United States
Environmental Protection
Agency
Health Effects Research
Laboratory
Research Triangle Park NC 27711
EPA-600/2-81-006
January 1981
Research and Development
Handbook
Quality Assurance
Guidelines for
Environmental
Health Research
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EPA -600/2-81 -006
January 1981
QUALITY ASSURANCE GUIDELINES FOR
ENVIRONMENTAL HEALTH RESEARCH
by
P. A. Cunningham
K. W. Gold
T. J. Hughes
L. E. Myers
C. E. Tatsch
EPA Contract No. 68-02-3226
EPA Project Officer: Ferris B. Benson
RTI Project Leader: C. E. Tatsch
Health Effects Research Laboratory
U.S. Environmental Protection Agency
Research Triangle Park, North Carolina 27711
November 1980
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DISCLAIMER
This report has been reviewed by the Health Effects Research Labora-
tory, U.S. Environmental Protection Agency, and approved for publica-
tion. 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 en-
dorsement or recommendation for use.
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FOREWORD
The U.S. Environmental Protection Agency's Health Effects Research
Laboratory located at Research Triangle Park, North Carolina, conducts an
extensive research program to evaluate the human health implications of environ-
mental factors related to contemporary society. The purpose of this research
is to develop appropriate information for formulating sound environmental
policies for protecting and improving public health and welfare while enhancing
the nation's productivity. To this end, the Laboratory conducts a comprehensive
environmental research program in toxicology, epidemiology, and research on
human subjects under controlled laboratory conditions.
The quality of the data resulting from this research is an overriding
factor in determining their usefulness to EPA. In recognition of the importance
of data quality assurance, our Laboratory instituted an active, comprehensive
program to coordinate the development and implementation of effective quality
assurance planning into all research within the Laboratory. More recently,
the Administrator has required quality assurance for all environmentally
related measurement activities supported by the Agency. This substantially
enhances the quality assurance aspects of our own research measurements.
This document represents the current statement of our effort. I am
confident that full implementation of our data quality assurance policy, with
the help of the guideline manuals and the increased application of quality
assurance principles, will enhance the scientific merit of our research program.
F. Gordon Hueter, Director
Health Effects Research Laboratory
HI
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CONTENTS
1 ABSTRACT .............................. 1
2 INTRODUCTION ........................... 2
2.1 Laboratory Objectives .................... ^
2.2 Background .......................... ^
2.3 Definitions ......................... ]?
2.3.1 Quality ........................ •*
2.3.2 Quality Assurance ...................
2.3.3 Quality Control
2.3.4 Environmental Measurements ............... ~|
2.3.5 Task .......................... 4
2.3.6 Protocol ........................ 4
2.3.7 QA Program Plan .................... 4
2.3.8 QA Project Plan .................... 5
2.3.9 Project Officer .................... 5
2.3.10 QA Officer ....................... 5
2.3.11 Contracting Officer .................. 5
2.3.12 QA Performance Audit .................. 5
2.3.13 QA Systems Audit .................... 5
2.4 References .......................... 6
3 MANAGEMENT POLICY ......................... 7
3.1 Quality Assurance Program Goals ............... 7
3.2 Quality Policies ....................... 8
3.2.1 Scope of the Laboratory QA Program .......... 8
3.2.2 Quality Assurance Project Plan Design ......... 9
3.2.3 Quality Assurance Project Plan Review and
Approval ....................... 10
3.3 Quality Assurance Program Organization ............ 10
3.3.1 Organizational Structure for Quality Assurance ..... 11
3.3.2 Assignment of Responsibilities ............. 11
3.4 References .......................... 17
4 GUIDELINES FOR PROJECT OFFICERS .................. 18
4.1 General Approach ....................... 21
4.1.1 Intramural Tasks .................... 21
4.1.2 Cooperative Agreements ................. 22
4.1.3 Contracts ....................... 23
4.1.4 Interagency Agreements ................. 28
4.2 Project Planning ....................... 28
4.2.1 Research Plan ..................... 31
4.2.2 Personnel ....................... 32
4.2.3 Facilities, Services, Equipment, and Supplies ..... 33
4.2.4 Recordkeeping ..................... 39
4.2.5 Chain-of-Custody Procedures .............. 41
4.3 Sample Collection and Analysis ................ 45
4.3.1 Sample Collection ................... 45
4.3.2 Sample Analysis .................... 45
4.4 Data Management ........................ 47
4.4.1 Data Collection .................. . . 47
4.4.2 Data Storage and Backup ................ 43
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CONTENTS (continued)
4.4.3 Data Transfers 49
4.4.4 Data Reduction 50
4.4.5 Software 50
4.4.6 Data Analysis 51
4.4.7 Reporting 52
4.5 Quality Control 54
4.5.1 Internal Audits 54
4.5.2 Preventive Maintenance 56
4.5.3 Calibration 57
4.5.4 Documentation Control 63
4.5.5 Configuration Control 64
4.5.6 Data Validation 65
4.5.7 Feedback and Corrective Action 66
4.6 References 67
Appendixes 70
5 EXTERNAL QUALITY ASSURANCE FOR RESEARCH PROJECTS 93
5.1 Systems Audits 93
5.2 Performance Audits 94
5.3 References 95
6 GUIDELINES FOR ATMOSPHERE GENERATION AND MONITORING 96
6.1 Introduction 96
6.2 Atmosphere Generation 95
6.2.1 General Considerations 97
6.2.2 Particulate or Aerosol Atmospheres 93
6.3 Sample Collection and Analysis 100
6.3.1 Introduction IQQ
6.3.2 Sample Representativity
6.3.3 Physical Characterization of the Atmosphere
6.3.4 Sample Quantity 102
6.3.5 Sample Handling and Storage 103
6.3.6 Recommendations for Sampling and Analysis of
Selected Pollutants 103
6.4 Pollutant Standards and Traceability H2
6.5 References H2
7 QUALITY CONTROL/QUALITY ASSURANCE GUIDELINES FOR RESEARCH
INVOLVING ANIMALS 115
7.1 Introduction 115
7.2 Animal Research Program Quality Control 117
7.2.1 Animal Facility Design Quality Control 117
7.2.2 Animal Husbandry Quality Control 120
7.3 Investigator Quality Control 126
7.3.1 Experimental Laboratory Environment 126
7.3.2 Experimental Compound Testing 127
7.3.3 Data Reporting 131
7.4 Summary 131
7.5 References 132
Appendixes 133
VI
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FIGURES
Number Page
3-1 Functional management structure, HERL-RTP 12
3-2 Interactions of the QA organization with other
HERL-RTP management 13
4-1 Project Officer QA/QC checklist for planning and
evaluating QA project plans 19
4-2 Summary of EPA's proposed GLPs for health effects .... 29
4-3 Example of a network chain-of-custody record 42
4-4 Minimum technical report content for EPA health
effects tests 53
7-1 Sample quality assurance inspection checklist for
an animal care facility 116
7-2 Sample QC checklist for animal care 118
7-3 Sample QC checklist for investigator using animals. . . . 119
TABLES
6-1 Summary of Measurement Methods for Selected
Pollutants 105
7-1 Space Recommendations for Laboratory Animals 122
7-2 Maximum Concentrations of Feed Contaminants 124
7-3 Monthly QC/QA Tests Performed for HERL/LAS 125
7-4 Recommended Temperature and Relative Humidity
for Common Rodents 126
7-5 Average Daily Nutrient Requirement in Percentage
of Whole Diet 128
7-6 Biologically Effective Concentrations of Selected
Heavy Metals 129
7-7 Biologically Effective Concentrations of Selected
Organic Feed Contaminants 130
Vll
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SECTION 1
ABSTRACT
This document is a statement of the Quality Assurance (QA) policy of
the Health Effects Research Laboratory, U.S. Environmental Protection Agency,
Research Triangle Park, North Carolina (HERL-RTP). It describes the HERL-RTP
QA organization and the QA responsibilities of both management and technical
research personnel in relation to the mandatory Agency QA policy and task
data quality requirements. It provides guidelines for managers in the
implementation of Agency QA policy and evaluation of research documentation,
and presents guidelines for project officers for (1) development of QA
Project Plans for intramural research and support tasks, (2) preparation of
requests for proposals, (3) preaward QA evaluation of proposals, and
(4) review and evaluation of QA Program or Project Plans for extramural
tasks. Aspects of research tasks that must be considered by project officers
in the development or review of QA elements are treated in detail. Specific
guidelines for atmosphere generation, dose monitoring, and animal research
are also included. These guidelines are reviewed and revised annually by
the HERL-RTP QA officer.
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SECTION 2
INTRODUCTION
2.1 LABORATORY OBJECTIVES
The Health Effects Research Laboratory, Research Triangle Park, North
Carolina (HERL-RTP), conducts animal and human studies under controlled
conditions and performs studies on human populations to assess the health
hazards of environmental pollutants. The effects of environmental pollut-
ants, both singly and in combination, are investigated; pollutants studied
include air pollutants, pesticides, toxic substances, and nonionizing radia-
tion. Controlled laboratory studies are conducted to determine effects of
pollutants on normal biological function as measured by clinical, chemical,
biochemical, physiological, histopathological, growth, reproduction, and
other parameters. In addition, HERL-RTP develops, evaluates, and improves
analytical chemical methods and biological screening techniques for direct
and indirect measurement of exposure to environmental toxicants. It also
serves as a resource for information on the health effects of environmental
pollutants and coordinates health-related programs with international organi-
zations.
2.2 BACKGROUND
Because of an increased awareness of the serious health effects of
environmental pollutants and of the need for adequate data quality to sup-
port risk assessment and control strategies, HERL-RTP management initiated a
formal Laboratory-wide data quality program in May 1976 with the issuance of
a "Quality Assurance Plan."1 Subsequently, a Quality Assurance Officer was
appointed as chairman of the Quality Assurance Committee for the purpose of
designing and implementing a Laboratory-wide QA Program. Quality assurance
guidelines have since been developed for management policy,2 research task
planning,3 and environmental pollutant measurements.4 5 6 Guidelines for
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quality assurance in selected areas of biological research are currently
being developed to complete the Quality Assurance Program at HERL-RTP.
The HERL-RTP QA Program is further supported by EPA's recent commitment
to a mandatory Agency-wide QA Program. Agency policy initiated by the
Administrator in memoranda of May 30, 1979,7 and June 14, 1979,8 requires
participation in a centrally managed Quality Assurance Program of all EPA
Laboratories, Program Offices, Regional Offices, and those monitoring and
measurement efforts supported or mandated through contracts, regulations, or
other formalized agreements.
The Office of Research and Development (ORD) is responsible for develop-
ing, coordinating, and directing implementation of the Agency QA Program.
ORD has delegated this responsibility to the Quality Assurance Management
Staff (QAMS) of the Office of Monitoring Systems and Technical Support.
To implement Agency policy, EPA Laboratories, Program Offices, and
Regional Offices are required to prepare QA Program Plans covering all
intramural and extramural monitoring and measurement activities that generate
and process environmentally related data for Agency use. A QA Program Plan
for HERL-RTP has been developed and submitted to QAMS for approval.
In addition, every project officer is required to prepare a comprehen-
sive Quality Assurance Project Plan for each project under his control, in
accordance with Agency criteria and guidelines.3 9 This document presents
guidelines for Laboratory managers and project officers for developing QA
Project Plans.
2.3 DEFINITIONS
The American Society for Quality Control has carefully defined terms
that apply to quality.10 The Quality Assurance Handbook for Air Pollution
Measurement Systems4 provides similar definitions of quality terminology
applicable to air pollution data collection systems. Several terms related
specifically to health research data quality are defined below as they are
used in these guidelines.
2.3.1 Quality
Quality is the totality of characteristics of research data that bear
on their ability to satisfy previously specified criteria. For labora-
tory measurement systems, accuracy; precision, and representativeness
are of major importance. Completeness is appropriately applied to
larger systems, such as air monitoring networks.
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2.3.2 Quality Assurance
Quality assurance is a program of planned, systematic actions that are
necessary to ensure that specified data quality criteria are achieved.
QA planning is necessary at the management level in the development of
QA policy, and at the project level in the development of research
protocols by project officers (see Section 2.3.6). QA activities
consist of: (1) quantitative measurements, such as inter!aboratory
tests or performance audits; and (2) qualitative measures, such as site
visits or systems audits, to evaluate the capability of a total measure-
ment system for providing specified quality data. Quality assurance,
in planning and execution, is a management function independent of task
operating personnel.
2.3.3 Quality Control
Quality control (QC) is a system of activities designed to achieve and
maintain a previously specified level of quality in data collection,
processing, and reporting. Quality control is performed by the task or
project personnel. QC activities include control or correction for all
variables affecting data quality (see Section 4.5).
2.3.4 Environmental Measurement
The term "environmental measurement" applies to all field and labora-
tory investigations that generate data involving the measurement of
chemical, physical, or biological parameters in the environment, such
as determining the presence or absence of priority pollutants in waste
streams; health and ecological effect studies; clinical and epidemio-
logical investigations; engineering and process evaluations; studies
involving laboratory simulation of environmental events; and studies or
measurements on pollutant transport and fate, including diffusion
models.
2.3.5 Task
A task is any project, intramural or extramural, that produces or uses
environmentally related data.
2.3.6 Protocol
The term protocol includes all task or project planning documents.
Specifically included are research plans, support activity procedure
statements, contractors' work plans, scopes-of-work, and plans for
total task quality assurance, i.e., QA Project Plans.
2.3.7 QA Program Plan
A QA Program Plan is a written document that presents in general terms
the overall policies, organization, objectives, functional responsibil-
ities (within the organization), etc., designed to achieve specified
data quality goals of a particular organization (e.g., EPA Laboratory,
Program Office, Regional Office, contracting organization).
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2.3.8 QA Project Plan
A QA Project Plan is a written document that details the policies,
organization, objectives, functional activities, and specific QA and QC
activities designed to achieve data quality goals or requirements of a
specific project. The QA Project Plan describes procedures to be used
to routinely assess data precision, accuracy, completeness, representa-
tiveness, and comparability of the measurements involved.
2.3.9 Project Officer
The project officer is that individual who is assigned overall respon-
sibility for a project from inception through completion. This respon-
sibility covers both technical and QA aspects of the project.
2.3.10 QA Officer
The QA officer is that individual who is assigned the responsibility
for overview and guidance of the QA program for an organization or for
a specific project. The QA officer should be in a position to provide
independent and objective evaluations and assessments of the effective-
ness of the QA program and to provide timely feedback and recommenda-
tions.
2.3.11 Contracting Officer
The contracting officer is that individual who is assigned the respon-
sibilty for ensuring that contracting is done as authorized by law and
regulation.
2.3.12 QA Performance Audit
A QA performance audit is a quantitative analysis or check with a
material or device with known properties or characteristics to deter-
mine the accuracy of a measurement system. Performance audits may
require either the identification or the quantisation of specific
elements or compounds or both.
2.3.13 QA Systems Audit
A QA systems audit consists of a systematic onsite qualitative review
of facilities, equipment, training, procedures, recordkeeping, data
validation, data management, and reporting aspects of the total measure-
ment system. This may be required to assess the capability of a measure-
ment system to generate data of the required quality or to determine
compliance of a project with specified QA requirements.
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2.4 REFERENCES
1. U.S. Environmental Protection Agency, Health Effects Research Labora-
tory, Quality Assurance Plan, Research Triangle Park, NC, May 1976.
2. U.S. Environmental Protection Agency, Health Effects -Research Labora-
tory, Management Policy for the Assurance of Research Quality, EPA-600/
1-77-036, Research Triangle Park, NC, 1977.
3. U.S. Environmental Protection Agency, Health Effects Research Laboratory,
Development of Quality Assurance Plans for Research Tasks, EPA-600/1-78-
012, Research Triangle Park, NC, 1978.
4.
U.S. Environmental Protection Agency, Quality Assurance Handbook for
Air Pollution Measurement Systems. Vol/ I - Principles, EPA-600/9-/b-
005, Research Triangle Park, NC, March 1976.
5. U.S. Environmental Protection Agency, Quality Assurance Handbook for
Air Pollution Measurement Systems, Vol. II - Ambient Air Specific Methods,
EPA-600/4-77-027a, Research Triangle Park, NC, May 1977.
6. U.S. Environmental Protection Agency, Quality Assurance Handbook for
Air Pollution Measurement Systems, Vol. Ill - Stationary Source Specific
Methods, EPA-600/4-77-027b. Research Triangle Park, NC, August 1977.
7. U.S. Environmental Protection Agency, Environmental Protection Agency
(EPA) Quality Assurance Policy Statement, Administrator's Memorandum,
May 30, 1979.
8. U.S. Environmental Protection Agency, Quality Assurance Requirements
for All EPA Extramural Projects Involving Environmental Measurements,
Administrator's Memorandum, June 14, 1979.
9. U.S. Environmental Protection Agency, Guidelines and Specifications for
Preparing Quality Assurance Project Plans, QAMS-005/80, Office or
Research and Development, Washington, D.C., October 1980.
10. The American Society for Quality Control, Glossary and Tables for Statis-
tical Quality Control, Jackson, J. E., and R. A. Freund, eds., Milwaukee,
WI, 1973.
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SECTION 3
MANAGEMENT POLICY
This section describes HERL-RTP management policies and goals and the
QA organization responsible for development and implementation of the Labora-
tory Quality Assurance Program.
All QA activities within HERL-RTP will be carried out in accordance
with Agency mandates and guidance specified by the Quality Assurance Manage-
ment Staff (QAMS) of the Office of Research and Development (ORD). Planning
for the incorporation of suitable QA measures into measurement activities is
the responsibility of project officers. It is the responsibility of manage-
ment to ensure that all project-related documents or plans incorporate
adequate QA measures. It is also management's responsibility to ensure that
QA Project Plans are implemented and that project data are of adequate and
documented quality. The HERL-RTP QA organization, consisting of a Quality
Assurance Committee chaired by the QA officer, is available to all Laboratory
technical and management personnel for consultation or active participation
in development and review of QA Project and Program Plans.
3.1 QUALITY ASSURANCE PROGRAM GOALS
The goal of the HERL-RTP Quality Assurance Program is to ensure, assess,
and document the quality of laboratory and field data used by EPA in assess-
ing the human health effects of pollutants and in developing adequate control
strategies.
Specific objectives of the HERL-RTP QA Program are to:
1. Establish a QA organization within HERL-RTP having responsibility
and authority for developing and implementing a Laboratory QA
Program.
2. Develop a program to familiarize all HERL-RTP personnel with the
basic concepts of quality assurance.
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3. Establish and maintain written QA guidelines to assist HERL-RTP
personnel in the logical development of general and specific QA
plans for current and future HERL-RTP research.
4. Provide criteria for evaluating proposed and ongoing tasks for
appropriateness to the data quality requirements of HERL-RTP.
5. Ensure that all data reported include data quality estimates of
representativeness, precision, accuracy, comparability, and, when
appropriate, completeness.
6. Implement procedures to review quality aspects of data currently
being collected or data collected in the past, as deemed appro-
priate.
7. Encourage the use and development of methods of analysis and data
treatment that are capable of meeting the data quality requirements
of HERL-RTP.
8. Develop and implement a comprehensive Laboratory QA audit program
covering all routine measurement methods in use.
9. Monitor the operational performance of HERL-RTP through appropriate
intralaboratory and interlaboratory quality evaluation programs.
10. Ensure that protocols with approved QA Project Plans are developed
and implemented by HERL-RTP investigators and contractors.
11. Develop mechanisms for identifying data quality problem areas,
alerting management to them, and evaluating the proposed solutions
to such problems.
3.2 QUALITY POLICIES
It is the policy of HERL-RTP that the program of quality assurance and
quality control will be appropriate to ensure that all data collected are of
known and documented quality. QA Program requirements cover all activities
supported or required by HERL-RTP that generate environmentally related
measurement data.
3.2.1 Scope of the Laboratory QA Program
The HERL-RTP QA Program covers all funded tasks—intramural and extra-
mural, contract, cooperative, and interagency agreement; it requires that
quality control considerations be included in all requests for proposals
(RFP's), research proposals and evaluations, work plan approvals, project
plans, and project reports. All measurement activities planned or conducted
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within HERL-RTP must have a QA Project Plan incorporated into the research
protocol and approved by the QA officer.1 Specifically, the QA Project Plan
ensures that:
1. The level of data quality needed will be determined and stated
before the data collection effort begins.
2. All data generated and reported will be of the quality and integ-
rity established by each QA Project Plan.
3.2.2 Quality Assurance Project Plan Design
Project officers are responsible for the design of QA Project Plans for
intramural tasks and for incorporation of these plans into research protocols.
QA Project Plans for tasks conducted under cooperative or interagency agreement
or contract are called for in the RFP and must be prepared by the cooperator
or contractor.
QA Project Plans must include all QA/QC activities appropriate to the
project data quality requirements and to the methods of data collection and
data processing employed. The following aspects of project data quality
should be addressed in all QA Project Plans and are individually discussed
in detail in Section 4 of this document:
Experimental Design
Personnel
Facilities
Services
Equipment
Supplies
Recordkeeping
Chain of Custody
Sample-Col lection
Sample Analysis
Internal Audits
Preventive Maintenance
Calibration
Documentation Control
Configuration Control
Data Validation
Feedback and Corrective Action
Data Processing and Analysis
Report Design
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3.2.3 Quality Assurance Project Plan Review and Approval
All QA Project Plans must be reviewed and approved by appropriate
HERL-RTP management prior to project funding. Specifically, approval by the
section chief, branch chief, division director, and QA officer is required.
Additionally, the project officer must review and approve all QA Project
Plans for extramural tasks under his supervision. Periodic review of QA
Project Plans by the project officer and the QA officer throughout the
project term provides the means by which management may assess if suitable
data quality has been obtained in a cost-effective manner. Assistance from
the QA organization is available to management for the evaluation of QA
Project Plans and the effectiveness of their implementation.
Systems and performance audits and interlaboratory/interfield compari-
son studies will be conducted on measurement projects within HERL-RTP as
planned by the QA officer and the appropriate division director to assess
the adherence to, and adequacy of, the approved QA Project Plans and to
assess the need for corrective action.
3.3 QUALITY ASSURANCE PROGRAM ORGANIZATION
In planning QA programs for particular tasks, project officers should
attempt to identify all variables that may affect the quality of data to be
produced and include appropriate QA/QC measures in QA Project Plans. As a
result of an increasing awareness of the need for quality assurance in
Agency programs, EPA has developed comprehensive QA guidelines.2"5 In
addition, Federal standards for nonclinical laboratories have been promul-
gated,6 health effects test standards have been proposed for testing under
the Toxic Substances Control Act,7 8 and guidelines for quality assurance
practices in health laboratories have been published by the American Public
Health Association.9 Project officers should refer to these, when appropriate,
in developing QA Project Plans.
To support project officers and management in the development and
implementation of QA Project Plans, the quality assurance organization is
interwoven with the existing HERL-RTP management. The structure of the QA
organization, the functional responsibility of QA personnel, and the lines
of communication for achievement of a cost-effective QA Program are discussed
below.
