
TV TT
dwk
TT TT
md
ocd rx
TT TV
hos
num
76—
Name:
0 ±
rs- re-
id .5 1 0 1 2 1 pd / T ill /O ^ dur
T ^-s—r~7—s—r nrrr rz-rrnr
con 0 dil 0 D dwk 0 O md O ocd' O rx / hos £>
TT 18 IS TTT ZT" . TT TT TV ST5"
num
Name:
id 3 1 U 1 ^ 1 pd / I ill / v / dur <9 A
"T ~T ~6 7" 8 9 TT TT TT TT TT TT TT
con -D dil 0 O dwk (DO md O ocd 0 rx (9 hos £> nirn /
TT TTTT 2o5T' 5T .TT TT TT. -jg-
Name:
id I. 2- JL .9L 1_ U_
it 56789
con ,dil
TT TT TT
pd r*r ill O O O dur .
TTTT TTTTTT TTTT
dwk md ocd rx ' hos
TT TT TT TT- TV
IF
Name :
id
2 3 8 1 1 0 pd / ^ ill O O O dur
TT ~T T T ~T ~T TTTTT TTTTTiT
TT T6-
con dil
TT TT TT
dwk
TT TT
md ocd rx hos
TT TT TT
num
Name:
id 1 2 1 0 1 0 • pd / ^ ill / O / dur
— -—---- -
T ^T ~r ^ s y Tir TT TT TT TV TT TT
con £> dil O O dwk & "2— md \ ocd C) rx
TT TT TT TT TT TT TT
/ hos O num /
TT TT ~^-
Name :
1 1 0 1 0
ill / ^ / dur /
id pd
— — — e ~T ~~ 6 ~T TTTT TTTTTir. TTTT
con D dil / 3 dwk O O md O ocd / rx O hos <9 num I
TT Ta TT To" 2 i TT TT TT TT -
26
J-6
-------
UTSA - CART CLINICAL ANALYSES REPORT
SwRI Project 01-M97
HEALTH EFFECTS STUDY FOR THE LUBBOCX LAND TREATMENT PROJECT
Reoort Date '.?/£ -i IfTi.
Control Numoer
Last Name
First Name
Samole Collection Date
Shioplng Problems
Virus Presence
Sent For electron Microscopy
Data Interoretatlon Comments
3101,;
31 32 JJ jo
1.
2.
3.
4.
6.
7.
3.
9.
1.0.
11.
12.
13.
H.
MICROORGANISMS ISOLATED
. (>(.
GROWTH SIGNIFICANT
H ^'6
J^
-C.-a-
CCM^NTS:
21J
;T7T
F c c L. 4-
-------
UNIVERSITY OF ILLINOIS
SEROLOGY REPOR7
SwRI Project Ot-6097
HEA1.TH EFFECTS STUDY FOR THE LUBBOCK LAND TREATMENT PROJECT
Report Date _j2rJ^
Data Collection Period
ID Number
Sansple Taken
AGENTS TESTED
1. POLIO 1.
2. POLIO 2
3. POLIO 3
4. COXSACKIE B5
5. ECHO 9
6. tCHO j
7. ECHO 11
s. COXSACKIE A9
9.
COMMENTS:
AGENT
IT'-H "IT "IT %
£_ _.9_ _L_ _2_ =
25 26 2^ 28 2_
39 40 41 42 43
C B 0 5 *-
53 54 55 56 57
ICO T0|
I_2-J
E C 0 9 <
E C 0 5 -
24 25 26 27 28
E C 1 1 t
"38 39 "40 41 "42
C A 0 9 =.
52 53 54 ' 55 ~56'
66 67 68 69" 70
TITER
O O S
16 17 18
o C5 SL
"30 TT-~32"
~3ff~5T~60
15 16 .17
29 30 31
/ & i-i
*— ' O e>*- Cp o ^^
60 61 62 63 64 65
7"4 75 76 77 78 79
2
. "80~
I.r.b Dlroctor
-------
GLOSSARY
AND INDEX
-------
GLOSSARY
infection episode: The observation in the study population of a number of
similar infection events (either serologically or in serial clinical
specimens) within a restricted interval of time. [Episodes will be
statistically analyzed for association with wastewater exposure when
the infectious agent(s) was(were) found(or can be presumed) to be
present in the wastewater that was sprayed during that period.]
