United States
Environmental Protection
Agency
Environmental Monitoring and
Support iwmN MENTAL
Research and Development
933
>EPA Research
Priorities for
Monitoring
Viruses in the
Environment
DALLAS, TEXAS
LIBRARY
28 OCT 1983
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EPA-600/9-83-010
September 1983
Research Priorities for
Monitoring Viruses in the Environment
by
Joseph V. Karaganis, Edward P. Larkin, Joseph L. Melnick,
Pasquale V. Scarpino, Stephen A. Schaub, Charles A. Sorber,
Robert Sullivan, and Flora Mae Wellings
Cochairmen of Working Group
Robert S. Safferman
Chief, Virology Section
and
Gerald Berg
Technical Advisor
U.S. ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF RESEARCH AND DEVELOPMENT
ENVIRONMENTAL MONITORING AND SUPPORT LABORATORY
CINCINNATI, OHIO 45268
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NOTICE
This document has been reviewed in accordance with U.S. Environmental
Protection Agency policy and approved for publication. Mention of trade names
or commercial products does not constitute endorsement or recommendation
for use.
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Foreword
Environmental measurements are required to determine the quality of
ambient waters and the character of waste effluents. The Environmental
Monitoring and Support Laboratory - Cincinnati conducts research to:
• Develop and evaluate methods to measure the presence and concentra-
tion of physical, chemical, and radiological pollutants in water, waste-
water, bottom sediments, and solid wastes.
• Investigate methods for the concentration, recovery, and identification of
viruses, bacteria, and other microbiological organisms in water; and, to
determine the responses of aquatic organisms to water quality.
• Develop and operate an Agency-wide quality assurance program to
assure standardization and quality control of systems for monitoring
water and wastewater.
• Develop and operate a computerized system for instrument automation
leading to improved data collection, analysis, and quality control.
This report was prepared at the invitation of the USEPA and is consistent
with the USEPA's commitment to provide the scientific public an opportunity to
be heard on the major scientific issues that face the Agency and to offer to the
Agency appropriate recommendations. To this end, non-EPA leaders in
environmental virology focused on research priority needs in monitoring for
viruses in the environment; they addressed the direction and rationale of these
needs. It is the expectation of the USEPA that the results of their efforts will
prove useful to regulatory, regional and research planners.
Robert L. Booth, Acting Director
Environmental Monitoring and
Support Laboratory - Cincinnati
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Contents
Page
Foreword ill
I Introduction 1
II. Summary and Recommendations 3
A. Summary - 3
B. Recommendations 3
1. High Priority Research Needs 3
2. Priority Research Needs 4
III. Rationale 5
A. Monitoring 5
B. Indicators 6
C. Methods Development 8
D. Quality Assurance 10
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Working Group on Research Priorities for
Monitoring Viruses in the Environment
September 22, 1982
These recommendations were prepared by:
seph'V. Karaganis, J.C
Karaganis and Gail Ltd.
Attorneys-At-Law
Chicago, Illinois
Dr. Edward P, Larkin*
Chief, Virology Branch
Division of Foods
U.S. Food & Drug Administration
Cincinnati, Ohio
L
Dr. Josep'h L. Melnick
Head, Department of Virology & Epidemiology
Baylor College of Medicine
Houston, Texas
V. Scarpino
Department of Civil & Environmental
Engineering
University of Cincinnati
Cincinnati, Ohio
Dr. Stephen A. Schaub*
Environmental Biology Branch
Environmental Quality Division
U.S. Army Bioengineering R&D Laborat
Ft. Oetrick, Frederick, Maryland
Dr. Charles A. Spptfer
Associate Oedtr^
College of Engineering
University of Texas
Austin, Texas
Robert Sullivan*
Virology Branch
Division of Microbiology
Bureau of Foods
U.S. Food & Drug Administration
Cincinnati, Ohio
^£«-r>;-
e We4Hngs
r ^
Dr. Flora Mae
Administrator
Epidemiology Research Center
Tampa, Florida
*Thi$ document does not necessarily reflect the agency views of participant.
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I. Introduction
On May 29 and 30,1980, the USEPA invited a group of experts to participate
in a Conference On Monitoring Viruses In the Environment.1 Following that
conference, EPA convened a smaller working group of seven persons2 to
prepare specific recommendations setting research priorities for monitoring
viruses in the environment. The working group met on September 22, 1982.
This document is their report recommending research priorities for monitoring
viruses in the environment
This report is organized in two sections:
1. Summary and Recommendations
2. Rationale for the Recommendations3
1 The following persons participated in the 1980 Conference: Georges Belfort, Gabriel Bitton, Kerby
Fannm. Charles Gerba, John Herrmann, Joseph Karagams, Edward Larkin, Joseph Melnick,
Theodore Metcalf, Bernard Sagik, Pasquale Scarpino, Stephen Schaub, James Smith, Mark Sobsey,
Charles Sorber, Robert Sullivan, James Vaughn, Craig Walhs and Flora Mae Wellings.
2 Joseph Karaganis, Edward Larkin, Pasquale Scarpino, Stephen Schaub, Charles Sorber, Robert
Sullivan, Flora Mae Wellings and Joseph Melnick, Chairman.
3 In writing the rationale for the research priorities, the working group drew heavily on the 1980
conference. However, the 1982 group has expressly focused on research priorities.
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II. Summary and Recommendations
A Summary
The following facts have been established about human viruses in the
aqueous environment.
1. Viruses of public health significance are abundantly present in wastewater,
and can be found even after activated sludge treatment and chlorine
disinfection.
2. Viruses are concentrated in wastewater sludges which may be ultimately
used on land as fertilizers and soil conditioners.
3. Viruses are present in surface waters, especially rivers which many com-
munities use as sources of potable water.
4. In addition to intentional wastewater reclamation, water is being recycled
inadvertently as one community pollutes the water source of a second
community.
5. Viruses have been detected in the treated water supplies of large and small
communities in a number of countries, including the United States.
6. Increasing numbers of communities in different parts of the country are
turning to land application of wastewater and sludges. Soils vary greatly in
their capacity to adsorb viruses, and thus, to preclude groundwater
contamination.
7. Enteric viruses can remain viable for months in water and probably longer
when associated with solids found in water.
8. Waterborne dissemination of infectious viruses may occur without being
detected.
Research should be conducted in several important areas designed to address
the problems described above.
B. Recommendations
1. High Priority Research Needs
a. Monitoring and evaluating the effectiveness of water and wastewater and
sludge treatment unit processes for virus removal are essential to develop-
ing appropriate virus control strategies. Process evaluations should be
accomplished through use of present methods and new methods as they
are developed.
b. Current methods often fail to detect viruses of human origin present in
water and wastewater. Emphasis should be placed on:
(1) improving the sensitivity of existing methods especially in the presence
of particulates;
(2) developing relatively simple methods or adapting existing methods for
viruses of particular significance such as hepatitis A virus, rotaviruses
and the Norwalk-like gastroenteritis virus group; and
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(3) developing a quality assurance program that includes round robin
testing for existing and newly proposed methods.
c. Currently available methods for monitoring shellfish for the presence of
viruses fail to detect hepatitis, Norwalk, or rotavirus and gastrointestinal
viruses. Thus, there is a need to develop more sensitive, quantitative
shellfish methodology to detect these and other viruses of importance to
human health.
2. Priority Research Needs
a. Develop improved, sensitive, quantitative methods for the detection of
viruses in sludges and soils associated with the land application of
wastewaters and sludges.
b. Search for more suitable indicators of the presence of viruses in water,
wastewater, sludges and soils. Some of this effort can be accommodated
through research appropriately designed to accomplish the high priority
research recommendation stated in 1.a. above.
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III. Rationale
Public health scrutiny of disease causing agents -- be they pathogens or toxic
chemicals -- is always beset with unanswered questions. Indeed, that is the
raison d'etre for pursuing and focusing on public health research.
The agents of concern to this working group are enteric viruses -- particularly
those viruses associated with the digestive tract and typically excreted in human
wastes. Enteric viruses are responsible for a wide range of serious illnesses
ranging from hepatitis (liver disease) to myocarditis (heart disease) to pleurodynia
(chest disease) to central nervous system disorders (e.g. polio), to acute
gastroenteritis (intestinal cramps, diarrhea), to death.
While much remains unknown about numerous aspects of viruses -- just as
much remains unknown about asbestos, vinyl chloride, polychlorinated biphenyls
(PCBs) and other toxic chemicals-- there are many known facts which allow us to
recommend research priorities at this time. The known facts about viruses in the
water environment which form the factual predicate for the working group's
research priority recommendations are set forth in the Summary, above.
Based on the eight facts set forth in the Summary, the working group has
identified certain health hazards and research priorities. These research priorities
and recommendations are an outgrowth of the working group's analysis of
research needs in four areas:
1. monitoring for viruses in the environment;
2. the development and identification of indicator organisms or tests to serve
as surrogates for direct measurement of viruses;
3. improvement of analytical methods for virus concentration and assay; and
4. improvement and development of quality assurance procedures for viral
analysis.
These four areas are closely interrelated and are chosen for analytical
convenience. The following discussion under these four areas collectively
presents the rationale for the working group's research recommendations listed
above.
A Monitoring
Given the health risk presented by viruses, it is essential to develop more
information on the nature and extent of viral contamination in our nation's
waters. This information can only be provided through increased monitoring of
each major pathway leading to the deposition of viruses into the nation's waters.
In establishing a virus monitoring program, several factors ought to be
considered:
Site Selection. The selection of monitoring sites around the country should be
made under the combined guidance of EPA and other scientists.
Laboratory Procedures — Round Robin Analysis. A procedure should be
developed for gathering, allocating and distributing the samples to various
laboratories for round robin testing.
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Standard Protocols. In performing viral analysis, all laboratories should follow
standard sampling and operational protocols agreed upon by the round robin
participants. However, each laboratory should also be free to use and compare
other methods of their own choosing.
Parallel Biological and Chemical Analysis. The participating laboratories
should also monitor biological indicators of pollution (for example, fecal
coliforms, fecal streptococci, mycobactena), and the chemical and physical
constituents of the water being sampled which are significant.
Media to be Sampled. The monitoring effort should encompass sampling at
all locations where viruses may be present including sewage, water, shellfish
and aerosols. In each medium, the following factors should be considered:
1. Sewage
a. Untreated raw wastewater (influent). These samples are usually rich in
viruses and results of their analyses should serve as a control of the
sensitivity of the virus concentration and virus enumeration method.
These samples also serve to indicate the virus load requiring treatment
b. Effluent Effluent from the wastewater treatment process before and
after dilution in the receiving water (recreational water) should be
monitored.
c. Sludge. Sludge monitoring is essential where sludge may come in
contact with humans or their food crops.
2. Water
a Untreated source water. This includes river, lake or ground water
immediately prior to treatment
b. Potable water immediately after treatment.
c. Potable water at the tap. Selected locations should be sampled to
determine if defects exist in the distribution system.
3. Shellfish
Monitoring shellfish should be the responsibility of the federal and state
agencies concerned (EPA, FDA and NOAA and the state agencies).
4. Aerosols
The emphasis at this stage of methods development should be on water
that forms the aerosol.
B. Indicators
Ideally, isolation of the viruses themselves is the most appropriate means of
virus detection. However, at this time, widespread direct testing for virus is
hampered by such factors as the long time required to obtain test results,
variations in the precision and accuracy (i.e. detectability) of various virus types,
the shortage of competent personnel, and the high cost of viral analysis.
Consequently, it is desirable to identify if possible, reliable indicator organisms
and analytical methods to serve as surrogates for the presence of viruses. The
use of such biological or chemical indicators is often used in public health. For
example, public health and environmental health practitioners currently use fecal
coliform bacteria as an indicator organism for fecal bacterial pathogens.
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No universal indicator presently exists that is suitable for detection of all
viruses. Virus occurrence in the environment is sporadic and there are
differences between viruses and indicators in survival capabilities, ease of
detection responses to environmental stress, and susceptibility to disinfection.
In addition, there are differences in the response to environmental stresses
between enteric viruses and candidate indicators.
Indicators may be drawn from bacterial, yeast and viral groups. Candidate
bacterial indicators may be the coliform group (total and fecal), fecal streptococci,
anaerobic spore formers (clostridia species) and non-spore formers (bifido-
bacteria), acid fast forms, and standard plate counts. Candidate viral indicators
may be either bacteriophages or selected enteric viruses.
Conceptually, any of the above may serve as a surrogate indicator for the
viruses. Certain candidate indicators may prove more useful than others in a
given situation, such as in the examination of sludges, soils, leachates, or water.
The choice of the surrogate virus indicator should be determined by the nature of
the environmental sample to be tested.
Candidate chemical indicators such as the fecal sterols (coprostanol) have also
been suggested as surrogate indicators of fecal pollution.
Historically, coliforms have gained wide acceptance as indicators of fecal
pollution. The acceptability of members of the coliform group as indicators of
viruses has been based upon the argument that there are many more coliform
bacteria present in sewage than viruses. While some general relationship may
exist between indicator and virus numbers in grossly polluted waters, dis-
crepancies may occur in high quality waters perhaps due to differences in
sample sizes used in tests for viruses and coliform bacteria (e.g., 400 liters vs 50
to 100 ml, respectively for drinking water) or perhaps due to the lack of nutrients
to sustain bacterial life as opposed to the inert viruses.
There are other limitations on the use of bacterial indicators. Viruses generally
are more resistant to disinfection than coliform bacteria. Moreover, the greater
persistence of viruses in receiving water and sediment may lead to a further
disproportionate relationship between surviving bacteria and the presence of
viruses. In addition, some members of the total coliform group such as Klebsiella
may manifest regrowth which is impossible for viruses.
Fecal streptococci are more resistant to disinfection than coliform bacteria.
Some strains of fecal streptococci persist for days in irrigation waters, sludges
and landfill leachates, but fecal streptococci do not multiply in the environment.
Some fecal streptococci biotypes are also ubiquitous in aquatic environments.
Since fecal streptococci do not multiply in the environment, they would appear in
some circumstances a priori to be more suitable indicators of enteric viruses
than fecal coliforms (e.g. in sludges).
The spore-forming clostridia have the disadvantage of much greater per-
sistence than other bacteria indicators and viruses in the environment. Conse-
quently, at best, certain clostridia may be used as tracers of remote pollution
rather than as indicators of viruses.
Although some species of bifidobactena are specifically associated with
human fecal pollution and although they are less likely to regrow in the
environment, they may be subject to environmental stresses, especially the
presence of oxygen. Bifidobacteria are unsuitable as indicators because they
are difficult to detect and because their relationship to enteric viruses is
unknown.
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Two acid fast bacteria (Mycobacterium phlei and M. fortuitum] and a yeast
(Candida parapsilosis) have demonstrated their usefulness as indicators of
disinfection effectiveness. Their usefulness is related to their greater resistance
to chlorine disinfection than Escherichia coli. Salmonella typhimurium and
poliovirus type 1. Two disadvantages are associated with the use of these
indicators. The minimum incubation time required for growth of mycobacteria is
3 days, and commercially prepared growth media are unavailable.
Standard bacterial plate counts have been suggested as an indicator of the
quality of reclaimed water. However, only limited information exists on the
suitability of this heterogeneous group of bacteria as an indicator of viruses.
Coliphages such as f2, MS2 and members of the T series may have a
restricted, yet useful, indicator function. These phages may serve as laboratory
or field models to assess the rate or extent of virus removal in water and
wastewater treatment plants, and may possibly be used as tracers. Two areas of
investigation remain unanswered. One concerns the ecological relationship that
exists between phages and bacterial host cells (i.e., multiplication of phages in
the test samples). The second concerns the relationship between the rate of
survival of phages and enteric viruses. There have been conflicting results in
comparative studies conducted on the survival of phages and enteric viruses,
which casts further doubt on the usefulness of phages as indicators of enteric
viruses.
A vaccine strain of poliovirus (type 1) has been suggested as a potential
indicator for other enteric viruses. This vaccine strain is shed in large numbers by
vaccinated individuals, is relativley safe to handle (in seeding studies), and may
be more readily detected in environmental samples than wild enteric viruses.
One of the most serious objections to the use of a single virus indicator, such as
poliovirus type 1, is that neither poliovirus type 1 nor any other enteric virus is
always found in fecal samples.
At present, the entire enteric virus group itself is the most meaningful, reliable
and effective virus index for environmental monitoring.
In summary, the search for the most suitable surrogate indicator for the
presence of enteric viruses should continue. Candidate indicators should be
evaluated as to their similarity to viruses on such qualities as regrowth, patterns
of survival and disinfectant resistance.
C. Methods Development
There are currently several analytical laboratory methods for the detection and
quantification of enteric viruses in environmental samples. There is general
agreement that when such methods indicate the presence of virus such an
affirmative finding is reliable. However, there is serious concern that existing
methods underestimate the quantity of virus or alternatively produce false
negative results when viruses are actually present in the sample.
Several factors have been identified which may account for these under-
estimation or false-negative problems. These include, but are not limited to, the
following:
1. The development of most of the current methods has emphasized the
recovery of members of the human enterovirus group (i.e., polioviruses,
echoviruses, coxsackieviruses). In addition, most development studies
have utilized laboratory strains of the respective virus types. Several recent
studies have suggested that such strains may not always represent an
appropriate model for naturally occurring viruses.
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2. The myriad of sample types and qualities under consideration (e.g., "dirty"
water, "clean" water, sludges, soils, marine sediments, etc.) lead to a wide
variability in technique efficiencies. This variability is further noted in
samples of similar types taken from different locations and from the same
location at different times of the year.
3. Current methods may not be appropriate for all of the viruses of public
health significance (e.g. at present, assay techniques are not generally
available for the recovery and enumeration of Norwalk-like agents, human
rotaviruses, most of the Coxsackie A viruses and hepatitis A viruses in
environmental samples).
4. Some current methods may not be adequate for the detection of solids-
associated or solids-occluded viruses.
5. Methods for enumeration of viruses in samples may exhibit variability
among different testing laboratory groups, which may result from the use
of different host-cell systems and differing methods of enumeration.
6. Methods have not yet been standardized, quality control is limited,
personnel involved in methods application are often not well-trained. No
single method is sufficiently meritorious to be put forth as a fully adequate
method at this time.
Specific areas for the application of research in improved virus recovery
techniques are:
1. Water
a. The type and quality of water (i.e., marine, fresh, wastewater) sampled
has a profound effect on the efficiency of a particular recovery technique.
In spite of this, several methods have been developed and successfully
used for the recovery of indigenous viruses.
b. The method most widely used for recovering viruses from aquatic
systems is adsorption-elution-reconcentration.
c. Tangential flow ultrafiltration-reconcentration has also been introduced
but has not yet been widely adopted.
d. The present methods do not detect or quantify all indigenous viruses
present in water.
2. Sludge
a Most methods for extraction of viruses from sludges involve elution
followed by concentration which does not address solids-occluded
viruses. A major problem with these and other methods has been the"co-
concentration" of a variety of chemical compounds which have proven
toxic to the assay tissue cultures and possibly to the viruses under test
b. The reduced recoveries noted in sludge-seeding studies correlate with
viral toxicity and also with viral complexing to components of the sludge.
To date, recovery methods have not addressed the variation posed by the
different types of sludge (e.g., aerobically and anaerobically digested,
raw, etc.).
3. Aerosols
a. Application of procedures for the recovery of viruses from wastewater
aerosols lags some years behind comparable methods development for
water. In addition, the physical problems inherent in the collection of
virus particles from large sample volumes of air are a complicating factor.
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b. An added sampling problem results from variability in meteorological
conditions.
c. The methods used have not been developed for the express purpose of
recovering viruses from wastewater aerosols, but rather have been
adapted from techniques designed for sampling for microorganisms and
other types of pollutants.
d. Present methods involve collection of airborne viruses into a liquid
medium which must then undergo some form of reconcentration.
4. Shellfish
a. Quantitative extraction of viruses from shellfish meats has proven
difficult.
b. At present, no single method can be used with the assurance of
recovering viruses from all shellfish types.
c. Present methods have cytotoxicity problems and sometimes are unable
to contain bacterial contamination (which further compromises an
already stressed tissue culture assay system).
d. While many methods have been described in the literature, most used
currently involve some combination of clarification, extraction and
reconcentration steps.
e. In addition to the need for efficient methods, there is a need to define
representative sampling procedures.
5. Solids
The full significance of the relationship between viruses and solids (soils
and sediments) has only begun to be evaluated. Proposed methods involve
elution followed by concentration. Definition of sample collection methods
is required.
In light of the foregoing discussion, specific research needs have been
identified within the general area of virus methods development. They are:
1. The development of more quantitative methods for assessing the role of
viruses in the variety of environmental systems is essential.
2. Increased emphasis must be placed on methods applicable to the recovery
of a wider spectrum of indigenous viruses of public health significance.
3. Recognizing the limitations of present methods, an emphasis should be
placed on further development of current methods and the development of
new practical methods for all environmental systems.
4. Methods to be used for practical monitoring must be rapid, sensitive,
reproducible, simple and economical. They must be capable of processing
sample volumes large enough to be significant with regard to detection of
expected indigenous viral concentrations.
5. A mechanism should be developed, preferably under EPA sponsorship, for
the comparative assessment by a variety of laboratories of the recovery
capabilities of different virus monitoring methods.
D. Quality Assurance
Consistent with the need for improved viral detection methods is the need for
standardization of quality assurance procedures in viral sample collection and
laboratory analysis.
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Quality assurance should apply to the collection of all environmental virus data.
With respect to virus monitoring data, the following statement is endorsed by
the working group:
"These data must be scientifically valid, legally defensible, representative,
comparable, complete, and of known precision and accuracy."
(From: The Quality Assurance Program, An Overview, USEPA, Office of
Research and Development Washington, D.C. 20460, March 1980.)
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The objectives and activities of a quality assurance program for monitoring
human enteric viruses in environmental samples should include:
1. the provision of methods and materials;
2. the evaluation of monitoring performance; and
3. the enhancement of monitoring performance capabilities.
There are a number of considerations which should be given to a quality
assurance program associated with the monitoring of viruses in the environ-
ment. Specific points for consideration follow:
1. General
a. General quality assurance criteria, including statistical considerations,
should be developed for the operation of an environmental virology
laboratory.
b. Quality assurance should apply to both reference and candidate methods
and procedures.
c. Quality assurance for enteric virus monitoring should consider:
(i) the variability in types, amounts and state of viruses present in field
samples;
(ii) the characteristics, quality and size of the sample; and
(IN) each step of all stages of virus recovery including sample collection,
processing, assay procedures and data analysis.
d. Laboratory procedures should be developed with respect to facilities,
operators, and sample handling in order to assure the validity of results
by prevention of extraneous virus contamination.
2. Laboratory Evaluation of Virus Monitoring
Methods
a. There should be two types of test samples for intra- and inter-laboratory
methods comparison, one containing laboratory-grown viruses and,
when feasible, another containing indigenous viruses.
b. The types, quantities and states (e.g., free, solids-associated) of viruses
used in test samples should reflect those that would be expected in field
samples.
c. Size and quality of test samples should be consistent with the size and
quality of field samples.
d. The number and frequency of quality assurance samples (includng
positive and negative test samples) should be governed by statistical
considerations. An important reason for including negative controls is to
assure the integrity of the test system with respect to extraneous and/or
cross-contamination.
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3. Field Monitoring
The following should be compatible with the field monitoring objectives:
a. the location, placement and number of monitoring stations;
b. environmental conditions, sample size and frequency of sample collection;
c. quality assurance procedures for collecting, processing, handling, trans-
porting, preserving and storing field samples, and all aspects of virus
procedures; and
d. consideration should be given to the development and use of internal
controls (markers, tracers) in field samples for quality assurance of field
monitoring.
4. Coordination of Effort
There should be coordination of virus monitoring quality assurance activities
among environmental laboratories and with other organizations involved in
similar efforts.
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•&U. S. GOVERNMENT PRINTING OFFICE: 1983/659-095/0/
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