xvEPA
United States
Environmental Protection
Agency
Office of
Drinking Water
Washington, D.C. 20460
EPA-570/9-80-002
March 1981
Water
Viruses, Organics, and Other
Health-Related Constituents
of the Occoquan Watershed
and Water Service Area
Part II Viruses
March 1981
WASHINGTON, D.C.
VIRGINIA -^ "'A.
-.' "OCCOQUAIS
> WATERSHED
MARYLAND
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VIRUSES, ORGANICS, AND OTHER HEALTH-RELATED
CONSTITUENTS OF THE OCCOQUAN WATERSHED
AND WATER-SERVICE AREA
PART II:. VIRUSES
by
Robert C. Hoehn and Clifford W. Randall
Department of Civil Engineering
Virginia Polytechnic Institute and State University
Blacksburg, Virginia 24061
Project No. 68-01-3202
Project Officer
Frank A. Bell, Jr.
Office of Drinking Water
Washington, D.C. 24060
March, 1981
Criteria and Standards Division
Office of Drinking Water
U. S. Environmental Protection Agency
Washington, D. C. 20460
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CONTENTS
Foreword. vi
Preface vii
Executive Summary ix
Figures xv
Tables xvi
Abbreviations and Symbols xix
Acknowledgments xx
1. Introduction 1
General Description of the Study 1
Background Information 1
The Watershed 2
Description of FCWA's Water Treatment Facilities . 2
Characteristics of Raw and Finished Water 4
Sampling Sites 7
References 10
2. Conclusions 11
3. Recommendations 14
4. Materials and Methods 15
Sampling and Assay Procedures 16
Basic Virus Concentration Procedures 16
Basic Viral Assay Procedures 16
The Carborundum Company's Procedures . 18
EPA Virus Group's Procedures 23
Comparative Studies: Field Sampling and Analysis. . 26
Additional Field Studies 27
Studies Associated With a Water Treatment Plant
Filter 27
Study of Filter Media 27
Sampling Frequencies 28
Occoquan-I 28
Natural Samples 28
Seeded Samples . 28
Occoquan-I 1 28
Supplemental Water-Quality Data. . 31
11
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Comparative Study with Coded, Seed-Virus Samples. . . 32
Overview . 32
Sample-Handling Procedures 33
Role of California State Health Department .... 36
Preparation of Pasteurized Dilution Water 38
Sterilization of Storage Reservoir 39
Mixing Study 41
Pasteurization Procedure 41
Description of Typical Experiment 41
Procedures Following Initial Concentration Step. . 43
References 44
5. Results 45
Occoquan-1 45
Environmental Monitoring 45
RCT-Marker Studies 45
Seeded Samples 45
Occoquan-II 49
Environmental Monitoring: The Carborundum Company 49
Comparative Studies: Environmental and Analysis . 49
Additional Field Studies 55
Studies Associated With Water Treatment Plant
Filters 55
Study of Filter Media 55
Comparative Study With Coded, Seed-Virus Samples. . . 55
Personnel Surveillance 60
6. Discussion 62
Occoquan-1 62
Environmental Monitoring 62
Finished-Water Isolates 62
RCT-Marker Studies 63
Reservoir and Tributary Isolates 64
Seeded Samples 66
Occoquan-II 66
Environmental Monitoring 66
Additional Field Studies 68
Comparative Study With Coded, Seed-Virus Samples. . . 68
Relative Efficiencies 68
,/-The Question of Contamination 71
Appendices
A. Supplemental Data Regarding Virus Sampling and Analysis,
June - August, 1975 78
B. Supplemental Data Collected on Each Sampling Date During
Occoquan-I, 1975-1976 82
C. Field and Laboratory Data Pertaining to Occoquan-II
Environmental Sampling Program for Viruses 100
111
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Comparative Sampling Program: Code Numbers Assigned to
Concentrates Prepared by California State Department of
Health Virus and Rickettsial Laboratory
Results of Swab Tests .................. 113
IV
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DISCLAIMER
This report has been reviewed by the Office of Drinking Water, U. S.
Environmental Protection Agency, and approved for publication.
Approval does not signify that the contents necessarily reflect the
views and policies of the U. S. Environmental Protection Agency, nor
does mention of trade names or commercial products constitute endorse-
ment or recommendation for use.
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FOREWORD
The Office of Drinking Water has broad interests in all aspects
of rendering a safe drinking water for the American public. These
interests run from questions of wastewater and urban impacts on raw
water quality to the effects of treatment and the quality of distributed
water.
The part of the "Occoquan" project reported herein addresses this
broad range of interests with respect to viruses in detail and depth,
with respect to a single, urbanizing reservoir and water service area.
Since this project involved not only environmental studies but also a
major basic testing of comparative virus sampling, concentrating and
analytical schemes, its results will be of interest not only to water
supply engineers but also to virologists and other scientists concerned
with virus measurement technology and assessments. Within the limi-
tations of the measurement technology, it also provides information
with respect to the impact (or lack of impact) of wastewater and
urbanizing factors on virus levels in natural untreated waters.
The Office of Drinking Water also wishes to recognize the extensive
cooperation and participation of EPA's Office of Research and Development
virus research group in Cincinnati, Ohio, without whom the entire
project could not have been completed.
Joseph A. Cotruvo, Ph.D.
Director, Criteria and Standards Division
Office of Drinking Water
VI
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PREFACE
The project discussed in this report was begun soon after the
passage of the Safe Drinking Water Act in 1974 (P. L. 93-523) and the
completion of the EPA's National Organics Reconnaissance Survey (NORS)
involving 80 cities in the United States. Late in 1974, the public
began to be made aware that drinking water supplies may be contaminated
with hazardous organic chemicals contributed by industries and created
during the disinfection process. Concern also was expressed about the
potential impact of urbanization and treated wastewater discharges on
virus levels in raw sources for drinking water supplies. Extensive
coverage by the news media fostered public interest in the subject,
and because the data were so limited at the time, EPA immediately
responded by conducting in-house and extramural research to determine
the extent of the problem and how best to solve it. Since then, much
has been accomplished.
This project, referred to as the Occoquan Report, carried out in
cooperation with the Fairfax County Water Authority, was conceived
initially as an opportunity to study extensively a water system that
serves a large population (approximately 640,000) in a rapidly urbanizing
area of Northern Virginia. Of special interest were viruses and
several chemical constituents of raw and finished waters. Provisions
were made to monitor the chemical constituents at varying frequencies
during all seasons of two consecutive years. Virus monitoring, which
was carried out during the first year, resulted in reported isolates
from finished waters which evoked expressions of concern on several
fronts concerning the reliability of presently available techniques
for detecting viruses. As a result, virus monitoring was increased
during the second year of the project, and in addition, provisions
were made for the contracting party to be joined by EPA's virus-
monitoring research group in a collaborative program of comparative
sampling and methods evaluation. The entire project is truly unique
in that, to date, more data concerning both raw and finished water
concentrations of toxic substances and other constituents exist for
FCWA's system than for any other in the United States.
The site for this project was especially attractive because there
was an ongoing monitoring program (the Occoquan Watershed Monitoring
Program, OWMP) to provide weekly water quality data for the Occoquan
Reservoir and its tributaries which could be used in studies of possible
correlations between raw and finished water quality. Too, during the
period of this project an advanced waste treatment (AWT) facility was
under construction which would effectively remove pollutants contributed
to the reservoir by the discharge of approximately six million gallons
per day of secondary treated sewage to Bull Run, one of the major
tributaries. The Occoquan Project enabled an expansion of the monitoring
effort of the OWMP to include organics, heavy metals, and viruses as
part of the preconstruction data base. The AWT plant, owned and operated
VII
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by the Upper Occoquan Sewage Authority went on-line in late June,
1978, approximately one year after the monitoring provided for by this
contract was completed.
The Occoquan Project discussed in this report involved sampling
the reservoir, a major tributary, and several sites in the distribution
system. The report appears in two parts: Part I Cissued September,
1979) discusses the data pertaining to trihalomethanes, pesticides,
and metals, and Part II is concerned solely with the virus portion of
this study.
Vlll
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EXECUTIVE SUMMARY
In 1975, a project to monitor viruses and chemical contaminants of
water was begun in the Occoquan watershed and water-service area of the
Fairfax County Water Authority (FCWA) located in northern Virginia near
Washington, D. C. The virus-monitoring portion of the study was designed
to supplement other virus monitoring being conducted by the FCWA and the
Occoquan Watershed Monitoring Program (OWMP). The OWMP, ongoing since
1972, is administeTed~by"ViTginia Polytechnic Institute and State Universit)
(VPI^SU) under the auspices of the Virginia State Water Control Board.
Financing is supplied by political subdivisions that lie within the
Occoquan watershed.
This report is the second of two parts and deals solely with the
virus studies that were conducted over a 2-year period, ending May 1977.
The first part (published September 1979, EPA-570/9-79-019) presented
data concerning the chemical contaminants (heavy metals, pesticides, and
trihalomethanes) of natural waters and FCWA's drinking water which it
supplies to approximately 640,000 residents.
Occoquan-I
During the first year of the project (June 1975-May 1976, referred
to as Occoquan-I), the emphasis was on virus monitoring: 1) in a major
tributary stream - (two points - one upstream and one downstream of
sewage treatment discharges on Bull Run), 2) from the source of FCWA's
water supply (the Occoquan Reservoir), 3) the finished water at FCWA's
New Lorton facility (one of three plants), and 4) at two points in the
distribution system, one in Fairfax County and the other in Alexandria,
Virginia. Two major objectives were 1) to determine the background
levels of viruses in Bull Run and at the FCWA's raw-water intake as a
means of assessing sewage discharge and urbanization effects and 2) to
evaluate the capacity of conventional clarification-purification processes
for removing viruses and the possible occurrence of viruses in the
distribution system. The sampling events provided for by this contract
were scheduled to supplement the biweekly schedule for virus-monitoring
in raw and finished water already being carried on by FCWA and the OWMP.
During Occoquan-I, finished water at the three sites was monitored
on 22 occasions, beginning in June 1975, and lasting through August
1975, under the provisions of the contract. The field procedures, which
involved passing approximately 100_g,allQns of water through The Carborun-
dum Company's Aquella Virus Concentrator, were carried out by OWMP
personnel and field technicians employed by The Carborundum Company, who
was VPI&SU's subcontractor. The samplings by FCWA and OWMP personnel in
the ongoing program of virus monitoring also involved the Aquella
concentrator and the schedule called for biweekly samples of both raw
and finished water at FCWA's New Lorton facility. The concentration
procedures were basically those listed as a tentative standard method in
IX
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14th edition of Standard Methods for the Examination of Water and
Wastewater.
Once the field-concentration procedures were completed, the concen-
trates were frozen and shipped by air express to the University of New
Hampshire's (UNH) Jackson Estuarine Laboratory (JEL) where they were
assayed for enteric virus content by inoculating them onto animal-cell
cultures—Buffalo Green Monkey (BGM) cells and African Green Monkey
Primary Kidney Cells (PMK) . The JEL was used exclusively for assay of
field concentrates; to minimize the risk of contamination of field
samples no laboratory-propagated virus strains were ever brought into
the facility.
On four occasions out of the 22 sampling events during Occoquan-I,
low numbers of a single enteric virus serotype (poliovirus type 1) were
recovered from «ae-^f the finished water sites—twice at the treatment
it'll"' plant and once at each of the two distribution system locations. No
( viruses were reported in any of the 26, drinking water samples collected
during the separate study by FCWA. On each occasion when virus recoveries
were reported, the free chlorine residuals were greater than 1.0 mg/1
with probable contact times of several hours, and the pH was between 7.0
and 7.5, conditions normally regarded as adequate for virus inactivation.
Coliforms were absent from samples taken during this period. Because of
the unusual nature of these findings, the data, along with all field and
laboratory procedures, as well as the water treatment processes, were
closely reviewed. In addition, the isolates themselves underwent rigorous
scrutiny at both UNH and the Center for Disease Control (CDC) . In
summary, the virus was characterized as a non-vaccine-like avirulent
poliovirus type 1 strain. This extensive review failed to uncover any
objective evidence to either refute or substantiate the findings. The
Carborundum field sampling and procedures were reviewed, however, and
were found to need improvement with respect to contamination prevention.
The Carborundum Company did not have a program of routine personnel
surveillance wherein throat- and rectal-swabs from the field technicians
were routinely taken, so there was no way to determine if the isolates
could have been the result of a chance contamination by a field team
member who was actively shedding virus.
One would expect that viruses could be recovered more frequently
from untreated natural waters than finished waters, especially if the
natural waters were receiving effluents from municipal sewage-treatment
plants, as was true of Bull Run, but such was not the case during the
Occoquan Project. During Occoquan-I, there were 44 sampling events from
natural waters; viruses were recovered on only three occasions. Six
additional samples, processed by the OWMP during this period from
FCWA's raw water intake, were also negative for viruses. The reasons
for the low percentage of recoveries are not known. Either viruses were
not there at all or were present in concentrations too low to be recovered
by the standard procedure when approximately 100 gallons of water were
processed. During Occoquan-I, the first clarifying filters, which could
have retained viruses trapped in debris, were not eluted, a fact which
might explain some of the failures to recover virus. However, in the
OWMP virus sampling, the clarifying and adsorbing filters were eluted in
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processing their raw water samples, and no viruses were recovered from
either filter type. The puzzling aspect of these data is that viruses
were not found more often in untreated natural waters when, in the same
time frame, viruses were recovered from finished drinking water samples
on four occasions.
Occoquan-II Design
Environmental Monitoring--
Because the reported virus isolations during Occoquan-I were un-
expected and apparently unusual, the EPA had an immediate interest in
continuing and expanding the virus monitoring program. A second year's
effort was designed which was more extensive than the first, and, in
addition, provisions were made for the EPA Viral Diseases Group (Health
Effects Research Laboratory; Cincinnati, Ohio) to periodically sample
side-by-side with The Carborundum Company's field team. During these
events, the final concentrates were to be subdivided, and each of the
respective assay laboratories were to examine a portion of the other
team's sample concentrates. In all, 13 such events were planned,
including two at a local sewage treatment plant. The remaining 11
events included 9 at the 3 finished water sites and 2 at FCWA's raw
water intake. In all, 5£ individual sampling events from finished water
and 19 from natural waters (Occoquan Reservoir at the raw water intake
and Bull Run) were included in the environmental monitoring p^otion of
Occoquan-II.
Prior to the beginning of Occoquan-II, The Carborundum Company
critically reviewed its field procedures and made many modifications in
order to eliminate as many opportunities as possible for a chance
contamination of samples during execution of the field procedures. An
enclosed truck, and later a van, was used to house the sampling equip-
ment. The personnel worked inside the vehicle and disinfected it each
day after use. In addition, a routine personnel surveillance program,
involving rectal and throat swabs, was instituted to provide additional
data in the event an isolate was reported in a sample.
Special Studies
Two special studies were planned for Occoquan-II. One consisted of
a series of eight sampling events of water from FCWA water treatment
plant filters immediately before, after, and during backwash. The
rationale for this study was that clarification processes concentrate
viruses in the floe, and because filters accumulate considerable quan-
tities of the floe over a period of one or more days, the likelihood of
recovering viruses should be greater at such a location during the times
mentioned. The second study involved collecting samples of the unwashed
filter-media (anthracite) and returning them to UNH where they were
examined for the presence of viruses by the routine elution and subsequent
assay procedures. Concentrates were divided for analysis by both EPA
and Carborundum. No viruses were isolated from any of these samples.
XI
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Comparative Sampling and Analysis with Coded, Seeded Samples--
A final aspect of Occoquan-I I / a truly unique one, was the design and
execution of a rigorously controlled study designed to evaluate the
sampling and analysis procedures used for virus monitoring by the EPA and
The Carborundum Company. The plan included the involvement of an
unbiased third party, the California State Health Department (CSHD) Viral
and Rickettsial Diseases Laboratory, which, under separate contract with
EPA, was to prepare blind, coded vials, some containing viruses and some
containing only sterile diluting medium (blanks). The virus vials
consisted of four different sets including polio 1 and polio 2 at four
different titers i.e. 53, 250, 300 and 2.9 x 1 O6 PFU (for 25 test runs)
and sterile medium blanks (for 10 test runs).
A rather elaborate system for pasteurizing finished water was designed
and constructed, and a plan was devised for the two field teams to sample
on 35 separate occasions. On each occasion, the contents of a coded vial
were added to the pasteurized test water, either through the thiosulfate
reservoirs of the respective parties' concentrator system and thereby
dosed into the water through the proportioner pump or added directly to
the large, disinfected tank containing 250 gallons of the pasteurized
test water. After 100 gallons of the water had been passed through their
respective concentrators, the two parties followed their established
protocols for eluting the filters and concentrating the eluates. Then
each party divided its final concentrate into two, equal portions and
returned them to FCWA for receding. Each assay laboratory received one
of the receded samples prepared by the other field team and one prepared
by their own team. For this portion of the study each lab used a common
cell line (BGM, furnished by UNH) in order to minimize a possible source
of error. After each party had completed all analyses and the results
had been reported, the data were decoded, tabulated and sent to the
principals in the project for analysis.
Occoquan-II Results
Environmental Monitoring and Special Studies —
On only one occasion during Occoquan-II was virus isolated out of 81
sampling events from drinking water (26 of these conducted by FCWA). On
the date &f the sole reported recovery, the rectal swab taken from the
principal Carborundum field technician was positive for viruses: six
poliovirus-1 and one Coxsackie B-4 were recovered from the specimen. The
drinking water isolate was an avirulent, no n- vaccine- like type 1
poliovirus, as were the poliovirus isolates from the rectal swab.
Recovery of viruses from the rectal swab, while not definitive, did raise
doubt regarding the validity of the environmental virus isolate from the
distribution system.
In none of the 1 1 comparative sampling events from drinking water
sites did either the EPA or The Carborundum Company report finding any
viruses. Neither were any recovered during the special studies involving
the filters and the backwash water. Both groups successfully recovered
viruses in comparatively large numbers from two samplings of the
xii
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sewage treatment plant effluent.
Viruses were recovered from very few natural water samples during
Occoquan-II, as was true during Occoquan-I. Only three (one at each
station) of 19 samples were positive for viruses, even though both the
clarifying and adsorbing filters were eluted each time. An additional
26 samples were taken from FCWA's raw water intake for virus analysis
as part of the OWMP, and no viruses were recovered from 52 subsamples
(26 eluates each from the clarifying and adsorbing filters). As
mentioned previously, the reasons for the low recoveries from natural
waters of the Occoquan watershed during both years of the study are
not known, but it does appear, based on the failure to find viruses in
143 of 149 subsamples from 95 different sampling events (including
those by the OWMP) using state-of-the-art procedures, that viral
contamination of FCWA's raw water source is not a significant problem,
thereby failing to indicate 'a significant impact on virus levels from
sewage treatment plant discharges or urbanizing activities.
Comparative Studies with Coded, Seeded Samples
The results of the comparative studies with coded, seeded, samples
were quite revealing in several aspects. First, in the series of 25
experiments when the viruses were added to the thiosulfate reservoirs,
the EPA procedure was more effective than the Carborundum procedure in
concentrating the viruses from the water and in detecting them through
the assay. The ranges of recoveries by the two groups during these
studies were 36 to 59 percent for EPA and 0 to 20 percent for Carborundum.
The percentages were calculated from inputs based on the initial seed
sample titrations performed by CSHD. Second, in the series of experiments
when viruses were added directly to the 250 gallons of pasteurized
water, the performance of both procedures were about equal, but the
efficiencies 0-15 percent and 0-18 percent for EPA and Carborundum,
respectively^ were notably lower than during the other tests. The
reason is nou known, but one possibility is that the viruses adsorbed
to the walls of the tank or were otherwise removed before they reached
the virus concentrators.
The third revealing aspect of the study concerned the matter of
contamination. The EPA group reported on three occasions the presence
of a type virus which had not been present in the coded input-vial.
An analysis of their own records and procedures led the EPA group to
conclude that inadequate equipment disinfection probably accounted for
one of the three contaminations. No tenable hypothesis could be put
forth to explain the other two contamination events. None of the 40
subsample analyses from the 10 experiments dosed with sterile medium
(blanks) revealed the presence of viral contaminants.
The results of the comparative study underscore the need for
additional research to devise procedures that will insure contamination-
free results. They also demonstrate that a practical test for virus
monitoring as a routine, water quality assessment procedure is not yet
available.
This report was submitted in fulfillment of Contract No. 68-01-
Xlll
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3202 by the Research Division of VPI£SU and its subcontractor, The
Carborundum Company, under the sponsorship of the EPA. This report
covers the period from May 23, 1975 through May 31, 1977 and work was
completed as of Feb. 1, 1978.
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FIGURES
Number Page
1 Map showing the raw and finished-water sampling
stations in the Occoquan Watershed and Fairfax
County Water Authority's distribution system 3
2 Schematic of virus sampling, Occoquan study, 1975-1977 8
3 Sequence of steps to recover and demonstrate the
presence of virus 17
4 Test procedures used for virus analyses of Occoquan
samples 22
5 Schematic for providing virus input concentrates for
each experiment 34
6 Schematic for processing: Steps in the comparative
sampling, concentration, analyses, and reporting of
data 35
7 Diagram of the water-pasteurization system and
storage reservoir 40
8 Virus titers in frozen concentrates on four occasions
estimated by interpolation and based on an assumption
that the observed decrease in titer was linear between
the initial and final assay by the California State
Health Department Laboratory 72
xv
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TABLES
Number
1 Averages and Ranges of Finished Water Production
Rates at Fairfax County Water Authority During
Project Period
2 Average Characteristics of Raw and Finished Water
at the Fairfax County Water Authority's New Lorton
Treatment Facility During Project Period, May 1975-
May 1977 6
3 Occoquan Project Sampling Site Locations and
Descriptions 9
4 Numbers of Samples Assayed for Enteroviruses, Reo-
viruses, and Adenovirus During Occoquan-I, June-
August, 1975 29
5 Numbers of Environmental Samples Assayed for Entero-
viruses During Occoquan-II, June 1976-March 1977
by the Carborundum Company 30
6 Contents of Vials Prepared by California State Health
Department Laboratory for use in Comparative Sampling
Study 37
7 Summary of Virus Recoveries and Supplemental Water
Quality Data During June, through September, 1975 . . 46
8 Results of the Reproductive Capacity Temperature Marker
Tests Performed on Occoquan Isolates 47
9 Results of Seeded-Virus Recovery Experiments .... 48
10 Laboratory and Field Data Associated with Environment-
al Samples on Days When Viruses were Recovered at
Sites A, B, C, and E, Occoquan-II, June 17, 1976-
March 25, 1977 . 50
11 Results of Comparative Virus Sampling Program, Phase
2-A, Involving Concentrates of Samples Taken From
Northside Sewage Treatment Plant by Carborundum and
Assayed in the Laboratories of Both the Carborundum
Company and the EPA's Health Effects Research Labs 51
xvi
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Number Page
12 Results of Comparative Virus Sampling Program,
Phase 2-A, Involving Concentrates of Samples Taken
From Northside Sewage Treatment Plant by the Virus
Research Group at EPA's Health Effects Research Labs
and Assayed in the Laboratories of Both the EPA and
the Carborundum Company 52
13 Results Reported by the Carborundum Company for
Assays of EPA/HERL Concentrates Taken During Phase
2-A in Comparative Sampling and Analysis Experiments,
July 1976. 53
14 Results Reported by EPA for Assays of Concentrates
Prepared by EPA and the Carborundum Company's Virus
Groups During Phase 2-A in Comparative Sampling and
Analysis Experiments, July, 1976 54
15 Physical, Chemical, and Biological Data Collected
During Special Studies (Phase 1-B) to Attempt Enteric
Virus Recoveries from Finished, Filtered Water
Immediately Preceding and Following Filter Backwash
and From the Backwash Water Itself in the Pipe
Gallery of the New Lorton Facility, Fairfax County
Water Authority, June, 1977 56
16 Virus Recoveries by the Carborundum Company and the EPA's
Virus Groups During the Comparative Sampling-and-Assay
Study with Coded Samples Supplied by California State
Health Department (Thiosulfate Reservoir Experiments). 57
17 Virus Recoveries by the Carborundum Company and the
EPA's Virus Group During the Comparative Sampling-and-
Assay Study with Coded Samples Provided by California
State Health Department (Diluted in 250 Gallons of
Water) 59
18 Virus Titers Reported After Direct Analyses of Coded
Vial Contents Stored at FCWA During the Comparative
Samp ling-and- Analysis Program 61
19 Results of Strain Characterization Tests Provided by
the Center for Disease Control for the Occoquan
Isolates 65
20 Antigenic and RCT Characteristics of Occoquan-II Virus
Isolates from Finished Water and a Rectal Swab. ... 67
21 Summary of Virus Recovery Data (Based on California
State Health Department's Initial Titer) Obtained
During the Comparative Sampling-and-Assay Study in
Which Viruses were Injected in the Thiosulfate Reserv-
voirs 69
xvi i
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Number Page
22 Summary of Virus Recovery Data (Based on California
State Health Department's Initial Titer) Obtained
During the Comparative Sampling-and-Analysis Study
in Which Viruses were Added to the 250 Gallon Tank. . 70
23 Revised Recovery Efficiencies of Viruses Based on an
Assumption of a Linear, Virus-Titer Reduction in
Frozen Concentrates (Viruses Diluted in Thiosulfate
Reservoir) 73
24 Revised Recovery Efficiencies of Viruses Based on
an Assumption of a Linear, Virus-Titer Reduction
in Frozen Concentrates (Viruses Injected into a
250-Gallon Tank) . 74
xvi 11
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LIST OF ABBREVIATIONS AND SYMBOLS
ABBREVIATIONS
BGM
°C
CPE
CSHD
EPA
FCWA
ft
g
gpm
HC1
HEK
JEL
km
a
M
pm
MCS
mg/1
MGD
ml
N
OWMP
PEG
PFU
PMK
prr
3TP
STS
UNH
UOSA
SYMBOLS
A1C1
Buffalo Green Monkey
degrees Celsius
Cytopathic effect
California State Health Department
Environmental Protection Agency
Fairfax County Water Authority
feet
gram(s)
gallons per minute
hydrochloric acid
human embryonic kidney
Jackson Estaurine Laboratory
kilometer
liter
Molar
micrometer
membrane coating solution
milligrams per liter
million gallons per day
milliliter
Normal
Occoquan Watershed Monitoring Program
polyethylene glycol
plaque-forming unit(s)
primary monkey kidney
sewage treatment plant
sodium thiosulfate
University of New Hampshire
Upper Occoquan Sewage Authority
aluminum chloride
sodium carbonate
sodium thiosulfate
mean
xix
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ACKNOWLEDGEMENTS
A special debt of gratitude is due the Fairfax County Water Author-
ity for making this project possible and for being willing to allow their
water system and operations to be scrutinized so thoroughly. Special
thanks are due Mr. Craig Cameron, the Laboratory Director at the treat-
ment facilities, for his interest and unwavering spirit of cooperation.
The authors gratefully acknowledge also the following people for
their contributions to the research discussed in this report: Dr. Elmer
Akin, Chief of EPA's Viral Diseases Research Group (Health Effects Re-
search Lab) in Cincinnati, Ohio, and his associates who participated in
the comparative sampling-and-analysis program and contributed to the
data analysis; Dr. Edwin H. Lennette, Chief of Biomedical Laboratories
and Dr. Nathalie J. Schmidt, Research Specialist in the Viral and
Rickettsial Disease Laboratory, California State Department of Health
in Berkeley, California who prepared the coded virus samples used during
this project; Dr. Milford H. Hatch, Chief of the Center for Disease
Control, Enteric Virology Branch (Virology Division, Bureau of Labora-
tories) in Atlanta, Georgia, who characterized the virus isolates ob-
tained during the environmental sampling portion of this project;
Dr. Theodore G. Metcalf, Professor of Microbiology and his associates
at the University of New Hampshire in Durham, New Hampshire, who were
responsible for all the subcontractor's viral assays and identification;
and to Ms. Kathleen Saunders, Research Associate at the Occoquan Water-
shed Monitoring Laboratory in Manassas Park, Virginia, who had the prime
responsibility for assisting the subcontractor (The Carborundum Company,
Niagara Falls, New York) in obtaining viral concentrates from the field
and for obtaining all supplemental data required by the contract.
Finally, the authors acknowledge the excellent support of Dr. Peter
T. B. Shaffer and Mr. Robert A. Fluegge, who coordinated the handling and
analysis of the samples and also provided technical input to the design
and execution of the viral program and to the analysis of the data which
resulted.
xx
-------
SECTION 1
INTRODUCTION
GENERAL DESCRIPTION OF THE STUDY
In June, 1975, an intensive monitoring effort, referred to as "The
Occoquan Project," was begun in the Occoquan, Virginia, watershed and
water-service area of the Fairfax County Water Authority (FCWA). The
study, which began within six months after EPA's "eighty-city survey" (1)
and the enactment of the Safe Drinking Water Act (PL 93-523), was
designed to determine the concentration range and variability of a variety
of health-related constituents of both the treated water from FCWA's
distribution system, which serves approximately 640,000 residents of
several northern Virginia communities, and the raw water source (the
Occoquan Reservoir), including one of two of its major tributaries (Bull
Run). The water constituents which were of interest were: a variety of
pesticides; haloforms (principally the trihalomethanes); selected heavy
metals, many of which are toxic to humans; and enteric viruses. The re-
sults concerning the chemical constituents have been presented in Part I
of this report, (EPA-570/9-79-019, September 1979).
The Occoquan Project was conducted by Virginia Polytechnic Institute
and State University (VPISSU) and its subcontractor, The Carborundum
Company, as a supplement to a larger, ongoing monitoring effort—the
Occoquan Watershed Monitoring Program (OWMP)--that is funded by the
several political jurisdictions that lie within the watershed and is con-
ducted under the auspices of the Virginia State Water Control Board.
That program, begun in 1972, is conducted by VPI^SU and provides weekly
data concerning water quality at several points within the Occoquan
Reservoir and along its major tributaries. The Occoquan Project provided
for an expansion of the water-quality data base compiled by the OWMP be-
fore an advanced waste treatment facility, owned and operated by the Upper
Occoquan Sewage Authority (UOSA), went on-line in June, 1978. The OWMP,
in addition to its routine monitoring, has been the nucleus for several
projects involving water-quality assessments, urban runoff, and other
related subjects. Reports of several of these studies have appeared in
the literature (2, 3, 4, 5).
This portion of the report contains a presentation and discussion of
data concerning the virus-monitoring phases of the project during the
first and second years of the project (hereafter referred to as Occoquan-
I and Occoquan-II, respectively) and a special methods-development study
in which the Viral Diseases Group of EPA's Health Effects Research Lab-
oratory (HERL), Office of Research and Development, participated. The
viruses of interest included three enteroviruses--polio, Echo, and Cox-
sackie--adenovirus, and reovirus. During the first year of the project,
which began in late May, 1975, only routine monitoring for viruses from
-------
untreated and treated waters was involved. The purpose of this monitoring
effort was to determine background virus levels in the raw water and to
evaluate the capacity of conventional clarification-purification processes
for removing viruses during the treatment of water prior to its distribu-
tion to consumers. The schedule was arranged so that the monitoring pro-
vided by the contract would supplement that being conducted routinely by
FCWA and as part of the OWMP.
During the second year (Occoquan-11), the environmental monitoring
effort was continued, but at less frequent intervals, and, during that
year, The Carborundum Company's virus group and EPA's virus group partici-
pated in several side-by-side studies which involved monitoring of both
raw and finished drinking water and, on one occasion, treated sewage. In
addition, a rather elaborate, controlled study was designed and executed
to evaluate the methods of virus sampling and analysis that were then
being used by the two groups. That study, which will be described in
considerable detail later in this report, involved the recovery of enteric
viruses, whOse concentrations and identities were not known by the
participants, from large volumes of heat-sterilized water. Through this
study, an evaluation of currently used virus-concentration and assay pro-
cedures was made possible.
The contract also provided for additional analyses during both years
which would help characterize the water quality at the time of sampling.
Included were: bacterial analyses [fecal and total coliforms plus stan-
dard plate counts (SPC)], temperature, pH, physical appearance, and a
qualitative assessment of odor if any unusual condition was noted.
BACKGROUND INFORMATION
The Watershed
The watershed (area approximately 580 square miles), as shown in
Figure 1, consists of portions of four counties: Fairfax, Fauquier,
Loudoun, and Prince William. Two of the major tributaries join to form
the Occoquan Reservoir, which was impounded in 1957. At full pool, the
reservoir holds 9.8 billion gallons. The headwaters of the watershed are
in forested and agricultural areas. Cedar Run and Broad Run drain areas
(approximately 191 square miles) that are almost entirely rural and
undeveloped from an urban perspective. On the other hand, Bull Run
flows between and adjacent to Fairfax County and the Manassas area, two of
the most rapidly urbanizing regions in the United States. Urban runoff and
effluents from sewage treatment plants [about 5.4 million gallons per day
(MGD)] flow into Bull Run and its tributaries, eventually reaching the
reservoir. Construction of an advanced watewater treatment plant, under the
auspices of the Upper Occoquan Sewage Authority (UOSA), was completed
during 1978, greatly reducing one source of pollution in the reservoir.
The urbanized areas, constituting approximately twenty percent of the
total watershed, are mainly along Bull Run (bordering Manassas), along
Flat Branch (draining Manassas to Bull Run), and along Big Rocky Run (a
tributary to Cub Run)
Description of FCWA's_ Water Treatment Facilities
Fairfax County Water Authority has three interconnected treatment
-------
RAW WATER STATIONS -^ ^
A. BULL RUN. UPSTREAM
B. BULL RUN, DOWNSTREAM OF SEWAGE TREATMENT DISCHARGES /
C. OCCOOUAN RESERVOIR INTAKE FOR LORTON PLANT
FINISHED WATER STATIONS Q
D. EFFLUENT, LORTON WATER TREATMENT PLANT
E. FAIRFAX COUNTY
F. ALEXANDRIA IFIRE STATION)
G. DUMFRIES (FIRE STATION)
—k
SCALE IN KILOMETERS
Figure 1. Map Showing the Raw and Finished Water Sampling Stations
in the Occoquan Watershed and Fairfax County Water
Authority's Distribution System.
-------
plants: the Occoquan Plant, the Old Lorton Plant, and the New Lorton
Plant. The latter was dedicated in 1973, and it employs conventional
sedimentation-filtration units. The other two plants utilize aldridge
sedimentation-filtration units. The design capacities of the three
plants, in million gallons per day (MGD), are 20, 26.4, and 16, respective-
ly, although the operating rates allowed by the Virginia State Health
Department have been increased to permit production rates of 111.6 MGD
with increased monitoring of turbidity in the finished water. The
Occoquan Plant employs tube sedimentation and high-rate-filtration at
rates of 4-5 gallons per minute per square foot (gpm/ft ). The other
two plants employ conventional filtration at rates of 2 gpm/ft and
higher.
Water is taken from the Occoquan Reservoir through a 72-inch main.
Pretreatment is usually chlorination only, but provisions are made for
the addition of powdered activated carbon. Alum is the principal coagulant,
but ferric salts are used occasionally. After flocculation, settling,
and filtration, additional chlorine is added, and lime is added to
increase the pH to approximately 7.5. Backwash water is sent to a
reclamation basin, and from there the settled water is recycled to the
head of the New Lorton Plant. Subsequent to the field sampling period
covered by this project, the chlorination points in all three plants was
moved in 1977-1978 to the settling basin discharge.
During the project period, the average finished-water production
rate varied from 56.5 to 75.1 MGD (See Table 1). The approximate contri-
bution by each of the three plants, as a percentage of the total during
1975-1976, were as follows: Occoquan, 45 percent; Old Lorton, 25 percent,
and New Lorton, 30 percent.
Characteristics of Raw and Finished Water
Table 2 shows the average raw and finished-water characteristics
recorded at the New Lorton facility during the project period. The data
are shown as average quarterly values and include most of the routinely
monitored constituents. Periodically, comprehensive analyses of the
waters are performed, and concentrations of selected heavy metals, total
organic carbon (TOC), carbon chloroform extract (CCE), chlorides, sulfates,
phosphorus, nitrates, and total solids are determined. In summer, 1977,
a monitoring program for trihalomethanes (THM's) was begun by FCWA
personnel to evaluate the effectiveness of varying treatment processes
in reducing finished-water THM concentrations.
As can be seen from Table 2, the raw water is low in alkalinity
(and dissolved solids, as reflected by the low specific conductivity),
and addition of coagulants reduces the pH to less than 7.0, necessitating
lime addition to increase the buffering capacity of the finished water.
Powdered activated carbon is added periodically to control tastes and
odors generated primarily by algae in the reservoir. Copper sulfate is
applied at several points in the reservoir, usually from May through
October. In 1975, 78.7 tons were applied, 69 percent of which was in
July and August. In 1976, only 52.7 tons were applied, approximately 52
percent being added in July and August. Since 1971, the Authority has
successfully improved raw water quality at the intake by forcing air
through perforated, plastic pipes that lay on the bottom of the reser-
voir extending from the dam for several hundred feet upstream.
-------
TABLE 1. AVERAGES AND RANGES OF FINISHED WATER PRODUCTION RATES
AT FAIRFAX COUNTY WATER AUTHORITY DURING PROJECT PERIOD
Time Period
Finished Water Production Rate, MGD
Average Range
June-Sept. 1975
65.9
52.5 - 89.5
Oct.-Dec. 1975
56.5
51.6 - 63.2
Jan.-March 1976
57.7
53.0 - 63.5
April-June 1976
72.5
60.0 - 99.0
July-Sept. 1976
75.1
60.7 - 97.0
Oct.-Dec. 1976
63.7
56.7 - 74.2
Jan.-March 1977
63.6
56.2 - 74.3
April-May 1977
72.4
61.3 - 98.4
MGD-million gallons per day. To convert to cubic meters per day, multiply
values shown by O.J785.
-------
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SAMPLING SITES
During the first year of the study, there were six sampling sites:
two on Bull Run upstream and downstream of major discharges of treated
sewage and urban runoff, two at the FCWA's New Lorton Water Treatment
Facility (both raw and finished waters) located near the reservoir high
dam, and two at distant points in the distribution system. During the
second year, the Bull Run sites were sampled only once, and an additional
site in the distribution system was added to the sampling schedule. A
detailed description of these sites is given in Table 3.
The particular location for Site F was selected originally because
an open-storage reservoir is located between it and the water treatment
plant. During the day, when the demand is low, the reservoir fills.
Between 4:00 p.m. and midnight, when the demand is high, the reservoir
is used to supplement the flow from the FCWA plant. However, during
this study, most all samples were taken at times when there was no
contribution from the open reservoir.
Figure 2 provides a schematic of the virus sampling stations
selected (also shown on the map in Figure 1) and the possible expected
results. First, Site A above the sewage treatment discharges was
selected to provide low background data. Next, Site B was selected
downstream of sewage outfalls to reflect possibly increased virus
levels. The last raw water site (C) was selected to show the virus
levels presented for treatment by the New Lorton Treatment Plant; virus
levels here would likely be lower than at Site B. Finally, four treated
water sites were selected; one of the New Lorton Plant effluent (D) and
three at distant points in the distribution systems (E, F, G). Site G
was the most distant from the treatment plant. Interestingly, the study
results failed to confirm many of the original expectations.
Three additional sites were selected for short-term studies in
which both the virus groups from EPA and The Carborundum Company partici-
pated. These sites were: 1) the effluent from the secondary clarifier
and the effluent from the subsequent storage lagoon (approximately 30-
days detention) at a local sewage treatment plant (Northside Sewage
Treatment Plant, a trickling-filter operation); 2) the open-storage
reservoir used by the Virginia American Water Company in Alexandria,
Virginia, to store treated water from FCWA to augment flow during peak
demand periods; and 3) below the filter gallery at FCWA's New Lorton
facility. Water was sampled immediately preceding backwash of a filter,
again during the backwash,, itself, and, finally, immediately after the
backwashing cycle was completed.
The sewage treatment plant was selected because it was expected
that the virus concentration would be quite high, and recoveries by both
sampling parties could be virtually assured. The open-reservoir in
Alexandria was sampled to determine if such a method of water storage
might possibly provide an opportunity for chance contamination of potable
water. The studies involving the filter backwashing process were con-
ducted to determine if the filter itself might store viruses or if the
disruption of the filter by backwashing might be conducive to virus
breakthrough immediately after the backwashing was complete.
-------
SITE A
UPSTREAM, LITTLE POLLUTION.
EXPECTED LOW VIRUS LEVELS
SEWAGE
TREATMENT 1ST)*
WASTE
DISCHARGES 3
SITE B
(BELOW ST
WASTE DISCHARGES
EXPECTED HIGH
VIRUS LEVELS)
.0 -
'INTAKE (EXPECT VIRUS
LEVELS TO FALL
BENEATH THOSE
AT SITE B)
VIRUS LEVELS
EXPECTED TO
BE MINIMAL OR
ELIMINATED
BY TREATMENT
DISTRIBUTION
V SYSTEM
POINTS
Figure 2. Schematic of Virus Sampling, Occoquan Study. 1975 - 1977.
-------
TABLE 3. OCCOQUAN PROJECT SAMPLING SITE LOCATIONS AND DESCRIPTIONS *
Site
Location
Conditions
*A On Bull Run, 23 miles upstream of the con-
fluence of Bull Run and Occoquan Creek.
Sice la designated "Cacharpln" and is ac
a point where Rt. 705 crosses Bull Run.
8 On Bull Run, 3.1 miles below confluence
with Cub Run and 2.3 miles below the dis-
charge of the last of 11 sewage treatment
plants. The site is 14 miles below Site
A, approximately 9 miles above the con-
fluence of Bull Run and Occoquan Creek.
Approximately 1000 feet below the Occoquan
Reservoir dam from the raw vacer main to
the Fairfax County Water Authority's (FCWA)
water treatment plant. Samples were taken
from inside the "carbon house". The site
is approximately 18 miles from Site B and
9 miles from confluence of Bull Run and
Occoquan Creek.
Pump Station Ho. 2 of the Lorton High Ser-
vice Plant at FCWA water treatment plant.
Within the distribution system in Fairfax
County at the Fairfax County Storage Yard
on Hwy 29 near County Road 645.
The Prince Street Fire Station in Alexan-
dria, 7a. At times, water from an open
storage reservoir is rechlorinated and
used to supplement flow from FCWA.
The Dumfries-Triangle Volunteer Fire De-
partment, Inc. 18329 Jefferson Davis
Highway.
Forested and agricultural
areas. Represents condi-
tions upstream of major
sewage treatment plants and
urban drainage.
Average flow approximately
75 cfs. Bull Run at this
point has received approxi-
mately 5.4 MOT of treated,
chlorinated sewage from 11
plants (6 from Prince Wil-
liam Co. and 5 from Fairfax
Co.) as well as urban and
agricultural drainage.
Raw water as received by the
Fairfax Co. Water Authori-
ty's (FCWA) three treatment
plants, combined capacity
approximately 60 MOT.
A mixture of finished waters
from the three treatment
plants of the FCWA.
Finished water approximately
10 hours distant from the
FCWA water treatment plants
at average flow.
Finished water approximately
3 hours distant from FCWA
water treatment plants at
average flow. The system
belongs to the Virginia
American Water Company.
Finished water approximately
13 hours distant from FCWA
water treatment plants at
average flow. The water going
to this site is derived totally
from the Occoquan Treatment
Plant.
*Sita A is sampled routinely as part of the OWMP. The other sites listed are
separata from those sampled by OWMP personnel.
**This site was sold in February, 1978, and is no longer under FCWA's control.
-------
REFERENCES
1. Symons, J. M., Bellar, T. A., Carswell, J. K., DeMarco, J.,
Kropp, K. L., Robeck, G. G., Seeger, D. R., Slocum, C. J., Smith,
B. L., and Stevens, A. A. National Organics Reconnnaissance
Survey for Halogenated Organics in Drinking Water. J. Am. Wtr.
Wks. Assoc., 67 (11): 634-647, 1975.
2. Randall, C. W., Garland, J. A., Grizzard, T. J., and Hoehn, R. C.
Characterization of Urban Runoff in the Occoquan Watershed of
Virginia. In: Proceedings of the Conference on Urbanization and
Water Quality Control, Am Wtr. Resources Assoc., pp. 62-69. Also
in: Urbanization and Water Quality Control, William Whipple, Jr.,
ed., American Water Resources Assoc., Minneapolis, Minnesota, 1975.
pp. 62-69.
3. Grizzard, T. J., Randall, C. W., and Hoehn, R. C. Data Collection
for Water Quality Monitoring in the Occoquan Watershed of Virginia.
In: Proceedings of the Conference on Environmental Modeling and
Simulation, U. S. Environmental Protection Agency Offices of
Research and Development and Planning and Management, Cincinnati,
Ohio, April 19-22, 1976, pp. 819-823. (Also, EPA 600/9-76-016,
July, 1976).
4. Randall, C. W., Garland, J. A., Grizzard, T. J., and Hoehn, R. C.
The Significance of Stormwater Runoff in an Urbanizing Environment.
Progress in Water Technology 9: 547-562, 1977.
5. Grizzard, T. J., Randall, C; W., and Hoehn, R. C. Runoff Sediment
Loads from Urban Areas. In: Proceedings of the International
Symposium on Urban Hydrology, Hydraulics and Sediment Control.
University of Kentucky, Lexington, Kentucky. July, 1977. pp. 317-
324.
6. Randall, C. W., Grizzard, T. J., and Hoehn, R. C. Monitoring for
Water Quality Control in the Occoquan Watershed of Virginia, USA.
In: Proceedings of the International Workshop on Instrumentation
for the Control of Water and Wastewater Treatment and Transport
Systems, International Association on Water Pollution Research,
London/Stockholm, May, 1977. pp. 4-20-1 to 4-20-6, 1977.
10
-------
SECTION 2
CONCLUSIONS
The following conclusions are based on the results of the virus
studies conducted during the Occoquan Project, June, 1975 through May,
1977.
1. During the first year of this project (Occoquan-I)t finished
drinking water was monitored for viruses on 22 occasions during the
summer of 1975 under the provisions of this contract and an additional
26 times on a bi-weekly basis during 1975, by the Fairfax County Water
Authority (FCWA) in conjunction with the Occoquan Watershed Program
(OWMP - not part of this contract). Recoveries of single, non-vaccine-
like, avirulent poliovirus isolates (type 1) were reported on four
occasions, which represents an inordinately high percentage of the
total samplings based on current knowledge of water treatment efficiency
for virus inactivation. This rate of recovery was significantly
higher than for the untreated natural waters, an anomalous result (see
Figure 2 in the Introduction Section). A review of the field data,
along with several subsequent investigations into the nature of the
virus isolates, the laboratory procedures, and treatment plant operations,
failed to uncover any objective evidence to either refute or firmly
substantiate the conclusion that viral isolates originated from the
finished water. No personnel surveillance program was conducted
during the first year's effort, however, and the lack of such data
limited the extent to which the possible sources of the isolates could
be explored.
2. During the second year (Occoquan-II) improved field techniques
were instituted, a program of personnel surveillance was established,
and provisions were made for comparative sampling and analysis with
the Cincinnati-based, EPA Viral Diseases Group (Health Effects Research
Laboratory). On one occasion a virus isolate was reported out of 81
sampling events from finished water (55 provided by the contract, 26
by FCWA). The single isolate (also a non-vaccine-like, avirulent
poliovirus, type 1) was recovered on a day when the principal field
technician's rectal swab was positive for virus (6 type 1 poliovirus
and 1 coxsackie B-4 virus), a fact which casts doubt on .the actual
source of the finished-water isolate-
3. Whereas one would expect to recover viruses quite frequently
from natural, .untreated waters contaminated by sewage, the number of
occasions when that occurred were quite small. During Occoquan-1,
there were 44 sampling events from natural waters; viruses were recovered
only on three occasions, once at each natural water station (upstream,
downstream"of sewage treatment plant discharges and at the FCWA's raw
water intake). During Occoquan-Il, only three of 19 samples were
positive (again, one a,t each natural water station), even though both
11
-------
the clarifying filters and the adsorbing filters (parts of the virus
concentrator) were eluted. An additional 38 samples were taken for
virus analysis at FCWA's raw water intake as part of the ongoing OWMP
with no viruses being recovered from 76 subsamples (adsorbing and
clarifying filter eluates). The reasons for the low recoveries of
viruses from natural waters, assuming they were present, lie in the
limitations of the current environmental virus recovery procedures.
Taking these limitations into consideration, the results (6 recoveries,
149 concentrates from 95 sampling events) do, however, appear to show no
significant virus contamination of the FCWA's raw water source, thereby
failing to indicate a significant impact on virus levels from sewage
treatment plant discharges or urbanizing activities.
4. During Occoquan-II, both laboratory groups demonstrated their
ability to recover and analyze viruses in limited, joint, environmental
sampling from a sewage treatment plant effluent. In eleven joint-
sampling events of finished drinking water, no viruses were found.
5. Attempts to recover viruses from water immediately before,
after, and during the backwash of water treatment plant filters were
unsuccessful on eight occasions. No viruses were found in the final
sample concentrates, half of which were assayed by each participant in
the comparative program, though one would expect that sampling at such
a location and at those times would provide the greatest opportunity for
recovery of viruses which might have persisted during the water-treat-
ment process.
6. A carefully controlled, large-scale, double-blind experimental
study was conducted in order to evaluate both the efficiency and relia-
bility of currently used methods for recovering and detecting small
numbers of viruses from large volumes of potable water. The results
showed the following:
a. When viruses were added to the thiosulfate reservoir (part
of the concentrator equipment) the .EPA Viral Diseases Group's perform-
ance (based on the percent recoveries reported by both parties that
participated in the study) was superior, both in concentrating the
viruses from water and in detecting them during laboratory assays of the
samples. The ranges of recoveries reported by the two groups (EPA and
The Carborundum Company) were 36-59 percent and 0-20 percent, respec-
tively, based on the original titer levels determined by the independent
laboratory which supplied the viruses..
b. When viruses were added directly to a large tank containing
250 gallons of water (a situation more closely approximating conditions
in a community water supply), the performance of both groups was about
equal (0-15 percent and 0-18 percent for EPA and The Carborundum Company,
respectively). These levels, much lower than the thiosulfate reservoir
recovery efficiencies, raise basic questions about accepted practices of
testing for virus recovery efficiencies and about the effective recovery
efficiencies being obtained in virus sampling of clean finished drinking
waters.
7. There were substantial, detectable differences in the efficiencies
12
-------
of both the virus-concentration and virus-assay procedures employed by
two, independent laboratories that were following currently accepted
techniques for virus recovery from drinking water. These findings,
together with the low level of tank test recoveries, point up the need
for more developmental research into the most desirable procedures
involved with virus monitoring, from the collection of samples to
shipping and handling procedures and, finally, for the assay itself.
Current methods, at best, are conservative in their potential for
reliably detecting the presence of low levels of viruses (e.g. at
levels of one in 100 gallons, the standard suggested by some).
8. Whereas contamination was suspected to have been responsible
for the reported virus isolates from drinking water during Occoquan-I
and II, the comparative study described in Conclusion 6 demonstrated
beyond all doubt that the possibility of contamination is an ever-
present danger whenever monitoring for low-level virus contamination of
drinking water is attempted. The EPA virus-research group reported
contaminants in three of the 70 subsamples which they analyzed. Among
the several plausible explanations, the most likely seemed to point to
improper equipment sterilization in one instance; no tenable hypothesis
could be put forth to explain the other two contamination events. None
of 40 subsample analyses from 10 experiments dosed with sterile medium
(blanks) revealed the presence of viral contaminants. Control tests
were routinely conducted in both assay laboratories to guard against
reports of false positives in water samples. In the light of all the
expertise, concern, and care put into this project, the report of contam-
ination serves to emphasize the complexities involved in effective
virus monitoring. The results of this study underscore the need for
additional research to devise procedures that will insure contamination-
free results. They also demonstrate that a practical test for virus
monitoring as a routine water quality assessment procedure is not yet
available.
13
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SECTION 3
RECOMMENDATIONS
1. The results of this study indicate that the present day, virus
monitoring technology, while extremely well developed compared to that of
a decade ago, still is not sufficiently precise to permit routine
surveillance of drinking water supplies for the reliable detection of one
or two viruses in a large volume of water, say 100 gallons. Therefore,
further methods-development research is needed under conditions that
simulate actual environmental conditions as nearly as possible.
2. While those concerned with environmental monitoring for viruses
have always been aware of the need for attention to contamination
prevention during sampling, the results of this study have clearly shown
that contamination can occur even when stringent precautions are being
taken to avoid it. Therefore, it is recommended 1) that a thorough
review of all existing procedures, both field and laboratory, be
undertaken to identify those operations where even the remotest
possibility of contamination exists and then, 2) that a detailed protocol
for contamination control and quality assurance be developed, tested and
established for inclusion in any future standard method.
3. As initially proposed, this project envisaged two parts - before
installation of the Upper Occoquan Advanced Wastewater Treatment (AWT)
Plant and after the plant was put in operation. In view of the low levels
of virus found in raw waters and the expected higher efficiency of virus
removal in the AWT plant, as compared to the secondary plants, further
study in an "after installation" mode would not appear to be fruitful.
However vigilance must be maintained regarding the possible virus
contamination of natural waters by any waste discharge.
4. Until there is further development of procedures to improve virus
sampling and analysis, including strict protocols for contamination
prevention during sampling, it is recommended that current procedures
still be applied to research investigations where it is felt that virus
contamination of untreated or treated drinking water may be a problem.
14
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SECTION 4
MATERIALS AND METHODS
A project such as this one would not have been possible fifteen
years ago before the technology existed to make possible the recovery
of viruses when only a few were present in large volumes of water.
The techniques, which will be described in considerable detail, were
essential because viruses do not exist in large enough numbers in most
waters to be detectable by direct assay. There first must be a
concentration step.
To give some idea of how dispersed viruses in nature are, Sproul
(1) has estimated that surface waters in the midwestern United States
contain only approximately three "plaque-forming units" (PFU's) per
gallon. (The PFU is used to designate one infective unit, whether one
or several viruses in a cluster, which causes a single lesion to
appear in a laboratory-cultured animal-cell sheet.) He also estimated
that a properly operated water treatment plant might reduce this level
of viruses to one PFU per thirty million gallons.
The contamination of public water supplies by viruses which are
of public health significance most often occurs because human sewage,
whether treated or untreated, has been discharged to those supplies.
There are more than one hundred known human enteric viruses, and
because they cannot survive outside living cells, their numbers, after
they are discharged to the environment, invariably decrease. Because
they are present in such low numbers, the task of recovering them is
quite difficult. Shaffer et^ al. (2) have put the problem in perspective.
They calculated that one virus in one gallon of water is equivalent to
only one part in 10 parts, whereas, by contrast, one part per billion
(ppb), which is considered to be a microquantity in chemical analyses,
is one part in 10 parts.
The importance of a human ingesting a single virus particle
capable of infecting a cell is difficult to ascertain. Plotkin and
Katz (3) have postulated that the minimum infective dose, i.e. that
which is required to initiate infection, is only one PFU. Others
apparently agree, though the subject is debatable (4). Melnick (5)
has proposed that any future virus standard for drinking water should
be "one detectable infectious unit per one hundred gallons."
While the technology which will permit detection of viruses at
such low levels is reasonably well advanced, only the larger water
utilities in this country would be able to undertake the task of virus
monitoring, both because the cost is extremely high and the technology
is quite complex. Hill et_ al_. (6) have speculated that an alternative
microbial indicator system or minimal treatment criteria may be required
15
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in the future-to assure that drinking water is "virologically safe."
The methods used by both the EPA Viral Diseases Group and The
Carborundum Company's personnel to concentrate viruses from large
volumes of water were approximately equivalent to those which have been
proposed as tentative methods in the fourteenth edition of Standard
Methods (7). The specific procedures followed by both groups are
presented in detail in the following:
SAMPLING AND ASSAY PROCEDURES
Before the specific details of methods used by both participants in
this project are presented, a general overview of the basic procedures
involved in virus concentration and assay would be useful. While a
number of different concentration procedures have been developed with
varying degrees of success, (see Hill et^ al^., reference 8, for an
excellent review) only that one which is the tentative standard method
will be discussed. Several modifications to this basic procedure have
been suggested by others (e.g. Sobsey, reference 9) since this project
was conducted, but both participants in the Occoquan Project followed
approximately the same procedures which will be described.
Basic Virus Concentration Procedures
Figure 3, adapted from a presentation by Shaffer et_ al. (2), shows
in rudimentary detail the sequence followed in recovering viruses from
water. The discussion given by Shaffer et_ al. is summarized as follows:
First, the water is filtered, if necessary, to remove interfering
solids. Then chemicals are added to adjust the pH and cation concentration
to enhance viral adsorption on the next filter in the system. Next, the
submicron filters holding the adsorbed viruses are treated with an
alkaline solution which reverses the polarity of the virus particle,
permitting it to be eluted from the surface where it had been bound.
The eluted virus is further reconcentrated (by one of four methods which
will not be discussed here) to reduce the final volume to one which is
easily handled in laboratory assay procedures. Further steps include
neutralizing the reconcentrated eluate, adjusting the salt concentration
to promote virus stability, and adding a protective colloid. Finally,
the solution is sterilized (.e.g. by filtration through submicron mem-
brane filters or treatment with ether) to remove bacterial and other
microbial contaminants. The final concentrate may be frozen, if necessary,
until the actual assay procedure in the laboratory is begun.
Basic Viral Assay Procedures
Because viruses are obligate, intracellular parasites, the only way
to replicate them is to provide them with a host cell system of some
type. Live animals, chick embryos, or cell cultures are suitable
hosts, though the most common procedure for assaying waterborne viruses
involves cell culture inoculation.
This procedure, in brief, involves the following basic steps:
Tissues from a suitable donor organ (e.g. monkey kidney) are treated
with enzymes to separate the individual cells. A suspension of these
16
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Water Containing
Virus & Solids
Water Containing
Virus
Virus on Filter
Medium
Virus in
Eluate
Virus in
Concentrate
Virus Shown
to be Present
(1) Filter
a/
(2) Adjust Chemistry
(3) Adsorb Virus
(4) Elute
(5) Reconcentrate
(6) Assay
Figure 3. Sequence of Steps to Recover and Demonstrate the Presence
of Virus.(Reference 2)
a/
Not all virus sampling trains include a clarifying
filter. The Carborundum equipment did, but the EPA's
did not.
17
-------
cells is then placed in a small, glass bottle with a nutrient' medium
and incubated. The cells will attach to the glass and begin to reproduce,
eventually forming a monolayer cell sheet. Once the cell sheet has
formed, the concentrate containing the viruses is placed in the bottle
and gently dispersed over the cell layer. After a suitable time,
during which the viruses "infect" tho host cells, the liquid is drained
and replaced with a solid or semi-solid agar-based medium which covers
the host cells and prevents newly formed viruses from an originally
infected cell from spreading unchecked over the entire surface of the
cell sheet. A vital stain is also added. In time, viral activity can
be determined either by microscopically detectable changes in cell
morphology or by macroscopic "plaques" in the cell sheet.
Plaques .are formed when viruses produced inside the initially
infected cells spread to the surrounding cells, infecting them and
repeating the process. After a time, the area in which adjacent cells
have been killed is visible, appearing as a clear area in the cell
monolayer which does not take up the vital stain. Each plaque is
considered to have arisen from a single infective virus, though clumps
of virus may behave as a single unit. To ensure that plaques were
indeed viral induced and not the result of some other cytopathic
effect (CPE), such as chemical toxicity, the plaques can be "picked"
with an inoculating needle and the collected material is passed onto
new cell cultures. If the plaques form again, the investigator is
assured that they were caused by a virus.
The Carborundum Company's Procedure
Concentration Equipment and Field Technique--
The Carborundum Aquella virus concentrator was used throughout
this program for field processing of water samples. During Occoquan-
I, the concentrator was transported in a van by the field team to each
sampling site, and the concentration procedures were carried out with
the equipment exposed to the environment. During Occoquan-II, the
concentrator was housed inside a truck (later in a large van), and all
procedures were carried out inside the truck. The enclosed mobile
vehicles provided a cleaner, more reproducible environment than when
the concentrator was being used outside the vehicle at the various
sampling sites. A local power source was used when available but a
gasoline-powered generator was used in remote sites. The sample
concentration procedures, discussed in the following paragraphs, are
basically those found in Standard Methods (7).
Sterilization of equipment--The Aquella concentrator was sterilized
with dilute hydrochloric acid (HCL). All pumps, sampling lines,
filter holders, flow-measuring devices, and filter holders with the
appropriate filters in place were exposed to the dilute acid (pH 0.5
or lower) for at least thirty minutes. The sterilizing solution was
pumped through the adsorbing filter system first, then through the
remainder of the apparatus. Care was taken to ensure that the effluent
from the clarifying system did not contact the adsorbing filter media.
After the thirty-minute contact period, the concentrator wa,s rinsed
with the water to be sampled, and rinsing was continued until the
18
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influent and effluent pH were essentially the same. As before, the
effluent from the clarifiers was not permitted to pass through the
adsorbing filters. The exterior of the filter holders, the tubing, and
the quick-connect fittings were similarly treated daily to minimize the
chance of contamination.
Chemical additives--Routinely, two solutions were prepared and
simultaneously added to the clarified sample water via Johanson propor-
tioning pumps. The first, a solution of 0.03 molar (M) aluminum chloride
(Aids), was acidified with HC1 so that when it was added to the sample
water at a rate of one part solution to twenty parts, sample, the final
concentration of Aids was 0.0015 M and the pH was between 3.5 and 4.0.
The second solution was sodium thiosulfate (^28203) [6.74 grams (g) per
liter (£) for each milligram per liter (mg/£) of chlorine present in the
sample stream] was injected into the sample water stream with a second
Johanson pump at a dilution rate of 1:100. Dechlorination of the sample
stream was verified by testing with either an orthotolidine or DPD test
kit.
Clarifying filters—When the sample turbidity was high, as when
stream water or reservoir water were being sampled, clarification of the
influent was achieved by passing it through one or more fiber-wound
cartridge filters. These filters, 019R105 and 03R105 (Commercial Filter
Division; The Carborundum Co.; Lebanon, Indiana) were, respectively, 1.0
and 10.0 micrometer (ym) nominal porosity and constructed of Orion wound
on stainless-steel supports.
Adsorbing media--Viruses were adsorbed from the acidified water by
passing it first through a wound-fiber, glass filter (K27R105), then
through two flat filters (1.0 and 0.45 ym porosity) which were made from
epoxy-bonded, asbestos and fiberglass materials (Cox Instruments,
Detroit, Michigan).
Saline rinse--After the desired volume of clarified (if needed),
acidified water containing Aids was passed through the adsorbing filters,
a filter-rinse with saline at pH 3.5 was provided to remove any residual
chemicals which might otherwise find their way into the eluate and ad-
versely affect reconcentration. Typically, two 1.0 $, portions were
forced through the filters by air pressure and discharged to waste.
Glycine elution--After the saline rinses were completed 1.0 £ of a
glycine solution, pH 11.5, was forced into the filter holder, filling it
completely, and then through it at a rate slow enough to ensure a
contact time between the glycine and filters of one to two minutes.
During Occoquan-1, the glycine concentration was 0.55 M, but it was
increased to 0.10 M for Occoquan-II studies.
The elution step was repeated at least twice. The pH of the last
effluent from the adsorbing filters was checked to see if it was between
pH 11.0 and 11.5. If it was, the elution was considered completed; if
not, additional portions of alkaline glycine were passed through the
filters. -At times, these procedures resulted in large volumes of
eluate, but the elution procedure was not considered to be complete
until the final pH was 11.0-11.5.
19
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Neutralization--The eluates were neutralized with 0.05 M (later, 0.1
M) glycine, pH 2. The neutralization was performed quite quickly after
elution of the adsorbing filters. These neutralized eluates can be
stored for short periods at 0°-5°C.
Reconcentration--During Occoquan-I, the neutralized eluate was
reacidifed and passed through a small Cox filter for readsorption and
subsequent elution with a much smaller volume of alkaline glycine. The
final volume was often small enough that the neutralized eluate would be
placed directly on cell sheets for assay without further reconcentration.
During Occoquan-II, the procedure was changed to a "gel reconcentration"
procedure. This procedure is explained as follows:
The first eluate was adjusted to approximately pH 7 with the acidic
glycine solution, and sufficient A1C1, solution was added to achieve a
final concentration of 0.003 M. During this step, the pH may decrease
to 4.0-5.0 and a floe may form. The mixture was stirred and sufficient
1.0 M sodium carbonate (Na^CCL) was added slowly and constantly until
the solution pH stabilized at 7.2 for several minutes. Then the floe
was allowed to settle for thirty minutes. If the floe settled well, the
supernatant was decanted and wasted. Then, the remaining suspension was
centrifuged for ten minutes to further concentrate the floe. When the
floe did not settle well, the entire eluate volume was centrifuged.
Final concentration--When the gel reconcentration step was included,
the final step involved combining the pellets from the individual
centrifuge tubes with sufficient 0.10 M glycine, pH 11.5, to raise the
pH of the mixture to 9.5. Then, this suspension was centrifuged for ten
minutes. The centrifugate (liquid above the pellets) was recovered and
neutralized to pH 7.5 by addition of pH 2.0 0.10 M glycine. Then 10
percent (by volume) of heat-inactivated fetal calf serum was added, and
the sample was quickly frozen and stored in a freezer until it was
shipped to the assay laboratory.
Shipment, receipt, and storage.--All concentrates were shipped on dry
ice (-78 C) in polycarbonate centrifuge tubes (50-ml, Oak Ridge type)
enclosed in styrene shipping containers and packaged in a styrofoam
container wrapped in a heavy-cardboard carton (Polyfoam Packers Corp.,
Chicago). The common carrier from Washington National Airport was the
Airborne Freight Company which transferred packages to the C£J Limousine
Service, located at Boston Metropolitan Airport, who delivered them to
the University of New Hampshire (UNH) .at Durham. Care was taken to
ensure that the samples were not stored for a weekend at the airport or
lost because shipping information was lacking. Telephone contact was
made with personnel at The Carborundum Co. in Niagara Falls, N. Y. at
the time of shipping so that all parties could be alerted. All federal
and airline regulations were observed.
Upon receipt at UNH, each sample was assigned a number and placed in
a Revco freezer at -70°C or lower until the assays could be performed.
20
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Viral Assay Procedures--
Samples were assayed for viruses in the Jackson Estuarine Laboratory
(JEL), Durham, New Hampshire. This facility was reserved entirely for
the enumeration and identification of viruses in environmental samples.
No research was conducted there, so no laboratory viruses were ever
permitted into the facility to reduce the likelihood of contamination.
The JEL is a portion of a larger laboratory involved with non-virus UNH
research and is several miles from UNH's main campus where the virus
research laboratory of Dr. T. G. Metcalf is located. An overview of
the procedures to be discussed in the following sections is presented in
Figure 4.
Reconcentration--Whenever the volume of the field-concentrates was
too great to be placed directly on the cell lines, an additional concen-
tration by "hydroextraction" was performed at JEL. This procedure
actually involves dialysis. The concentrate was placed in a dialysis
membrane (Union Carbide), covered with solid polyethylene glycol (PEG
20M, Union Carbide), and placed in a refrigerator until the desired
volume reduction was accomplished. The hydroextracted concentrate was
recovered from the dialysis tubing, and the tubing itself was rinsed
with a three-percent beef extract (DIFCO) solution.
Virus enumeration and identification--Virus concentrates were
treated with diethyl ether (10-20 percent by volume, anesthetic grade)
for sixteen hours to inactivate bacteria, yeasts, and fungi. Entero-
viruses are ether resistant when treated under these conditions. Ether
treatment was selected in preference to antibiotic treatment or membrane
filtration because the former gave irregular inactivation of microorgan-
isms, while the latter caused significant losses of viruses in the
filters. Antibiotics were added to the cell sheets used during the
assays to provide yet another protection against loss of a culture
because of bacterial contamination.
When assaying for enteroviruses, samples were divided between two
types of all cultures--Buffalo Green Monkey (BGM) kidney cells and
African Green Primary Monkey Kidney (PMK) cells — in order to increase
the chances for virus recovery. Hela cells (human cervical carcinoma
cells) are required for assay of adenoviruses because the adenoviruses
do not respond to nonhuman cells. Hela cells are preferable on the
basis of price and availability but are somewhat less sensitive than
human embryonic kidney (HEK) cells which could be used.
Samples were inoculated onto the cell sheets contained in one ounce
bottles. The inoculum volume was from 0.1 to 0.2 ml per bottle. The
bottles were then gently rocked back and forth for approximately one
hour to allow the viruses to adhere to the cell sheets. Then, the cells
are covered with an agar overlay and incubated for the required time.
An entire sample was always tested, but only one-half was tested at
a time. The other half was kept frozen for two purposes: 1) to serve
as a safeguard against loss of an entire sample in the event of problems
(toxicity, contamination, etc.) encountered during the tests and 2) to
provide confirmation of virus recovery if any were found during the
assay of the first half of a sample.
21
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Plaques were cloned and propagated on additional cell sheets. The
cells were lysed and centrifuged, first at low speed to remove debris
and then at high speed to remove the viruses. The supernatant liquid
was removed, and the viruses were washed and recentrifuged. The purified
viruses were then subjected to the antisera-pools (Lira, Baenish-Melnick)
for typing.
Conventional serum neutralization tests (10) were used for typing.
The isolate was passed to large-area tissue cultures under liquid
overlay. After incubation at 37.5°G for up to several days after which
extensive CPE's were evident, the cells were lysed by repeated cycles of
freezing and thawing. The liquid, containing progeny virus from the
lysed cells, was freed of extraneous cell debris and other solids by
low-speed centrifugation.
The product was titered over a range of serial dilutions to deter-
mine its virus concentration. An aliquot (0.025 ml) of the appropriate
dilution was reacted with an excess of the appropriate antiserum pools
and transferred to tissue cultures in microtiter plates. These were
incubated and observed for CPE's. Observed patterns of neutralization,
positive and negative controls were recorded and compared to establish
the virus identity (10).
Reporting of data was on the basis of "CPE-positive" or "CPE-
negative" and by type or, more commonly, by the number of PFU's per unit
volume for each specific type of virus recovered. The minimum time
required for assay and typing was approximately one to two months.
RCT Marker Test Procedure--
Virulent strains of poliovirus type 1, such as the Mahoney strain,
replicate as well, or nearly as well, at 40°C as they do at 37.5°C.
Avirulrnt. strains such as LSc, replicate poorly, if at all, at 40°C. To
establish the "T-Marker" characteristic or, more appropriately, the
"reproductive capacity temperature" (RCT) characteristic, of a polio 1
virus, a single preparation is plated at several dilutions onto a
common lot of tissue cultures. A portion of these is exposed to 40°C
temperature before incubation at 37.5°C. The remainder is incubated at
37.5°C without the 40°C exposure. The rate of replication, shown as
the number of PFU's, is measured for each. A calculation is made (See
Table 8, Results Section) using Mahoney as the standard virulent type 1
poliovirus (R=100) and LSc as the avirulent standard (R=0). Calculated
R values from 0 to 30 are considered typical of avirulent strains,
whereas R values from 60 to 100 are considered typical of virulent
strains. Other values are simply regarded as intermediate.
EPA Virus Group's Procedures
Concentration Equipment and Field Techniques--
The equipment used by EPA's Virus Group is basically that shown in
Standard Methods, 14th edition (7), page 971. The specific details
regarding their equipment and basic operating procedures used for
23
-------
concentrating viruses from large volumes have been discussed in detail
by Hill et^ al_. (6). A summary of their procedures follows.
Sterilization of equipment--The virus concentrator (proportioner
unit, mixing chamber, hoses, and acid and sodium thiosulfate additive
containers) was decontaminated with acidic calcium hypochlorite. The
additive containers were filled with approximately 500 mg/1 chlorine
solution to which HC1 has been added to reduce the pH to <_ 6. This
solution was dosed into tap water through the virus-concentrator
proportioner and through a second proportioner placed between the tap
water faucet and the inlet hose of the virus concentrator. This
arrangement exposed the entire system to a decontamination solution of
5-10 mg/1 hypochlorous acid, the most virucidal form of chlorine. The
hyperchlorinated tap water is pumped through the system for 10 to 15
minutes, and then the ends of the hoses are covered with foil to
maintain the disinfected state.
Adjustment of the tap water pH--Immediately prior to a sampling
run, the volume of 0.2 Normal (N) HC1 needed to reduce 220 ml of the
water to be sampled to pH 3.5 is determined. For a standard 500
gallon water sample, a volume of 12 N HC1 equal to 100 times the
volume determined above is added to 22 liters of tap water in the acid
additive container. When this solution is proportioned into the water
sample at a 1:100 dilution, it achieves a pH of 3.5.
Neutralization of chlorine in water--A filter sterilized stock
solution of sodium thiosulfate (STS) (75.6 g/100 ml) is prepared. One
hundred and fifty ml of this stock solution is added .to 22 liters of
the water to be sampled in the second additive container. This mixture
is dosed into the water to be sampled at a 1:100 dilution with two
additive pumps so that STS is introduced with each stroke of the
proportioner. The STS added is sufficient to neutralize approximately
7 mg/1 chlorine.
Sampling procedure--The faucet from which the water sample will
be drawn is flamed and the decontaminated hose is attached. Water is
passed through the system to flush the residual chlorine remaining
from the decontamination procedure. Also the effluent water is checked
for sufficient pH adjustment (3.5 to 4.0) and chlorine neutralization.
When accomplished, the heat-sterilized, virus-adsorbing filter unit
(7-inch Balston filter with 8 ym porosity in a glass and stainless
steel housing) is inserted aseptically into the system via quick
disconnects. The flow is adjusted to 3-5 gallons per minute (gpm) and
the sampling is begun. The effluent water is checked periodically for
pH and chlorine neutralization. Corrections are made if needed.
Water is also collected before and after the run for turbidity determina-
tions.
Elution of virus adsorbing filter—At the end of the sampling
period, the filter unit is aseptically removed and connected to a
sterile pressure vessel which contains one liter of sterile glycine
buffer at pH 11.0 to 11.5. The glycine eluent is pushed through the
adsorbing filter and a smaller porosity clarifying filter with nitrogen
gas. The eluate is immediately lowered in pH to 8.0 to 9.0 with
24
-------
approximately 300 ml of glycine buffer pH 1.1. The eluate is collected
in a sterile, screw-cap, polypropylene bottle. The cap is sealed with
tape and the container is placed on wet ice until it is returned to
the laboratory.
Virus Assays--
Reconcentration of eluate--If the eluate is to be reconcentrated
within 12 to 16 hours after arrival at the laboratory, it is stored at
4°C; if not, it is stored at -70°C. Just prior to processing, the pH
meter is standardized with pH 4.0 standard buffer and the electrode
subsequently sterilized with 10 mg/1 Cl_ solution for 30 seconds and
neutralized with sterile sodium thiosulfate solution. Processing
begins by transferring sample to a 1,500-ml beaker with stir bar in a
laminar flow hood. The stirrer is started and glycine buffer at pH
1.1 is added slowly until pH 3.5 is reached. A 0.05 M solution of
A1C1- is added to achieve 0.005 M A1C1,.
The sample is then filtered by suction through a "sandwich" of 5
urn and 1 ym Cox Filters. After all the sample has passed, 25 ml of
sterile, physi'ological saline (pH 3.5) is passed to wash filters.
The filter assembly is transferred to a 125-ml, side-arm flask
and 14 ml of glycine buffer at pH 11.1 is passed under suction. The
eluate is caught in the side-arm flask which contains 2.6 ml of pH 1.1
glycine buffer, 2.0 ml of 1OX nutrient broth and 1.4 ml of 10X Hank's
basic salt solution. At no time in the elution or reconcentration
step was the sample allowed to remain at pH 11.0 to 11.5 for more than
five minutes (usually less than three minutes). The total volume of
approximately 20 ml is divided equally and placed in plastic centrifuge
tubes and immediately frozen at -70°C. The entire process is usually
accomplished within 20-30 minutes. This can vary depending upon the
ease at which the sample passes the filters. All operations are
carried out in laminar flow hoods.
Isolation and identification of viruses from the sample--The 20
ml, reconcentrated sample is frozen at -70"C until appropriate cell
culture is available. Generally, the sample is divided equally and
the entire sample is placed on monolayers of three cell types: Barren's
line of African green monkey kidney cells at passage number approximately
130 (BGM), McAllister's line of human rhabdomyosarcoma cells (RD) and
primary African green monkey cells obtained from Flow laboratories.
The cell monolayers are observed for 14 days for CPE. If no CPE is
visible by the 14th day, the cells and media are frozen and thawed and
five ml of this solution is passed to each of three new monolayers.
If no CPE develops in 14 days of the second passage, the sample is
considered negative for virus. If CPE is observed, the cell bottle(s)
is removed from the isolation lab and identification procedures begun.
Personnel Surveillance
During Occoquan-U, the Carborundum field personnel, and anyone
else entering the mobile laboratory used for the virus concentration
work, were examined periodically to determine if they were shedding
25
-------
viruses. Swabs, both rectal and throat, were taken with a sterile,
cotton-tipped swab, placed in a sterile, screw-capped test tube containing
1.0 ml of sterile nutrient broth and returned to UNH (Spaulding Life
Sciences Laboratory) for assay. The swab tests were performed at a
frequency which was practical (from the standpoints of both cost and
time-and-effort) for the purpose of assisting in the interpretation of
data in the event a concentrated sample was positive for virus.
During Occoquan-II, rectal swabs were collected monthly and sera
specimens were collected quarterly on each EPA participant in the
study. The swabs were stored at -70°C in 1.0 ml of sterile nutrient
broth containing 3X antibiotics. During the comparative seeded studies,
rectal swab collection was altered to coincide with the Initiation of
an experimental run. These stored specimens were assayed for virus or
specific antibody only when such data were needed to determine the
occurrence of sample contamination, i. e. when field samples yielded
virus isolations or aberrations occurred in the seeded study results.