10
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3.3.1 Organizational Structure for Quality Assurance
The HERL-RTP management structure is shown in Figure 3-1. All QA
management responsibilities are assigned to a Quality Assurance officer.
The independence and objectivity of the QA Program is supported by the QA
officer's organizational independence of all divisions involved in the data
generation process. The QA officer reports directly to the HERL-RTP Labora-
tory Director on QA matters and is primarily responsible for the design and
implementation of the Laboratory QA Program. The QA Committee, chaired by
the QA officer, is responsible for evaluating the effectiveness of the
Program throughout the Laboratory and for recommending improvements. The QA
Committee members act as liaison between the QA Committee and their divisions
or offices.
All divisions within HERL-RTP (shown in Figure 3-1) are covered by QA
Program requirements. Interfacing of the QA organization with various
levels of management on QA matters is shown in Figure 3-2.
3.3.2 Assignment of Responsibilities
Although each person involved in the generation of data is implicitly a
part of the QA Program, certain individuals have specific, assigned QA respon-
sibilities. Refer to Figure 3-2 in the following discussion.
3.3.2.1 HERL-RTP Laboratory Director--
The HERL-RTP Laboratory Director has overall responsibility for all
Laboratory activities, including quality assurance. Because the success of
the QA Program ultimately depends on the Director's full support of QA man-
agement, it is his/her responsibility to enlist and encourage the cooperation
of all HERL-RTP personnel in the program.
3.3.2.2. Quality Assurance Officer--
The QA officer has primary responsibility for all Laboratory QA activ-
ities and reports directly to the HERL-RTP Laboratory Director. His/her
responsibilities include the development, evaluation, and documentation of QA
policy and procedures appropriate to the Laboratory objectives. This includes
evaluation of the cost-effectiveness of QA programs and plans and recommenda-
tions for their improvement.
11
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HEALTH EFFECTS RESEARCH
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HERL/RTP
DIRECTOR
DIVISION
DIRECTOR
•RANCH
CHIEF
SECTION
CHIEF
QA
OFFICER
QA
STAFF
QA
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HERL-RTP
QA ORGANIZATION
PROJECT
OFFICER
—— Functional nuiugemiit authority
— — — QA authority/consulting
Figure 3-2. Interactions of the QA organization with other HERL-RTP management.
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As advisor to the Laboratory Director, the QA officer regularly reports
on the status of the Laboratory QA Program, identifies specific needs (e.g.,
methods development and problem areas), and recommends specific courses of
action for strengthening the Program.
As chairman of the QA Committee, the QA officer initiates development
of Laboratory-wide QA guidelines and procedures. He/she coordinates methods
development efforts for new QA procedures for specific HERL-RTP research
techniques, and assesses data provided by the Committee regarding evaluation
of the QA Program. He/she is also responsible for the development of special
audit programs for Laboratory-wide measurement techniques.
As QA consultant, he/she is available to consult with and recommend to
the HERL-RTP professional staff (project officers, investigators, etc.)
appropriate and necessary QA methods and plans for ensuring the quality of
the research data produced.
Motivation of personnel is a critical factor in the success of the
Laboratory QA Program. Hence, a major responsibility of the QA officer is
to ensure that all personnel have a good understanding of the Laboratory QA
Program, an understanding of their responsibilities, and an appreciation of
the importance of their roles to the overall success of the program.
3.3.2.3 Quality Assurance Coordinator/Committee--
Each Division Director designates QA coordinators to serve on the
HERL-RTP QA Committee. The QA coordinators recommend and review proposals
for improvements in QA policies and procedures, and report and evaluate
potential data quality problem areas. Within their respective divisions,
the QA coordinators consult on matters of quality assurance, serving as a
primary source of information on research quality assurance matters, review-
ing contract proposals for QA aspects, and helping to implement the Labora-
tory QA Program.
The QA Committee serves as an advisory committee to the Laboratory
Director. Specifically, the Committee's functions include assisting in the
evaluation and refinement of data quality objectives of the QA Program to
ensure that they meet the Laboratory needs with minimum disruption of exist-
ing workloads and procedures, reviewing recommendations presented to the
Committee, and assessing the effectiveness of the QA Program.
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3.3.2.4 Quality Assurance Staff--
The QA staff is composed of technical personnel; key members hold joint
appointments in other research areas at HERL-RTP. They are responsible for
providing the necessary technical expertise to ensure adequate implementation
and review of the Laboratory QA Program. In particular, they are available
to consult with project officers and management on the technical aspects of
specific task activities that affect overall data quality (e.g., methods
development) and to aid the QA officer in the development, implementation,
and evaluation of audit programs for both Laboratory-wide and task-specific
measurement techniques.
3.3.2.5 Quality Assurance Contractors/Consultants—
External QA contractors/consultants assist in implementing major parts
of the HERL-RTP QA Program. Their tasks may include development and/or
evaluation of Laboratory QA guidelines and plans, development and implementa-
tion of Laboratory-wide audit programs, methods development, and provision
of analytical services.
3.3.2.6 Decision Unit Coordinator--
Evaluation of the need for specific types of research is an essential
part of the Laboratory QA Program. The Decision Unit (DU) Coordinator is
the functional manager for each general program area, and, as such, is
responsible for distributing resources (i.e., funding and manpower) and for
evaluating the relevance of each proposal (and task) in his/her program area
to the broadly defined Laboratory and Agency goals. In addition, he/she is
responsible for identifying impending needs in specific areas of research
and encouraging proposals for new tasks in these areas, thus aligning the
production of research data with the overall Laboratory and Agency needs.
3.3.2.7 Project Officer-
As task manager, the project officer is responsible for fulfilling the
technical and administrative requirements of each task. It is his/her
responsibility to adequately ensure and document the quality of the task
product, including both the research data and conclusions. The project
officer draws upon his/her professional training and expertise, in collabora-
tion with the HERL-RTP QA organization, to determine which QA/QC techniques
most appropriately apply to a particular task and to develop task-specific
QA Project Plans.
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To ensure adequate data quality, the project officer should anticipate
events that might threaten data quality (e.g., slowly degrading reagents),
provide contingencies for anticipated failures and problems, and obtain
objective evaluation of task data quality as the task progresses. These
topics are discussed in detail in Section 4 of this document.
To adequately document QA plans and activities, the project officer
must include data quality considerations in all task-related documents and
collaborate with the HERL-RTP QA organization in external QA activities (see
Section 5).
3.3.2.8 Functional Managers--
Functional managers (e.g., division director, branch chief, section
chief) are responsible for ensuring the quality of research data produced
under their direction. This responsibility includes review and approval of
QA Project Plans and periodic evaluation of ongoing QA programs. Peer
review of QA techniques by the QA Committee may also be requested by manage-
ment for evaluation of task planning and execution. Critical aspects of
task data quality addressed in Section 4 of this document should be referred
to for guidance in evaluating proposals, plans, progress reports, and final
reports.
Management support of QA programs should be visible and active. Such
support may include:
1. Development and support of data evaluation techniques appropriate
to health effects research, coordinated through the QA organiza-
tion if appropriate.
2. Provision of information to project officers on interlaboratory
and intralaboratory testing programs and encouragement of their
participation in them.
3. Development of QA programs and techniques for health effects
research, using available standards and procedures.
4. Development of standards and QA procedures for new measurement
methods.
Such activities will demonstrate the commitment of HERL-RTP management to
the Quality Assurance Program.
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3.4 REFERENCES
1. U.S. Environmental Protection Agency, Health Effects Research Labora-
tory, Research Protocols, Research Triangle Park, NC, Director's Memoran-
dum, September 25, 1979.
2. U.S. Environmental Protection Agency, Health Effects Research Laboratory,
Guides for Quality Assurance in Environmental Health Research, EPA-600/1-
79-013, Research Triangle Park, NC, 1979.
3. U.S. Environmental Protection Agency, Health Effects Research Laboratory,
Development of Quality Assurance Plans for Research Tasks, EPA-600/1-78-
012, Research Triangle Park, NC, 1978.
4. U.S. Environmental Protection Agency, Quality Assurance Research Plan,
FY 1978-81, EPA-600/8-77-008, Washington, DC, July 1977.
5. U.S. Environmental Protection Agency, Quality Assurance Guidelines for
Biological Testing. EPA-600/4-78-043, Las Vegas, NV. August 1978.
6. Non-Clinical Laboratories Studies: Regulations for Good Laboratory
Practice, Federal Register, December 22, 1978, pp. 59985-60025.
7. Proposed Health Effects Test Standards for Toxic Substances Control Act
Test Rules, Federal Register, May 9, 1979, p. 27334.
8. Good Laboratory Practice Standards for Health Effects, Federal Register,
May 9, 1979, p. 26362.
9. Inhorn, S. L., ed., Quality Assurance Practices in Health Laboratories,
American Public Health Association, 1977.
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SECTION 4
GUIDELINES FOR PROJECT OFFICERS
By memoranda of May 30, 1979,J and June 14, 1979,2 the EPA Administrator
established the goal of the Agency Quality Assurance (QA) Program to ensure
that all environmentally related measurements funded or mandated by EPA and
interagency agreements be scientifically valid, defensible, and of known
precision and accuracy. As part of the mandatory Agency QA Program, HERL-RTP
requires that protocols for all projects producing or using environmentally
related data contain approved QA Project Plans.
This section presents guidelines for project officers for (1) develop-
ment of QA Project Plans for intramural research and support tasks, (2)
preparation of requests for proposals (RFPs), (3) preaward QA evaluation of
proposals, and (4) review and evaluation of QA Program or Project Plans for
extramural tasks. A detailed checklist of specific items that should be
considered in all QA planning or review of task-related documents is given
in Figure 4-1. The relative impact of each of these aspects on overall data
quality will vary between tasks; however, no element should ever be deleted
from consideration.
In the following discussion, as elsewhere in this document, the terms
"must" and "should" are used with very specific and distinct meanings:
"must" is used where there is an actual requirement as stated by the QAMS or
HERL-RTP management; "should" is used to denote a recommended or desirable
activity. It is understood that specific requirements apply to all HERL-RTP
projects; recommendations apply only to those projects or activities where
appropriate.
Aspects of research tasks that should be considered by the project
officer in the development or review of QA elements are detailed in Sections
4.1 through 4.5 under the following general areas:
1. General approach to quality control in research.
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Project Officer QA/QC Checklist
QA/QC Item
Identification Comments
( ) Title page with provision for approval and review
signatures
( ) Table of contents
Project Description
( ) Project organization and responsibility
( ) Objectives
( ) Hypotheses
( ) Experimental design
( ) QA objectives for measurement data
Personnel
( ) Training and experience
( ) Adequacy (numbers)
( ) Resumes of key task personnel
Facilities
( ) Appropriateness to task requirements
( ) Environmental aspects (e.g., temperature, lighting,
ventilation)
( ) Maintenance (preventive and corrective)_
( ) Inspection procedures
( ) Configuration control
Security_
( ) Safety provisions_
( ) Support services
Equipment
( ) Appropriateness to task requirements
( ) Maintenance (preventive and corrective)_
( ) Configuration control
( ) Safety provisions_
( ) Recordkeeping (i.e., documentation of calibration and
maintenance history)
Supplies
( ) Certification
( ) Acceptance screening_
Figure 4-1. Project officer QA/QC checklist for planning
and evaluating QA project plans.
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( ) Animal care and testing procedures
Storage_
( ) Stockroom tracking system_
( ) Recordkeeping
( ) Chain-of-Custody
Sampling Procedures
( ) Sample collection_
( ) Sample analyses
Data Management
( ) Collection
( ) Storage and backup_
( ) Transfers
( ) Reduction
( ) Software
( ) Analysis
( ) Reporting
Quality Control/Quality Assurance
( ) Specific procedures to be used to routinely assess data
precision, accuracy, and completeness
( ) Internal audits
( ) External audits
( ) Calibration
( ) Documentation control
( ) Configuration control]
( ) Data validation
( ) Feedback and corrective action
( ) Quality assurance reports to management_
Special Requirements
( ) Radiation safety form
( ) Toxic chemical form
( ) Request for Animal Procurement and Care
(V) Satisfactory
(U) Unsatisfactory
(NA) Not Applicable
NOTE: Specific checklists for animal facilities, animal husbandry, and
animal testing should be completed, if applicable (see Section 7).
Figure 4-1 (continued)
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2. Planning—experimental design, personnel, facilities, equip-
ment, supplies, recordkeeping, and chain of custody.
3. Experimental--sample collection and analysis.
4. Data quality control activities—internal audits, preventive
maintenance, calibration, documentation control, configura-
tion control, data validation, feedback, and corrective
actions.
5. Data processing, analysis, and reporting.
4.1 GENERAL APPROACH
The purpose of this section is to assist project officers in the uniform
implementation of QA requirements for intramural tasks, cooperative agree-
ments, contracts, and interagency agreements. Project officers should work
closely with the HERL-RTP QA officer on all QA matters.
4.1.1 Intramural Tasks
4.1.1.1 Research Tasks—
The project officer is responsible for preparation of QA Project Plans
and inclusion of these plans in the research protocol for each intramural
research task. To assist project officers in this effort, the HERL-RTP QA
organization has developed guidelines for preparation of intramural research
protocols (see Appendix A). These guidelines contain specific instructions
for preparing QA Project Plans and should be consulted. In particular, it
should be noted that all intramural support tasks that may be involved in
the proposed research task must be referenced in the research protocol by
task number and project officer and must have an approved protocol (includ-
ing a QA Project Plan) on file with the QA officer prior to approval of the
research protocol.
The QA officer may participate in the development of QA Project Plans
at the request of the project officer and must approve all QA Project Plans
prior to project funding. The project officer and the QA officer should
also review and evaluate the implementation of QA Project Plans throughout
the project term via periodic systems and/or performance audits and review
of regular QA project reports. Upon completion of the project, the project
officer and the QA officer should assess the actual performance of the
planned QA activities and the subsequent results. The final QA project
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report must contain the results of this assessment and must be approved by
the QA officer.
4.1.1.2 Support Tasks--
The project officer for each HERL-RTP support task is responsible for
development of an appropriate QA Project Plan and incorporation of this
plan into the task protocol. To assist project officers in this effort, the
HERL-RTP QA organization has developed specific guidelines for preparation
of intramural support protocols including QA Project Plans (see Appendix A).
In addition, project officers may consult with the QA officer, or his/her
staff, in the preparation of QA Project Plans.
The QA officer must approve all intramural support task QA Project
Plans prior to project funding. Since approval for funding of any intramural
research task supported by an intramural support task is contingent on
approval of the QA Project Plan for the support task, all intramural support
tasks must have approved QA Project Plans on file with the QA officer.
4.1.2 Cooperative Agreements
The originating project officer must notify the QA officer of all
cooperative agreements involving the generation or use of environmentally
related measurement data during the planning phase. It is the responsibil-
ity of the QA officer to ensure that QA requirements are met prior to award-
ing the cooperative agreement.
Preparation of QA Project Plans for cooperative agreements is the
responsibility of the cooperator. All cooperating institution QA Program
and Project Plans must be consistent with EPA criteria and regulations. QA
Program Plans must be included in the proposal and reviewed by the project
officer and the QA officer for approval prior to funding. Detailed QA
Project Plans may be required to be included in the original proposal or to
be submitted within a specified time after award for approval by the QA
officer. Where appropriate, final approval may be contingent upon success-
ful completion of a systems audit of the offerer, as directed by the QA
officer.
To facilitate systematic review of QA requirements for HERL-RTP projects
covered by cooperative agreements, ORD QAMS is preparing Guidelines and Speci-
fications for Implementing Quality Assurance Requirements for EPA Grants and
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Cooperative Agreements Involving Environmental Measurements.3 These should
be consulted when available. At present, project officers should consult
with the QA officer for specific guidance on QA requirements for cooperative
agreements to ensure compliance with QAMS criteria. The QAMS guidelines and
review forms for interagency agreements (reference 4, Appendix B) may be
referred to for general guidance.
The project officer and the QA officer must review and evaluate the
effectiveness of the QA Project Plan during the project and must assess the
quality of data generated and processed. Upon completion of the project,
the project officer and the QA officer must assess the overall effectiveness
of the planned QA activities and the study results. The final QA project
report must contain the results of this assessment and must be approved by
the QA officer.
4.1.3 Contracts
QA requirements for HERL-RTP contracts must be consistent with existing
EPA criteria as specified in the recently published QAMS Guidelines and
Specifications for Implementing Quality Assurance Requirements for EPA Con-
tracts and Interagency Agreements Involving Environmental Measurements.4
To assist project officers and QA officers in QA planning and decision-
making for their projects (contracts), QAMS designed QA Form QAR-C (see
Appendix B). Although project officers have responsibility for their proj-
ects and are the official contacts with the contracting officer, they should
consult with and obtain agreement from the QA officer on all QA matters. It
is the responsibility of the QA officer to ensure that QA requirements are
met prior to the awarding of the contract or interagency agreement.
4.1.3.1 QA Requirements in the Request for Proposal--
Prior to release of the purchase request to Procurement and Contracts
Management Division, the project officer should determine if the project
involves the generation of environmentally related measurement data. If so,
the project officer must delineate in the RFP the QA requirements, approved
by the QA officer, which each offerer must include in the proposal. The
statement of work should state as clearly as possible: (1) the objectives
of the project; (2) the data quality acceptance criteria, including minimum
requirements for precision, accuracy, representativeness, completeness, and
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comparability; and (3) the quality assurance requirements. The following
must be included as requirements in the special instructions, to be sub-
mitted as part of the offerers' proposals:
1. QA Program Plan—The offerer must include as part of the proposal the
following information concerning management of the QA Program for the
proposed project:
a. A statement of policy concerning the organization's commit-
ment to implementing a QA program to ensure generation of
measurement data of adequate quality to meet contract require-
ments.
b. An organizational chart showing the position of the designated
QA officer or coordinator within the organization. This QA
person should, if possible, be independent of the functional
groups that generate measurement data.
c. A delineation of the authority and responsibilities of the QA
person and the data quality responsibilities of the functional
groups of the organization.
2. QA Project Plan—The offerer may be required to submit, as part of the
technical proposal, a proposed QA Project Plan for the effort required
by the contract. The QA Project Plan must include the QA program of
proposed subcontractors. If not required as part of the technical
proposal, a QA Project Plan must be submitted as a separate, identifi-
able part of the awardee's work plan. The QA Project Plan must be
completed and approved by the QA officer before the awardee initiates
full-scale operations.
EPA's Guidelines and Specifications for Preparing QA Project Plans5
should be included by reference in the RFP.QA Project Plans must
address the following:
a. Title page, with provision for approval signatures
b. Table of contents
c. Project description (experimental design)
d. Project organization(s) and responsibilities
e. QA/QC objectives for measurement data, in terms of precision,
accuracy, completeness, representativeness, and comparability
f. Personnel (adequacy of training and experience)
g. Facilities, services, equipment, and supplies
h. Recordkeeping
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i. Chain-of-custody
j. Sample collection
k. Sample analysis
1. Data processing, analysis, validation, and reporting
m. Specific procedures to be used to routinely assess data
precision, representativeness, comparability, accuracy, and
completeness of the specific measurement parameters involved
(this section is required for all QA Project Plans)
n. Internal QC checks and frequency
o. QA performance and systems audits, and frequency
p. Calibration procedures, references, and frequency
q. Preventive maintenance procedures and schedules
r. Documentation control
s. Configuration control
t. Feedback and corrective action
u. QA reports to management
QA Performance Audits—The offerer must agree to participate in perform-
ance audits using selected QC reference samples or devices, or to docu-
ment recent performance on such samples. Such reference samples or
devices will be supplied by EPA at no cost to the offerers. The inclu-
sion of requirements for performance audits in the RFP and contract
will depend on the availability of QA reference materials or devices
for the measurements to be made. Performance audits are required where
samples are available, unless the offerer has previously met this
requirement to the satisfaction of the QA officer. In the event that
routine QC reference materials or devices are not available for the
measurements involved, consideration should be given to the use of
common or split samples for cross-comparisons of results from offerers
with those of EPA. (The requirement for preaward QA performance audits
may be limited to offerers in the competitive range.)
QA Systems Audit—The offerer must agree to permit a QA systems audit
by EPA of the offerer's facilities, organization, and operations as
part of the preaward evaluation and during the period of contract
performance. (The requirement for preaward QA systems audits may be
limited to offerers in the competitive range.)
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4.1.3.2 QA Evaluation of the Proposal--
The project officer and the QA officer are responsible for review of
the proposal for QA aspects. Each of the preaward requirements listed above
must be included in the RFP evaluation criteria. The offerer must ensure
that the requirements for QA Project Plans, QA performance audits, and QA
systems audits are met by any proposed subcontractors. The proposal should
contain a statement of the bidding institution's policy on QC/QA programs.
This should include a description of previous performance on similar projects
and program effectiveness in those projects. In particular, there should be
an explicit response to the QA requirements in the RFP.
Each proposal should be evaluated against the same set of criteria.
Section IV of Form QAR-C (Appendix B) is designed to assist in the evaluation
of the following QA aspects of the offerer's proposal:
1. QA Program Plan
2. QA Project Plan
3. QA Performance Audit
4. QA Systems Audit
The relative weight of each required QA factor is determined and entered on
the form so that the total of the required QA items is 100. In turn, the
relative importance of each QA item is specified in relation to the total of
all evaluation factors.
4.1.3.3 QA Requirements for Awarded Contracts--
In reviewing the QA requirements for the awarded contract, the project
officer must, in accordance with Section 4.1.3.1, specify the QA require-
ments of the project for the duration of the contract. The contractor is
required to ensure that the QA requirements of the contract are met by all
subcontractors. A complete and detailed QA Project Plan is required as a
part of the contract, or incorporated therein by reference, to be submitted
as a deliverable item. The QA Project Plan requires approval by the project
officer and the QA officer. Deviations from, or changes in, the approved QA
Project Plan during the project term must be documented and submitted to the
QA officer for review and approval prior to their implementation.
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4.1.3.3.1 QA requirements in the contract—A completed Form QAR-C
indicates that all preaward QA requirements have been fulfilled. The project
officer must forward the completed form to the contracting officer for
inclusion in the contract file.
4.1.3.3.2 QA reports—Provision for adequate QA reporting must be made
by the project officer and the QA officer. Contracts of short duration
(i.e., one year or less) may require only a final QA report. Contracts of
longer duration may require periodic (e.g., quarterly) QA reports. These QA
reports should be separate from other contractually required reports and
should contain such information as:
1. Status of QA Project Plan
2. Measures of data quality from the project
3. Significant quality problems, quality accomplishments, and
status of corrective actions
4. Results of QA performance audits
5. Results of QA systems audits
6. Assessment of data quality in terms of precision, accuracy,
completeness, representativeness, and comparability
7. Quality-related training.
4.1.3.3.3 Level of effort contracts—If the awarded contract is a work
assignment or technical directive contract, every work assignment (or techni-
cal directive) issued under the contract involving environmentally related
measurements must have a QA Project Plan approved by the project officer and
QA officer before measurements are initiated. As a minimum, the procedures
to be used to assess data precision, accuracy, representativeness, complete-
ness, and comparability of the specific measurement parameters must be
addressed.