new bacterial infection (event): 1) Isolation of a major enteric bacterial
pathogen (i.e., specific procedures were designed to attempt the
isolation and identification of any Salmonella species, Shi gel la
species, Campy!obacter fetus subsp. j e j u n i , or Yersim a
enterocolitica) at any level from an individual whose previous
specimen was negative for the respective pathogen; or 2) isolation of
a possibly significant organism (i.e., API Group I, Candida albicans,
Chrome-bacterium, Citrobacter, Klebsiella, Morganella, Proteus,
Providencia, Serratia, and Staphy1ococcus~aureus) at the heavy level
from an individual whose previous specimen was negative to light for
the respective organism; or 3) isolation of selected organisms
uncommon in feces but prevalent in wastewater effluent (i.e.,
Aeromonas hydrophila and the fluorescent Pseudomonas group) at
moderate or heavy levels from an individual whose previous specimen
was negative to light for the respective organism. Organisms in
categories 2 and 3 may be associated with enteric disease if isolated
in large numbers from stools.
new viral infection (event): The isolation of a virus (either a specific
type or unidentified) from the second of a pair of fecal specimens
which was not isolated from the first specimen.
new viral infection (event) by electron microscopy (EM): The detection of
a morphologically distinct class of virus-like particles in the second
of a pair of fecal specimens in the absence of laboratory isolation of
such particles from the same specimen. EM detection of the same class
of particles in consecutive specimens would be counted as a single
event.
serologic conversion: A serologic conversion (seroconversion) is defined
when paired sera from one participant are titrated for agent specific
antibody and the second serum has a fourfold or greater rise in titer
from the first serum.
serological negative: A participant is considered serologically negative
for a specified agent when his/her serum antibody titer to that agent
(a specific type, group, or strain) is less than (<) 8 or 10,
depending on the titration protocol.
titer: The reciprocal of the highest dilution at which a predefined
endpoint of reaction is observed.
-------
CROSS REFERENCE INDEX
Data Type
Hefttitdatt
Serdogy
Hepatitis A
Entero- and adenovirvses
Pdiovirus
Coxsackie and echoviruses
Adenovi ruses
Reovirvses
Potavirus
Norwalk virus
Influenza
Legkmella bacilli
Clinical bacteriology
Clinical virology
Electron microscopy
(fecal specimens)
Tuberculin test
Self-reported illness
Environmental data
Wastewater
Bacteria
Viruses
Wastewater aerosols
Background runs
Microorganism runs
ParMe size runs
Quality assurance runs
Virus runs
Dye runs
Flies
Drinking water
Expotundata
Activity diary
Purpose
3-4
18,35,71-77
72
33,74
74-75
75
75-76
76
76
76
77
18,39
18,39
94
18
14-15,18,36
11-15
14,19
18-20
19-20
15,20
20,42
20
20
20,48
20
20,51
20,60
20
4,11-15
39
Design
11-18
16,18,73
"
"
"
"
••
••
••
"
••
"
16
16
16
16,36
16
16
••
••
16
42,45-46
4849
61-62
16,269
148
16,152
Sample
Collection
35-40
35,71
"
•*
"
"
••
••
••
••
••
"
36,38-39
36,38-39
36,38-39
35
36,38
41-70
41-44
••
••
42-57
42-47,62
47-50,63,66
54-56,70
48-52,67
51-53,68
51-55,69
61-62
267-269
39-40
Sample
Analysis
71-95
77-83
83-84
"
"
••
84-85
85
85
86
86-87
87-92
91-94
94-95
95-115
95-112
95-105,110-112
104-110
112-114
112
112
54
112
112-114
54
114-115
Data
Calculation
132
82-83
132
••
"
••
••
••
••
••
87
57-60
57
57
58
57
58
60
148-153
151-152
Quality Data Statistical
Assurance Management Methods
117-124 147,153-169
125,129-135,146 " 155-163
129-133
132-135
" " "
" " "
•• •• "
•• " "
•• •• "
" " "
•• •• ••
" " "
136-141,146 163-165
141-142,146
141-142
165-166
125 " 166-167
143-145
143-144
125-130
125-130
125-130 127,129
148-153
117-124
Statistical
Results Analysis
171-218
181,209-218
215-218
209-216
209-214
215
180-199
203-209
209-212
209-213
178-187
219-271
219-247
219-247
219-247
247-265
252-255
255-261,264-265
247-252
125-130 125-130
261-265
247-248
267-268
267-271
272-277
183,267-276
Interpretation
167-169
199-203
264-267
"
"
"
------- |