Specimens were assayed in BGM and RD cells. The inoculated cultures
were observed for 14 days for CPE and blind passaged to fresh cells
and observed for an additional 14 days before declaring them negative
for virus.
COMPARATIVE STUDIES: FIELD SAMPLING AND ANALYSIS
During Occoquan-II, EPA's virus group participated on thirteen
occasions in a collaborative, field-sampling effort with The Carborundum
Company's virus group. The first studies were in July, 1978, when
eleven samples were collected by each group from the raw water and
finished water at FCWA's New Lorton treatment plant and at two sites
in the distribution system. In October, 1976, samples were taken from
a local sewage treatment plant. Two sites were selected: 1) the
effluent from the plant's secondary clarifier prior to its entrance
into a storage lagoon (detention time approximately thirty days) and
2) the chlorinated effluent from the lagoon itself.
Each field team processed approximately 100 gallons of treated
water from FCWA's system through their concentrators and completed the
necessary elution steps in the field. When the sewage treatment plant
studies were conducted, only a relatively small volume of the extremely
turbid effluent could be forced through "the concentrators (2.2 gallons
and 8.5 gallons for Carborundum and EPA, respectively), but more of
the lagoon effluent (50 and 100 gallons, respectively) could be concentrated.
After the elution and reconcentration steps were completed, each
party sent a portion of each of their concentrates to the other for
viral assay. The Carborundum Company's concentrates from the sewage
treatment plant study included one from the clarifying filter and one
from the adsorbing filter. All procedures, including those for storage
and shipping, were as previously described.
Prior to the initial comparative-sampling and analysis study, the
BGM cell line used at UNH was shipped to the EPA laboratories in
Cincinnati. The purpose was to eliminate one possible source of
variability between the two laboratories, namely the degree of susceptibility
of different cell cultures to any viruses which might be recovered.
26
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ADDITIONAL FIELD STUDIES
Two additional short-term studies were conducted in an attempt to
recover natural viruses from a public drinking-water supply. While
these studies were conducted only by the VPI/Carborundum group, portions
of the final concentrates were sent to EPA for virus analysis as needed.
It is known that alum flocculation will concentrate a majority of viruses
present in water and, because flocculation is a principal process in the
treatment of water for public consumption, it was believed virus recovery
might be possible from back-wash water collected from a water treatment
plant filter and from the unwashed filter media itself. It seemed
reasonable to assume that virus recovery from the water would be enhanced
by the concentration potential the alum floe and the filter media pro-
vided.
Studies Associated With a Water Treatment Plant Filter
A pipe-and-valve assembly was placed in the waste line from the
filter gallery that carried backwash water (including the surface wash
water) to waste and during backwashing of a filter, the water to be
concentrated was pumped into a tank. Because of difficulties getting
water out of the valve during backwash (Bernoulli's principle), it was
necessary to pump from the line to a large tank, and consequently, only
about 50 gallons could be collected before the backwash cycle was
completed. During the concentration step, the water in the tank was
mixed continuously with compressed air to keep floe suspended.
The tank used to collect the water prior to carrying out the
concentration step of the virus-recovery procedure was a 500-gallon,
polyethylene tank. It was initially sterilized with a high concentra-
tion (50-75 mg/1) of chlorine at an approximately neutral pH. There-
after, at the end of an experiment, the tank was thoroughly cleaned with
tap water having a residual of 3-4 mg/1 and then dechlorinated. The
rigorous sterilization procedure was not regarded as necessary between
experiments.
In addition to the backwash water, samples were taken also immediately
before and after a backwash cycle from pipes below the filters and
pumped directly into the Aquella concentrator. Two filter underdrains
collected water passing through the filter, one for each half of the
filter, and. consequently, the pre- and post-backwash samples were water
from only one-half of a given filter. When a filter was backwashed, the
wasteline carried washwater from the entire filter.
Study of Filter Media
A method was developed by The Carborundum Company to examine the
anthracite from one of FCWA's mixed-media water filters. Several
attempts were made to recover poliovirus (LSc-1) from seeded samples of
anthracite samples collected from an unwashed filter. The method which
was adopted allowed 67 percent recovery of the seeded virus.
A weighed sample (six to eight pounds) of the anthracite
filter medium was placed in a 4-liter beaker and elutea twice by
27
-------
covering it with pH 10.5, 0.5 M glycine. The decanted solution was
reconcentrated by floe formation with addition of 0.003 M A1C1-, followed
by neutralization with Na-CO^and centrifugation. The pellet was eluted
with pH 10.5 glycine, and neutralized, then fetal calf serum was added.
The final concentrate was divided equally for analysis by both EPA and
The Carborundum Company virus groups. The experiment was conducted
twice.
SAMPLING FREQUENCIES
Occoquan-I
Seeded Samples--
During Occoquan-I, a total of twelve field experiments, two at each
sampling site, were conducted to determine the effectiveness of methods
used by The Carborundum Company for concentrating and assaying for
viruses. In these tests, large numbers of viruses (10 -10 PFU, polio
type 1, adenovirus, and reovirus) were added to the thiosulfate reser-
voir (one virus type at a time) and were injected into the stream of
water taken from the sampling site as it was being forced through the
adsorbing filter. In most instances, from 85 to 100 gallons of water
were used at the finished-water sites. Much less (50-85 gallons) of the
stream samples could be forced through the filters because of the high
suspended solids in the water.
Natural Samples--
Table 4 shows the number of water samples collected for virus
analysis during a ten week period from June 2 through August 27, 1975.
The sampling was restricted to this period during Occoquan-I because it
is this time of year that viruses are shed by humans in large numbers.
Because treated sewage enters the reservoir (via Bull Run), the likeli-
hood of virus recoveries was regarded to be greatest during the summer
months.
After the concentration step was completed, the preparation of the
final concentrate for shipment to UNH was by the routinely used proced-
ures which have already been described.
Occoquan-II
Environmental Samples--
During Occoquan-II, the environmental sampling for viruses was
restricted to the enteroviruses (polio, Echo, and Coxsackie). (Reovirus
and adenoviruses were included in Occoquan-I.) Table 5 shows the
numbers and frequencies of the samplings from June through October,
1976, and in March, 1977. During July, 1976, the EPA virus group
participated in a joint sampling and analysis program at the sites at
the treatment plant and in the distribution system. They returned in
October, 1976 to jointly sample at the sewage treatment plant.
28
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Additional Field Studics--
On June 2, 3, 6, 7, and 10, 1977, samples were collected from the
filter backwash waste line and from clean water passing a filter at
FCWA's New Lorton facility. The anthracite sample was taken for
analysis during this period also. The EPA did not participate in the
sampling program during this period but did analyze the concentrates
of these samples provided by The Carborundum Company.
Seeded Samples--
Both The Carborundum Company and EPA jointly participated in
studies designed to evaluate the sampling, handling, and assay procedures
used by each group. These studies were conducted in the Number 2 Pump
House at the New Lorton facility from December 3 through December 21,
1976, and from February 15 through March 1, 1977. A total 35 samples
was collected by each group. A detailed description of this program
is presented in the following section.
SUPPLEMENTAL WATER-QUALITY DATA
Supplemental water quality data required by the contract for both
years of the study included chlorine residuals (free and total), pH,
temperature, bacterial counts (coliforms and total plate counts, the
latter on finished water only), and notes concerning any unusual
characteristics such as high turbidity and any unusual color or odor.
All methods were according to Standard Methods for the Examination of
Water and Wastewater f7). During the second year, monitoring of
finished waters for total organic carbon (TOC) was required, primarily
for use in interpreting trihalomethane data presented in Part I of
this report.
Chlorine Residuals
Both total and free residuals were determined by the DPD Ferrous
Titrimetric Method Sec. 409 E (7). The reliability to 0.1 mg/1 was
verified by testing aliquots of samples by the Amperometric Titration
Method, Sec. 409 C (7). The titrimetric method was suitable for
analysis of all but the most turbid samples, as are common to the
tributary streams of the reservoir after a rainstorm, but the residuals
there were less than 0.1 mg/1 on all occasions. The principal sites
where chlorine residuals were important were those where finished
water samples were collected.
pH Value
The pH of all samples was determined according to Standard Methods,
Section 424 (7). A portable pH meter was used and standardized before
each use with commercially available buffer solutions.
Temperature
Temperature was measured according to Standard Methods, Section
212 (7) with a mercury-filled Celsius thermometer calibrated in increments
of 0.1°C. The thermometer was placed in the sample immediately after
31
-------
collection and allowed to equilibrate before the reading was taken.
Bacterial Counts
Coliforms--
The Multiple-Tube Fermentation Technique, Section 908, was used for
both total coliforms (908 A) and fecal coliforms (908 C) (7). Both the
presumptive and confirmed tests were performed using five transplants of
aliquots from the serial dilutions. Occasionally, the completed test
was performed to verify the identification reliability. Both raw and
finished-water samples were analyzed for coliforms, and the dilutions
and media inoculations were performed in the field. The tubes were
returned to the laboratory for incubation within four to six hours.
Total Plate Counts--
The standard plate count procedure (Section 907) (7) was performed
only on finished waters. Plates were inoculated in the field at the
time samples were collected and returned to the laboratory for incubation
within four to six hours.
Total Organic Carbon (TOC)--
The TOG concentrations were determined (during Year 2) by analysis
with a Beckman Instruments 915A TOG Analyzer. Potassium phthalate
standards were used so that full-scale deflection on the strip chart
recorder was 20 mg/1, and the reproducibility of analyses of standards
was within 0.2 mg/1 of the true concentration. During the latter part
of the study period, the laboratory acquired a Dohrmann/Envirotech Model
54 TOC Analyzer, permitting analyses with precision to less than 50 yg/1
TOG.
COMPARATIVE STUDY WITH CODED, SEED-VIRUS SAMPLES
Overview
Because viruses were reported to have been recovered from finished
water during Occoquan-I (to be discussed in this report) a carefully
controlled, large-scale experiment was devised to evaluate the virus
recovery methods and control procedures involved in this study. The
study was designed so that procedures used by the virus monitoring and
assay teams from both the EPA and The Carborundum Company could be
evaluated in an objective manner. A third group from the California
State Department of Health's Viral and Rickettsial Disease Laboratory
(CSDH) prepared and coded the viral concentrates and "blanks" (virus-
free samples) to be used in the studies.
A system was designed which would provide large quantities of
virus-free water which could be held in a large, sterile reservoir. On
twenty-five occasions, a vial containing either a blank or a virus
concentrate (virus type and titer unknown to experiment participants)
was opened, diluted, and divided equally between the two parties for
32
-------
mixing in the thiosulfate reservoir associated with their concentrators.
On ten occasions, the contents of a coded vial was diluted directly in
the tank containing the heat-treated and cooled water and thoroughly
mixed with a large agitator (Lightnin1 Corporation). Each virus group
had the responsibility for passing approximately 100 gallons of the test
water through their concentrator and for preparing their respective
concentrates by their particular procedures. Each group's assay lab-
oratory was to receive one of these portions for virus assay.
The coding system devised assured that neither field party knew the
contents of any vial sent from CSDH, nor did their respective laboratory
personnel know which group had prepared the concentrates that they were
analyzing. After all data had been reported to the EPA Project Officer
in coded form, he and another party decoded it for analysis.
Sample-Handling Procedures
Figures 5 and 6 show schematically the flow of sample vials, field
concentrates, and data. Specific details are as follows:
1. Vials, containing either virus concentrates or blanks, were
prepared and coded by CSHD and sent frozen to the OWML director
(B, Fig. 5). They were stored at -70°C in a Revco freezer
(Model ULT-7100B, 7 cubic ft) at FCWA's New Lorton treatment
Plant. Before the actual samples were sent, a shipment of
blanks was made to verify that the established procedures
would be successful, i.e., that samples would be received
unbroken and frozen without delay. Samples (packed in dry
ice) arrived by air and were taken by truck to the Federal
Express Office in Alexandria, Virginia, where they were picked
up by the OWML director.
2. Samples were recoded (without thawing) by the EPA Project
Officer, the FCWA laboratory director, and the OWML director
(step 2, Fig. 5).
3. On the day of an experiment, the contents of one vial were
thawed and diluted into the thiosulfate reservoirs or added
directly to the heat-sterilized water reservoir by OWML
personnel (Step 3, Fig. 6). During the thiosulfate experiments,
each party added one-half of the diluted vial contents to
their reservoirs and measured the volume remaining at the end of
the run in order to determine the input virus titer.
4. The field concentrates were then sent to the field teams' res-
pective assay laboratories for further reconcentration and
separation into two aliquots which were then returned to FCWA
and stored in the Revco freezer.
5. The EPA Project Officer and the FCWA laboratory director recoded
the vials once again (Step 4, Fig. 6) and shipped back to each
assay laboratory one-half of a concentrate prepared both by its
own personnel and by those from the other laboratory (Step 5,
Fig. 6).
-------
A
CSHO: SOURCE
LAB. FOR
VIRUS
STEP1
CODED
VIALS
B
INDEPENDENT
PARTY
STEP 2
RECOOEO
VIALS
25 THIO
RESERVOIR
EXPERIMENTS
10-250
GALLON TANK
EXPERIMENTS
Figure 5. Schematic for Providing Virus Input Concentrates
for Each Experiment.
34
-------
COOED CONCENTRATE
(OR ANALYSIS
It CAROnUNDUNI AND
I EPAIOROI
K THIO
RESERVOIR
EXPERIMENTS
AND
ID 250 GAl
TANK
EXPERIMENTS
STEP;: PARTY g DECODES
AND REPORTS DATA
TO All PARTICIPANTS
It CARBORUNDUM AND
t IPAIOROI
Figure 6. Schematic for Processing: Steps in the Comparative
Sampling, Concentration, Analysis, and Reporting of
Data.
35
-------
6, The final results (PFU's per ml, total volume of concentrate, and,
on occasions, virus type when an isolate was recovered) were then
sent to the EPA Project Officer. He then decoded the data and
sent copies to each of the principals in the project for analysis
(step 7, Fig. 6).
Role of California State Health Department
CSHD was selected as the independent party in this project, both be-
cause their personnel are internationally known, extremely capable virolo-
gists and because of their total lack of personal bias in the project it-
self. It was necessary that some competent independent party be found that
would agree to provide the required number of ampules of viruses--correctly
titered and identified—and CSHD met these requirements. The work by CSHD
was performed under a separate contract with EPA.
Number, Types, and Titers Required--
The contract between EPA and CSHD required that vials of two poliovirus
types at three different titers be prepared and coded by CSHD. In
addition, replicate vials, identical to those which were to be shipped,
were to be retained by CSHD as a backup in the event samples were lost
in shipment or otherwise rendered unusable. Table 6 shows details of
the numbers and contents of vials to be sent and the intended purpose
of each. The listed titers were close to the target titers called for by
the contract, and indicated valid performance by CSHD.
The samples intended for direct analysis by both assay laboratories
were provided so that a comparison could be made at the end of the
study to the original titers as determined by CSHD.
Specifications for Preparation of Virus Materials--
The following instructions for preparing the virus materials were
given in the contract between EPA arid CSHD:
1. The viruses shall be grown on any appropriate cell system.
2. The viruses shall be titered on the BGM cell system furnished
from Dr. Metcalf's laboratory.
3. The stock virus should be harvested at the point of maximum virus
yield and rapidly frozen and thawed two times to release the virus.
This crude virus preparation should be refined and rendered mono-
dispersed by the following steps (modified after Ver e_t al^,, J_.
of Vir. 2: 21, 1968):
a. Preparation of Membrane Coating Solution (MCS)-Prepare a 10
percent solution of fetal calf serum in distilled water. Filter,
under lab pressure, this solution through a series of the
following filters: An AP20 prefilter, a 0.22 um and a 0.05 um
Millipore (MF) membrane. Make the filtrate isotonic with 10X
Earls balanced salt solution.
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b. Pretreatment of Filters with MCS - Using pressure filtration,
slowly pass 10 ml of MCS through two sets of a 47 mm, 0.22 ym, and
a 0.05 ym MF membrane in series. Rinse the membranes with 5 ml of
Tris buffer. Discard the filtrate and use these two sets of mem-
branes to filter the virus stock suspension. (Prepare these fil-
ters shortly before use).
c. Filtration of Virus Suspension - Centrifuge the stock virus
suspension at low speed ('v 2000 RPM) for 15 minutes. The supernate
should then be removed and adjusted to pH 8.0 with a nonbicarbo-
nate buffer, e.g., Tris. The supernate should then be pressure
filtered in the cold through one series, then the other series
of the 0.22 ym/ 0.05 ym treated membranes. (Filtration may
take several hours). Store the filtrate in small volumes in
sealed ampules at -708C until ready for use.
4. The virus dilution and suspension fluid shall be sterile nutrient
broth (Difco #003-01) prepared with sterile Hanks BBS, buffered to
pH 8.0, rather than distilled water.
5. The desired virus material vials shall be produced and titered in
following fashion:
a. The filtrate, produced according to step 3.C., will be
assayed by 10 replicate titrations. For example, this may be
accomplished by diluting the filtrate to give a range of 10-30
PFU/ml then by inoculating ten 25-cm cell monolayers with one
ml each of the diluting filtrate. Based on this titration appro-
priate pools can be prepared to give the desired titers. Five
ml each of the pools should be placed in 10-ml ampules and flame
sealed. They should be checked for leakage by an appropriate
procedure and should be frozen at -70°C.
b. For the desired 2.5 X 10 PFU titer, it is recognized that
this may prove impracticable to attain and therefore the highest
attainable will be acceptable.
c. To obtain official titers, 10 ampules at each virus concen-
tration level will be removed from the freezer at random.
(1) For the 2.5 X 10 PFU titer, the vial contents
may be diluted to give 10-30 PFU/ml and a I/ml sample
taken for assay.
(2) For the 250 PFU and lower levels, the entire vial
contents should be assayed.
(3) As a guide the contractor should try to have the
mean plaque count come within +_ 2 times the square root
of the desired number.
Preparation of Pasteurized Dilution Water
A rather elaborate system was designed for use during the comparative
38
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study to ensure that the test water was free of naturally occurring viruses.
This detail was of utmost importance because the contamination question is
always a concern in virus monitoring and assay research. In the following
paragraphs, the design and operation of the system will be discussed.
The Equipment--
A diagram of the units comprising the water-pasteurization system is
shown in Figure 7. The specifications for these units were as follows:
1. Packaged water chiller - Kold Wave Model LC500A, 5-ton unit.
Included freon compressor, shell-and-tube heat exchanger, electrical
box with motor starter and thermostat. The unit could be installed
either outdoors or inside.
2. Plate heat-exchanger - Chester Jensen Company, Model HTW with type
316 stainless-steel plates. Unit could be mounted either on the wall
or the floor. Heating and cooling surface area 15 square feet. Equal
flow on each side of the plates (hot and chilled water) was provided
to accomplish 80-83 percent regeneration. The head loss through the
unit at five gallons per minute was 30 pounds per square inch.
3. Water heater - Hi-Power Model 120-18, Commercial electric type,
120 gallon capacity, manufactured by Lochnivar. Included thermostat,
heavy duty tank, high-temperature cutoff, and insulation.
4. Storage reservoir - The pasteurized water was stored in a 250-gallon,
polyethylene tank (United Utensils Co., Model 260-S, Port Washington,
N. Y.) equipped with a loose lid and 3/4-inch, IPS, female, threaded
connection. Two take-off spigots were installed, one for each of the
two virus concentrators. The influent line terminated about one foot
above the surface of the filled reservoir, providing an air break
which ensured that there was no opportunity for back flow.
5. Mixer - A high-speed mixer (Model ND-1A Lightnin' Agitator, 1/4-
horsepower, carbon-steel shaft and 2-prop shaft) was mounted on the
tank to provide the mixing required for those studies (ten in all)
when the virus concentrate was added directly to the reservoir prior
to passage of the water through the virus concentrators.
Sterilization of Storage Reservoir--
Procedures for sterilizing the 250-rgallon tank before each of
the seeded experiments where virus were added to the tank itself were as
follows:
a) Fill tank, add one quart commercial sodium hypochlorite bleach,
adjust pH to less than 7.0 with HC1. Let stand overnight (calculated
dose, 220 mg/1 chlorine).
b) Add sodium thiosulfate to dechlorinate.
c) Drain tank and rinse with water from the pasteurization system.
d) Ensure chlorine residual is zero.
39
-------
t
e?
os w TO VIRUS CONCENTRATORS
VL
>, ^o 5
a H
m
CM
Figure 7. Diagram of the Water-Pasteurization System and Storage Reservoir.
All temperatures are design values.
40
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e) Fill4 tank with pasteurized water.
After each experiment when viruses were added to the thiosulfate (the
first 25 experiments), the superchlorination of the tank was not required,
so only a pasteurized-water rinse was used to prepare the tank for the next
day's experiment.
Mixing Study--
To ensure that viruses added directly to the storage reservoir prior
to the concentration step were completely mixed, a dye study was performed.
A stock solution of methylene blue was prepared and introduced into the
tank with the Lightnin1 Agitator operating. The dye was dispersed
rapidly and no dead spaces could be seen. Complete mixing appeared to
be accomplished within two minutes, but when seed viruses were added
directly to the tank, the mixing time was exactly five minutes.
Pasteurization Procedure--
Finished water (under line pressure) was passed through an in-line
flow-meter, at the rate of no more than four gallons per minute (gpm)
through the chiller (to cool it to 4-59C if necessary) to one side of
the heat exchanger. If the influent water temperature was less than
4°C, the chiller was bypassed. (The chilled water in the heat exchanger
was used to cool the heat-pasteurized water to 21°C before it was stored
in the 250-gallon tank.) In the heat exchanger, the water was warmed to
approximately 60°C before it entered the water heater. At a low of 4
gpm, the theoretical detention time in the heater was 30 minutes. From
the water heater, the water (approximately 77*C) flowed through the
other side of the heat exchanger where it was cooled to approximately
21°C. On occasions when the effluent from the water heater was not as
warm as desired, the flow rate was reduced below 4 gpm to provide greater
detention. The thermometer which registered the temperature in the tank
probably was several degrees higher than that recorded. The pasteurized
water was collected in the storage tank. The time required to fill
the tank in preparation for an experiment was from one to one and one-
half hours, depending on the flow rate.
Description of a Typical Experiment
The following instructions were followed by all field personnel in
the execution of the seeded-virus experiments.
Handling of Coded Vials Prior to Concentration Steps--
1. Rinse hands with water treated with HC1 to reduce pH to less than
1.0. Wear a sterile, gauze mask.
2. Select a coded vial from the freezer. Record its code number, then
permit it to thaw completely at room temperature. Rinse its outside
well with water, pH < 1.0.
41
-------
3. Break vial and withdraw contents with a sterile, 10-ml Luer-Lok
syringe equipped with an 18-20 gage needle. Dispense the contents
into a sterile, one-liter graduated cylinder containing pasteurized
water. Fill the vial several times with pasteurized water, with-
drawing each rinse with the syringe and dispensing to the graduated
cylinder.
If the diluted material is to be added to the thiosulfate
reservoirs of the two field teams, proceed to Step 4. If the material
is to be added to the 250-gallon tank, proceed to Step 5.
4. a. Fill the graduated cylinder to the mark with pasteurized
water from the water treatment system. Mix the contents well
by pouring back and forth into a second, sterile, graduated
cylinder. Keep the vessels that contain the diluted material
covered with aluminum foil when they are unattended.
b. Dispense exactly 500-ml of the diluted virus concentrate into
each of two, sterile containers having a capacity greater than
5.0 H. These containers will be the thiosulfate reservoirs.
The total volume of the thiosulfate in the reservoir should be
4.75 H ± 5 ml. Use pasteurized water when preparing the thio-
sulfate.
5. Add contents of the graduated cyclinder directly to the 250-gallon
tank containing approximately 250-gallons of pasteurized water.
Rinse graduated cylinder and empty rinse water into the tank. Mix
contents for five minutes with Lightnin1 Agitator.
Waste-Handling Procedures--
1. After each virus team has passed approximately 100 gallons through
their concentrator units, the excess water in the thiosulfate
reservoir or the water tank was chlorinated (approximately
100 mg/1) and allowed to stand for one hour.
2. The waste was pumped to a 55-gallon drum containing hypochlorite
solution. When the drum was full it was discharged to the sewer
and flushed with tap water.
Additional Precautions and Clean-Up Procedures--
1. A large catchment basin was placed under the tank tap to collect
any material that should leak out. If there was leakage and the
depth in the catchment exceeded one-half inch, the experiment was to
be terminated.
2. The catchment basin and 250-gallon tank were rinsed with a
strong hypochlorite solution at the end of each experiment.
3. The work area was sprayed with a hypochlorite solution.
42
-------
4. Responsible personnel remained in the area during an experiment
to pour hypochlorite onto spilled material in case of an accident.
5. All glassware to be used was soaked overnight in acidified water,
pH < 1.
Data Collection and Analyses Performed--
Prior to the initiation of the concentration step, and again at the
end, the temperature, chlorine concentration (free and total), and pH of
the pasteurized water were measured. Bacterial tests (only at the be-
ginning) included total and fecal coliforms and standard plate counts.
After the concentration step was completed, the remaining volume in the
thiosulfate reservoir was measured and recorded. This information was not
needed if the coded material had been added directly to the 250-gallon
tank.
Procedures Following Initial Concentration Step
The adsorbing filters were eluted on-site by the field personnel.
The Carborundum group carried out the gel-reconcentration procedure
before the samples were shipped to UNH for further reconcentration. At
UNH the concentrates were divided into two equal portions, refrozen, and
returned to FCWA where they were stored in the Revco freezer. Later, the
samples were assigned new codes by the Project Officer and FCWA Lab
Director, and one of the two portions of each concentrate were shipped to
the two assay laboratories to be put on the cell lines.
The EPA group returned its concentrates to Cincinnati where they were
reconcentrated and divided into two equal portions. The procedures from
that point on were identical to those just described.
Once the assay laboratories had received their samples, they followed
the protocols previously described. Due to a misunderstanding, the UNH
laboratory group failed to identify all the plaques they observed during
analysis of both halves of the first twenty-five concentrates (when coded
concentrates were added to the thiosulfate reservoir). Instead, only one
representative plaque was picked and subjected to the routinely used typing
procedures. The UNH group typed all plaques during analysis of the final
ten experiments (when coded concentrates were added directly to the
reservoir) except in those tests where the titers were 10 PFU. In
these, several plaques were picked at random for typing. EPA identified
all PFU except for the 10 PFU experiment.
Copies of all the data, code sheets, correspondence, etc., were de-
livered to each participating individual or team, and the entire series
was decoded and summarized.
At the end of the 35 experiments, the CSHD group, which had prepared
and titered the original concentrates, retitered the various virus lots to
determine if changes had occurred during storage. These data were for-
warded to the EPA Project Officer for use in calculations of recoveries by
both of the principal participants in the project.
4.3
-------
REFERENCES
1. Sproul, 0. J. Virus Removal and Inactivation During Water Treatment.
Journal New England Water Works Assoc., 89: 6, 1975.
2. Shaffer, P. T. B., Meierer, R. E., McGee, C. D. Virus Recovery From
Natural Waters. Journal American Water Works Association, 69: 528-
531, 1977.
3. Plotkin, S. A. and Katz, M. Minimal Infective Dose of Attenuated Polio-
virus for Man. American Public Health Association, 57: 1837, 1967.
4. Drinking Water and Health, Safe Drinking Water Committee, Advisory Cen-
ter on Toxiciology, Assembly of Life Sciences, National Research
Council, National Academy of Sciences, Washington, D.C. 1977. 939 p.
5. Melnick, J. L. Detection of Virus Spread by the Water Route, pp. 114-
125. In: Proceedings of the 13th Water Quality Conference. Virus and
Water Quality: Occurrence and Control, ed. V. Snoeyink, Univ. of
Illinois College of Engineering, Urbana, Illinois, February 15-16, 1971.
6. Hill, W. F., Jr., Jakubowskii, W., Akin, E. W., and Clarke, N. A.
Detection of Virus in Water: Sensitivity of the Tentative Standard
Method for Drinking Water. Applied and Environmental Microbiology, 31:
254-261, 1976.
7. American Public Health Association, American Water Works Association,
and the Water Pollution Control Federation. Standard Methods for the
Examination of Water and Wastewater, 14th edition, American Public
Health Association, Washington, D. C. 1976, 1193 p.
8. Hill, W. F., Jr., Akin, E. W., and Benton, W. H. Detection of Viruses
in Water: A Review of Methods and Applications. Water Research 5:
967-995, 1971.
9. Sobsey, M. D., Glass, J. S., Carrich, R. J. and Jacobson, R. R., and
Rutola, W. A. Evaluation of the Tentative Standard Method for Enteric
Virus Concentration From Tapwater. Proceedings American Water Works
Association Technology Conference Proceedings: New Laboratory Tools
for Quality Control in Water Treatment and Distribution, December 3-6,
1978. Paper 3B-1, 18 p.
10. Melnick, J. L., Rennick, V., Hampil, B., Schmidt, N. J., and Ho, H. H.
Lyophilized Combination Pools of Enterovirus Equine Antisera: Prep-
aration and Test Procedures for the Identification of Field Strains
of 42 Enteroviruses. Bulletin World Health Organization, 48: 263,
1973.
44
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SECTION 5
RESULTS
OCCOQUAN-1
Environmental Monitoring
During the first year of the study viruses were reported in seven
concentrates, four of which were from finished-water samplings. All the
isolates were identified as Polio 1. During that year, there was a total
of 22 finished-water samples (Sites D, E, and F) and 44 natural samples
(Sites A, B, and C) collected from June 2 through August 27, 1975 through
this project. A summary of the locations and dates, along with the
corresponding bacterial counts and chlorine residuals, is given in Table
7. All the isolates were from adsorbing filters. None of the clarifying
filters were eluted during the first year's study. In addition to the
samples provided for by this project, biweekly samples of raw and finished
water were sampled for viruses by the OWMP and FCWA as part of their
routine monitoring programs.
Selected data collected in the field during sampling and in the
laboratory during the assay procedure are presented in Appendix Table
A-l. Appendix Table B-l contains all the supplemental data required by
the contract.
RCT-Marker Studies
Because all the isolates were Poliovirus 1, there was concern that
they were contaminants from either a failure to properly disinfect the
Aquella concentrator or to improper handling of the sample after its
collection and during the assay. An additional characteristic of the
isolates—the ret-marker--was determined for all but one isolate (from
Site F, June 3) and compared with those of other viruses, including the
routinely used laboratory strains and that used in the seeding experiments
conducted before the first environmental sample was taken. The results of
the marker studies are presented in Table 8. These data are discussed
in Section 6.
Seeded Samples
Results of the twelve tests where viruses were added to the influent
water are presented in Table 9. As can be seen, the recoveries of polio-
virus were quite good, though the adenovirus recoveries were much lower.
Too, reovirus was never recovered. The reason is not known, and the
problem was an enigma because The Carborundum Company's field teams have
recovered reoviruses in other studies.
45
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OCCOQUAN-II
Environmental Monitoring: The Carborundum Company
During Occoquan-II, a total of 76 environmental samples were taken
from June 17, 1976, through March 25, 1977, including two at the North-
side Sewage Treatment Plant (STP), and from these, a total of 97 concen-
trates (from adsorbing and clarifying filters) were prepared and assayed
for viruses. This phase of the project was coded "2-A," a designation
which will be used in the discussion section. The majority (55, 72 per-
cent) were taken from finished-water sites. Viruses were recovered on
one occasion each at Sites A, B, C, and E and at both sampling points at
the STP. A summary of the data relative to these recoveries, exclusive
of those at the STP, is given in Table 10.
Note from Table 10 that three of the four virus recoveries were from
raw water. The one occasion when an isolate (Polio 1) was reported in a
sample taken from finished water (Site E) occurred on a day when viruses
were recovered from the rectal swab of The Carborundum Company's field
technician. Complete field and laboratory information concerning all the
environmental samples is presented in Appendix C, Table C-l. It should
be pointed out that no viruses were recovered from the open reservoir
(Site H) in Alexandria on two sampling occasions and only once out of
seventeen occasions from the raw water used by FCWA even though both the
clarifying filters and adsorbing filters were eluted each time. However,
a single virus was recovered each time a sample was taken in Bull Run
(twice), once above and once below the point where the last of the STP
effluents were entering at the time of sampling.
Comparative Studies: Environmental Sampling and Analysis
Tables 11 and 12 present an analysis of the data reported by both the
EPA and The Carborundum Company for the samples they analyzed after the
sampling event at the Northside STP in October, 1976. Personnel at each
laboratory sent the others a portion of the concentrates which they had
prepared after they had sampled the unchlorinated influent and chlorinated
effluent to the lagoon. The Carborundum Company prepared concentrates
from both their adsorbing and clarifying filters (Table 11), and EPA pre-
pared one concentrate from their adsorbing filter (Table 12). As can be
seen from both the tables, the EPA group reported higher virus recoveries
whether the concentrate they analyzed was their own or The Carborundum
Company'-s. However, from these data it appears that The Carborundum Com-
pany's concentrator more effectively concentrated viruses. [For example,
compare EPA concentrated/EPA analyzed (Table 12) value of 268.6 PFU per
gallon with Carborundum concentrated/EPA analyzed (Table 11) value of
3273 PFU per gallon.]
The EPA group sampled eleven additional times from Sites C, D, E, and
F at the same time the Carborundum group sampled. As has been described
previously, each group sent the other a portion of their respective con-
centrates to be assayed for virus. Table 13 lists the sites from which
the samples were taken and the results of The Carborundum Company's anal-
yses of EPA's concentrates. Table 14 shows EPA's results. Note that
both laboratories reported finding no viruses in EPA's concentrates. The
49
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TABLE 13. RESULTS REPORTED BY THE CARBORUNDUM COMPANY FOR
ASSAYS OF EPA/HERL CONCENTRATES TAKEN DURING
PHASE 2-A IN COMPARATIVE SAMPLING AND
ANALYSIS EXPERIMENTS, JULY .1976.
Sampling
Date
July 13
14
15
16
19
20
21
22
23
27
28
Site
D
E
C
D
F
E
F
C
D
F
C
UNH
Lab
No.
763
764
765
766
767
768
769
770
771
772
773
EPAa
Lab
No.
180
181
182
183
184
185
186
187
188
189
190
Assay
Date
(UNH)
8/25
8/25
8/25
8/25
8/25
8/25
8/25
8/25
8/25
8/25
8/25
Viruses,
BGM°
0
0
0
0
0
0
0
0
0
0
0
PFUb
PMKd
0
0
'0
0
0
0
0
0
0
0
0
rt
EPA reported no recoveries of virus for these samples
PFU = plaque-forming units
Buffalo Green Monkey Cells
Primary Monkey Kidney Cells
53
-------
TABLE 14. RESULTS REPORTED BY EPA FOR ASSAYS OF LIQUID-OVERLAY
CONCENTRATES PREPARED BY EPA AND THE CARBORUNDUM COMPANY'S
VIRUS GROUPS DURING PHASE 2-A IN COMPARATIVE SAMPLING AND
ANALYSIS EXPERIMENTS, JULY, 1976
Viruses, PFU, Recovered From Three Cell Types3
Sampling
Date
7/13/76
7/14/76
7/15/76
7/16/76
7/19/76
7/20/76
7/21/76
7/22/76
7/23/76
7/26/76
7/27/76
7/28/76
Carborundum Samples
Site
D
E
C-a
C-c
D
F
E
F
C-a
C-c
D
E
F
C-a
C-c
RD
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
BGM
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
PMK
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
EPA
RD
0
0
bo
0
0
0
0
bo
0
0
0
bo
Samples
BGM
0
0
bn
0
0
0
0
bo
0
0
0
bo
PMK
0
0
bo
0
0
0
0
bo
0
0
0
bo
Cells observed for two passages, 14 days each
Clarifier and adsorbent filters eluted and assayed together
54
-------
supplemental data pertaining to water quality on these dates and the re-
sults of Carborundum analyses of their own concentrates (all negative)
are found in Appendix C.
ADDITIONAL FIELD STUDIES
Studies Associated With Water Treatment Plant Filters
The results of these studies, designated as phase 1-B for future dis-
cussion, are presented in Table 15. As can be seen, the chlorine residuals
in all samples were quite high during the tests, the lowest being 1.2 mg/1
in one of the backwash water samples. No viruses were recovered during
any of the tests by either EPA or The Carborundum Company's assay laborator-
ies.
Study of the Filter Media
No viruses were recovered from either of the two samples of anthracite
(UNH Numbers 1731 and 1770, collected on August 3 and September 6, 1977,
respectively, and analyzed on October 4, 1977). The EPA viral assay lab-
oratory reported finding no viruses in the concentrates they received (195
and 100 ml, respectively).
COMPARATIVE STUDY WITH CODED, SEED-VIRUS SAMPLES
The results of the first twenty-five virus-recovery studies are given
in Table 16. The number of PFU's in each sample were known only after the
samples were decoded once all data had been reported. The numbers in the
column entitled "PFU Given to Each Group" were based on the assumptions
that the contents of each coded vial had been divided equally (after
dilution) between each of the field teams and that any viruses present
were monodispersed. Data in the column entitled "Fraction of Total PFU
Actually Used by Group" are the fractions of the initial thiosulfate vol-
umes which were injected into the pasteurized water by the proportioning
pumps during the first twenty-five experiments.
The virus recoveries by each group, expressed as a percentage, were
calculated on the basis of the initial titers determined by CSHD, and an
assumption was made that the numbers of plaques recovered by the two
assay laboratories would have been double those reported had the entire
volume of the final concentrate been analyzed. Recall that each labora-
tory analyzed one-half of each concentrate; therefore, the data (PFU) they
reported were multiplied by 2.0 before the percent recoveries were calcula-
ted. On two occasions, (tests numbered 2 and 12 in Table 16) the EPA
laboratory reported virus types which were not those provided by CSHD.
These are reported in the table as "contaminants." No contaminants were
reported by The Carborundum Company, but they picked only one represen-
tative plaque from each cell sheet. The EPA laboratory, on the other
hand, typed all of them, except when the titer was greater than 10 PFU's
(test 1).
Table 17 presents the virus recoveries reported by each laboratory,
both in PFU's and as percent recoveries, for the experiments in which the
concentrates were diluted in the large reservoir. The calculated percent
55
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TABLE 17. VIRUS RECOVERIES BY THE CARBORUNDUM COMPANY AND THE
ENVIRONMENTAL PROTECTION AGENCY'S VIRUS GROUPS DURING THE COMPARATIVE
SAMPLING-AND-ASSAY STUDY WITH CODED SAMPLES PROVIDED BY
CALIFORNIA STATE HEALTH DEPARTMENT CONCENTRATES ADDED TO 250 GALLONS
OF PASTEURIZED WATER
Test
No.
26
27
28
29
30
31
32
33
34
35
Virus Cone. ,
PFU/100 gal.
& Type
120
Polio 1
21.2
Polio 1
1.16 x 106
Polio 2
Blank
120
Polio 1
120
Polio 1
21.2
Polio 1
21.2
Polio 1
100
Polio 2
120
Polio 1
Actual Recoveries, PFU & Calculated Efficiency, Per Cent?
Carborundum -
Garb-Analyzed
2,3%
0
1.7 x 104
3%
0
0
3,5%
2,19%
3,28%
1,2%
1,2%
Concentrated
EPA Analyzed
14,23%
0
2.07 x 104
4%
0
1,2%
3,5%
0
^contaminated
6,12%
4,7%
EPA - Concentrated
Garb-Analyzed EPA Analyzed
0 1,2%
0 0
2.9 x 104 2.5 x 104
5% 4%
0 0
5,8% 4,7%
0 11,18%
1,9% 1,9%
0 0
5,10% 2,4%
5,8% 20,33%
Each team passed 100 gallons through its respective concentrator
Plaque forming uuits
CPercent calculated as follows: (PFU recovered x 2) divided by (PFU/100 gal)
x 100. PFU/100 gallon figure based on CSHD initial titers.
dl Polio-3 isolate
59
-------
recoveries were based on the same assumptions as those in Table 16. .
Both laboratories typed all viruses recovered except when more than 10
PFU's were recovered (test 28).
The original numbers assigned to the vials sent by CSHD and their
respective code and recode numbers assigned at FCWA are tabulated in
Appendix Table D-l.
Table 18 shows the results reported for the sets of vials stored at
FCWA during the course of the study and analyzed directly by EPA and
Carborundum at the end of the study. These analyses were planned so
that any deterioration in virus titer due to storage, shipping, etc.,
could be detected. As can be seen, the numbers recovered were less
than 40 percent in all vials and below 15 percent in the majority (based
on the initial titers reported by CSHD). In November, 1977, fourteen
months after the initial titers were first determined, the CSHD labora-
tory reanalyzed samples of the concentrates they had retained in their
laboratory. The titers had decreased from 61 to 79 percent (67 percent
average) of their original level. The implications of this observation
will be discussed in the discussion section (Section 6).
PERSONNEL SURVEILLANCE
The results of the analyses of throat and rectal swabs for the field
personnel and others who entered the Carborundum van during a series of
experiments are tabulated in Appendix Table E-l. The recovery of viruses
from one rectal swab on a single occasion has previously been mentioned.
No other analyses of swabs were positive. EPA's swab data are presented
in Appendix Table E-2.
6Q
-------
TABLE 18. VIRUS TITERS REPORTED AFTER DIRECT ANALYSES OF
CODED VIAL CONTENTS STORED AT FCWA DURING THE COMPARATIVE
SAMPLING-AND-ANALYSIS PROGRAM (PERCENT RECOVERIES BASED
ON CSHD INITIAL TITERS)
CSHD Lab
PJTJ TYPE
0 Blank
-
53 Polio-1
250 PoUo-2
PFO
0
0
0
0
-
8
6
7
6
_6
x-6.6
26
24
52
44
27
Carborundum Lab
Z Recovery
0
0
0
.0
-
15
11
13
11
11
x-nz
10
10
21
18
11
pro
0
0
0
0
0
3
10
10
13
_9_
x-10.
93
63
86
31
92
EPA Lab
Z Recovery
0
0
0
0
0
15
19
19
25
17.
X-19Z
37
21
29
27
37
x-34.6
X-14Z
x-31.4
x-IOZ
ax-2.5X10s
x-83
x-105.2
r-33Z
300 Polio-1
41
41
33
21
21
14
14
11
7
7
113
110
93
96
114
45
37
31
39
46
X-35Z
2.9X106 PoUo-2
55500/ml
61500 /ml
67330/ml
33500 /ml
28000 /ml
10
11
12
6
5
1.82X103
1.14X105
2.16X1Q5
1.72X105
2.00X105
6
4
7
6
7
S-1.77X10-3
-6Z
5 ml sample: Z Recoveries and Means (z) are based on PFU reported X 5.
61
-------
SECTION 6
DISCUSSION
OCCOQUAN-1
The report of viruses in finished water during Occoquan-I was the
impetus behind most of the activities that comprised the virus portion
of Occoquan-II. The first year's report prompted close scrutiny of the
Carborundum Company's field- and laboratory procedures—resulting in
some changes in the field procedures during Occoquan-II--and was directly
responsible for the involvement of EPA's virus-research group in the
second year's effort. Never before has a study such as the comparative
sampling-and-analysis study with coded samples been reported in the
United States. It provided a unique opportunity, not only to compare
the specific efficiency and reliability of field and laboratory procedures
used by EPA and The Carborundum Company, but also to evaluate the existing,
tentative standard method for virus concentration.
The discussion which follows focuses on the reported virus findings
during Occoquan-I, the follow-up studies which resulted from these find-
ings, and the studies during Occoquan-II which were planned because of
their potential for providing more insight into the interpretation of
the first year's results.
Environmental Monitoring
Finished-Water Isolates--
The four finished-water samples from which viruses were isolated
during Occoquan-I represents 18 percent of the total finished-water
sampling events. The chlorine residuals in the samples exceeded 1.0
mg/1, and coliforms were absent (Table 7). For these reasons, the
results were regarded as highly unusual, and prompted much comment and
discussion by all parties concerned. While it obviously would have been
difficult to determine after-the-fact that the isolates indeed were
present in the original 100 gallons of water taken from each site,
follow-up investigations involving several parties were made in an
attempt to provide some insight into the findings. It should be mentioned
that from November, 1974, through September, 1978, 101 samples (100
gallons each) of finished water at FCWA were all negative for viruses.
The samples were taken by FCWA and OWMP personnel but were assayed at
the Jackson Estuarine Laboratory (JEL) where all Occoquan-I samples were
assayed.
The first and most logical investigation was into the nature of the
recovered viruses themselves. This investigation involved the rct-
marker studies, mentioned previously, and, in addition, the progeny
viruses from the original isolates were sent to the Center for Disease
62
-------
Control in Atlanta for further characterization. Next, the EPA made an
inspection of FCWA's facilities to determine whether the routine operating
practices there could in any way be conducive to virus contamination.
In addition, an EPA virologist visited the JEL to review the laboratory-
assay procedures used by those who reported isolating the viruses from
the samples. These investigations are discussed in the following para-
graphs .
RCT-marker studies--The tests to determine the rct-characteristics
of all the natural isolates except the one from Site F (Table 8) showed
that none possessed the T+ marker. All of the R values (defined in
Table 8) fell within the range (0-30) typical of attenuated strains.
However, all but one of the R-values were greater than that calculated
for the LSc-1 attenuated strain which was used in the virus-seed exper-
iments during Occoquan-I.
The fact that all but one of the isolates survived at 40°C (T+) is
of particular importance because it gives some basis for distinguishing
them from seed viruses. Reproduction capacities ranging from 2.5 x 10^
to 1.2 x 105 PFU's were recorded for the natural isolates, whereas the
LSc-1 strain (control) did not survive. Although the T-marker tests
were not performed for the purpose of providing an experimental basis
for distinguishing among the isolates (i.e., for identifying each as a
distinctly different virus strain), the data do indicate differing
growth behavior as a function of temperature. Therefore, some of the
isolates appeared to differ from each other as well as from the seed
strain (LSc-1). It should be mentioned that virologists differ on the
significance of T-marker studies in general as a means of differentiating
between polio-virus strains.
Involvement of independent parties — Representatives from EPA's
Region III Water Supply Branch and Virginia's State Health Department
conducted a site survey of FCWA's three water treatment plants in January,
1976, to determine if there was any obvious operational procedure which
might account for the virus recoveries. Their report noted: 1) the
high chlorine residuals (average, 2.0-2.5 mg/1 free chlorine) and low
turbidities (0.1-0.2 units) in finished water during June-August, 1975,
when viruses were reported, and 2) the fact that no viruses had been in
bimonthly samples collected by FCWA personnel and analyzed at the JEL.
It also noted: 1) that the New Lorton plant was not equipped with
filter-to-waste lines (the Old Lorton and Occoquan plants were) which
required that filters be put in operation immediately after backwash is
completed and 2) that filtration rates at the New Lorton Plant were from
5 to 7 gallons per square foot per minute for a brief time after the
backwash cycle is initiated when the influent is shut off and the rate
controller is opened. In the opinion of the survey team, this was the
worst time for a rate increase because floe breakthrough into the
finished water is possible under these conditions. This comment was
responsible for the special studies during Phase 1-B of Occoquan-II
involving attempts to isolate viruses in filter effluent before, during,
and after the backwash cycle. The survey team also commented that
filter rates exceeding the rated capacity were noted at one or two of
the three plants on the days viruses were reported.
In February, 1976, the Chief of the Enterovirus Branch of the
63
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Center for Disease Control (CDC) (Public Health Service, Department of
Health, Education, and Welfare) was contacted and requested to examine
the Occoquan isolates. The six isolates from Site D (7/23 and 8/18/75)
were sent to CDC from UNH, and a report was made in April, 1976. The
results are presented in Table 19. The report stated:
"...It is not possible to draw firm conclusions as to the origin of
these viruses from these results. The viruses might be of vaccine
origin with antigenic characteristics which have 'drifted' to the non-
vaccine-like state observed (type 1 is known to change antigenically in
this manner during the course of multiplication in the human intestinal
tract). On the other hand, these viruses might be 'wild' strains with
the ret characteristics observed (about 30 percent of type 1 strains
isolated before oral polio vaccine was used, and therefore presumably
'wild' strains, show ret characteristics like those observed)."
It should be noted that neither The Carborundum Company's field
technician, any of his immediate family, or either of OWMP's two field
technicians who were responsible for the sample concentrations during
Occoquan-I had received polio vaccinations during 1975. However, the
precaution of routinely taking throat and rectal swabs and storing them
in the event a positive virus was found was not observed during Occoquan-
I. Therefore, whether one or more of the field personnel were shedding
viruses on the days recoveries were reported is purely conjecture.
In February, 1976, an EPA representative from the Cincinnati labora-
tories visited UNH and the JEL to evaluate laboratory procedures and
facilities for handling virus samples. Their report included an overview
of these procedures and concluded:
"...none of the procedures (which were) observed would account for
the reported isolation of the single type virus from various waters."
The writer continued: "The only plausible explanation would seem to be
a contribution from a carrier, someone along the line from samplers to
laboratory technicians, who might intermittently shed virus and thereby
contaminate a sample." The latter statement, of course, is highly
speculative, but it strengthened the position that if a contamination
had occurred, the assay laboratory most likely was not involved.
In summary, none of the studies succeeded in resolving the issues
surrounding these virus isolates. The greatest area of uncertainty was
with the actual field procedures used during Occoquan-I when the field
crew was not following a routine program of surveillance (swab tests),
and stringent precautions to protect the equipment and chemicals from
contamination in the field were not being observed. Whereas many argu-
ments can be put forth concerning both sides of the issue, the fact re-
mains that there was no consensus of opinion whether these isolates were
really in the drinking water or were present as contaminants. The issue
remains today and most likely can never be resolved scientifically.
Reservoir and Tributary Isolates--
Viruses were recovered from Bull Run (Sites A and B) and from the
Occoquan Reservoir at Site C on only three occasions, one at each site,
during June-August, 1975. Normally, one would expect to recover viruses
64
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TABLE 19. RESULTS OF STRAIN CHARACTERIZATION TESTS
PROVIDED BY THE CENTER FOR DISEASE CONTROL FOR
THE OCCOQUAN ISOLATES
Isolate
1/23/75.
91
112
in
8/18/71
#1
92
#3
3R-Value
5.8
13.9
9.3
4.6
2.2
4.5
Antigenic . RCT Characteristic
Characteristic 39.5"c 39.9°C
NVC ±
NVC ±
NVC ±
NVC -
NVC
NVC - CNT
3 Determined at UNH, Table 8
NVC » Nonvaccine-like determined by CDC's modified Wecker antigenic
differentiation test
c Not tested
65
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more frequently from waters that receive treated sewage, especially
immediately downstream of the discharges as was Site B, and certainly
one would expect more frequent virus recoveries from raw water entering
the water treatment plant if finished-water samples from that plant were
positive for viruses on several occasions. The failure to recover virus
from the natural waters more frequently cannot be explained. It should
be noted that during Occoquan-I, the first clarifying filters were never
eluted, but from November, 1974 through 1976, both the adsorbing and
clarifying filters were eluted and analyzed as part of OWMP's routine
monitoring program for viruses. Only twice were viruses reported in raw
water samples during that period. Through September 5, 1978, there had
been 100 samples taken from Site C (as part of the OWMP) and 167 concen-
trates from the adsorbing and clarifying filters analyzed. On only
three additional occasions, all in 1978, were virus recoveries from Site
C reported (Jan. 10--11 reovirus; Jan. 24--1 polio 3; and Oct. 16--1
Echo 7, 2 polio 3). The infrequency of virus recoveries indicates that
the impact of sewage treatment discharges and urban runoff on virus
levels was not significant. Any potential problem that might have
existed would have been further alleviated when the UOSA advanced waste
treatment plant went on-line in late June, 1978.
Seeded Samples
The reason for the failure to recover reovirus from seeded samples
at any of the six sites during Occoquan-I is not known. As was pointed
out, The Carborundum Company has recovered reovirus from natural samples
on numerous occasions. (In 1978, eleven reoviruses were recovered at
Site C in one sample). However, because seed virus could not be recovered,
the only conclusion one can draw is that the desired monitoring for
reovirus during Occoquan-I was not accomplished. Recoveries of the
adenovirus were erratic (0.9-25%, Table 9), but the numbers of viruses
recovered actually were large (103-105). Thus, the problems which
caused the failure to recover reovirus apparently did not interfere with
adenovirus recovery (nor with poliovirus either. See Table 9).
OCCOQUAN-II
Environmental Monitoring
Only once was an isolate reported from a finished-water site during
Occoquan-II, that being one Polio 1 at Site E (Alexandria, Va.) on Sep-
tember 2, 1976. A rectal swab taken this day also showed the only posi-
tive results of any swab examined during the program. Six Polio-1 and
one Coxsackie B-4 viral units were recovered. All the polio isolates
were shown to be avirulent by T-marker tests. The water-associated
isolate and five of the six poliovirus, swab-associated isolates were
sent to the CDC for examination. Tests confirmed that all these were
nonvaccine-like (Table 20). While the technician did not demonstrate a
history of shedding before or after this event (Appendix Table E-l) and
while the low numbers of isolates indicate that he was not actively
shedding, one still cannot completely rule out the chance, however re-
mote, that the isolate recovered from the finished-water sample was the
result of a chance contamination by the field technician.
As was true during Occoquan-I, viruses were recovered very seldom
66
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TABLE 20. ANTIGENIC AND. RCT CHARACTERISTICS OF OCCOQUAN-II
VIRUS ISOLATES FROM FIHISHED WATER AND A RECTAL SWAB
Sample
Finished Water,
Site E, 9/2/76
Rectal Swab,
9/2/76
LSc Control
Hahoney Control
ONE
Ho.
810-3
812-4
812-5
812-6
812-7
CDC
RCT
39.5'C
"Tteg.
Keg.
Neg.
Keg.
Keg.
Data
Antigenic
Test
bnvc
KVC
NVC
KVC
NVC
UNH
a
r
0.064
0.044
0.048
0.055
0.079
0.058
1.14
Data
Ra
0.55
0
0
0
1.94
0
100
See Table 3 (Results Section) for details
Konvaccine-like
Keg. - Kegative
67
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from natural waters during the second year's effort. A series of samples
taken the week of October 11, 1976, yielded most of the natural isolates,
A single Polio 2 isolate was recovered from the Occoquan Reservoir
(raw water (Site C) and a Polio 1 isolate was obtained from Bull Run at
Site A above all the STP discharges. Three isolates—one Polio 1 and
two Coxsackie B-4--were recovered from Bull Run below the STP discharges
(Site B). The isolates were obtained from the clarifying filters used
at Sites B and C and from the adsorbing filter at Site A.
The comparative sampling events when both EPA and The Carborundum
Company sampled natural waters were nonproductive because no viruses
were recovered. The results from the STP samplings served only to
demonstrate: 1) that both teams could recover virus from the same
location, 2) that Carborundum's sampler was more effective for virus
concentration on that date, and 3) that EPA's laboratory analyses re-
sulted in more virus isolations than UNH's.
Additional Field Studies
Viruses which enter a water treatment plant and survive treatment
should be concentrated in the floe which either settles in the clarifier
or is trapped within the filter. For this reason, the sampling events
associated with the filter gallery at FCWA were designed in the belief
that the chances for virus recovery there were much greater than at
finished-water sites. None were recovered, however, either from the
filter underdrains immediately before and following backwash, in the
backwash water itself, or from the anthracite filter media. In retro-
spect, a better opportunity for virus recovery from the backwash water
might have been provided had the floe remained in suspension during the
concentration step. Of course, less water would have been passed through
the concentrator, because the suspended floe would have plugged the
clarifying filter, but then, too, the likelihood would be greater that
viruses would be trapped within the concentrator. As it was, the floe
was allowed to settle in a large container and only the supernatant was
passed through the concentrator.
In retrospect, the failure to recover viruses from the anthracite
filter media is not particularly surprising. It has been shown at UNH
that the efficiency of virus recovery from adsorption sites is inversely
related to the time lapse before elution is attempted. Any viruses on
the anthracite most likely had been there for several hours before the
media was taken from the filter and several days before the media was
eluted. A better procedure, it seems, would be to perform on-site
elutions rather than attempt to store and transport the media to a
laboratory.
Comparative Study With Coded, Seeded-Virus Samples
Relative Efficiencies--
Tables 21 and 22 summarize the data presented earlier in Tables 16
and 17. The recovery percentages given in these tables were based on
the original titers reported by CSHD. The EPA virus research group per-
formance (EPA concentration/EPA assay) was obviously superior to that of
The Carborundum Company (Carborundum concentration/Carborundum analysis)
68
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TABLE 21. SUMMARY OF VIRUS RECOVERY DATA (BASED ON CALIFORNIA
STATE HEALTH DEPARTMENT'S INITIAL TITER) OBTAINED DURING THE COMPARATIVE
SAMPLING-AND-ASSAY STUDY IN WHICH VIRUSES WERE INJECTED
IN THE THIOSULFATE RESERVOIRS
Concentrator/ Avg. Recovery ± Std. Deviation. %. For Virus Levels Shown, a?FU
Assay Blank 2TT125- 1501.5 x 1
-------
TABLE 22. SUMMARY OF VIRUS RECOVERY DATA (BASED ON CALIFORNIA
STATE HEALTH DEPARTMENT'S INITIAL TITER) OBTAINED DURING THE COMPARATIVE
SAMPLING-AND-ANALYSIS STUDY IN WHICH VIRUSES WERE ADDED TO THE
250 GALLON TANK
Concentrator/ Avg. Recovery % ± Std. Deviation For Virus Cone. Shown,
Combination 3PFU/100 gal
Blank
EPA/EPA
No. of Samples
Avg. Recovery
± Std. Dev.
Carbo/Carbo
No. of Samples
Avg. Recovery
± Std. Dev.
Carbo/EPA
No. of Samples
Avg. Recovery
± Std. Dev
•
EPA/Carbo
No. of Samples
Avg. Recovery
± Std. Dev.
1
0
1
0
1
0
1
0
21.2 100
(Polio 1) (Polio 2)
3 1
3% 4%
(2 negative)
3 1
16% ± 14% 2%
(1 negative)
3b 1
0 12%
3 1
3% 10%
(2 negative)
120 1.2 x 10"
(Polio 1) (Polio 2)
4 1
15% ± 15% 4%
4 1
3% ± 2% 3%
4 1
9% ± 9% 4%
4 1
4% ± 4% 5%
( 2 negative)
aPFU = Plaque forming units. Contamination was noted in only one test run,
as is shown in note b.
Analysis revealed contamination by one polio 3 virus in a basic polio 1
virus test.
70
-------
when the viruses were injected into the sample stream from the thiosulfate
reservoir (36%-59% vs. 0%-11%). Furthermore, in every one of the first
twenty-five tests, the EPA assay laboratory recovered a higher percentage
of the viruses present regardless of who had prepared the concentrate.
(Compare EPA/EPA with EPA/Carbo and Carbo/Carbo with Carbo/EPA, Table
20.) The range of mean recoveries when EPA assayed the concentrates was
36-52 percent versus 8-20 percent for Carborundum's data.
When the viruses were added directly to the large reservoir of
pasteurized water, both teams' performances were about equal (Table 22),
but the efficiencies in each case were notably lower than when the viruses
were injected via the thiosulfate reservoir. The reasons for this
difference are not known, though one possibility is that the viruses may
have been lost by adsorption to the sides of the tank. In these tests,
The Carborundum Company's overall performance ranged from 0 to only 18
percent recovery, whereas EPA's performance ranged from 0 to 15 percent.
The percent recoveries expressed in Tables 21 and 22 would be
higher if calculated on the basis of CSHD's final titer results. It was
unusual that the final titers reported by CSHD for the concentrates,
which had been kept frozen throughout the study, were much lower (21-30
percent of the original) than those reported at the beginning of the
study. This was surprising because it is generally thought that virus
stocks are stable for periods of extended storage. One possible explanation
is that the diluent used in preparation of the concentrates was hyper-
tonic and did not contain stabilizing agents such as fetal calf serum.
Another is that the monodispersed viruses reassociated into clumps of
one or more viruses during storage, and each clump behaved as if it were
a single infectious unit during assay.
Figure 8 was prepared by drawing straight lines connecting the
initial and final titers reported by CSHD for each of the four levels
used in the studies. If the titer reduction is assumed to have occurred
linearly, the titers at the times when the experiments were conducted
can be estimated. For example, at the time when a vial originally
titered at 53 PFU was used in the tests involving dilution of the seed
in the thiosulfate reservoir, the titer estimated by interpolation (on
the lower line, Figure 8) was only 45 PFU. Thus, the percent recoveries
listed in Table 16 corresponding to 53 PFU should be increased by 53/45
or 1.18. Tables 23 and 24 summarize the data for the first 25 and the
last 10 experiments, respectively calculated in this manner. The orig-
inal interpretations of the relative virus-recovery efficiencies of EPA
and The Carborundum Company are not altered by this analysis, but the
absolute magnitudes of the recoveries increase somewhat. By this analy-
tical procedure the calculated recoveries during the tests in which the
viruses were added to the large reservoir would be increased by approxi-
mately 30 percent.
The Question of Contamination--
Uninoculated viruses were isolated from sample concentrates of
experiments number 2, 11 and 33. The experimental-procedures record
indicated that the desired temperature had been reached during the
pasteurization of the test water. Therefore, the source of these viral
71
-------
liter, Total PFU
72
-------
TABLE 23. REVISED RECOVERY EFFICIENCIES OF VIRUSES BASED
ON AN ASSUMPTION OF A LINEAR, VIRUS-TITER REDUCTION
IN FROZEN CONCENTRATES (SAMPLES DILUTED IN THIOSULFATE
RESERVOIR)
Concentrator/ Avg. Recovery ± Std. Deviation for Virus Cone. Shown, fPFU
Assay 26.5 US 150 1.5 x 10
Combination (Polio 1) (Tolio 2) (Polio 1) _(Polio 2)
EPA/EPA
No. of Samples 5 1 8 1
Avg. Recovery ± 62% i 25% 41% 68% ± 24% b
Std. Dev.
cEst. titer loss 15% 14% 13% 20%
Carbo/Carbo
No. of Samples 5 181
Avg. Recovery ±0 8% 9% ± 8% 5%
Std. Dev.
CEst. titer loss 15% 14% 13% 20%
Carbo/EPA
No. of Samples 5 181
Avg. Recovery ± 22% ± 13% 12% 17% ±7% b
Std. Dev.
cEst. titer loss 15% 14% 13% 20%
EPA/Carbo
No. of Samples 5 181
Avg. Recovery ± 15% ± 15% 21% 23% ± 18% 10%
Std. Dev.
cEst. titer loss 15% 14% 13% 20%
aPFU " Plaque-forming units
Avg. recovery ± standard deviation could not be calculated because the
virus titer was too numerous to count at dilution used.
CBased on interpolation of data shown in Figure 8.
73
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TABLE 24. REVISED RECOVERY EFFICIENCIES OF VIRUSES BASED
ON AN ASSUMPTION OF A LINEAR, VIRUS-TITER REDUCTION
IN FROZEN CONCENTRATES (VIRUSES INJECTED INTO 250-GALLON TANK)
Concentrator/
Assay
Combination
EPA/EPA
No. of Samples
Avg. Recovery ±
Std. Dev.
Est. titer loss
Carbo/Carbo
No. of Samples
Avg. Recovery ±
Std. Dev.
Est. titer loss
Carbo/EPA
No. of Samples
Avg. Recovery ±
Std. Dev.
Est. titer loss
EPA/Carbo
No. of Samples
Avg. Recovery ±
Std. Dev.
Est. titer loss
Avg . Recovery
21.2
(Polio 1)
3
4%
25%
3
21% ± 19%
25%
3
0
25%
3
4%
25%
± Std. Dev.
100
(Polio 2)
1
5%
23%
1
3%
23%
1
16%
23%
1
13%
23%
for Virus Cone.
12Q
(Polio 1)
4
19% ± 18%
21%
4
3% ± 3%
21%
4
12% ± 12%
21%
4
5% ± 5%
21%
Shown, a?JFU/100 gal
1.2 x 10 «>
(Polio 2)
1
6%
28%
1
4%
28%
1
5%
28%
1
7%
28%
a
PFU = Plaque-forming units.
Based on interpolation of data shown in Figure 8.
74
-------
isolates could not have been indigenous to the finished drinking water
used in these experiments. Also, a test of duplicate vials of the viral
inoculum for these experiments revealed that only the appropriate viral
type was contained in each vial. Therefore, it must be concluded that
the presence of an alien virus in these samples indicates that contamina-
tion had occurred at some point during equipment disinfection, sample
collection, sample concentration, or sample assessment.
Experiment No. 2 was conducted with poliovirus 1. During the
assay/ identification procedure, both laboratories isolated a viral
contaminant (poliovirus 2) from their half of the EPA-processed sample
concentrate. This finding suggested that contamination had occurred
prior to the assessment step.
Experiment No. 12 was conducted with poliovirus 2. The EPA labora-
tory isolated a contaminant (poliovirus 1) from their half of the sample
concentrate that had been processed by EPA. Because the Carborundum
laboratory identified the viral type of only one plaque (poliovirus 2)
isolated from their half of the EPA-produced sample, it is unknown
whether the contaminant was also present there. Therefore, it is imposs-
ible to narrow the likely point of contamination to either the collecting,
processing, or assessment procedure.
Experiment No. 33 was conducted with poliovirus 1. The EPA labora-
tory isolated a single plaque, identified as poliovirus 2, from its half
of the Carborundum-processed sample. Poliovirus 3 had not been an
inoculum in any of the 35 experiments conducted in this study; its
isolation unquestionably represented an exogenous contamination.
Because both laboratories had been involved in field surveys of
drinking water for viruses, considerable thought and planning had been
given previously to the development and use of contamination-prevention
procedures. A major objective of this study was to determine the occur-
rence of viral contamination and, if it occurred, to reveal the weak
point(s) in the control procedures.
The experimental design produced 140 subsamples for viral analyses
from the 35 experiments. A viral contaminant was revealed in 3 of the
140 subsamples (2%). On two occasions, the contaminant had been the
viral type used as the inoculum for the previous experiment. Therefore,
this would appear to be a possible source of the contaminant and would
implicate the equipment-disinfection procedure of EPA as a weak point in
the control procedures. This possibility seems very likely for ex-
plaining the contamination of experiment No. 2. The inoculum for
experiment No. 1 had been a large number of units of poliovirus 2 (2.4
x 106 PFU). The 45 PFU of poliovirus 2 isolated from experiment No. 2
could have represented viral units that remained viable in the equipment
after chlorine disinfection. Although this disinfection procedure had
been used during an EPA field survey where no contamination was revealed
in more than 200 samples, the procedure had never been challenged with
viral concentrations greater than 100 PFU.
The previous explanation for the source of the contaminant of ex-
periment No. 12 seems less likely. The viral inoculum for experiment
No. 11 had been 40 PFU of poliovirus 1. A total of 16 of the 40 PFU had
75
-------
been recovered from the viral concentrates, 14 by EPA and 2 by Carbor-
undum, leaving 24 viral units that had not been recovered. It seems
highly unlikely that these 24 viral units adhered to the equipment's
inner surfaces, were not flushed out or inactivated by the acidic chlorine
solution, and then were released in a viable form during during the next
experimental run where 10 PFU of poliovirus 1 were recovered in addition
to 36 PFU of the inoculum (polio 2). This conclusion is supported by
the absence of any contamination of samples from 10 experiments conducted
with sterile inocula (blanks), many of which followed experiments with
higher titers of virus. There is a remote possibility that the EPA
sampling equipment was not disinfected subsequent to experiments Nos. 1
and 11. Each were conducted on a Friday prior to weekends on which a
change in field personnel took place. The weather was very harsh during
the study period, and the discomfort could have resulted in a postponement
of the disinfection step. A failure to communicate to incoming personnel
the need for equipment disinfection prior to the Monday runs (Nos. 2 and
12) could have occurred. However, the field personnel were all confident
that such a break in the disinfection procedure had not occurred.
The most plausible sources of the experiment No. 12 contaminant
seemed to be a viral shedder among the field and laboratory personnel or
other experiments in the laboratory with the same type of virus. However,
no viruses were isolated from the rectal swabs collected from project
personnel 15 days before and 31 days after the conducting of experiment
No. 12 (Appendix Table E-2). None of the personnel was ill during this
period nor had they received a live polio vaccination. In addition, no
other projects were ongoing in the laboratory with poliovirus 1. Although
this evidence was circumstantial, it did not support the above mentioned
possibilities for the origin of the contaminant. Of incidental interest
was the finding that the contaminant was equally distributed among the
three cell culture flasks used in the assay of the sample, thereby
indicating that it had been well mixed into the sample or that contamin-
ated labware had been used in the assay procedure.
The contamination of experiments Nos. 2 and 12 is directly assoc-
iated with the EPA procedure/personnel. However, the contamination of
experiment No. 33 was not as clearly associated with either laboratory.
Because poliovirus 3 was not an inoculum for any experiment in this
study, its introduction must have been associated with the sample pro-
cessing (Carborundum) and/or assay (EPA) procedure. However, as in
experiment No. 12, an evaluation of personnel and laboratory activity
did not reveal a likely source of the contaminant. Further, there were
no other findings of poliovirus 3 in the cross-check sampling/analysis
conducted by either EPA or Carborundum. Thus, no additional evidence was
provided to aid in identifying the source of the contaminant. An addi-
tional possibility was also explored, i.e., the erroneous identification
of the viral type. A repeat of the neutralization test with a new vial
of monospecific antiserum again identified the single isolate from this
sample as poliovirus 3.
The failure to obtain any supportive evidence or to advance a
tenable hypothesis to explain the source and cause of two of the three
sample contaminations is of major concern. Even though this study has
been the most extensive one of its type to date, no conclusions as to
the necessary control procedures that must be included in a viral field
76
-------
survey can be made. It is obvious that the protocol used for this study
was not adequate to prevent contamination. Whether the mechanism of
contamination was unique to the circumstances of this study or common to
all field studies of this type is unknown. However, it is clear that
investigators who are attempting to recover viruses from environmental
samples, where positive findings may have major importance, must be
extremely diligent in maintaining the integrity of their samples.
77
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.APPENDIX A
Summary of Data Relative To Virus
Sampling and Analysis
June - August, 1975
78
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APPENDIX B
Supplemental Data Collected on. Each. Sampling
Date During Oceoquan-I, 1975-1976
Key to Table Abbreviations:
OCP = Organochlorine Pesticides
DPI » Organophosphorus Insecticides
CPH = Chlorophenoxy Herbicides
VO = Volatile Organics
KM = Heavy Metals
MPN = Most Probable Number of Coliforms per 100 ml
TPC = Standard Total Plate Count per ml
Key to Observations (Last Column):
1 = Low Turbidity, No Unusual Odor or Color
2 = Moderate Turbidity; No Unusual Odor or Color
3 = High Turbidity; No Unusual Odor or Color
4 = Clear, Colorless, No Unusual Odor
Key to Sites:
A = Catharpin, upper Bull Run Above Treated Sewage Discharges
B = Bull Run Below Treated Sewage Discharges
C = Intake Water at the Fairfax County Water Authority's Water
Treatment Facility
D = Finished Water at the Fairfax County Water Authority's Water
Treatment Facility
E = Distribution System at the Fairfax County Water Authority's
Storage Yard
F = Distribution System in Alexandira, Virginia
G = Distribution System in Dumfries, Virginia
82
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110
-------
APPENDIX D
Comparative Sampling Program:
Code Numbers Assigned to Concentrates
Prepared by California State Department
of Health Virus and Rickettsial Laboratory
111
-------
TABLE D-l.CODE NUMBERS ASSIGNED TO VIALS SENT BY CALIFORNIA STATE HEALTH DEPARTMENT
(CHSD), RECODE NUMBERS GIVEN UPON RECEIPT AT FAIRFAX COUNTY WATER AUTHORITY,
AND SECOND RECODE NUMBERS ASSIGNED FOR CONCENTRATES SUPPLIED BY THE TOO VIRUS
GROUPS INVOLVED IN THE COMPARATIVE STUDY (EPA AND THE CARBORUNDUM COMPANY)
CSHD
Code
No.
1-24
1-46
1-83
1-52
1-72
2-21
2-49
2-80
1-32
2-59
1-81
1-54
1-12
1-90
1-97
1-15
1-02
1-05
2-06
2-22
2-50
2-75
1-56
2-94
1-19
1-04
1-95
1-70
1-26
1-42
1-44
1-76
1-92
2-47
4-84
Virus Titer, Re-
Total PEU & Code
Virus Type No.
53 181
Polio 1 146
150
110
120
291
200
229
250 107
Polio 2 284
300 103
Polio 1 159
169
117
199
114
157
191
273
265
236
219
2.9 X 106 148
Polio 2
222
Blank 101
160
162
153
196
119
164
116
111
207
468
Test
No.
11
17
6
5
22
27
32
33
12
34
2
21
4
9
24
3
23
20
26
30
31
35
1
28
15
16
18
19
7
13
10
3
14
29
25
Carborundum
Concentrate.
Number Assigned at FCWA
Carbo.
Analyzed0
2063
1166
2498
2206
1606
1069
1706
1065
2210
1705
2428
1549
2905
2738
1927
2119
1411
1688
1377
1565
1818
1168
2544
1100
1703
1581
1612
1782
2310
2585
2134
2956
1379
1751
1635
EPA
Analvzed c
1400
2079
1807
1243
2600
2143
2843
2533
1749
2566
1858
2969
1309
1537
2903
1944
2542
2955
2789
2531
2537
2784
1556
2366
2019
2705
2887
2922
1163
1775
1415
1421
2952
2281
2083
EPA Concentrate
Number Assigned at FCWA
Carbo.
Analyzed
2213
1560
2872
2999
1721
1071
1599
1515
2691
1219
2809
1684
2564
2440
1012
2418
1006
1130
921
1399
1602
1182
2394
842
1997
1867
1191
1604
2167
2412
2884
2272
1375
1277
1461
EPA
Analyzed0
1849
2721
1856
1616
2451
2426
2782
2112
1179
2586
1629
2014
1306
1713
2425
1053
2584
2686
2579
2810
2740
2494
1334
2110
2197
2463
2620
2945
1763
1816
1700
1679
2962
2174
2230
Test Nos. 1-25: Vial contents diluted in thiosulfate reservoir; Nos. 26-35, in
260-gallon dilution tank filled with pasteurized water.
No. assigned after concentrates returned to FCWA by the respective laboratories
Carborundum labs at Univ. New Hampshire; EPA labs in Cincinnati
112
-------
APPENDIX E
Results of Swab Tests
113
-------
TABLE E-l. VIRUS DATA ON PERSONNEL
Date
1976
6/21
8/13
8/16
9/2
9/8
9/22
9/30
10/5
10/14
10/15
12/3
12/9
Lab No.
703
704
705
706
775A
775B
774A
774B
8123
812A
813B
813A
817
818
893
892
897
896
894
895
899
898
891
890
389
b!058-59
b!060-61
1062
1063
1064
1065
1066
1067
1068
1069
1070
1071
1072
1073
1074
1075
From
McGee
McGee
Saunders
Saunders
George
George
Thacker
Thicker
McGee
McGee
Saunders
Saunders
McGee
McGee
McGee
McGee
Saunders
Saunders
McGee
McGee
Thacker
Thacker
McGee
McGee
Saunders
Saunders
Saunders
McGee
McGee
Thacker
Thacker
Hoehn
Hoehn
Grizzard
Grizzard
Saunders
Saunders
McGee
McGee
Thacker
Thacker
Swabs
Description
Rectal
Throat
Rectal
Throat
Throat
Rectal
Throat
Rectal
Rectal
Throat
Rectal
Throat
Throat
Rectal
Rectal
Throat
Rectal
Throat
Throat
Rectal
Rectal
Throat
Rectal
Throat
Throat
Rectal
Throat
Rectal
Throat
Rectal
Throat
Rectal
Throat
Rectal
Throat
Rectal
Throat
Rectal
Throat
Rectal
Throat
-Continued-
Virus
BGM
0
0
0
0
0
0
0
0"-
a7
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
, PFU
PMK
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
114
-------
TABLE E-l (continued)
Swabs
Dace
12/17
1977
2/15
3/1
3/21
6/6
6/7
tab No.
1076
1077
1078
1079
1080
1081
1243
1244
1247
1243
1173
1172
1245
1246
1197
1196
1194
1195
1241
1242
1239
1240
1519
1520
1521
1522
1523
1524
From
McGee
McGee
Saunders
Saunders
Thacker
Thacker
Thacker
Thacker
McGee
McGee
Cameron
Cameron
Saunders
Saunders
Thacker
Thacker
Saunders
Saunders
Thacker
Thacker
Saunders
Saunders
McConnaghy
McConnaghy
Thacker
Thacker
Saunders
Saunders
Description
Rectal
Throat
Rectal
Throat
Rectal
Throat
Rectal
Throat
Rectal
Throat
Rectal
Throat
Rectal
Throat
Rectal
Throat
Rectal
Throat
Rectal
Throat
Rectal
Throat
Rectal
Throat
Rectal
Throat
Rectal
Throat
Virus
BGM
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
, PFU
PMK
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 Polio 1 and 1 Coxsackie B-4
Duplicate Samples
115
-------
TABLE E-2. VIRAL ASSESSMENT OF RECTAL SWABS
COLLECTED FROM EPA PERSONNEL ASSIGNED
TO THIS PROJECT
Specimen
Name Collection Cell Type
Date PMK BGM
Akin 11/29/76 a-
Brashear 11/29/76
1/17/77
Mayhew 11/29/76
1/14/77
Stetler 11/29/76
1/14/77
Waltrip 12/03/76
1/17/77
no viruses isolated by CPE method over 7-day
observation period
116 «U.S. GOVERNMENT PRINTING OFFICE: 1981 341-082/230 1-3
-------
-------
United States Official Business
Environmental Protection Penalty for Private Use
Agency $300
Washington DC 20460
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