4.1.3.3.4 QA review of contracts—The QA officer and the project
officer must review and evaluate the effectiveness of QA Project Plans
throughout the project term. Upon completion of the project, the QA officer
and project officer must assess the actual performance of the planned activi-
ties and subsequent results. The QA officer must review and approve the
final QA project report.
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4.1.4 Interagency Agreements
Although interagency agreements do not involve RFPs, the QA requirements
are similar to those for awarded contracts. Therefore, the discussions and
considerations included under Section 4.1.3 should be referred to. Form QAR-
IA4 (see Appendix B) should be used by project officers in QA planning and
review of interagency agreements.
4.2 PROJECT PLANNING
The quality of research data is strongly affected by the "weak link"
phenomenon. If experimental design, equipment maintenance, and data analysis
are excellent, but quality of the sample analysis is poor, the overall
quality of task data is lowered. Similarly, competent technical skills,
excellent facilities, or documented procedures cannot compensate for poor
experimental design. It is therefore of paramount importance that all
aspects of the project that may impact on data quality be considered in
project planning. QA Project Plans should address all items in the project
officer's QA/QC Checklist (Figure 4-1), as detailed in Appendix A of this
section.
A comprehensive QA program should also include Good Laboratory Practice
(GLP) concepts where appropriate. The Food and Drug Administration's (FDA)
Good Laboratory Practice Regulations6 apply to nonclinical studies performed
on animals, plants, microorganisms, or subparts thereof. The EPA's proposed
Good Laboratory Practice Standards7 (shown in Figure 4-2) are for use in
development of data on the health effects of chemical substances and mixtures
tested in accordance with Section 4 of the Toxic Substances Control Act.
The proposed EPA GLPs generally apply to all animal bioassay laboratory
health effects studies conducted by or on behalf of manufacturers of chemical
substances.
It should be emphasized that compliance with the data quality require-
ments outlined in either the FDA or proposed EPA GLPs does not in itself
constitute an adequate QA Program or necessarily ensure the production of
high quality research data. An effective QA program must also have the
active support of both management and task personnel.
During all phases of protocol development, the project officer should
consult with HERL-RTP support personnel (e.g., statisticians, animal care
28
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1. The proposed GLPs apply to studies relating to health and safety evalu-
ations conducted under Section 4 of the Toxic Substances Control Act,
whether conducted by the sponsor or under contract or grant. Fourteen
terms are defined in this section.
2. Test and control substances must be characterized by strength, purity,
composition, and stability before initiation of a study. Their containers
must be labeled by name, chemical abstract number or code number, batch
number (expiration date), and storage conditions requirements. Handling
procedures must be used that ensure proper identification and minimize
contamination, deterioration, or damage. Mixtures must be suitably ana-
lyzed to characterize their uniformity, concentration, and stability:
expiration date is that of the earliest expiring component.
3. An ample number of personnel having adequate and documented education,
training, and/or experience must be available to the s^udy. Their personal
habits, health, and clothing must be appropriate for their assigned
duties. The designated study director ensures that all provisions of the
GLPs are fulfilled for the study. The QA unit independently assures
management that the facilities, equipment, personnel, methods, practices,
records, and controls conform with the GLPs, in each phase of the study,
at no more than 3-month intervals.
4. Facilities must be of suitable size, construction, and location to facil-
itate proper conduct of the study. For animal studies, animals must be
properly separated, isolated, and quarantined. Separate areas are required
for:
biohazardous substances;
diagnosis, treatment, and control of known or suspected laboratory
animal diseases;
sanitary disposal;
feed, bedding, supplies, and equipment;
handling of test and control substances and their mixing;
routine procedures;
administrative and personnel use; and
secure archival of raw data and specimens.
5. Equipment must be suitably designed and located for operation, inspection,
cleaning, maintenence, and calibration according to written procedures;
written records are kept to document these operations.
6. Testing facility operation must be by written standard operating proce-
dures (SOP) for (as a minimum):
animal room preparation;
animal care;
test and control substance handling;
test system observations;
lab tests;
(continued)
Figure 4-2. Summary of EPA's proposed GLPs for health effects.7
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handling of moribund/dead animals;
necropsy;
specimen collection and identification;
histopathology;
data handling, storage, and retrieval;
equipment maintenance and calibration; and
transfer, placement, and identification of animals.
All deviations must be authorized by the study director and documented in
the raw data. Each lab must have immediately available, suitable lab man-
uals and SOPs, both active and historical. Reagents and solutions must be
labeled to indicate identity, concentration, storage requirements, and
expiration date. SOPs for animal care include housing, feeding, handling,
care, receiving quarantine, health parameters, and identification. In
addition, periodic feed and water analysis must be documented as part of
the raw data; cages and racks must be cleaned at appropriate intervals.
Bedding, cleaning materials, and pest controls must be documented as
noninterfering in the study.
7. Minimum protocol specifications are given (as in the HERL QA guidelines
document). The conduct of the study is detailed in terms of the protocol,
specimen identity, records, and data recording.
8,9. Reserved.
10. Minimum contents of the final report are outlined (as in the HERL QA
guidelines document). Archival of all raw data, protocols, specimens, and
final reports is detailed. Indexed, orderly, and secure storage is required
for at least 10 years.
11. Inspection of the testing facility must be permitted to an employee of
EPA or FDA at reasonable times and manner: for records and specimens, not
including QA records.
Appendix A
Additional guidelines are given as follows:
Handling of test substances: DHEW's "Guidelines for the Laboratory Use of
Toxic Substances Posing a Potential Occupational Carcinogenic Risk" and lARC's
"A Manual on the Safety of Handling Carcinogens in the Laboratory."
Handling of radioactive materials: NRC's Title 10 of CFR.
Administrative and personnel facilities: OSHA's Title 29 of CFR.
Animal care and handling: HEW's "Guide for the Care and Use of Laboratory
Animals."
Animal care facilities: HEW's "Guide for the Care and Use of Laboratory Ani-
mals," and 9 CFR Part 3.
Figure 4-2 (continued)
30
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coordinator, human exposure chamber project officer) who will be involved in
the project. The research protocol, when completed, should include statis-
tical design of the experiment, the data collection and analysis procedures,
and appropriate QC and QA measures.
4.2.1 Research Plan
It is not a QA function to evaluate the merit of the proposed approach
to research. However, it is necessary that a well-defined protocol be
provided to determine if the QC efforts are properly placed and timed. In
any HERL-RTP task that involves data collection and analysis, it is important
to consult a statistician during the initial planning phases of the study
and through data collection and analysis. An analysis plan, no matter how
ingenious, cannot compensate for a poor experimental design.
A written draft of the protocol for the study should be prepared,
including:
1. A statement of objectives and hypotheses to be tested;
2. A description of the experiment, covering the variables to be
measured, sample sizes, experimental materials, conditions,
and instruments; and
3. An outline of the method of data analysis to be used.
4.2.1.1 Objectives and Hypotheses to be Tested--
A clearly written statement of the research objectives allows precise
formulation of the specific hypotheses to be tested. The statement should
be specific, avoiding vagueness or excessive ambition. It is advisable to
prioritize objectives. The reference population to which the results are to
apply should be clearly defined.
4.2.1.2 The Experimental Design—
The experimental design should produce a clear definition of all the
variables to be considered, the size of the testing program, the exper-
imental subjects (e.g., animals, cell cultures, humans) and exactly what
data are to be collected. A well-designed testing program should answer the
following questions:
1. Are all the relevant intrinsic factors (e.g., age, size,
weight, sex, reproductive condition) and extrinsic environ-
mental factors (e.g., temperature, duration of exposure,
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light-dark cycle, chemical form of the pollutant tested,
synergistic interactions) being considered?
2. Are the effects of the relevant variables adequately distin-
guishable from the effects of other variables, or would it be
preferable to smooth over the effect of one or more variables
by choice of design (e.g., factorial or randomized design)?
3. Has the possibility of interaction between variables been
anticipated and accounted for?
4. Is the experimental design as free of bias as possible?
5. Is the experimental design consistent with the stated objec-
tives? Will the project yield enough data (degrees of free-
dom) to estimate the main effects and interactions of inter-
est, with precision sufficient for effects to be statistically
significant? Are sample sizes justified on the basis of
precision using historic or conjectured estimates of variances?
6. Is the experimental design cost effective? Would a more
limited design provide equivalent information at lower cost?
7. Does the design make adequate provision for controls (negative,
positive, and solvent comparison groups)?
8. Is the design logistically sound? (Are adequate time, space,
personnel, etc., available to properly perform the checks
necessary to ensure the specified data quality?)
4.2.1.3 Data Analysis—
The research protocol should cover in some detail the proposed method
of statistical analysis and the assumed underlying mathematical/probabilistic
model. The quality assurance aspect of data analysis primarily involves
assessment of accuracy of mathematical operations. Data analysis QA is
essentially reduced to software QA and should encompass all means of data
manipulation and analysis, whether by hand, calculator, or computer.
4.2.2 Personnel
All personnel participating in research-related activities under the
auspices of HERL-RTP should possess experience and knowledge adequate to
perform the technical tasks assigned. Personnel qualifications should be
reviewed and evaluated by the project officer and may be reviewed by the QA
officer as well. Professional resumes of key task personnel should be
available for these evaluations.
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Where appropriate, personnel will be expected to participate regularly
in certification programs, including external audit programs for performance
evaluation and/or accredited training courses in their areas of specializa-
tion. All task personnel should keep abreast of current developments in
their fields of expertise. Periodic meetings during task implementation may
be helpful in information exchange and lead to improved quality control.
Bench-level personnel should also be involved in the feedback and
corrective action loop (Section 4.5.7). This involvement should begin early
in the task with a briefing on the overall task goals, methods to be employed,
and personnel roles in data quality assurance.
Another aspect relating personnel and data quality is the work attitude
of task personnel. Appropriate workloads prevent excessive mental and
physical fatigue, and useless effort is avoided with optimum facility and
equipment configurations. Proper management techniques result in maximum
productivity and data quality. The project officer is in the position to
recognize and address those aspects of quality control that create a healthy,
motivating atmosphere for task personnel.
4.2.3 Facilities, Services, Equipment, and Supplies
Evaluation of task-specific facilities, support services, equipment,
and supplies is the responsibility of the project officer in cooperation
with the HERL-RTP QA organization. The QA officer may, at his/her option or
at the request of the project officer, inspect and evaluate or request an
audit by qualified personnel of facilities, support services, equipment, and
supplies used by laboratories performing HERL-RTP-supported work.
4.2.3.1 Facilities-
All HERL-RTP-supported facilities should be capable of producing accepta-
ble data quality in an efficient, cost-effective manner with minimum risk to
personnel.
The suitability of a facility for the execution of both the technical
and QA aspects of a task may be assessed prior to use through a systems
audit by qualified technical and QA personnel. These audits should deter-
mine if facilities are of adequate size, with satisfactory lighting, venti-
lation, temperature, noise levels, and humidity, and if they are operationally
consistent with their designed purpose. Satisfactory personnel safety and
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health maintenance features should also be present. HERL-RTP requires that
all facilities meet acceptable safety and health standards.8
Facility security should be tailored to the task research needs and to
personnel safety requirements. Security may range from areas available for
common use by nontask personnel to restricted areas accessible only to
authorized task personnel.
Authorization and documentation of all changes in facility configura-
tion should be limited to a single professional staff member (e.g., project
officer) who is qualified to ensure that necessary modifications will not
jeopardize data quality or personnel health and safety within the facility.
4.2.3.2 Support Services--
The reliability of required support services is of primary importance
in evaluating the task facilities. Numerous measurement processes depend on
routine services (i.e., gases, electricity, heat, steam, or water) and loss
of these may cause significant deterioration of data production or quality.
Adequate provisions for backup support services should therefore exist.
4.2.3.3 Equipment--
All equipment should be evaluated prior to use for its applicability to
the HERL-RTP task. Under the HERL-RTP QA Program, the relationships of all
measurement methods and the variables to be monitored should be well charac-
terized and documented before being approved for use. Similarly, the design
and performance of equipment should be thoroughly evaluated with the aid of
a professional who has both a theoretical and a practical understanding of
the specific instrument operation. In some cases, such as for atmospheric
analyzers, comparative studies of different manufacturers' equipment have
been conducted by EPA or its contractors. These data should be taken into
consideration in light of precision and accuracy requirements for the task.
Definitive statements about the performance of different manufacturers'
equipment cannot be based reliably on examinations of single pieces from
each manufacturer. Acceptance testing for new equipment should be performed
on an item-by-item basis and documented for comparison with future testing.
All testing programs should be designed to determine the optimum operating
range of the equipment. Equipment performance should be evaluated periodi-
cally by systems and performance audits.
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To ensure consistently high data quality in the HERL-RTP program, a
plan for routine inspection and preventive maintenance (PM) should be devel-
oped and followed for all equipment. Scheduling of a particular PM program
should be based on the identification of critical components that are most
likely to fail and the overall effect of equipment failure on data quality.
All maintenance activities should be performed by suitably qualified
technical personnel using accepted, documented procedures according to the
PM plan. The desirability of full- or part-time equipment operator and/or
maintenance support should be considered. Frequently, sophisticated instru-
mentation performs poorly or not at all when many occasional users have
access to it. On the other hand, minor but frequent maintenance often keeps
an instrument operating at peak performance. In such cases, the cost of a
full-time dedicated operator is justified.
Documentation of all scheduled and unscheduled maintenance is essential
to monitoring and documenting data quality. Permanent records of the main-
tenance histories of all equipment, including detailed descriptions of all
adjustments made, parts replaced, etc., should be kept in individual bound
notebooks, dated, and signed by the proper authority.
4.2.3.4 Supplies--
A well-documented acceptance testing program for all incoming expend-
able supplies should be adhered to. This acceptance screening ensures that
supplies not meeting task specifications are not used. The results of a
successful acceptance test confirm: (1) that the substance fully corresponds
to the manufacturer's specifications; and (2) that known or suspected inter-
ferents are absent. Acceptance screening under the HERL-RTP QA Program
involves two classes of consumables: chemicals and biological materials.
4.2.3.4.1 Chemicals—The screening of chemicals or reagent commodities
involves verification of assay and examination for impurities. Such screen-
ing should be performed on a batch basis using accepted, documented analyti-
cal methods. For example, it is necessary to characterize all incoming
cylinder gases containing pollutants as to the pollutant concentration and
the composition of the diluent gas(es). Following successful completion of
the acceptance test, an expiration date should be permanently marked on each
container; containers should be stored on a first-in first-out basis.
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Permanent labels should be attached to the container with the following
information:
Date received:
Date tested:
Date expired:
Storage conditions that will protect the integrity of the material and
protect personnel from harmful exposures should be observed. In particular,
parameters such as temperature, light, and humidity should be considered.
Recertification should be performed routinely to characterize changes
in concentration, formation of new species, or loss of original species to
prevent them from degrading task data quality. When possible, the integrity
of the substance should be checked prior to each use.
A permanent record of all certification procedures, dated and signed by
the appropriate authority, should be kept in a bound laboratory notebook
that is filed with the project officer and is accessible to the QA officer.
A well-designed central stockroom tracking system will facilitate rapid
reference to the identity of other users of a substance. This is useful for
informal sharing of information of interest as well as for rapid identifi-
cation of users if specific problems (e.g., degradation or contamination)
are detected with a particular substance.
Since many chemicals tested at HERL-RTP are potentially hazardous to
human health, task personnel should be protected from exposure at all times.
When chemicals are known to be toxic, mutagenic, carcinogenic, or teratogenic,
the project officer should identify where potential personnel health and
safety problems may arise during the completion of the task. Task personnel
should be advised of the specific hazards and proper handling procedures for
all potentially hazardous chemicals.
4.2.3.4.2 Biological materials—The majority of HERL-RTP research
tasks involve the use of biological systems to analyze environmental samples
for mutagenicity, toxicity, carcinogenicity, and biochemical response. The
biological screening of test materials involves J_n vjtro microbial and
tissue culture assays, j_n vivo animal assays, and human subjects. These
systems present special problems, as biological systems inherently possess a
36
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high degree of variation. Because of this inherent variation, QA in this
area of research is still in a developmental stage.
In Vitro Microbial and Cell Culture Lines
Ir\ vitro microbial strains and cell culture lines that have been quite
thoroughly characterized are available for research purposes. The American
Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland, 20852,
provides:
1. Certified animal cell lines (and is a depository for new
animal cell lines),
2. Animal viruses and antisera,
3. Chlamydiae,
4. Richettsiae,
5. Certified pathogenic bacteria.
In addition, the following cultures may be obtained from specific
research laboratories:
Ames/Salmonella reverse mutation assay
Dr. Bruce Ames
Dept. of Biochemistry
University of California at Berkeley
Berkeley, CA 94720
E. coli/pol A DNA damage assay
Dr. Herbert Rozenkrantz
Dept. of Microbiology
NY Medical College
Valhalla, NY 10595
Mouse lymphoma mammalian cell culture forward mutation assay
Dr. Donald Clive
Burroughs Wellcome
Research Triangle Park, NC 27709
Chinese Hamster Ovary (CHO) Cells forward mutation assay CHO/HGPRT
Dr. Abe Hsie
Oak Ridge National Laboratory
P.O. Box Y
Oak Ridge, TN 37830
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CHO/V79-cell transformation and forward mutation assay
Dr. Elie Huberman
Oak Ridge National Laboratory
P.O. Box Y
Oak Ridge, TN 37830
Health hazard assessment of environmental chemicals evaluates the poss-
ible health hazards associated with those chemicals. The approach HERL-RTP
has taken is to use j_n vitro and j_n vivo bioassays in a battery of tests, each
test measuring a different endpoint.9 These tests include microbial, tissue
culture, plant, sperm, and whole animal testing. Endpoints measured include
mutagenicity, toxicity, carcinogenicity, genetic damage in chromosomes (both
somatic and genetic), neurological damage, and related health effects. l£i
vitro tests are cost- and time-effective and can identify substances that
may then be screened further for possible health effects in more time-
consuming and costly in vivo animal testing.
In Vivo Animal Testing
Intermediate between microbial cultures and human subjects are animal
subjects (primarily mammals). Inbred animal strains have been quite well
characterized for generations and correlate closely with certain aspects of
human health. For example, C3H/HeJ mice have been selected for their ability
to convert polycyclic aromatic hydrocarbons to their active carcinogenic
form.10 A large portion of HERL-RTP testing involves experimentation with
animal subjects.
Specific screening procedures for intramural HERL-RTP animal studies
should be developed with the assistance of the HERL-RTP Laboratory Animal
Staff (LAS). Adherence to accepted animal handling procedures and animal
facility accreditation by the American Association for Accreditation of
Laboratory Animal Care (AAALAC) are considered minimum requirements for all
HERL-RTP animal studies. Animal selection should be based on awareness of
the animal strain's genetically determined immunities, as well as the specific
dose-response relationship to be investigated. The research protocol should
clearly state the basis for selection of a particular species and strain.
Acceptance testing, or prescreening and surveillance, should be sufficiently
comprehensive to ensure that only suitable animals are used as experimental
subjects and controls. Although the added expense of such testing may limit
38
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the quantity of animals used, the increase in data quality will generally
more than compensate for this loss.
Comprehensive HERL-RTP guidelines for research involving animals are
presented in Section 7. The animal care support facility has a QA Project
Plan on file with the QA officer.
Human subjects come from diverse and largely unknown backgrounds.
Variability among human subjects can be minimized, but not eliminated, by
careful pretest screening and questioning to determine medical history, work
history, personal habits, and present health status. Project officers
supervising tasks involving human subjects must comply with the existing
regulations and guidelines on the protection of human subjects.11
4.2.4 Recordkeeping
The cardinal principle of recordkeeping for scientific research is that
all raw data must be retained in a manner that is secure and that expedites
validation and access. Complete, permanent, and chronologically organized
records of all project activities should be maintained. All information
that might be useful in data analysis and interpretation should be recorded.
This includes, in addition to raw data, explicit identification of equipment,
reagents and other supplies, experimental subjects (e.g., animals), protocol
modifications, and quality control activities. The exact organization of
the project records should be specified in the protocol and is subject to
approval by the project officer.
A cross-referencing system should be used if the data are to be easily
accessible following their initial use. Such a system may be of various
levels of complexity, depending on the amount of data collected and their
potential applications. Rules for nonclinical laboratory reports and records,
and their generation, storage, retrieval, and retention on a long-term basis
are available.12 When data are logged by computers, it is important that
adequate provision be made for redundant and physically separate long-term
storage of such records (Section 4.4.2).
All technical personnel should be provided with a personal notebook in
which they chronologically record all data in dark permanent ink, from
weights and temperatures to calculations and general observations. Formats
for data should be standardized for the project and not left to individual
39
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discretion. Efforts should be made to encourage the entry, not only of
specific data (e.g., weights, absorbances, volumes, atmospheric or meteoro-
logical conditions, status of instruments, etc.), but also of anecdotal data
and comments. Erroneous or invalidated data should be indicated in such a
way that the entry is flagged, but remains legible. Drawing a single line
through the entry, so that the value is still readable, is an acceptable
indication; this flag should be initialed. The reason for suspicion of the
datum should be recorded in the comments column. Such information may
become extremely valuable in subsequent evaluation of a completed experiment
or in initial planning of a related one.
It may be advisable to provide station, laboratory, or task data note-
books, in addition to individual notebooks or project data notebooks, to
follow the relationship between project components. Such records will
generally take the same form and adhere to the same recommendations as
personal notebooks. Related notebooks should be cross-referenced.
Instrument logbooks contain all data relating to a particular piece of
equipment. This log maintains, in one location, a chronological record of
instrument operation, calibration, maintenance, failures, and idiosyncrasies.
Such a record is often useful in determining trends, spare parts inventories,
etc. A specific format should be used for such data to minimize the possi-
bility of omission of important procedures or data.
The project officer should check that data logging forms for measured
parameters have been designed to ensure complete data, high productivity of
technical personnel, and ease of reading the raw data. Data coding forms
should be designed in consultation with personnel who must record and evalu-
ate the data. In some cases, a data transfer can be avoided by designing
forms in consultation with keypunchers or other data entry personnel.
Computerized data acquisition systems have many advantages, but require
close monitoring and frequent auditing for erroneous or stray electrical
signals. Many systems are able to concurrently produce printed output as
well as computer-readable output (usually magnetic tape); where possible, it
is advisable to employ both.
High quality recordkeeping serves at least two useful functions:
(1) it makes possible the detailed reanalysis of a set of data at a future
time when the model has changed significantly, thus increasing the cost-
40
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effectiveness of the data; and (2) it may be used in support of the experi-
mental conclusions if various aspects of the study are called into question.
This latter point goes to the heart of scientific research: objectively, it
is often possible to interpret data in more than one way and the raw data
must be available for evaluation by qualified professionals; subjectively,
when recordkeeping is careless, suspicion is quickly aroused that all other
aspects of the research are of similarly poor quality.
4.2.5 Chain-of-Custody Procedures
All projects involving health effects .research should document and
implement a chain of possession and custody of any sample collected, whether
or not the resulting data are to be used in enforcement cases. Such proce-
dures ensure that the samples are collected, transferred, stored, analyzed,
and destroyed only by authorized personnel.
4.2.5.1 Field Custody Procedures--
The following chain-of-custody procedures are specifically applicable
to large-scale monitoring programs involving shipment of samples from the
sampling site to the analysis laboratory.13 They are intended to be compre-
hensive and may be used as guidelines for all measurement or research pro-
grams. Depending on the specific scope and nature of a project, the project
officer should tailor the chain-of-custody procedure to the project. The
most important concern is that any chain-of-custody procedure be properly
documented and followed for the duration of the task.
1. Samples must be accompanied by a chain-of-custody record that
includes the project title, collectors' signatures, collection
site, date, time, type of sample, sequence number, number of
containers, and analyses required. (An example of a chain-of-
custody record is shown in Figure 4-3. Note: Standardized
formats should be tailored specifically to each project and
used consistently for the duration of the task.) When turning
over possession of samples, the transferor and transferee
sign, date, and time the record sheet. This record sheet
allows transfer of custody of a group of samples from a
collection area to the central analysis laboratory. When a
custodian transfers a portion of the samples identified on
the sheet to the laboratory, the individual samples must be
noted in the column with the signature of the person relin-
quishing the samples. The laboratory person receiving the
samples acknowledges receipt by signing in the appropriate
column.
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CHAIN OF CUSTODY RECORD
STATION
NUMBER
STATION LOCATION
DATE
fe
TIME
^
%*'
Relinquished by:^™^™;
Relinquished by: is.s™u«i
Relinquished by: is,,™^/
Received by: is^auni
Dispatched by:ts,,wu*i
Date
SAMPLERS __
SAMPLE TYPE
Water
Comp.
4
Grab.
P
Air
SEO.
NO.
NO. OF
CONTAINERS
ANALYSIS
REQUIRED
Received by: tsisiawei
Relinquished by: is,s^ia,,i
Received by: is.Sn,,urd
Received by Mobile Laboratory for field
analysis: «,s™,0«;
/Time
Received for Laboratory by:
Method of Shipment:
Date/Time
Date/Time
Date/Time
Date/Time
Date/Time
Distribution: Or g. -Accompany Shipment
1 Copy— Survey Coordinator Field Files
Figure 4-3. Example of a network chain-of-custody record.12
42
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2. The collector custodian has the responsibility of packaging
and dispatching samples to the laboratory for analysis. The
dispatch portion of the chain-of-custody record must be
filled out, dated, and signed.
3. To avoid breakage, samples should be carefully packed in
shipping containers such as ice chests. The shipping con-
tainers are padlocked for shipment to the receiving laboratory,
Special shipping precautions are necessary for toxic or haz-
ardous materials and must conform to Federal regulations.14 17
4. Packages must be accompanied by the chain-of-custody record
showing identification of the contents. The original record
must accompany the shipment. A completed copy is retained by
the project officer after completion of the analysis.
5. If sent by mail, register the package with return receipt
requested. If sent by common carrier, a Government bill of
lading should be obtained. Receipts from post offices and
bills of lading should be retained as part of the permanent
chain-of-custody documentation.
6. If delivered to the laboratory when appropriate personnel are
not there to receive them, the samples must be locked in a
designated area within the laboratory so that no one can
tamper with them, or they must be placed in a secure area.
The recipient must return to the laboratory, unlock the
samples, and deliver them to the appropriate custodian.
4.2.5.2 Laboratory Custody Procedures--
Suitable laboratory chain-of-custody procedures include:13
1. The laboratory should designate a sample custodian and an
alternate custodian to act in his/her absence. In addition,
the laboratory should set aside a sample storage security
area. This should be a clean, dry, isolated room with suffi-
cient refrigerator space that can be securely locked from the
outside.
2. Samples should be handled by the minimum possible number of
persons.
3. Incoming samples should be received only by the custodian,
who will indicate receipt by signing the chain-of-custody
record sheet accompanying the samples and retaining the sheet
as a permanent record. Couriers picking up samples at the
airport or post office shall sign jointly with the laboratory
custodian.
4. Immediately upon receipt, the custodian places the samples in
the sample room, which will be locked at all times except
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when samples are removed or replaced by the custodian. Only
the custodian should have access to the sample storage room.
5. The custodian should ensure that all samples are properly
stored and maintained under appropriate environmental condi-
tions (i.e., temperature, humidity, light intensity).
6. Only the custodian should distribute samples to task personnel
who are to perform analyses.
7. In the laboratory notebook or analytical worksheet, the
analyst records information describing the sample, the proce-
dures performed, and the results of the analysis. The notes
should be dated, should indicate who performed the tests, and
should include any abnormalities that occurred during the
testing procedure. The notes should be retained as a perma-
nent record in the laboratory. In the event that the person
who performed the tests is not available as a witness at the
time of a trial, the Government may be able to introduce the
notes in evidence under the Federal Business Records Act.
8. Approved methods of laboratory analyses should be used and
documented on all samples.
9. Laboratory personnel are responsible for the care and custody
of a sample once it is handed to them and should be prepared
to testify that the sample was in their possession and view
or secured in the laboratory at all times from the moment it
was received from the custodian until the analyses were
completed.
10. The laboratory area should be maintained as a secured area
and should be restricted to use by authorized personnel only.
11. Once the sample analyses are completed, the unused portion of
the sample, together with identifying labels and other docu-
mentation, should be returned to the custodian. The returned,
tagged sample should be retained in the custody room until
permission to destroy the sample is received by the custodian.
12. Samples should be destroyed only upon the order of the project
officer in consultation with the QA officer and only if it is
certain that the sample is no longer required. The same
destruction procedure is true for tags and laboratory records.
4.2.5.3 Evidentiary Considerations--
As accurate and reliable environmentally related measurements become
increasingly important in documentation of environmental conditions of
public health concern, organizations collecting these data must address
evidentiary considerations.
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Recording all chain-of-custody procedures and promulgated analytical
procedures in writing will facilitate the admission of evidence under Rule
803(6) of the Federal Rules of Evidence (Public Law 93-575). Under this
statute, written records of regularly conducted business activities may be
introduced into evidence as an exception to the hearsay rule without the
testimony of the person(s) who made the record. Although it would be prefer-
able, it is not always possible for the individuals who collected, kept, and
analyzed samples to testify in court. In addition, if the opposing party
does not intend to contest the integrity of the sample or testing evidence,
admission under Rule 803(6) can save a great deal of trial time. For these
reasons, it is important that the procedures followed in the collection and
analyses of evidentiary samples be standardized and described in an instruc-
tion manual, which, if need be, can be offered as evidence of the regularly
conducted business activity followed by the laboratory or office in generat-
ing any given record.13
4.3 SAMPLE COLLECTION AND ANALYSIS
4.3.1 Sample Collection
Collecting a sample that properly represents the environmental condi-
tions present may be the most technically difficult, hazardous, and time-
consuming part of the analytical task. Sampling may range from collecting a
representative volume of emission gases from a smoke stack to obtaining
tissue samples from rats or biological samples from human subjects. In some
special cases, extra data may be required to document the collection proce-
dure. In some research systems, separate control samples must also be ob-
tained.
Requirements for optimal data transmission during sample collection
include:
I. Correctly identifying the material or subject to be sampled.
2. Having appropriately trained personnel perform the collection.
3. Having a feedback loop to the project officer or principal
investigator regarding problems of sample collection.
Written instructions for sample collection and handling are essential
and should include:
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1. Project title.
2. Type of sample required.
3. Amount of sample required.
4. Description of sampling procedure.
5. Special processing or handling steps to avoid sample degrada-
tion.
6. Test subject preparations or environmental site preparations
and precautions.
These instructions should be systematically documented and should be acces-
sible to all personnel.
For the laboratory phase of sample handling, various in-house specimen
processing steps (i.e., handling, preservation procedures, precautions, and
distribution to analysts) should be documented. In addition, criteria for
rejecting inadequate or inappropriate samples should be defined at the
initiation of the task.
4.3.2 Sample Analysis
Sample analysis often involves a repeated sequence of similar, docu-
mented operations by technical personnel and/or automated instrumentation.
For this reason, sample analysis is amenable to the use of quality control
techniques. The best available operating procedures used by trained techni-
cal personnel are the norm in a laboratory research context. Quality control
activities require planning by the project officer and may include the
analysis of blind samples on a regular basis, samples spiked with known
amounts of the analyte to serve as a check on analytical bias, split-sample
aliquots analyzed by different analysts at different times using a different
set of reagents, and frequent calibration checks using standard samples and
blanks.
QC measurements requiring highly developed subjective evaluations
(e.g., pathological evaluation of tissue) may require side-by-side or round-
robin analysis to establish the quality of the data. The project officer
should choose the QC activities appropriate to a given task that will provide
highest quality data given the existing analytical limitations.
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4.4 DATA MANAGEMENT
Following the collection, storage, backup, and validation of raw data,
a series of transfers generally occurs prior to data reduction and preparatory
to data analysis, interpretation, and reporting. Each of these aspects of
the data analysis/processing regime must be addressed in the project plan,
together with associated QA measures and their documentation. Validation of
computerized data against raw data is discussed in Section 4.5.6; the remain-
ing aspects of data management are discussed below.
4.4.1 Data Collection
A clear description of the manner in which raw data are collected is
essential to QA planning. These data, representing the actual measured
parameters in chronological sequence, subsequently may be flagged as invalid
but are never destroyed or deleted. Manually collected data are frequently
monitored by the person recording the data. However, computerized data
acquisition systems do not have the potential for this treatment and are
known to pick up false voltage transients, which may bias the data. In more
complex systems, careful analysis of data trends and of the relationships
between various parameters may be used to establish windows or intervals
within which valid data are expected to occur. It must be recognized,
however, that evaluating data to determine if they lie in an expected range
does not alone constitute adequate validation. Clearly, data can appear in
such an interval, but still involve considerable error.
The use of computerized data acquisition systems is increasing. This
frequently permits a statistically acceptable, cost-effective extension of
the control chart concept for real-time data validation. There are several
advantages to using such a system. Raw data are transformed into tabular or
graphical form, thus minimizing human error.18 Similarly, the capability of
rapidly and automatically comparing experimental data against recent values
of similar data can serve as a real-time check on data validity.
In all cases, methods for assessing the validity of the recorded raw
data as compared with measured or observed data must be established prior to
task initiation and documented in the project report (see Section 4.5.6).
It is a QA function to evaluate the adequacy of these methods with respect
to time, place, and documentation.
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4.4.2 Data Storage and Backup
Raw data must be stored in such a way that they are not degraded or com-
promised and that any datum (value) desired may be retrieved (uniquely
identified). For computerized raw data, there must always be at least one
copy that is off-line and not machine mounted. It is a common practice of
large computation centers to provide this service with regularly scheduled
backups for users renting on-line disk space. The user should know when
such backups are performed. Duplication of user-owned tapes is usually the
responsibility of the user.
Raw task data must be securely archived. Such aspects as storage media
(e.g., paper, punched cards or paper tape, magnetic tape or disk), conditions,
and location must be addressed. Access by authorized personnel and retention
time must also be addressed in the QA Project Plan. For certain types of
studies, EPA's proposed Good Laboratory Practices7 are quite explicit.
The storage media, conditions, and locations should be selected based
on task-specific criteria. For example, storage location and condition may
be inalterably determined for administrative reasons, which would then imply
storage media requirements. Another example might be the use and "exercising"
of computer files (tape or disk) due to their rather high volatility.
Physically separate storage of duplicate raw data sets should be considered.
The retention time will vary, but it should be clearly stated at the outset
of any study.
Access to the archived data should be described. The fewer persons
allowed access, the less chance there is of losing the data. This may
conflict with the need to disseminate data to a wide audience. In such a
case, copies of data may be provided rather than permitting free access to
the unrecoverable raw data.
Another aspect of data storage that should be addressed is data inviol-
ability. Raw data must never be altered. It may be copied, and the copy
altered in such a way that an audit trail is generated. An audit trail is
an account of the data (values, pages, keypunch forms, keypunch cards, etc.)
and a verification after each operation on the data that the number of data
items fed into the process is reliably reflected by (usually equal to) the
number of data items resulting from the process.
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It is not necessary that all data sets created from the raw data set be
saved or backed up. In fact, if a second or later generation data set is
retained without sufficient documentation to explain how it was created,
then that data set can be of little or no value for QA purposes. Thus, more
important than retention of intermediate data sets or analyses is the adequate
documentation of the procedures used. All computer code (including control
language), which accomplishes objectives planned in the protocol, or results
to be cited in the final report should be saved.
4.4.3 Data Transfers
Data transfer changes the form or location of a data set, but not its
content. Thus, a transferred data set may be used to fully reconstruct its
originating data set. If data transfer is error free, then no information
is lost in the transfer and the input is completely recoverable from the
output. Examples of data transfer are copying the raw data from the notebook
onto a data form for keypunching, converting a written data set to punched
cards, or copying from a computer tape to disk.
A good general rule is to minimize the number of data transfer steps in
the data processing, since the overall probability for errors increases with
the number of such transformations. Often this can be influenced by judi-
cious design or choice of data forms. For instance, the reliability of
keypunchers and other data entry personnel is highly dependent upon the form
and legibility of the data they receive. They should be consulted in advance
and, insofar as possible, forms should be designed to accommodate them;
standardized 80-column Fortran coding forms (6X28-7327-6 IBM) are often
desirable. In some cases, it is possible to initially record the raw data
on the same form used for data entry (keypunching) or in computer-readable
form, which is highly desirable.
As part of the study design, an overall admissible transfer error rate
should be specified. The purpose of data validation is to test whether this
error rate has been exceeded. If the transfer process has several components,
their individual error rates are not of particular concern, as long as the
composite error rate is below the desired level. Data validation is discussed
in Section 4.5.6.
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4.4.4 Data Reduction
Data reduction includes all processes that transform one data set to
another in such a way that the original data set cannot be recovered from
the reduced data set. It is distinct from data transfer in that it entails
a reduction in the size (or dimensionality) of the data set and an associated
loss of information. Assumptions about the distribution of the observations
are implicit in data reduction, making it a data analysis activity. For
instance, if repeated measurements of a quantity are made in the laboratory
and summarized as a mean and standard deviation, then statistical theory can
be invoked to justify the sufficiency of these two measurements, i_f the data
follow a normal distribution.
If the data are reduced before analysis, the study documentation or
data management analysis scheme must clearly define the mathematical or
other processes used to obtain the reduced data set from the raw data set.
Quality assurance should address the accuracy of the mathematical operations
used in the reduction process.
Permanent data reduction, resulting in irretrievable loss of raw data,
must not occur on EPA projects.
4.4.5 Software
The objective of software quality assurance is to ensure that calculator
and computer programs perform accurately and as planned. Such operations
should introduce no more than negligible error (e.g., I percent or less)
relative to the intrinsic variation in the measured processes. For manual
calculations, an example should be given in which actual raw data are trans-
formed and can be checked by reviewers. If a programmable calculator is
used in this process, a copy of the programs used should be provided.
Computer programs should be designed to expedite validation. Programs
should be modular, structured, well documented, logical, and should liberally
employ comment statements. The use of widely available statistical analysis
packages such as SAS, BMD, SPSS, and MINI-TAB is recommended, as opposed to
writing analysis programs in FORTRAN, BASIC, or PL/I code. Such packages
are heavily used, so that errors have been largely eliminated, and standard
documentation is widely available.
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The following minimal documentation is sufficient for computerized data
manipulation or analysis:
1. Reference to system documentation (some software packages
supply this automatically);
2. A copy of the calling program and resulting output;
3. A concise, clearly written description of the operand data
set and how it derives from the raw data and the operation or
analysis to be performed; these may be embedded in the begin-
ning of the program as a comment statement; and
4. A data dictionary defining the variables as they pertain to
the operation or analysis as described in item 3 above; the
data dictionary may be embedded in 3.
Compliance with items 1 through 4 has no implications for validity of
the analyzed data (see Section 4.5.6) or appropriateness of the statistical
methodology employed; they must each be addressed separately (see Section
4.4.6).
4.4.6 Data Analysis
Data analysis involves statistical comparison of a model, be it a
linear model, a dispersion model, an infectivity model, or a legally defined
pollutant dose-response threshold, against observed data. It frequently
includes computation of summary statistics and their standard errors, confi-
dence intervals, tests of hypotheses about the parameters, and model valida-
tion (goodness-of-fit tests). The QA Project Plan should outline a tentative
approach to data analysis, enumerate potential problems in the data analysis
schemes, and suggest methods of addressing the problems.
It is unlikely that any real-world phenomenon can be perfectly repre-
sented by a simple model. Neglected factors, noise, round-off error, incom-
petence, and other contaminations can degrade data quality. Often a data
set can be perfectly summarized by a function, just as some polynomial can
be found to pass through data pairs at different values of the independent
variable; the predictive value of such a function is, however, questionable,
since it essentially just summarizes the data. Typically, there are so many
functions available for fitting a given data set that it is impossible to
decide among them on the basis of goodness-of-fit alone. A reasonable
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reduction of the problem is to first restrict attention to models that are
scientifically based, in that the model parameters are identified with
essential substantive features of the phenomenon. Even then, the same
danger exists with scientifically based models if they contain so many
parameters that they allow (near) perfect fit of any data set. Thus, the
objective of the modeling process should be to arrive at a scientifically
plausible model that has a limited number of parameters.19
Some common numeric and statistical problem areas are:
Treatment of missing and partially missing data (e.g., cen-
sored data).
Selection of a small number of most informative variables
from among an unmanageable number of variables (e.g., stepwise
regression, principal components).
Multiple comparisons and resulting inflated significance
levels (e.g., two independent hypothesis tests, each at an
individual .05 = 1 - .95 significance level, have together a
composite .10 = 1 -(.95) (.95) significance level).
Outliers and other model inaccuracies.20
Numeric problems (e.g., attempting to invert an ill-conditioned
matrix involving highly correlated variables, or using itera-
tive procedures to solve systems of equations).
The project plan should specifically address any of these problems
relevant to the project.
Data analysis QA consists of determining whether the analyses proposed
in the protocol were in fact carried out and, if so, if they were performed
correctly. Complete study documentation is required for this. The model-
building process for a large-scale study may be so involved that a statisti-
cian functioning in a QA capacity cannot check on all planned analyses. In
such a case, sampling inspection principles may be applied, leading to
review of a subset of the study documentation.
4.4.7 Reporting
The most visible product of a research task is the report of important
findings. Publication guidelines applicable to the HERL-RTP research reports
are available21 22 and minimum technical contents for nonclinical laboratory
reports and health effects research have been promulgated7 and are shown in
Figure 4-4.
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1. Name and address of the facility performing the study and the dates on
which the study was initiated and completed.
2. Objectives and procedures stated in the sponsor-approved protocol,
including any changes in the original protocol including justification(s).
3. Statistical methods employed for analyzing the data.
4. The test and control substances identified by name, chemical abstract
(CAS) number or code number, strength, purity, and composition or other
appropriate characteristics.
5. Stability of the test and control substances under the conditions of
administration and storage.
6. A description of the methods used.
7. A description of the test system used. Where applicable, the final
report must include the number of animals used, sex, body weight range,
source of supply, species, strain and substrain, age, and procedures
used for identification.
8. A description of the dosage, dosage regimen, route of administration,
and duration.
9. A description of all circumstances that may have affected the quality
or integrity of the data.
10. The name of the study director, the names of the other scientists or
professionals, and the names of all supervisory personnel involved in
the study.
11. A description of the transformations, calculations, or operations
performed on the data, a summary and analysis of the data, and a state-
ment of the conclusions drawn from the analysis.
12. The signed and dated reports of each of the individual scientists or
other professionals involved in the study.
13. The locations where all specimens, raw data, and the final report are
to be stored.
14. The statement prepared and signed by the quality assurance unit.
Figure 4-4. Minimum technical report content for
EPA health effects tests.7
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The report should be concise, complete, and consistent with standard
EPA formats.21 22 Discussion of the important technical aspects of the
research should be adequate to permit qualified professionals to duplicate
results. Adequate data should be included to permit at least partial calcu-
lation of important results. The conclusions, drawn from the data, and the
rationale behind those conclusions should be clearly stated. Graphical and
illustrative data correlation with supporting tables should be used whenever
possible. Well-defined error estimates should be included with all quanti-
tative values reported.
The presentation of results should delineate the functional relationship
linking the data and the tables or graphs and be understandable to nonstatis-
ticians. Since any scientific study falls short of realism, useful conclu-
sions usually require generalizations that tend to lie outside the realm of
strict statistical justification. Thus, the reader of the technical report
should be informed of the amount of statistical and physical justification
supporting each conclusion. The purpose(s) and conclusion(s) of the research
should be stated clearly. The estimated errors, as well as the limits of
applicability of results, should be stated in such a way as to minimize
misinterpretation. Application of the results to alternative theories
(models-) should be provided, with indication of the rationale used in reach-
ing the stated conclusions rather than the alternative conclusions.
Quality control and quality assurance activities should be detailed to
permit the specialist and nonspecialist alike to assess correctly the level
of the QA effort invested in the research. Subjective evaluation of the
validity and accuracy of the reported results and conclusions should be
possible from the data presented.
4.5 QUALITY CONTROL
4.5.1 Internal Audits
The ability of the total data system to produce data of a specified
quality should be regularly evaluated to determine if corrective action (see
Section 4.5.7) is needed. Internal audits, conducted by the operating group
or organization, are used to obtain data for this evaluation.
The Environmental Protection Agency defines two types of audits that
perform these functions.23 24 A quantitative measure of the quality of the
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data produced is usually obtained through a performance audit. A qualitative
assessment of the ability of a system to produce data of the specified
quality is evaluated by means of a systems audit.
Performance audits should be performed by qualified technical personnel
not routinely involved in the specific task measurement process being audited.
For example, for the Ames/Salmonella assay, an audit could be performed by
introducing several mutagens of known response into the assay system. The
identity of the test sample or samples would be known only to the auditor.
The same principles should also be applied to laboratory measurement instru-
ment systems. Frequently, the performance audit can only be designed to
evaluate a part of the total data system, such as sampling, analysis, and/or
data reduction. In this case, the audit should be designed to evaluate each
subsystem to the fullest extent possible. In either case, the audit values
are compared with those generated by the data system(s), and conclusions are
drawn as to the quality of the data being generated by the total system.
Tools available for use in performance audits generally fall into one
of four categories:
1. Reference materials, for accuracy determinations, are availa-
ble from several sources,25 26 2' most notably the National
Bureau of Standards. These may be included for analysis in
various types of measurement systems at relatively low cost
with little interference to the normal laboratory routine and
with the highest possible degree of confidence.
2. Reference devices may be obtained for which the critical
parameters are known to the auditor but not the analyst.
These may be more disruptive of laboratory operations and
there is no possibility of anonymity of the sample; however;
the final result is still a measure of the performance of the
total analytical system, including the operator.
3. Cooperative analysis, such as round-robin analysis is useful
for estimating the precision of measurement among several
different operators and/or laboratories. Accuracy of the
measurement can only be assessed if the analyte is a reference
material.
4. Side-by-side analysis, or collaborative analysis, may be used
if important variables are not controllable in the sample.
Systems audits consist of an evaluation of the various components of a
research operation principally through onsite visits by qualified profes-
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sionals. Professional and technical evaluation, resulting from observation
and discussion, is made of the capability of a data system (including instru^
ments, personnel, organization) to produce the specified data quality. Use
of checklists or written questionnaires will allow more complete, objective
assessment during the systems audit. Questions answered on the basis of
such observations include:
Are written sampling and analysis procedures being used?
Are written calibration procedures used as frequently as
necessary?
Is a defined preventive maintenance schedule followed?
Are data reduction, validation, and reporting techniques
completely documented and routinely used?
It is important to emphasize that the purpose of an audit is to evaluate
constructively measurement process data quality (not personnel) and to
identify areas where improvements can be made. If this intent is followed
by project officers and made clear from the beginning, personnel will be
more likely to cooperate in audit and corrective action cycles.
In either situation, the program and rationale for internal audits
should be designed based on individual components of the specific measure-
ment process and clearly planned for and budgeted into the task plans. By
using internal audits, the project officer will be able to objectively eval-
uate data quality as the task progresses.
4.5.2 Preventive Maintenance
To ensure long-term data quality in a cost-effective manner, a rational
preventive maintenance (PM) program must be followed. This assumes impor-
tance roughly in proportion to the amount of instrumental data recorded.
Reference 23 contains a discussion of preventive maintenance, especially as
related to routine measurements (e.g., air quality monitoring). In particu-
lar, preventive maintenance will increase the completeness of data from
continuous monitoring systems, which is an important measure of quality in
such systems.
In a laboratory research environment, PM has the real but less visible
benefit of minimizing and controlling equipment downtime and therefore
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extending the completeness of the data. Preventive maintenance can be
budgeted and scheduled based on historical failure analysis data or recommen-
dations of the manufacturer. Extended laboratory use of specific items can
be scheduled with higher reliability than if maintenance occurs only follow-
ing equipment failure.
The laboratory equipment PM program should include scheduling, perform-
ance, and recordkeeping. Scheduling of PM should be developed based on the
effect of equipment failure on data quality, any relevant site-specific
effects, and equipment reliability. This schedule should be available to
the person or group responsible for performing the maintenance as well as
the person or group using the equipment so that use of the equipment may be
scheduled accordingly.
Preventive maintenance should be performed by qualified technicians,
using accepted, documented procedures. The specific service should be based
on the considerations noted in the preceding paragraph and should be known
to both the user and maintenance groups. A predefined set of data should be
obtained before and after the maintenance activities to permit equipment
performance evaluation. Calibration (see Section 4.5.3) should be performed
following all maintenance activities.
Documentation of all maintenance activities—scheduled or not—is
essential to monitoring and documenting data quality. A bound notebook (see
Section 4.2.3.3) should be kept with each instrument as a record of its
maintenance history. A detailed description of all adjustments made and
parts replaced should be recorded. If the notebook is the multicopy type,
one of the copies should be kept by the maintenance group for analysis.
This analysis may include such considerations as mean time between failure
(MTBF) for specific components, MTBF analysis for total systems (individual
and laboratory-wide), and development of an onsite spare parts inventory to
cost-effectively reduce equipment downtime. Where possible, checklists
should be used to ensure and document thorough maintenance activities.
4.5.3 Calibration
Calibration is the process of establishing the relationship between the
output of a measurement system and that of a known input; it allows different
instruments to be correlated with each other and with a specified reference
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standard.28 Calibration is an integral part of the measurement process and
is a major factor in controlling the accuracy of results. Since the reported
accuracy of the measurement method can be no better than the accuracy of the
calibration system, calibration is also a limiting factor.
A sound calibration system should include provisions for:
1. Selection of the highest quality calibration standard.
2. Detailed documentation of calibration procedures including
specifications for reagents, materials, support equipment,
and pertinent environmental conditions.
3. Construction of a calibration curve or a corrective table to
determine appropriate correction factors.
4. Maintenance of a record of calibration histories for instru-
ments, support equipment, and standards including identifica-
tion of instruments and standards, dates of calibration, and
calibration results.
5. Determination of calibration frequency needed to ensure
quality data collection; identification of acceptance limits
in terms of precision and accuracy, and corrective action to
be taken when limits are exceeded.
4.5.3.1 Calibration Standards-
Calibration standards should be of the highest quality available and
fully characterized. In the United States, the National Bureau of Standards
(NBS) holds the position of final authority in the preparation of many
reference materials. The NBS Standard Reference Material (SRM) series is
generally regarded as producing the best standards of each type available.
Investigators should check on the availability of SRMs applicable to
their measurement needs. NBS has been rapidly developing suitable SRMs for
environmentally related measurements. The National Bureau of Standards
provides information on available standards in the regularly revised Special
Publication 260,25 regular publicity releases, and in a special mailing list
for newly issued SRMs. In addition, a new monthly column in American Labora-
tory entitled "Reference Materials," edited by the Deputy Chief of the
Office of Standard Reference Materials, is an excellent source of current
information on NBS-SRMs. Considering the rapidity with which SRMs are being
developed, and the pressing need to compare data to standards of known high
quality, this column should be reviewed regularly by each investigator.
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Examples of SRMs currently available from NBS that may be applicable to
environmental measurement systems include:
Hydrocarbon Blends
Primary Working and Secondary Standard Chemicals
Microchemical Standards
Metallo-Organic Compounds
Isotopic Reference Standards
Radioactivity Standards
Industrial Hygiene Standards
Trace Element Standards
Clinical Laboratory Standards
Environmental Standards
Biological Standards
Certified Physical Properties Standards.
Selected examples of SRMs are shown in Appendix C.
Use of NBS-SRMs completely fulfills the requirement of high quality and
full characterization. However, because SRMs are not mass produced and are
individually characterized by lot, they are expensive and often in short
supply. Therefore, it is generally desirable to employ secondary standards
as the actual calibration standards, maintaining an SRM as a final high
quality calibration standard for the secondary working calibration standard.
Whenever a secondary standard is employed in a calibration, it is
necessary that traceability to a quality primary standard be established and
maintained. EPA regulations now require traceability of calibration standards
to NBS-SRMs where possible,29 30 and it is likely that this requirement will
appear in all future regulations.
Unfortunately, for many common measurement processes routinely used at
HERL-RTP, there are no SRMs available. In these cases, the investigator
must use the best available calibration standard or devise a standard. Such
standards must also meet the requirements of high quality and complete
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characterization applicable to SRMs. Careful characterization of such
standards involves rigorous testing to establish the true value of the
reference material within known limits of precision. Such rigorous testing
may include repeated analysis of the standard material by more than one
analyst or technique, or round-robin interlaboratory analyses, to establish
the true value.
4.5.3.2 Calibration Procedures--
Written calibration procedures describing each step in the process are
required. Calibration procedures may be prepared in-house by qualified
personnel, derived from instrument or process manufacturer's instructions,
or obtained from sources such as the American Society of Testing and Mater-
ials31 or the National Bureau of Standards.26 Only the most current and
acceptable procedures available for the specific calibration should be used.
In addition, only personnel familiar with the measurement process and the
calibration procedure should perform the calibration.
An aspect of the calibration operation that is often overlooked is the
calibration of reagents, materials, and support equipment. Most calibration
procedures use equipment and/or reagents in addition to the standard(s).
All such reagents, materials, and support equipment should have been subject-
ed to recent calibration prior to use in the standard calibration procedure.
Even for authoritative standards, such as NBS-SRMs, sample integrity
may be questionable if proper storage and handling procedures are not ob-
served. Maintenance of environmental conditions should be appropriate to a
specific calibration measurement being conducted. Documented environmental
conditions must be maintained during the entire calibration procedure. The
use and handling of the calibration standard should be of particular concern
as potential problems in calibration may arise with mistreatment of otherwise
valid calibration standards.
The following list delineates some of the more common problems associ-
ated with the use of some standards:
1. Permeation devices should be used and stored under carefully
specified environmental conditions of humidity,32 and tempera-
ture,33 and should be protected from possible environmental
contaminants.32
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2. Certain gases in pressurized cylinders require special proce-
dures for routine installation to prevent cylinder and regula-
tor contamination with atmospheric oxygen or moisture.
3. Electronic standards frequently require periods of several
hours for stabilization of output.
4. Most solid standards require conditioning at a specified
humidity prior to weighing.
These examples illustrate that users of standards should be familiar
with specified environmental conditions pertinent to handling of each stan-
dard. It is imperative that this be recognized if high quality data are to
be obtained from the measurement process.
4.5.3.3 Analysis of Calibration Data--
Data from a calibration should be summarized as a function,
y = f(x) + error
where x denotes correct value and y denotes measured value. A linear rela-
tion is often assumed:
y = a x + b + error
where a is the slope, and b is the intercept of the line. The inverse,
x = g(y) = y~ , serves as a correction function.
a
Data from a calibration should first be plotted and judged as to their
linearity. Ideally, tests for quadratic or higher order effects would be
performed. Tests of the hypothesis that the correlation coefficient is at
least as large as a prespecified value (R) at a certain significance level
(s.l.) may be performed as a partial check on linearity. The value of R and
s.l. should be selected by the project officer in consultation with a statis-
tician. If the assumption of linearity is valid, the following statistics
should be computed: correlation coefficient, slope, intercept, standard
error of estimate (square root of estimated error variance, a.), and standard
error of slope and intercept. Raw data from calibrations, as well as these
summary statistics, should be recorded in the instrumentation history. If
the data exhibit nonlinearity in the working range, then the data should be
fit with another more appropriate model, or alternative methods should be
considered.
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For data interpretation, it is useful to plot the estimated slope,
intercept, and standard error of estimate on control charts plotting parame-
ter estimates as functions of time.28
4.5.3.4 History of Calibration--
Documentation of each calibration and the full history of all calibra-
tions performed on a measurement system must be recorded. Control charts
for slope, intercept, and standard error constitute partial graphical his-
tories of results. The history of calibration for a measurement system
should include:
1. Dates of calibration;
2. Identification of the standards used;
3. Support equipment, reagents, and devices used;
4. Personnel performing calibration;
5. Pertinent environmental conditions;
6. Results of calibration (raw data and summary statistics); and
7. Corrective action.
4.5.3.5 Corrective Action--
To characterize the dynamics of the system, each measurement system
should undergo an initial intensive audit phase in which it is frequently
calibrated. The object is to investigate trends in parameters such as slope
(at), intercept (bt), and the error variance (at). The time subscripts have
been included here to indicate the possibility of changes in the measurement
system over time. Two important uses of this information are: (1) to
determine if corrective action (adjustment or replacement of equipment) is
necessary; and (2) to schedule future maintenance and calibration.
Exceeding control limits calls for corrective action, which should be
specified by QA personnel. In some cases, these limits follow from an a
priori accuracy requirement. In other cases, they may result from observa-
tion of the measurement process over a period of time (e.g., the intensive
audit phase). If the measurement process is judged to have performed satis-
factorily, then the average parameter estimates (e.g., slope, intercept,
standard error of estimate), plus or minus three of their standard errors,
may serve as future control limits.
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4.5.3.6 Calibration Frequency--
As indicated, it is possible to study the behavior of a measurement
system over time and to define the limits of acceptable performance. It is
thus possible to estimate the probability of unacceptable performance during
a time interval of arbitrary length between calibrations, """he time between
successive calibrations can then be chosen as the maximum time for which the
probability of exceedance is acceptably low, as defined by QA.
Another possible approach is to form a loss function that incorporates
cost of calibration and loss due to inaccurate measurement. The first term
increases, while the second decreases, as a function of calibration frequency.
Assuming that calibration resets the error of measurement to zero, the
frequency of calibration may be chosen to minimize the expected overall cost
function.28
These procedures will not anticipate shocks to the measuring system as
opposed to trends. Perceptions of operating personnel are the best source
for this information and any suspicion that such a shock has occurred should
be followed by recalibration.
4.5.4 Documentation Control
A QA program should include a system for updating formal documentation
of all operating procedures. One system frequently employed uses a standard-
ized indexing format and provides for convenient replacement of pages that
may need to be changed within the technical procedures descriptions.28
The indexing format should include, at the top of each page, the follow-
ing information: section number, revision number, date (note that the date
given is the date of revision), and page number as shown below:
Section No. 2.12
Revision No. 0
Date September 27, 1977
Page 1 of 5
A digital numbering system identifies sections and subsections within the
text. New subsections should begin on a new page. This format groups
together the pages within a functional subsection to allow for its expansion.
Each time a new page is added or expanded within a section, the number of
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the preceding page should be included, and a letter added to it. A record
of the revisions made to a document must be maintained to derive full benefit
of documentation control techniques.
The most important elements of a quality assurance program to which
document control is applied include procedures for: sampling, calibration,
analysis, data collection and reporting, auditing, sample shipping and
storage, computational and data validation (including programmed checks when
data processing is computerized), and preventive maintenance.
Full control should be maintained of the distribution of documents. A
file should be established within the organization showing such pertinent
information as: document number, title, branch originating the document,
latest issue date, change number, list of persons to whom document is sent,
and signatures of persons acknowledging receipt. Whenever a revision is
made, the group responsible should issue the revision, together with a
revision notice. Obsolete documents should be removed from all files and
points of use, returned to the group responsible, and logged in and destroyed.
The group responsible should have sole authority to destroy obsolete documents
except for one set of originals and revisions.
Revisions may be promulgated by the issue of entire new documents or of
individual replacement pages. In the case of minor revisions, pen-and-ink
posting on the original document with the action noted on the revision
notice is sufficient. The quality control coordinator should be responsible
for distributing documents and/or revisions and for obtaining the required
signatures.28
4.5.5 Configuration Control
An adequate program of equipment/hardware configuration control (e.g.,
equipment location, environment, component alteration and/or replacement)
will readily permit tracking all changes that are made to a data-producing
system that may affect data quality. This applies to individual instruments
as well as to entire data acquisition systems.
Authorization for configuration changes should be limited to one person,
preferably the project officer, to ensure that all changes to the facility
are properly documented. This documentation is essential for understanding
and explaining shifts in data patterns following such changes. All personnel
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involved in the task should be notified of changes so that any special
procedures required by the changes (e.g., recalibration of analyzers) will
be initiated. Finally, configuration control will provide a convenient way
of ensuring that all preventive maintenance procedures are performed on
schedule.
For monitoring systems, any sampling site changes or monitoring instru-
ment replacements should be recorded in a bound, page-numbered notebook
reserved for this purpose. Calibration should follow all such changes.
Configuration control for the laboratory environment is fully as impor-
tant as for extensive monitoring systems. It includes instrument location
in the laboratory as well as modifications that affect measurement data.
Temporary and/or permanent equipment configuration changes should be made
only when the effect is well characterized and has been demonstrated to
improve data quality.
4.5.6 Data Validation
Data validation has been defined by EPA as: "the process whereby data
are filtered and accepted or rejected based on a set of criteria."24 This
process may include any form of manual or computerized checks, but it clearly
involves specified criteria. The QC plan should clearly indicate that
raw data are not altered and how subsequent data sets are generated, with a
clearly defined audit trail.
Validation criteria may include evaluation of the data with respect to
physically determined checks (e.g., a record indicating a negative weight is
not reasonable). Similarly, as the sophistication of the model increases,
relational checks between measured parameters may also be used.
The data set to be statistically analyzed should be compared with the
first recorded form of the data to estimate the error rate. The QA officer
should request both the raw data and the analysis data set. QA functions
involve definition of error and specification of allowable error rates. In
the case of a small data set, quality control may consist of item-by-item
verification that the error rate does not exceed the allowable limit. For
large data sets, data validation should be considered as a hypothesis-testing
problem, with type I and II error probabilities34 chosen by appropriate QA
personnel. A random sample of size sufficient to achieve the desired signif-
65
-------
icance level and power should be drawn. This subsample is then compared
item by item with the corresponding raw data to determine if the error rate
is acceptable. QA requirements may include documentation in the form of a
hard copy of the subsample and access to the raw data. Dodge-Romig tables
may be employed to determine sample size, if 90 percent power is acceptable.35
4.5.7 Feedback and Corrective Action
For each task, a system for detecting, reporting, and correcting prob-
lems that may be detrimental to data quality must be established. The
feedback and corrective action system chosen should accommodate the need for
quick response and thorough communication and documentation of the problem
and its solution. If a solution is not immediately apparent, direct contact
between the project officer and the involved technician is the best approach
to corrective action.
An important aspect in improving the potential for effective feedback
and corrective action by task personnel is a quality assurance briefing.
The purpose of this briefing is to make the task personnel aware of how
their individual contributions to the task affect overall data quality.
Such briefings should take place during the initial phases of the task and
should be continued at specified intervals throughout the task.
The project officer should hold regular summary briefings with technical
personnel to discuss problems encountered in the research task. Monthly
progress reports and quarterly summary documents also aid in communication
of ideas and therefore improve overall data quality.
These activities provide an excellent opportunity to establish and
maintain an active employee-management feedback loop. Since bench-level
personnel should be the best observers of routine task operations, they are
the most likely to detect disturbances that may affect data quality. With
an effective feedback loop in operation, management can quickly become aware
of fluctuations that might otherwise go undetected. In general, it is
important to impress on task personnel that their daily conduct controls
total data quality.
Additional feedback systems should be established. For example, the
discovery of an impure substance by one investigator should be communicated
at once to all other users of the particular substance. This can be facili-
tated by the use of central stockroom records.
66
-------
A description of all problems detected, the solutions devised, and
estimates of the effect of the problems on data quality should be made
available to appropriate management on a regular basis.
4.6 REFERENCES
1. U.S. Environmental Protection Agency, Environmental Protection Agency
(EPA) Quality Assurance Policy Statement, Administrator's Memorandum,
May 30, 1979.
2. U.S. Environmental Protection Agency, Quality Assurance Requirements
for All EPA Extramural Projects Involving Environmental Measurements,
Administrator's Memorandum, June 14, 1979.
3. U.S. Environmental Protection Agency, Guidelines and Specifications
for Implementing Quality Assurance Requirements for EPA Grants and
Cooperative Agreements Involving EnvironmentalMeasurements, in prepara-
tion, Office of Research and Development, Washington, D.C.
4. U.S. Environmental Protection Agency, Guidelines and Specifications
for Implementing Quality Control Requirements for EPA Contracts and
Interagency Agreements Involving Environmental Measurements, QAMS-002/80,
Office of Research and Development, Washington, D.C.
5. U.S. Environmental Protection Agency, Guidelines and Specifications for
Preparing Quality Assurance Project Plans, QAMS-005/80, Office of
Research and Development, Washington, D.C., October 1980.
6. Nonclinical Laboratory Studies. Good Laboratory Practice Regulations.
Federal Register, December 22, 1978, p. 59986.
7. Good Laboratory Practice Standards for Health Effects, Federal Register,
May 9, 1979, p. 26362.
8. (a) Public Law 91-596, Occupational Safety and Health Act of 1970
(Dec. 29, 1970).
(b) Occupational Safety and Health Manual, U.S. EPA (Jan. 8, 1976).
(c) Safety Management Manual, U.S. EPA, TN 1440.1 (Dec. 4, 1972).
9. U.S. Environmental Protection Agency, Health Effects Research Laboratory,
Environmental Assessment--Short-Term Tests for Carcinogens, Mutagens and
other Genotoxic Agents, EPA-625/9-79-003, Research Triangle Park, NC,
July 1979.
10. Green, E. L. (ed.), Biology of the Laboratory Mouse, 2nd Edition, New
York: McGraw-Hill Book Company, 1966.
11. (a) Federal Register, Vol. 40, No. 50, March 13, 1975.
(b) Declaration of Helsinki, Recommendations Guiding Doctors in Clini-
cal Research, Jour, of American Med. Assoc., 197 (11):32, Septem-
ber 12, 1966.
67
-------
(c) The Institutional Guide to PHEW Policy on Protection of Human
Subjects, U.S. Government Printing Office, 1972, #0-445-427.
12. Non-Clinical Laboratories Studies: Regulations for Good Laboratory
Practice, Federal Register, December 22, 1978, pp. 59985-60025.
13. U.S. Environmental Protection Agency, Environmental Monitoring and
Support Laboratory, Handbook for Analytical Quality Control in Water
and Wastewater Laboratories, EPA-600/4-79-019, Cincinnati, OH, March
1979.
14. Title 49, Code of Federal Regulations.
15. Federal Express Service Guide, 2nd Quarter, 1980.
16. Guide for Handling Hazardous Materials, United Parcel Service, March
1980.
17. Domestic Mail Manual, Section 124.28.
18. Agee, W. S., and R. H. Turner, Application of Robust Statistical Methods
to Data Reduction, Technical Report No. 65, White Sands Missile Range,
1978.
19. Box, G.E.P., Robustness in the Strategy of Scientific Model Building,
in Robustness in Statistics. R. Launer and G. Wilkinson (eds.), New York:
Academic Press, 1979.
20. Huber, P. J., Robust Statistical Procedures, No. 27 in SIAM Regional
Conference Series, 1977.
21. U.S. Environmental Protection Agency, Handbook for Preparing Office of
Research and Development Reports, EPA 600/9-76-001, 1976.
22. U.S. Environmental Protection Agency, Health Effects Research Laboratory,
Health Effects Research Laboratory Procedures for Publishing Office of
Research and Development Technical and Scientific Materials, Research
Triangle Park, NC, July 1977.
23. U.S. Environmental Protection Agency, Quality Assurance Handbook for
Air Pollution Measurement Systems, Vol I - Principles, EPA 600/9-76-005,
Research Triangle Park, NC, March 1976.
24. U.S. Environmental Protection Agency, Quality Assurance Handbook for
Air Pollution Measurement Systems, Vol II - Ambient Air Specific Methods,
EPA-600/4-77-027a, Research Triangle Park, NC, 1977.
25. National Bureau of Standards, Catalog of NBS Standard Reference Mate-
rials, NBS Special Publication 260, U.S. Department of Commerce, Wash-
ington, DC.
68
-------
26. National Bureau of Standards, NBS Standard Reference Materials for
Environmental Research Analysis and Control, U.S. Department of Com-
merce.
27. World Health Organization, Biological Substances: International Stand-
ards, Reference Preparations, and Reference Reagents, Geneva: World
Health Organization, 1977.
28. Inhorn, S. L. (ed.), Quality Assurance Practices for Health Laboratories,
American Public Health Association, 1978.
29. Measurement principle and procedure for the measurement of nitrogen
dioxide in atmosphere (gas phase chemi luminescence), Title 40, Code of
Federal Regulations, Part 50, Federal Register, December 2, 1976,
p. 52688.
30. National Archives and Records Service, Traceability requirements for
calibration gases, Title 40, Code of Federal Regulations, Part 60.13
31. American Society for Testing and Materials, Annual Books for ASTM
Standards, Philadelphia, PA, annual publication.
32. Scaringelli, E. P., A. E. O'Keefe, E. Rosenberg, and J. P. Bell, Prepa-
ration of known concentrations of gases and vapors with permeation
devices calibrated gravimetrically, Analytical Chemistry, 42 (8):
July 1970.
33. Federal Register, December 14, 1977, p. 62971.
34. Steel, R.G.D., and J. H. Torrie, Principles and Procedures of Statistics,
New York: McGraw-Hill, 1960.
35. Dodge, H. F. , and H. G. Romig, Sampling Inspection Tables, New York:
John Wiley & Sons, 1959.
69
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APPENDIX A
GUIDELINES FOR PREPARATION OF HERL-RTP PROTOCOLS
A.I Research Tasks
A. 2 Support Tasks
A.I. GUIDELINES FOR PREPARATION OF PROTOCOLS FOR HERL-RTP
RESEARCH TASKS
These guidelines provide a standardized format for use by project
officers in preparation of all HERL-RTP research task protocols. Research
protocols must include:
1. Cover sheet
2. Summary sheet
3. Technical research plan, including QA Project Plan
Research protocols for all HERL-RTP research tasks must be submitted for
review and approval as indicated on the cover sheet. A copy of the approved
protocol must be filed with the QA officer.
70
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A.1.1 RESEARCH PROTOCOL COVERSHEET
TASK NO.
Nine-Digit Task Number
RESEARCH PROTOCOL
HEALTH EFFECTS RESEARCH LABORATORY
Research Triangle Park, North Carolina
EPA
•"Routine Procedures on Reverse***
Extramural Research Intramural Research
Project Institution:
Principal Investigator:
DU Name: Reporting Division/Branch:
PROJECT TITLE:
PROJECT OFFICER: /s/ Date:
Original Submission
Revision =
i
SAFETY PROTOCOL(s) Approved Copy Attached
Not Required/?/ Data-
Division Director
REVIEWERS:
f=RO -Safety Offio
-------
- HERL Research Protocol, when required, must include the Health and
Safety Protocol.
- The combined protocols will be reviewed as a single entity.
ROUTINE PROCEDURES:
1. Project Officer submits combined protocols to all reviewers.
2. Within 3 weeks, all reviewers will send approved protocols to the
Protocol Coordinator (MD-70) who certifies reviewers' signatures and
routes through Administrative channels.
3. Protocol Coordinator distributes approved protocols to the Division
Directors, Branch Chiefs, Project Officers, and ERC Safety Office.
72
-------
A.1.2 SUMMARY SHEET
A one-page summary should be prepared to provide the reviewer with a
brief overview of the objectives and approaches of the task. This informa-
tion should be presented under the following headings.
1. Overall Objective: State clearly and concisely the overall
objective of the proposed study.
2. Proposed Means of Meeting Objective: Describe in summary
form the means by which the objective will be met. List the
components or subtasks that will make up this study.
73
-------
A.1.3 TECHNICAL RESEARCH PLAN
1. Introduction: State the overall objectives of the study and summarize
briefly the approach to be taken to meet these objectives. Normally a
project will be divided into subtasks. List these subtasks and describe
each under the following headings: Hypothesis, Proposed Means of
Testing the Hypothesis, Experimental Design and Statistical Methodology,
and Quality Assurance Project Plan.
2. Hypothesis: State clearly the hypothesis to be tested for the subtask.
Include a concise discussion of the facts and/or observations upon
which this hypothesis is based, and any alternative hypotheses.
3. Proposed Means of Testing the Hypothesis: Describe clearly the method
or methods by which the hypothesis'will be tested. Describe each
experiment to be performed in sufficient detail to illustrate the
relationship of the experiment to the hypothesis. Describe the vari-
ables that are to be controlled.
4. Experimental Design and Statistical Methodology: Describe the statis-
tical basis for the collection of data and/or the testing schedule.
Determine (estimate) differences in results between test and control
measurements that would be accepted as significant; refer to previous
work whenever possible to substantiate decisions regarding these differ-
ences. Describe measurement design, numbers of measurements, numbers
of exposures (i.e., animals to be tested), level of exposure, time of
exposure, measurement conditions, etc., that would permit identification
of significant differences between test and control measurements in a
reasonable period of time and/or in a cost-effective manner.
5. Quality Assurance Project Plan: Describe all QA/QC activities that
will be conducted to ensure that the data produced in the task are of
adequate and documented quality. The project officer must consider all
aspects of the study that may introduce significant variability or are
critical to the success of the task, and determine appropriate QA/QC
measures and their scheduling. QA Project Plans must address the
following:
a. QA/QC objectives for measurement data, in terms of precision,
accuracy, completeness, representativeness, and comparability
b. Personnel (adequacy of training and experience)
c. Facilities, services, equipment, and supplies
d. Recordkeeping
e. Chain-of-custody
f. Sample collection
g. Sample analysis
74
-------
h. Data processing, analysis, validation, and reporting
i. Specific procedures to be used to routinely assess data precision,
representativeness, comparability, accuracy, and completeness of
the specific measurement parameters involved. (This section is
required for all QA Project Plans.)
j. Internal QC checks and frequency
k. QA performance and systems audits, and frequency
1. Calibration procedures, references, and frequency
m. Preventive maintenance procedures and schedules
n. Documentation control
o. Configuration control
p. Feedback and corrective action
q. QA reports to management
QC activities may also be described in other parts of the protocol and
should be clearly identified as such.
Support Tasks and Other Activities Required to Successfully Complete
This TasT:Support tasks and other major resources that will be re-
quired to successfully complete the study should be described. These
might include exposure measurements, animal care, consultation with
regard to statistical treatment of data, and testing the agreement of
various models with the data collected. Support tasks should be identi-
fied by task number, title, and project officer.
75
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A.2 GUIDELINES FOR PREPARATION OF PROTOCOLS FOR HERL-RTP SUPPORT TASKS
These guidelines provide a standardized format for use by project
officers in preparation of protocols for all HERL-RTP support tasks. Sup-
port task protocols must include:
1. Cover sheet
2. Summary sheet
3. Technical project plan, including QA Project Plan.
Protocols must be submitted for review and approval as indicated on the
sample protocol cover sheet. A copy of the approved protocol must be filed
with the QA officer. Protocols must be revised annually and approved for
continued funding.
76
-------
A.2.1 SUPPORT TASK PROTOCOL COVERSHEET
TASK NO.
Nine-Digit Task Number
SUPPORT TASK PROTOCOL
HEALTH EFFECTS RESEARCH LABORATORY
Research Triangle Park, North Carolina
EPA
•••Routine Procedure! on Reverse***
Extramural Support
Project Institution:
Principal Investigator:
DU Name:
Intramural Support
Reporting Division/Branch:
PROJECT TITLE:
PROJECT OFFICER: /s/ Date:
Original Submission
Revision #
SAFETY PROTOCOL(s) Approved C
Not Require
REVIEWERS:
ERC Safety Off ica
Pear Raviawar
opy Attached '
«H/«/ DatB!
Division Director
natn:
DatB!
Lah Animal Support J^*V DatB:
NX
Duality Assurance >"% l^S^ Data:
Statistics/Data P">OW?ing ^^J Ratn;
OU Coordinator
APPROVALS:
Rranch Chi»f
Division Director
Laboratory Director
Data-
Data-
Data-
Date:
KEY WORDS
77
-------
- HERL Support Task Protocol, when required, must include the Health and
Safety Protocol.
- The combined protocols will be reviewed as a single entity.
ROUTINE PROCEDURES:
1. Project Officer submits combined protocols to all reviewers.
2. Within 3 weeks, all reviewers will send approved protocols to the
Protocol Coordinator (MD-70) who certifies reviewers' signatures and
routes through Administrative channels.
3. Protocol Coordinator distributes approved protocols to the Division
Directors, Branch Chiefs, Project Officers, and ERC Safety Office.
78
-------
A.2.2 SUMMARY SHEET
A one-page summary should be prepared to provide the reviewer with a
brief overview of the objectives and approaches of the support task. This
information should be organized as follows:
1. Overall objective: State clearly and concisely the overall
objective or purpose of the support task.
2. Proposed means of meeting objective: Briefly summarize the
means by which the support task objective will be met. List
the specific areas in which support will be maintained and the
resources (e.g., facilities, equipment, personnel) available.
79
-------
A.2.3 TECHNICAL PROJECT PLAN
1. Introduction: State the overall objective or purpose of the support
task and summarize briefly the resources available and approach(es) to
be taken to meet the objective. When a support task can be divided
into subtasks, list these separately and discuss each under the follow-
ing headings: Experimental Design and Statistical Methodology, and
Quality Assurance Project Plan.
2. Experimental Design and^Statistical Methodology: Describe the experi-
mental design or analytical methods to be used to meet the task objec-
tives. Documentation of facilities, equipment, and personnel should
include identification and statement of capabilities.
Where applicable, describe the statistical basis for data collec-
tion and/or testing schedules. Specify data quality estimates in terms
of accuracy and precision, and determine (estimate) differences in
results between test and control measurements that would be accepted as
significant. Provide documentation of previous performance whenever
possible to support statistical rationale and data quality projections.
3. Quality Assurance Project Plan: Describe all quality assurance/quality
control activities that will be conducted to ensure that the data
produced are of adequate and documented quality. The project officer
must consider all aspects of the support task that may introduce sig-
nificant variability or are critical to the success of the associated
research tasks and determine appropriate QA/QC measures and their
scheduling. QA Project Plans must address the following:
a. QA/QC objectives for measurement data, in terms of precision,
accuracy, completeness, representativeness, and comparability
b. Personnel (adequacy of training and experience)
c. Facilities, services, equipment, and supplies
d. Recordkeeping
e. Chain-of-custody
f. Sample collection
g. Sample analysis
h. Data processing, analysis, validation, and reporting
i. Specific procedures to be used to routinely assess data precision,
representativeness, comparability, accuracy, and completeness of
the specific measurement parameters involved. (This section is
required for all QA Project Plans.)
j. Internal QC checks and frequency
80
-------
k. QA performance and systems audits, and frequency
1. Calibration procedures, references, and frequency
m. Preventive maintenance procedures and schedules
n. Documentation control
o. Configuration control
p. Feedback and corrective action
q. QA reports to management
QC activities may also be described in other parts of the protocol and
should be clearly identified as such.
4. Other Activities Required to Successfully Conduct This Task: Other
major resources that will be required to successfully conduct the
support study should be described.
5. Research Tasks Requiring This Support Task. Identify all research tasks
by task number, title, and project officer that are known to be supported
by this task.
81
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APPENDIX B
QUALITY ASSURANCE REVIEW FORMS FOR EXTRAMURAL PROJECTS
B.I Contracts (QAR-C, QAMS-002/80)
B.2 Interagency Agreements (QAR-IA, QAMS-002/80)
B.1 QUALITY ASSURANCE REVIEW FOR EXTRAMURAL PROJECTS
(CONTRACTS)
I. GENERAL INFORMATION
1. Descriptive Title:
2. Sponsoring Program Office:
3. Approximate Dollar Amount:
4. Duration:
II. DESCRIPTION OF WORK
This contract requires the generation of environmental measurements
Yes No
III. QUALITY ASSURANCE (Projects requiring environmental measurements)
Yes No
1. Submission of a written QA Program Plan (commitment of the
offerer's management to meet the QA requirements of the scope ^
of work) is to be included in the contract proposal.
2. Submission of a written QA Project Plan is to be incluH' i the
contract proposal.
3. A written QA Project Plan is required ?<• ^» *
4. Audit samples or devices are a*'-' ^(^ |S^^ *" -nt
to this project (see list on revei •<""\V^J^^
5. Performance on available audit i \S .cquired as part
of the evaluation criteria (see list .,
-------
QA Performance Audits or Split Sample Comparisons
QC Reference Split Samples FREQUENCY
Sample or Device for
Measurement Available Cross-Comparison Preaward During Contract
1.
2.
3.
4.
5.
6.
7.
8.
9.
0.
Planned Date for QA Systems Audit Planned Audit Team
Preaward During Contract
QA Reports Schedule
Progress Reports
Final Report
Before submissioft of the RFP to the Contracts Office, the signatures below verify that the QA
requirements have been established.
QA Officer: Project Officer:
Signature Dan Signature Date
After signatures, a copy of this form should be mailed, with the RFP, to the Contracts Office.
After the selection of the awardee, , the signatures
below verify that the preaward requirements have been satisfactorily fulfilled.
QA Officer: Project Officer:
Signature Date Signature Date
After signatures, one copy of the completed form should be mailed to the Quality Assurance Manage-
ment Staff and one copy to the Contracts Office.
QA Form QAR-C
83
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B.2 QUALITY ASSURANCE REVIEW FOR EXTRAMURAL PROJECTS
(INTERAGENCY AGREEMENTS)
I. GENERAL INFORMATION
1. Descriptive Title:
2. Sponsoring Program Office:
3. Approximate Dollar Amount: Total:
4. Duration:
II. DESCRIPTION OF WORK
This agreement requires the generation of environmental measurements .
Yes No
III. QUALITY ASSURANCE INTERAGENCY AGREEMENT REQUIREMENTS
(Projects requiring environmental measurements)
Yes No
^
1. A written detailed QA Project Plan is requir'
in the interagency agreement or identifi'
interagency agreement (required)
2. Audit samples or devices are available i .neters
relevant tr this project (see list on reversv
3. Performance on available audit samples or devices is required:
Prior to agreement
During project performance
(See schedule on reverse side)
4. An on-site evaluation of performing agency's facilities will be made
to determine that a QA system is operational and the capability
exists for successful completion of this project:
Prior to agreement
During project performance
(See schedule on reverse side)
5. Periodic and final QA Reports are required
QA Form QAR-IA
84
-------
QA Performance Audits or Split Sample Comparisons
QC Reference Split Samples Planned Schedule
Sample or Device for During
Measurement Available Cross-Comparison Interagency Agreement
1.
2.
3.
4.
5.
6.
7.
8.
9.
0.
Planned Date for QA Systems Audit Planned Audit Team
Preaward During Agreements
QA Reports Schedule
Progress Reports
Final Report
QA Officer: Project Officer:
Signature Date Signature Date
After signatures, one copy of this completed form should be mailed with the Interagency Agreement
request to the Contracts Office, and one copy to the Quality Assurance Management Staff.
QA Form QAR-IA
85
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APPENDIX C
SELECTED NATIONAL BUREAU OF STANDARDS STANDARD
REFERENCE MATERIALS
Source: National Bureau of Standards, Catalog of NBS
Standard Reference Materials for Environmental
Research Analysis and Control
Clinical Laboratory Standards
These SRM's are intended for use in calibrating apparatus and validating analytical methods used in
clinical and pathological laboratories, and to assist manufacturers of clinical products in meeting the chemical
and physical specifications required for clinical chemicals. (For details on SRM's 930D and 93Ib, see
Spectrophotometric Filters, page 65.)
SRM
900
91 la
912
913
914
915
916
917
918
919
920
921
922
921
924
925
926
927
928
929
910D
93lb
932
913
934
935
936
937
1968
Type
Antiepilcpsy Drug Level Assay
Cholesterol
Urea
Uric Acid
Crcatinine
Bilirubin
Potassium Chloride
Sodium Chloride
Cortisol . ....
Tris (hvdroxymethyl) aminomethane
Tris (hydroxymethyl) aminomethane HCI
VMA (4-hydroxy-3-methoxymandclic acid)
Bovine Serum Albumin (Powder)
Bovine Serum Albumin (7% Solution)
Lead Nitrate
Magnesium Cluconate
Glass Filters for Spectrophotometry ....
Liquid Filters for Spectropholomctry .
Quart/ Cuvette for Spectrophotometry . . ... ....
Clinical Laboratory Thermometers ....
Clinical Laboratory Thermometer
Crystalline Potassium Dichromate .
(UV Absorbance) Standard
Quinine Sulfate Dihydratc
(Fluorescence)
Iron Metal
Gallium Melting Point
Purity %
998
997
99.7
998
999
990
999
99.9
99.9
998
989
99.9
99 7
100.0
99.4
•*
•»
10000
IN PRF.P
+
+
+
t
tt
(99 972)'**
(98 2)***
99 90
+++
Wt/Unil
Set of 4 vials
2 K
25 n
10 e
10 e
20 K
100 mg
25 e
30 g
30 g
50 K
1 a
25 g
35 8
30 E
1 g
5 g
10 vials. 2.15 mLea.
30 e
10 g
Set of 3
3 sets of 4
Sel of 3
15 e
I I!
50 R
1 ea
*SRM 915. Calcium Carbonate, was used to develop the first referee method of analysis in clinicnl chemistry. This work is described
in NBS Special Publication 260-36. A Referee Method for the Determination of Calcium in Serum. (Sec inside of hack cover for
ordering instructions.)
+Certified for optical properties (see p. 65.)
flndividually calibrated at 0°C and either 25. 30, or 37 °C.
ttlndividually calibrated at 0, 25, 30. and 37 °C.
••Conforms to NCCLS specification ACC-I.
"•Apparent purity, certified for optical properties.
+++Melting Point Certified at 29.7723 °C. (See p. 61.)
86
-------
Biological Standards
These SRM's are intended for use in the calibration of apparatus and methods used in the analysis of
biological materials for major, minor, and trace elements.
(Values in parentheses are not certified, but are given for information only.)
SRM
l<66
156"
I'^S
1569
I5r0
Type
Ovster Tissue IN PREP
Wheat Flour
Rice Flour
Brewers Yeast
Spinach. Trace Elements
Wt/llnit
(prams)
80
SO
50
60
1571
1573
1575
1577
Pine Needles
75
70
70
50
Content in pg/g (or where noted, wt %)
ELEMENT \ SRM/ 1566
Beryllium
Bromine
Chlorine
Cobalt
Fluorine
Lead . •
Molybdenum
Nickel . . .
Phosphorus
Sodium
Sulfur
Thallium
Thorium
Zinc
—
1567
(0.006)
(9)
0.032
0.019%
2.0
18.3
8.5
0.001
(0.4)
(0.18)
0.136%
(1)
LI
8.0
(5=0.002
10.6
1568
0.41
(1)
0.029
0.014%
0.02
2.2
8.7
20.1
0.0060
(1.6)
(0.16)
0.112%
(7)
0.4
6.0
O0.002
19.4
1569
2.12
1570
870
(0.04)
0.15
(30)
(54)
(1.5) .
1.35%
4.6
(1.5)
12
(0.02)
550
(0.37)
1.2
165
0.030
(6)
(5.9%)
0.55%
3.56%
12.1
(0.16)
87
(0.03)
0.12
0.046
50
1571
2.9
10
(44)
0.027
(O.I)
33
(10).
0.11
2.09%
(0.04)
(690)
2.6
iO.2)
12
(4)
(0.08)
(0.17)
300
45
(0.6)
0.62%
91
0.155
0.3
1.3
2.76%
0.21%
1.47%
12
0.08
82
37
(1900)
(0.01)
0.064
0.029
25
1573
<0.12%)
0.27
(30)
(26)
(3)
3.00%
(1.6)
4.5
(0.6)
II
(0.04)
690
(0.9)
6.3
(0.7%)
238
(0.1)
(5.0%)
0.34%
4.46%
16.5
(0.13)
44.9
(0.05)
0.17
0.061
62
1575
545
(0.2)
0.21
(9)
«0.5)
0.41%
(0.4)
2.6
(O.I)
3.0
(0.006)
200
(0.2)
10.8
675
0.15
(3.5)
(1.2%)
0.12%
0.37%
11.7
(0.03)
4.8
(0.05)
0.037
0.020
1577
(0.005)
0.055
(0.017)
0.27
124
(0.27%)
0.088
(0.18)
193
(0.05)
(0.18)
268
0.34
604
10.3
0.016
(3.4)
10.6%
(1.1%)
0.97%
18.3
I.I
(17)
(0.06)
0.243%
(0.14)
(0.05)
(0.0008)
130
87
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Environmental Standards
Analyzed Gasas
These SRM's are intended for the calibration of apparatus used for the measurement of various compo-
nents in gas mixtures, and in some cases for particular atmospheric pollutants. Each SRM is accurately
certified and is primarily intended to monitor and correct for long-term drifts in instruments used. Each
cylinder (except 1609) contained 870 liters at STP prior to certification, and thus contains somewhat less than
870 L (SRM 1609 contained 68 liters). All cylinders conform to the appropriate DOT specifications.
SRM
1609
1638
1639
1660
1661
1662
1663
1664
I665a
1666a
I667a
I668a
!669a
1673a
1674a
1675a
I677b
I678b
I679b
I680a
168la
1683a
1684a
I685a
1686a
1687a
2613
2614
2619
2620
2621
2622
2623
2624
2625
2626
Type
Oxygen in Nitrogen
Methane in Air
Sulfur Dioxide in N2
Sulfur Dioxide in N2
Sulfur Dioxide in N3
Sulfur Dioxide in Nj
Propane in Aif
Propane in Air
Propane in Air
Carbon Dioxide in Nitrogen
Carbon Dioxide in Nitrogen
Carbon Monoxide in Nitrogen
Nitric Oxide in Nitrogen
Nitric Oxide in Nitrogen
Nitric Oxide in Nitrogen
Nitric Oxide in Nitrogen
Carbon Monoxide in Air
•Carbon Dioxide in N2
Carbon Dioxide in Nj
Carbon Dioxide in N,
Nominal Concentrations
Oj, 20.95 mole percent
CH4, 0.951 ^mol/mol (ppm)
CH4, 9 43 //mol/mol (ppm)
CH4 4 10 p mol /mol (ppm)
C3H,, 0.976 jimol/mol (ppm)
SOj, 480 /imol/mol (ppm)
SO2, 942 ^mol''mol (ppm)
SOj 1497 /imol'mol (ppm)
SOj 2521 ji mol/ mol (ppm)
CjHj, 50 PPm
C,H8 100 ppm
CjH8. 500 ppm
CO; 1 0 mol <%
CO,, 7.5 mol %
CO2, 150 mol ^
CO, 10 ppm
CO 50 ppm
CO 100 ppm
CO 500 PPm
CO 1000 PPm
NO, 50 ppm
NO 100 ppm
NO 250 ppm
NO 500 ppm
NO 1000 ppm
CO, 18.1 /imol/mol (ppm)
CO 41 0 44 mol'' mol (ppm)
COj 0 5 mol percent
CO; I 0 mol percent
COj 2 0 mol percent
COj, 2 5 mol percent
CO2. 1 0 mol percent
CO2 3 5 mol percent
CO-,. 4.0 mol oercent
88
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Analyzed Liquids and Solids
These SRM's are intended for use in the analysis of materials for elements of interest in health or environ-
mental problems. See also: Clinical SRM's page 39, and Industrial Hygiene SRM's page 44.
SINGLE ELEMENT
Concentrations:
Weight percent — boldface
Microgram per gram — light face
Nanogram per milliliter — italics
SRM
1579
1620
1621
I622a
I623a
1624
1610
I64la
I642a
Type
Powdered Lead Base Paint
Sulfur in Residual Fuel Oil
Sulfur in Residual Fuel Oil
Sulfur in Residual Fuel Oil
Sulfur in Residual Fuel Oil
Sulfur in Distillate Fuel Oil
Trace Mercury in Coal
Mercury in Water (/ug/mL)
Mercury in Water (ng/ mL)
Unit Size
35 g
IN PREP
100 mL
IN PREP
IN PREP
100 mL
50 g
IN PREP
950 mL
Lead
11 87%
Sulfur
1 05%
211%
Mercury
Oil „ at o
SRM
1636
1637
1638
Type
Lead in Reference Fuel
Lead in Reference Fuel ...
Lead in Reference Fuel
Element
Certified
Pb
Pb
Pb
Nominal
Concentration
12 20 28 and 773 /ig/g*
12 20 and 28/ig'g*
773 ug'E*
Vol/Unit
(ml.)
12 vials
•Equivalent grams per gallon are: 0.03, 0.05, 0.07, and 2.0 g/gal, respectively.
MULTI-ELEMENT
Concentrations:
Weight percent — boldface
Microgram per gram — light face
Nanogram per gram — italics
SRM
I632a
I633a
1634
1635
I643a
1645
1646
1648
Type
Urban Paniculate
Unit Size
75 g
IN PREP
100 mL
75 g
IN PREP
70 g
IN PREP
2?
Al
(1.64%)
—
As
9.3
(0.095)
.42
_
115
Be
_
(
-------
Industrial Hygiene Standards
Organic Solvents on Charcoal
These SRM's consist of charcoal tubes to which have been added known quantities of the specified organic
solvent. Each SRM consists of eight tubes, two each of four solvent levels (except 2661a). SRM 266la consists
of nine tubes, three each of three solvent levels. Each tube is color coded for both the solvent and the solvent
level.
SRM
266 la
2662
2663
2664
2665
2666
2667
Solvent \ Solvent Color Code
\
\
m-Xytene
p-Dioxane
1,2-Dichloroethane
Trichlorocthylene
red
blue
green
white
yellow
black
gold
Solvent level, mg per tube
(Solvent Level Color Code)
1
(red)
16*
0.040
.016
.098
.147
.286
.033
II
(blue)
30*
0.293
.112
.381
.516
1.03
0.114
III
(green)
54*
1.79
0.996
1.56
2.14
4.09
0.414
IV
(black)
8.38
6.49
5.80
6.87
15.4
1.58
•>ig per tube
Freeze-Dried Urine
These SRM's consist of two bottles of freeze-dried human urine, one containing a low and one an elevated
level of the element certified.
SRM
2671
2672
Element
Low Level*
(mg-Ll
0835
00042
Elevated Level*
(nig/L)
7 14
0 294
•When reconstituted with 50 mL water.
Materials on Filter Media
These SRM's consist of potentially hazardous materials deposited on filters to be used to determine the
levels of these materials in industrial atmosphere.
SRM
2675
2676a
2679
Type
Beryllium on Filter Media
Metals on Filter Media
Quart? on Filter Media . . ...
Material Certified
Beryllium
Cadmium
Lead
Manganese
Zinc
Quartz
Clay
1
0.052
1.02
6.96
1.97
9.86
3.8
(400)
Quantity
(nn.it
II
0.26
2.50
15.23
9.89
49.52
29.9
(370)
Certified
'liter)
III
1.00
10.18
29.64
19.70
99.22
76.1
(320)
IV
193.2
(200)
90
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Trace Element Standards
The SRM's listed below were designed for trace chemical analysis, specifically for calibrating instruments
and checking analytical techniques and procedures used to determine trace elements in various inorganic
matrices. In addition many SRM's certified for chemical composition have one or more constituents certified at
or below the 100 n g/g level.
Trace Element Standards (Nominal Concentrations)
Element
Boron
Cobalt
Cooper
Dysprosium
Gadolinium
Gold
Iron
Lead
Molybdenum
Nickel
Rubidium
Silver
Thallium
Uranium
Zinc
607
(ppm)
523.90
65.485
610-611
(ppm)
(351)
(398 51
(390)
(444)
(25)
458
426
485
(422.6)
458.7
(461)
(49.43)
425.7
(254)
515.5
(61.8)
(447)
457.2
(437)
461.5
(433)
612-613
(ppm)
(4H
(32)
(39)
(37 8)
(35 5)
(377)
(35)
(39)
(36)
(39)
(5)
51
(36)
3857
O96)
(36.94)
(36)
38.8
(64)
(6.67)
31.4
(39)
22.0
78.4
15.7
(44)
37.79
(50.1)
37.38
(42)
614-615
(ppm)
(1 06)
(1 30)
(0 55)
(099)
0 71
1 34
(099)
(1 3)
(05)
(0 75)
13.5
(0 83)
2.32
(1.41)
(0.79)
(0.95)
30
(0.17)
0.855
(0.59)
0.42
45.8
0.269
(0.74)
0.748
(3.1)
0.823
(2.43)
616-617
(ppm)
(0 078)
(020)
(065)
(0 23)
(0 18)
(0 26)
(II)
(0 034)
1.85
062
29
(0.004)
0.0998
(0.026)
41.72
(0.0082)
(0.025)
0.0252
(2.5)
0.0721
In addition to the.36 elements listed above, the Glass SRM's contain the following 25 elements: As, Be. Bi. Cs. Cl, K. Ge, Hf. Hg. Li,
Ui. Mg. Nb, P, Pr, Se, S, Te, Tb, Tm, Sn, W, V, Y, and Zr.
91
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CERTIFIED PHYSICAL PROPERTIES STANDARDS
Ion Activity Standards
These SRM's are intended for use in the preparation of solutions for the calibration of specification
electrodes. This includes the pH and pD measuring systems.
pH Standards
These SRM's are furnished as crystals for the preparation of solutions of known hydrogen ion con-
centration "for calibrating and checking the performance of commercially available pH materials and
instruments. They are furnished with certificates giving directions for preparation of the solutions and
tables of pH values at various temperatures.
SRM's 186Ic and 186IIc, 191 and 192, and 922 and 923, are certified for use in admixture only. At an
equimolar (0.025 molal) mixture of SRM's 186Ic and 186IIc, a pH(S) of 6.863 at 25 °C is obtained. Direc-
tions also are furnished for the preparation of a physiological reference solution from 186lc and 186Mc
having a pH(S) of 7.415 at 25 °C.
SRM
185e
I86Ic
I861!c
I87b
188
189
191
192
922
923
Type
Potassium dihydrogen phosphate 1
Disodium hydrogen phosphate /
Sodium bicarbonate!
Sodium carbonate /
Tris(hydroxymethyl)aminomethane \
Tris(hydroxymethyl)aminomethane hydrochloride/
pH(S)
(at 25 °C)
4.004
( 6.863 l
17.415 f
9.183
3.557
1.679
10.01
7.699
WfUnit
(grams)
60
30
30
30
60
65
30
30
25
35
pD Standards
These SRM's are furnished as crystals for preparation of solutions of known deuterium-ion concentra-
tion for the calibration and correction of pH indicating equipment to indicate pD data. SRM's 21861 and
218611, and 2191 and 2192, are certified for use in admixtures only.
SRM
21861
218611
2191
2192
Type
Potassium dihydrogen phosphate')
Disodium hydrogen phosphate J
Sodium bicarbonate!
Sodium carbonate J
pD(S)
Values
741
10.74
Wt/Unit
(grams)
30
30
30
30
Ion-S«l»ctiv8 Electrode*
These SRM's are certified for the calibration of ion-selective electrodes and have conventional ionic
activities based on the Stokes-Robinson hydration theory for ionic strengths greater than O.I mole per liter.
SRM
2201
2202
2203
TyF*
Potassium Chloride
Potassium Fluoride
Certified Property
pNa, pCl
pK, pCl
pF
Wt/Unit
(grams)
125
160
125
92
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SECTION 5
EXTERNAL QUALITY ASSURANCE FOR RESEARCH PROJECTS
Every measurement activity supported by HERL-RTP must be performed in
accordance with an approved research protocol, including a QA Project Plan
and the research hypothesis, on file with the Quality Assurance officer.
Objective evaluation of task performance (i.e., application of Good Labora-
tory Practices (GLPs)) and adherence to QA Project Plans is accomplished
through periodic audits by nontask personnel. Such audits may be initiated
at the request of the project officer, the QA officer, or the QAMS. They
may be conducted on new projects requisite to funding, or on ongoing projects
either in response to identified problems in performance or as part of a
routine evaluative program. The following discussion focuses on these
external quality assurance aspects of the measurement task.
5.1 SYSTEMS AUDITS
Systems audits consist of an evaluation of the various components of a
research operation, principally through inspection. The first step of a
systems audit is an investigation of the laboratory's activities via inspec-
tion of protocols, standard operating procedures, proposals, reports, and
scientific publications. After inspection of these materials and contact
with key laboratory personnel to clarify questions, an onsite inspection may
be performed. The onsite systems audit consists of inspection of facilities
and operations, interviews with laboratory personnel, and reviews of key
operations and documentation. The systems audit may be scored using a
checklist comparing actual laboratory practices with some standard such as
FDA or proposed EPA GLPs.1 2 Scoring schemes may be devised to quantify
results of a systems audit.
The objective of the onsite qualitative systems audit is to assess and
document: (1) facilities; (2) equipment; (3) personnel; (4) recordkeeping;
(5) data validation and management; (6) operation, maintenance, and calibra-
93
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tion procedures; and (7) reporting aspects of the total quality control
program for a project. The review should:
1. Identify existing system documentation; i.e., maintenance
manuals, organizational structure, operating procedures;
2. Evaluate the adequacy of the procedures as documented; and
3. Evaluate the degree of use of and adherence to the documented
procedures in day-to-day operations, based on observed condi-
tions and a review of applicable records on file.
From qualitative measures of data quality, an auditor(s) independent of
the task organization can assess the suitability of the facilities and
operations to meet project goals and identify specific areas where corrective
actions may be implemented.
5.2 PERFORMANCE AUDITS
Quantitative measurements and comparisons provide the best objective
estimates of data quality. The performance audit consists of evaluating the
measurement aspects of the laboratory research operation being audited. The
laboratory is usually given samples to be analyzed or to be used in some
test and their results are compared to expected results and judged for
accuracy and precision. A pivotal issue in the proper interpretation of
audit results is whether or not high quality reference standards are avail-
able.
The National Bureau of Standards has developed a series of environmen-
tally related Standard Reference Materials (NBS-SRMs).3 4 A current catalog
of NBS-SRMs may be obtained from:
Office of Standard Reference Data
National Bureau of Standards
Washington, DCT 20234.
In addition, the World Health Organization maintains information on world-
wide sources of biological standards.5
Appropriate use of available reference materials by the auditor can
provide an objective measure of specific parameter data quality. A variety
of techniques, all of which should be designed as blinds (i.e., with opera-
tion personnel unaware of the nature of the reference sample), are available.
Direct analysis of the reference material and routine duplicate analysis of
the samples (one of which is "spiked" with a known amount of the reference
94
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material) are two possible uses of reference materials in performance audits
of analytical systems.
Unfortunately, NBS-SRMs do not exist for some environmentally related
measurements (e.g., known mutagens for audit testing in the Ames/Salmonella
reverse mutation assay). In such cases, techniques should he devised for
probing the quality of the task measurement methods. Round-robin analysis
of aliquots of a single sample may be performed by any number of laboratories
Although accuracy (i.e., deviation from a true value) cannot be measured, an
estimate of analytical variability (precision) is available. For labile
samples, collaborative (side-by-side) analysis may be used. This is equiva-
lent to the round-robin test, but is performed at one location and at approx-
imately the same time. To give a measure of various research system compo-
nents' variability, interlaboratory and intralaboratory analysis/measurement
programs may be designed. In this case, it is important that the statistical
design of such testing recognizes such aspects as operating shift changes,
diurnal biological changes, and other nonrandom variability in the sample(s)
and total measurement system.
After completion of the performance audit, it is useful to present the
results to the audited laboratory for review. This review can be a starting
point for cooperative identification of sources of measurement weakness and
subsequent corrective action.
5.3 REFERENCES
1. Department of Health, Education, and Welfare, Food and Drug Administra-
tion Nonclinical Laboratory Studies Good Laboratory Practice Regula-
tions, Federal Register, 43 (247), December 22, 1978, p. 59986.
2. U.S. Environmental Protection Agency, Proposed Good Laboratory Practice
Standards for Health Effects, Title 40, Code of Federal Regulations,
Part 772, Federal Register, 44 (91), May 9, 1979, p. 27362.
3. National Bureau of Standards, Catalog of NBS Standard Reference Mate-
rials, NBS Special Publication 260, U.S. Department of Commerce, Wash-
ington, DC.
4. National Bureau of Standards, NBS Standard Reference Materials for
Environmental Research Analysis and Control, U.S. Department of Com-
merce.
5. World Health Organization, Biological Substances: International Stand-
ards, Reference Preparations, and Reference Reagents, Geneva: World
Health Organization, 1977.
95
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SECTION 6
GUIDELINES FOR ATMOSPHERE GENERATION AND MONITORING
6.1 INTRODUCTION
In the HERL-RTP exposure facilities and in population studies, the effects
of various atmospheric pollutants on test subjects are evaluated. These
pollutants are in gaseous and/or aerosol form. The exposure facilities are
used to study the effects of synthetic atmospheres on humans and other test
subjects. Population studies evaluate the effects of the ambient atmosphere
on humans.
The generation of synthetic atmospheres and the monitoring of both arti-
ficial and ambient atmospheres are extremely complex tasks. Guidelines for QA
planning for air pollution measurement systems1 and ambient air methods2 have
been developed by EPA. The goal of these tasks is to produce high quality
exposure effects data, hence the details of generation, sampling, and analysis
techniques must be considered within the QA Project Plans. In human exposure
studies, QA planning begins with subject safety, continues in the experimental
tests, and finally provides the basis for estimating the confidence limits on
the exposure-effect relationships.
6.2 ATMOSPHERE GENERATION
The test atmosphere in an exposure chamber must be well-characterized, in
terms of both the composition and the concentration of the components. Expo-
sure experiments may run for a few hours or for several consecutive days. The
total dose, as well as the instantaneous concentration level, is important in
such experiments. Therefore, it is essential that the exposure source output
be stable over the exposure period. Since synergistic effects can complicate
interpretation of the experiments, care must be taken to ensure that the
desired species are present and that interferents are controlled and/or moni-
tored. Changes in the atmospheric composition due to loss of specific species
or generation of another species by physical or chemical reactions must be
monitored and documented.
96
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6.2.1 General Considerations
Specific test pollutants (gaseous and/or particulate) are produced by a
source, mixed with diluent gas, and then introduced into the space surrounding
the test subject. Gaseous pollutants are usually obtained from high pressure
gas cylinders, although some are produced iji situ (e.g., ozone from ultraviolet
irradiation). Occasionally, pollutants in ambient concentrations are obtained
by dilution of mass emissions from permeation tubes (e.g., S02, N02, H2S,
CH3SH). Aerosols may be generated by nebulizing a solution or a suspension of
known composition. Aerosols in solid form may be obtained from a dust-feeder
type of apparatus such as the Wright Dust-Feeder.3
To ensure the composition of the atmosphere, the source and the back-
ground atmosphere' into which the pollutant is released must be stable and
well-characterized. Reactivity of the pollutant with the test chamber, in-
cluding the delivery system, must be characterized and documented.
Since atmospheres are generally prepared by introduction of a specific
amount of pollutant into a known volume of background air, knowledge of the
quality of this background air is vital. Particulate matter, organic vapors,
and other gases should be removed by appropriate filters or adsorbents prior
to pollutant introduction. A schedule should be established for the periodic
replacement of these filters and adsorbent elements.
One frequently neglected gas is water vapor. The humidity of the test
atmosphere is an important variable, especially when atmospheres containing
particulate matter are being generated. Surface reactions on particulates and
aerosol composition are strongly dependent on the amount of water vapor present.
All moisture should be removed from the background air using a mechanical
dryer and absorbent. The air can then be rehumidified to a specified level by
the addition of steam or a water spray. Test subject humidity requirements
must be considered in determining the relative humidity.
Since the test atmosphere is prepared by mixing pollutants and air in
proportions determined by the ratios of their volumetric flow rates, the
accurate measurement of each of these flow rates becomes critical. Flow
measuring devices should be properly calibrated and operated. The pressure
and temperature of gases at the flow rate measuring devices must be stable and
known. Small absolute errors in the measurement of low pollutant flow rates
97
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result in large errors in pollutant concentration. In addition, after pollu-
tant and air flows have been combined, it is important to provide for good
mixing of the two components to ensure a homogeneous atmosphere.
Finally, it is important to characterize the test atmosphere as it en-
counters the test subject, i.e., spatial as well as temporal characterization.
This characterization provides data concerning the actual exposure conditions.
It is also helpful to characterize the atmosphere as it leaves the source
since this information is useful in the early detection of harmful levels of
pollutants resulting from source malfunctions. Early detection allows diver-
sion of the defective atmosphere before it reaches the test subject. Interac-
tion between individual components of test atmospheres, always a potential
source of error in atmosphere generation, can be minimized by careful atten-
tion to parameters such as composition, concentration, and residence time.
Interaction between the atmosphere and conduit or chamber walls can also be a
source of error. This is especially true for aerosols and reactive gases such
as ozone and sulfur dioxide. Even the test subject may interact with the
atmosphere in an unexpected and undesirable manner (e.g., NH3 from animal
excreta). For valid data to be accumulated from an experiment, each of the
interactions that may occur must be carefully examined and controlled by the
investigator.
6.2.2 Particulate or Aerosol Atmospheres
Atmospheres containing generated particulate matter or aerosols exhibit
so many specialized problems that they warrant separate discussion. Since the
dose of inhaled particles is mass- and size-dependent, knowledge of both the
mass of the particles and their size distribution is necessary to characterize
the dose level. If the aerosol atmosphere is a mixture of several particulate
components, the size distribution of each should be characterized.
Aerosols may be obtained by nebulization of a solution or a suspension of
known composition. Deviations in the aerosol characteristics may result from
inadequate flow control, excess loss of solvent, and cooling of the solution
due to solvent evaporation. Circulation of the solution from an external large
reservoir may be used to avoid the problems due to solvent evaporation. In the
Wright Dust-Feeder, lack of homogeneity in the powder plug may produce devia-
tions in aerosol output.3
98
-------
Characteristics of an aerosol may change due to particle-gas or particle-
particle interactions. The particle-gas interactions in hygroscopic aerosols
result in evaporation or growth of particles.4 In salt aerosols, humidities
above 75 percent will generally result in growth of the particles. This
growth process is extremely rapid and can lead to a several-fold particle size
increase at high humidities (>90 percent). In acid aerosols, humidities below
20 percent can produce a decrease in particle diameters due to evaporation. As
a general rule, and dependent on test subject health parameters, humidity
should be maintained constant between 20 and 70 percent to avoid particle
growth or evaporation losses.
Particle-particle interactions resulting in coagulation are dependent
upon particle size and concentration. Coagulation can lead to significant
errors for dense aerosols. In general, if the concentration is less than 105
particles/cm3, coagulation may be neglected. Other factors leading to coagu-
lation are turbulent mixing and extreme polydispersity. Charges on particles
also significantly influence aerosol behavior. An aerosol generated by nebu-
lization may require charge neutralization. This will avoid the uncertainty
of the effect of charge on particle-particle interaction and deposition on
surfaces.
Methods for characterization of aerosol size and concentration are based
on a variety of principles. Interconversion between two methods is not usually
possible without introduction of significant errors. If an aerosol is used
for an inhalation study, the aerodynamic size distribution based on mass is
appropriate. To obtain this information, inertial classification of particles
by a method such as impaction is necessary. However, in various size ranges,
other methods based on electrical mobility, microscopic, or light scattering
analysis may be needed to characterize the aerosol. Conversion of data from
these methods into aerodynamic size should follow recognized procedures.5 An
estimate of the errors involved should also accompany the conversion.
After the aerosol has been generated and characterized at the source, it
is delivered to the test subject. Certain precautions and pretests should be
taken to prevent significant change in the atmosphere before it reaches the
subject.
Loss of the aerosol component en route to the exposure chamber can be
significant. The most common cause of particle loss is deposition on conduit
99
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walls. This surface deposition of particles is due to sedimentation, inertia,
and diffusion processes: the extent and nature of particle loss is size-
dependent. In polydisperse aerosols, deposition loss will affect the particle
size distribution of the delivered aerosol as well as the concentration. In
general, large particles over a few microns aerodynamic diameter are preferen-
tially lost by sedimentation and inertia. These effects can be minimized by
using high flow velocities and by avoiding bends or sudden transitions in
conduits (e.g., changes in tubing diameter). Because of this tendency toward
deposition, it is extremely important that aerosol atmospheres be finally
characterized immediately before they encounter the test subject.
If the atmosphere contains particles larger than I |jm in diameter, lack
of homogeneity in the chamber may be significant. Segregation may occur due
to sedimentation or bypassing the inlet and outlet. Distribution of the
incoming test atmosphere over as broad an area as possible would minimize the
flow channeling problems. Sedimentation effects may be minimized by a verti-
cally downward movement of the test atmosphere. Even with these precautions,
segregation may occur. Again, this tendency necessitates characterization of
the aerosol at the test subject. The sampling position for this characteriza-
tion must represent the same location and elevation in the chamber as the test
subject. This will assure the characterization of that portion of the atmos-
phere that actually reaches the subject.
6.3 SAMPLE COLLECTION AND ANALYSIS
6.3.1 Introduction
Collection of a representative sample is of utmost importance in any
measurement process as noted in Section 4.3.1. The analytical results may be
of excellent quality; however, if the sample is contaminated, degraded, or is
otherwise not representative of the area or population under study, the rela-
tionship between the measured pollutant concentration and the response of
exposed subjects will not be valid.
It is important to recognize that obtaining a representative sample is
difficult, especially when low concentrations of components of the ambient air
are measured. For this reason the procedures for sample collection and analysis
should be included in the experimental design (see Section 4.2). Sampling
methodology and the number of samples required should be established prior to
beginning the task.
100
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Ambient air studies frequently deal with large populations and extended
airshed areas, which cannot be thoroughly monitored. Thus, statistical sam-
pling techniques are generally required. The number and size of "blanks,"
control groups, and samples taken from the background should be evaluated care-
fully. Calibration, instrument spanning, and audits also have an impact
on sample collection and analysis efforts.
In most studies, more than one pollutant or parameter will be measured.
During the experimental design phase, the requirement for measurement of
co-occurring pollutants should be addressed. Important parameters such as
humidity, temperature, and atmospheric pressure are also commonly measured.
6.3.2 Sample Representativity
A representative sample of ambient air to which plants and animals
may be exposed is difficult to obtain. Spatial and temporal aspects of sam-
pling should be considered carefully prior to locating the sampling stations.
A thorough background study in support of an ambient air monitoring program
should include a study of source inventories, historical meteorology of the
area, local topography, and examination of data from any preexisting air
monitoring stations.
The point in space from which a sample is taken is an important variable.
The sample should be collected in a location that is clearly representative of
the air space being characterized. For example, if the objective of the study
is to assess the effects of air quality on children, the sampling point might
be located 1 to 1.5 meters above the ground in a schoolyard or playground.
The inlet to the sampling probe must be located so that it is protected from
possible damage by the elements or by vandals, yet is out of the microenviron-
ment of the sampling equipment. The use of mobile sampling equipment is often
helpful in locating proper sampling points and in surveying a large area at
minimal expense.
The time frame in which a sample is taken also has a bearing on sample
representativity. Generally, the longer the period of sampling, the better
the sample will characterize the environment. However, the final decisions
concerning sampling duration and frequency must be made with respect to the
objectives of the task (see Section 4.2). If continuous or semicontinuous
analyzers are used, concentration trends and any unusually high or low values
101
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will appear when the overall data are examined. On the other hand, if grab
samples are collected for short periods of time, it is possible that very high
or very low concentrations will be obtained that do not represent the subject's
average exposure. If the experiment is well controlled (such as a captured
air mass in a chamber), periodic grab sampling can be used. However, even
here with certain pollutants the use of grab samples is discouraged due to
potential chemical degradation of the sample during transport and storage.6
Sample integration is helpful when collecting an air sample for later
analysis. In this process the sampling vessel is slowly filled with an air
sample over a period of time. Again, sample integration must consider the
stability of the sample with time, as well as the averaging of concentration
fluctuations.
6.3.3 Physical Characterization of the Atmosphere
To obtain an accurate intercomparison of samples taken in various cities
or air regimes, it is necessary to know some of the physical characteristics
of the ambient or enclosed air mass. Such characteristics include temperature,
barometric pressure, relative humidity, and perhaps wind speed, wind direction,
and solar radiation. Additionally, knowledge of the temperature, pressure,
and humidity within the analytical laboratory is necessary for correction of
gas flow rates to standard temperature (25° C) and pressure (1 atmosphere).
This is particularly important during the calibration and operation of analyz-
ers and impinger systems.
6.3.4 Sample Quantity
A sufficient volume of air must be collected or passed to an instrument
to obtain valid data. In the case of continuous analyzers, an excess volume
of sample generally flows through a glass sampling manifold and the instrument's
sampling line is attached to this manifold. An initial flow rate at least
50 percent in excess of that required by the analyzer(s) is recommended. If
the sample flow is less than that demanded by an analyzer, the analyzer or
sampling device will pull in room air and the sample will be diluted. Suffi-
cient sample quantity is also needed during calibration. The rate of sample
flow to a continuous analyzer should be identical to the flow rate established
during calibration. That is, if an analyzer samples 200 cmVmin during cali-
102
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bration, it should sample 200 cm3/min during analysis of ambient air. The
same is true of impinger samples in which air is bubbled through a chemical
solution. In the case of impingers containing chemical solutions, a sufficient
volume of sample must bubble through the solution to achieve a reliably detect-
able spectrophotometric or other response. For particulate collection devices
(high volume samplers, dichotomous samplers, cascade impactors, etc.) the flow
rate must be that specified to achieve the entrainment of the desired particle
sizes. The particle sampler must sample long enough to build up sufficient
deposit for accurate weighing and/or chemical analysis.
6.3.5 Sample Handling and Storage
Since many of the pollutants in ambient air are highly reactive, unstable
species, they cannot be reliably collected and stored for later analysis.
Ozone, oxides of nitrogen, peroxyacetyl nitrate (PAN), sulfur dioxide, and
other sulfur species should be delivered directly from the ambient air to the
analyzer or impinger through Teflon or glass tubing.
Other less reactive pollutants, such as carbon monoxide and hydrocarbons,
may be stored for periods of several days prior to analysis. Teflon or Tedlar
bags are adequate for carbon monoxide samples. Stainless steel or glass
sampling containers are better for hydrocarbons. There may be no clear con-
sensus in the scientific community as to the reactivity of a specific pollutant.
In such cases, it is essential that it be determined and documented as part of
the study if the conclusions are to be valid and defensible.
Particulate samples collected on glass fiber or other types of filters
are often weighed and analyzed at a later date. For reproducible weight
determinations, the filters must be conditioned at a constant relative humidity
for a specified period prior to weighing for tare and gross weights. Care
must be taken to avoid sample loss during filter handling.
In all cases, stored samples should be protected from unusually high
temperatures and intense light. Some samples are preferably stored under
refrigeration in the dark.
6.3.6 Recommendations for Sampling and Analysis of Selected Pollutants
Recommendations for the sampling and analysis of selected pollutants
commonly found in ambient atmospheres follow. Included are suggestions and a
103
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summary (Table 6-1) for the six EPA criteria pollutants (S02) N02, 03, CO, TSP
and Pb) as well as other species of current interest to HERL-RTP personnel.
This is not an exhaustive list of pollutants or of sampling and analysis meth-
ods. Rather, it is a list of some pollutants of current interest, the most
acceptable analysis methods to be used, major interferences, and calibration
concepts for additional pollutants.7
6.3.6.1 Sulfur Dioxide (S02)--
The EPA reference method for determination of ambient levels of sulfur
dioxide is the pararosaniline method. This manual, wet chemical method is a
complex sampling and analysis procedure. EPA has accepted an automated ver-
sion of this method that reduces the complexity of the analysis.8 In addi-
tion, other continuous instrumental methods are currently available that are
officially considered equivalent to the EPA reference method. These methods
include coulometric, flame photometric, pulsed fluorescent, and second deriva-
tive spectroscopic detection of sulfur dioxide. The coulometric, pulsed
fluorescent, and second derivative spectroscopic methods are specific for S02.
The flame photometric method detects sulfur-containing species (e.g., S02,
H2S, R-SH); it can be made specific for S02 by inserting a scrubber cartridge
into the sample inlet line. A technical assistance document on the use of the
flame photometric method in ambient air measurements has been published by
EPA.9
Some nonsulfur compounds do interfere with these methods. It is reported
that differences between carbon dioxide concentration in the calibration/zero
matrix and the sample matrix interfere with certain flame photometric detec-
tors.10 Hence, the C02 concentration in the calibration gas for this instru-
ment should be matched to the C02 concentration expected in the sample. The
pulsed fluorescence method will respond to certain aromatic hydrocarbons
unless a special scrubber (e.g., a hydrocarbon cutter) is placed in the sample
inlet line.
Calibration of these instruments is usually accomplished against an S02
permeation device. Gas cylinders of S02 in N2 or air (widely used for calibra-
tions of source level monitors) may also be used for calibration and span
checks of ambient air instruments if the cylinder concentration is approximately
50 ppm or higher, if the concentration is checked versus a higher standard
104
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TABLE 6-1. SUMMARY OF MEASUREMENT METHODS FOR SELECTED POLLUTANTS
Pollutant
Ammonia (NH3)
Carbon monoxide (C0)a
Hydrocarbons (THC)
Hydrogen sulfide
(H2S)
Lead (Pb)a
Nitrogen dioxide
(N02)a
Ozone (03)a
Peroxyacetyl nitrate
(PAN)
Total suspended
particulates (TSP)
Measurement
method
Indophenol method
Nitrite method
NDIR
(40 CFR, App. C)
Methanation/GC-FID
GC-FIO
(40 CFR, App. E)
GC-MS
Methylene blue
ALSO FPO
Flame atomic
absorption
Chemi 1 umi nescence
of NO with 03
(40 CFR, App. F)
UV photometry
Chemi 1 umi nescence
03-ethylene
03-rhodamine-B
GC-EC
High volume sampler
(40 CFR, App. B)
Interferents
Certain particulates
Particulates containing
ammonium salts
H20, C02 in high cone.
and other IR_absorbers,
near 2165 cm"
Nonmethane hydrocarbons ,
depending on scrubber
efficiency
Nonlinear FID response
to increasing carbon
number
None
Strong oxidizing or
reducing agents
(e.g., S02)
Light scattering
C02, H20b PANC
High humidity, at low
Og concentrations
High humidity at low
03 concentrations
Varying sample/standard
moisture content
Secondary particle
formation
Gas-filter reactions
Calibration
Standard solution of NH4
or permeation devices
CO in N2 (cylinders)
Methane cylinders
Propane cylinders
Standard cylinders
of pure gases, and
mixtures
Permeation system
Standards prepared daily
Cylinder NO in N2
and gas phase titration
to N02
UV photometry or accept-
able transfer standard
UV photometry or accept-
able transfer standard
Photoanalysis of ethyl
nitrite in oxygen
High volume sample flow
rate is calibrated with
ReF device or orifice plates
Generation
Cylinder and dilution
Cylinder and dilution
Cylinder gas dilution
with clean air
system
system
Dilution of working standard
Oxidation of NO (from
by 03
N02 permeation tube
03 (UV) generator
03 (UV) generator
Dilution of generated
standard
cylinder)
See footnotes at end of table.
(continued)
-------
TABLE 6-1 (continued)
o
o
Pollutant
Sulfates (S04)
Sulfur dioxide
(S02)a
Sulfuric acid
(H2S04) mist
Measurement
method
Interferents
Calibration
Generation
Barium methyl thymol
blue
Ion chromatography
FPD with S02 denuder
Anions complex!ng barium Standard solution of S04
Standard solution of S04
Pararosaniline
(40 CFR, App. A)
Pulsed fluorescence
Flame photometric
Effects of the principal
known interferences have
been minimized or elimi-
nated as follows: (1)
oxides of nitrogen elimi-
nated by sulfamic acid, (2)
ozone eliminated by time
delay, (3) heavy metals
eliminated by EDTA &
phosphoric acid
Aromatic hydrocarbons
("cutter1' prevents inter-
ference)
Any sulfur-containing
species (H2S scrubber
beneficial in removing
this interferent)
S02 permeation system
Mixing of high-concentration
S02 with clean air.
S02 permeation system
S02 permeation system
Mixing of high-concentration
S02 with clean air.
Mixing of high-concentration
S02 with clean air.
Coulometric
Oxidizing (e.g.
N02) species
Reducing (e.g. ,
species
Olefins
, Oa,
H2S)
S02 permeation system
S02 permeation system
S02 permeation system
Mixing of high-concentration
S02 with clean air.
Mixing of high-concentration
S02 with clean air.
Mixing of high-concentration
S02 with clean air.
Ion chromatography
SO,
EPA criteria pollutants.
Mercer, T. T., Aerosol Technology in Hazard Evaluation, Academic Press, New York, 1973.
cCooper, C., G. Langer, and J. Rosinski, Air Pollution Control Assn. J., 18:57, 1979.
Ellis, E.G., Technical Assistance Document for the Chemiluminescence Measurement of Nitrogen Dioxide, EPA Environmental Monitoring
Series, EPA-600/4-75-003, Environmental Protection Agency, Research Triangle Park, NC, December 1975.
federal Register, December 14, 1977, p. 62971.
-------
every 6 months, and if the tank's contents are carefully diluted with clean,
S02-free air.
6.3.6.2 Nitrogen Dioxide (N02)--
The EPA measurement method for the determination of ambient nitrogen
dioxide (N02) is based on the chemiluminescence produced by the oxidation of
NO by ozone. The method is instrumental and continuous. These analyzers
detect NO directly and total oxides of nitrogen (NO, N02, and other N-
containing compounds that can be reduced to NO) after passage through a thermal
converter. A readout of N02 concentration is provided indirectly by electronic
subtraction. One automated and two manual wet chemical methods have recently
been accepted by EPA as equivalent to the reference method.11 Neither the
Christie (arsenite) method nor the TGS-ANSA method is affected by the inter-
ferences listed above, but they suffer from the difficulties inherent in all
manual sampling and analysis methods.
Recently published research has indicated that the chemiluminescent
method is subject to interference from third-body quenching reactions includ-
ing those with carbon dioxide and water vapor.12 Research has also shown that
the thermal converter (used in this method to reduce N02 to NO) can reduce
nitrogen-containing compounds to NO. PAN is also converted with relatively
high efficiency.13 The efficiency of this converter should be determined
frequently, especially when high concentrations of nitrogen dioxide are being
analyzed.
Calibration of the NO and NO channels of the instrumental method is
/\
generally accomplished using bottled standards of nitric oxide in nitrogen.
The N02 channel is calibrated by oxidizing some of the NO calibration standard
to N02 before the gas is introduced into the instrument. This oxidation is
accomplished by ozone gas phase titration (GPT). Calibration of this channel
may also be accomplished by use of an N02 permeation device. Much helpful
information on the calibration and use of chemiluminescence N0-N02-N0 analyz-
X
ers is available in an EPA technical assistance document.14
6.3.6.3 Ozone (03)~
Ozone (03) is the most commonly measured photochemical oxidant. Wet
chemical methods can be employed for measurement of ozone. However, the EPA
reference and equivalent methods are the instrumental methods based on ultra-
107
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violet photometry, chemiluminescence from the reaction between ozone and
ethylene, or chemiluminescence from the reaction between ozone and rhodamine-B.
Calibration is accomplished using a stable ozone source in conjunction
with an ultraviolet photometer or a certified transfer standard such as an
ozone generator. The response or output of the transfer standard should be
determined by ultraviolet photometry. Technical assistance documents are
available that discuss the calibration of various ambient air ozone analyzers
employing the principle of UV photometry15 and the methods for establishing
certified ozone transfer standards for use in calibration and auditing of
monitors.16
6.3.6.4 Carbon Monoxide (CO)—
The EPA measurement method for continuous monitoring of carbon monoxide
in the atmosphere is nondispersive infrared spectrometry (NDIR). The principle
is based on the absorption of infrared radiation by carbon monoxide in a
nondispersive spectrophotometer. Another method is based on catalytic conver-
sion of carbon monoxide to methane by hydrogenation. The methane is then
sensed by a flame ionization detector.
The infrared absorption spectrum of water is sufficiently similar to that
of CO to interfere with NDIR measurements. In source level concentrations
(i.e., 2000 ppm), C02 is also an interferent.
Calibration of such analyzers is by injection of carbon monoxide from
standard cylinders. Steel cylinders have a tendency to react slowly with
carbon monoxide, forming iron carbonyl. Because of this tendency, standards
should be verified every 4 to 6 months by comparison to an NBS-traceable
standard.
6.3.6.5 Total Suspended Particulates (TSP)--
The EPA reference method for total suspended particulate (TSP) is the
high volume sampler method. Air i's drawn into a covered housing and through a
filter by means of a high-flow-rate blower (1.0 to 1.7 m3/min). This flow
rate allows suspended particulates having aerodynamic diameters of less than
100 urn to pass to the filter surface. Accurate control of the flow rate is
critical to obtaining a valid sample. The collection period for ambient air
is generally 24 hours. The filter is conditioned to a fixed relative humidity
and weighed before and after sampling. The net weight and total volume sampled
108
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are used to estimate average suspended particulate matter in terms of micrograms
per cubic meter.
For experiments where smaller volumes of air are available for sampling,
low flow rate filters and impactors may be useful. Because the emphasis here
will probably be on chemical analysis and not weight, care must be exercised
in selecting the filter media or impaction surface to avoid artifact formation
and background interference. The possibility of interferences in the analysis
should be examined through background and blank analyses. Optical particle
counters are available for continuously monitoring the number of particles
and, in certain models, the size of particles. Manufacturers of such counters
and size discriminators should be able to show how calibration was achieved.
6.3.6.6 Lead (Pb)~
The EPA reference method17 for the determination of ambient levels of
lead is atomic absorption spectrometry using an air-acetylene flame. Lead in
particulate matter is solubilized by extraction with nitric acid facilitated
by heat or by a mixture of nitric and hydrochloric acids facilitated by ultra-
sonication.
No major chemical interferences have been reported. If the analyst
suspects an interference due to the sample matrix, the interference can be
verified and corrected for by using the method of standard additions.
A second type of interference, light scattering, may be observed when
using the 217.0 nm lead absorption line. This may be corrected for instru-
mental^, as described in the Federal Register Method, or by use of the ammonium
pyrrolidinecarbodithioate-methylisobutyl ketone (APCD-MIBK) chelation-solvent
extraction technique18 of sample preparation.
Calibration is accomplished using standards prepared daily by dilution of
the working standard with the same acid matrix. Standards, in the same acid
concentration as the samples, should be selected to cover the linear absorp-
tion range indicated by the instrument manufacturer.
6.3.6.7 Hydrocarbons (THC)—
The EPA measurement method for determination of hydrocarbons corrected
for methane is based on gas chromatography with flame ionization detection.
The method is designed to measure both total hydrocarbons (THC) and methane so
that methane can be subtracted from the hydrocarbon analysis. No reference
109
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instruments are currently designated because of problems resulting from an
inefficient methanator and a nonlinear detector. The instrument is usually
calibrated on the basis of methane or propane supplied from low-level standard
reference tanks.
If analysis for specific hydrocarbons is sought, the chromatographic
column-flame ionization detector approach is preferred. The specific compound
is distinguished from others by introducing a known concentration of this
hydrocarbon and determining its column retention time. The signal strength
from the detector is correlated with concentration by introducing varying
known concentrations of the hydrocarbon of interest. Permeation tubes con-
taining certain hydrocarbons may be used to generate standards. Mixtures of
hydrocarbons in air or other gases may also be purchased in cylinders.
The possibility of peaks from one or more compounds overlapping during
chromatographic analysis increases with the complexity and similarity of the
molecules. The extent of this problem should be investigated using several
column packings over a range of operating conditions.
Hydrocarbon samples may be collected in Teflon or Tedlar bags for later
analysis. However, glass or stainless steel containers are preferred. Cer-
tain higher molecular weight hydrocarbons and other organic compounds may be
adsorbed on columns of polymeric material such as TENAX-GC and volatilized
onto a chromatographic column at a later time.
For maximum reliability in identification of hydrocarbons (and other
organic species), the method of choice is the combination of gas chromato-
graphy and mass spectrometry.
6.3.6.8 Peroxyacetyl Nitrate (PAN)--
PAN is a photochemical oxidant often found in smoggy atmospheres. Meas-
urement is generally by a gas chromatographic procedure employing an electron
capture detector.19 This method may be subject to interference from low
sample moisture content unless the relative humidities of the samples and
standards are controlled.20 21
Standards may be synthesized by the photolysis of ethyl nitrite in oxy-
gen.22 The synthesized standard, however, is not a primary one and must be
verified (e.g., by infrared spectroscopy).
no
-------
6.3.6.9 Sulfuric Acid Mist (H2S04)--
Sulfuric acid mist may be collected on glass fiber or Teflon membrane
filters if it is at low concentration levels (<50 mg/m3) and no sulfur dioxide
is present. The sample can then be extracted with deionized water and analyzed
by ion chromatography or other acceptable methods. When sulfur dioxide is
present, it may be oxidized to sulfate in the presence of water vapor or by
oxidants on the filter surface, thus interfering with the acid mist analysis.
6.3.6.10 Sulfates (SO*)--
Airborne sulfates may be measured by analysis of the particulate matter
taken from high volume on dichotomous filters. The technique is generally as
described for the measurement of total suspended particulate matter (Section
6.3.6.7). Analysis of the collected particulate for sulfate is then performed
using one of several available analytical techniques (e.g., turbidometry, ion
chromatography). The analysis method usually recommended is the automated, wet
chemical method based on the detection of the barium-methyl-thymol blue chelate.23
To avoid unwanted formation of sulfates on the filter by reaction of S02, the
pH of the filter must be controlled during manufacture to around pH 5.
6.3.6.11 Benzene (C6H6)—
A tentative method for the determination of benzene in the atmosphere by
24-hour integrated sampling has been established and used by EPA.24 In this
method, a known-volume of ambient air is drawn through a tube (NIOSH) contain-
ing charcoal at 200 cmVmin for 24 hours; benzene vapors are adsorbed on the
charcoal. The charcoal is subsequently transferred to a stoppered container
where trapped vapors are desorbed by carbon disulfide. An aliquot of the
extract is injected into a gas chromatograph and benzene is quantified by
comparing the peak retention time and area to standard solutions of benzene in
carbon disulfide. The method's sampling efficiency has been determined to be
in excess of 90 percent over a concentration range of 2 to 100 ppb.
6.3.6.12 Vinyl Chloride (C2H3C1)—
A tentative method for the determination of vinyl chloride in the atmos-
phere (24-hour integrated sampling) has been established by EPA. In the
method, vinyl chloride is adsorbed from ambient air onto charcoal packed in
glass tubes. The sample is drawn through the tube at a flow rate of 200 cm3/
min for 24 hours. The charcoal is extracted with carbon disulfide. An aliquot
in
-------
of the extract is injected onto a 2.5-m length of 0.4% carbowax 1500/carbopak A
column. Vinyl chloride is identified and quantified by comparing peak reten-
tion times and peak heights to those determined for standards using the flame
ionization detector.
6.4 POLLUTANT STANDARDS AND TRACEABILITY
Special emphasis should be placed on the need to characterize all incom-
ing cylinder gases containing pollutants in specified concentrations. The
characterization should also include identification of cylinder contents with
reference to both pollutants(s) and matrix. It is well known that problems
concerning the identity of cylinder contents and accuracy of the specified
concentrations are commonplace. Even the best known and most reliable gas
suppliers occasionally supply faulty materials. In addition, after the cylin-
der contents have been initially verified, experience indicates that over a
period of time the contents degrade. Therefore, regular recertification must
be performed to characterize changes in concentration, formation of new species,
or loss of original species to prevent them from degrading task data quality.
Because of these considerations, all HERL-RTP gas cylinders should be subjected
to a rigorous program of initial, and regularly recurring, certification of
contents and concentrations. The reader is referred to the traceability
protocol for establishing true concentrations of gases used for calibrations
and audits contained in Volume II of the Quality Assurance Handbook for Air
Pollution Measurement Systems.2
The Environmental Monitoring Systems Laboratory, Quality Assurance Divi-
sion, maintains a laboratory in Research Triangle Park, NC, for use in valida-
tion of cylinder and permeation tube concentration outputs, as well as veri-
fication of flow rate apparatus and pollutant generating/dilution systems.
For further information on pollutant verification or standards, contact Mr.
Thomas Clark, EMSL/QAD, EPA, Research Triangle Park, NC 27709.
6.5 REFERENCES
1. U.S. Environmental Protection Agency, Quality Assurance Handbook for Air
Pollution Measurement Systems, Vol I -Principles.EPA 600/9-76-005,
Research Triangle Park, NC, March 1976.
2. U.S. Environmental Protection Agency, Quality Assurance Handbook for Air
Pollution Measurement Systems, Vol II - Ambient Air Specific Methods,
EPA-600/4-77-027a, Research Triangle Park, NC, May 1977.
112
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3. Messrs. L. Adams, