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
-------
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



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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

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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

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               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

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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

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STUDY SITE

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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

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       -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.

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          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

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   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.
                                     33

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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

-------
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

-------
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

-------
     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

-------
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

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                                                                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.

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                 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)

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LABORATORY ANALYSIS

-------
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

-------
              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

-------
         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

-------
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

-------
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

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                                                         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

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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

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                                      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

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       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

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(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

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  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

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          (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

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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

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   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

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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

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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

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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

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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

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                                      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

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                    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

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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

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                 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

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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

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       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

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      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

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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

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                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

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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

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                  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.

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      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

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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

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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

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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

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  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

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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

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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

-------
        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

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                        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

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              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)

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          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

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                 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

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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
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PO
oo
                                    — TRICKLING FILTER
                                    -PIPELINE
J J

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      1981
FMAM JJAS

   1982
                   SAMPLING MONTH

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                                           J  *
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 1982
                                                                                                   JJ
                                                                                                   1980
JFMAMJJAS

    1981
                                                                                                         FMAMJJASO
                                                                                                            1982
                                                      SAMPLING  MONTH
                                                                                             SAMPLING MONTH
   tu
   vt
   3
   K


   O
   CC.
   O
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                                   	TRICKLING FILTER
                                   	PIPELINE
                                                       q
                                                       10
                                           in

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      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
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    (0
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    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
<
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250

200


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100


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O
-

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-
V
1 1 1 1 1
 1982

   SAMPLING MONTH
MJJAS

 1982

   SAMPLING  MONTH
                                                                                                    ^   250
                            ~   200

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                            o

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                                                                                                    o
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                            a.
                            CO
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                            U>




                            I
250-




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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
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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

-------
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                                            275

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                        irrigation period

-------
                              REFERENCES
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Camann, D. E., D.  E. Johnson, H. J. Harding,  and C. A.  Sorber.   1980.
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Carnow, B., R. Northrop, R. Wadden,  S. Rosenberg, J. Hoi den,  A. Neal ,  L.
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                                   279

-------
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                                  280

-------
Flewett, T. H.   1978.  Electron Microscopy in the Diagnosis of  Infectious
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Goldman, D. A.  1981. Bacterial Colonization and Infection in the Neonate.
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                                  281

-------
Hart,  C.  A. and  M.  F. Gibson.   1982.  Comparative Epidemiology of
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Haverkorn, M. L.  and M. F. Michel.  1979.  Nosocomial Klebsiellas I.
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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
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     p. 9-47.
                                  282

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                                   283

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Walker, S. and D.  B.  Duncan.   1967.  Estimation of the Probability of an
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     54:167.
                                   284

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Wilkinson, H.  W., D.  D. Cruce, C.  V.  Broome.  1981.  Validation  of
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     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

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           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

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      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

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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

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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

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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

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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

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                                                                                 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

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                                                                                  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

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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

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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

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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

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     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

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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

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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

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  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 
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                   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

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                        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

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       APPENDIX D



ACTIVITY  DIARIES AND MAPS

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                               .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  
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     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

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                                    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

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j
^ 3.0
! ...
UNION
^

D-4

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                iS	1 I* 10  :
                 iy. LJL^ i
-1-         	

 •••'•••••.••••••••a

                                  ,
                     r'-i — k'.isrsis!  «*a s i — s-~\
                                    '"
      i iy""*  i*.  •„•'<»•*  i   ['•
                D-5

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                                 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

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COORD/MATE  MAP
                          D-7

-------
        ' ;~ 3 ••'••••_ iS ' ; i-; S  i ! - :5

         '• •-.'•'•• -•'>_:_:! [ .. i^-J i"il.]! ..,


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^            ^p  inn pi m            EH

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                            :^O^=r, i	_ — (— oj---' r—"v -i 	r—:
                            ==-2i=3 '—"< H I—"— :—2~H t—*>"":
                            ervr—., —-% —. i—-— —-r——. ———
Ofer
                                      -r"^tt—^i— (  ••.11 .'.- i i v"i 1 '• i-1 •_ !.j! '., I i ;:.]i j...

                        •a ^   -J «    »o  iii'*^"	' r^ti—•—•;


                        11"?"  i^^r
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                                                             Ti
                               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

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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

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     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

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     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

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                APPENDIX F

PROCEDURE FOR WASTEWATER SAMPLE COLLECTION
       WILSON IMHOFF TANK EFFLUENT

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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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Figure 1.2.   Operating voltage versus relative collection efficiency
                      (LVS/AGI ratio) (1982 NBL data)
                                  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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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
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                                                                      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_
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con      ,dil
    TT        TT TT
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id
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con £>   dil  O O   dwk  & "2—  md  \   ocd C)    rx
    TT       TT TT       TT TT      TT       TT
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id                      pd
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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
"
"


"






-------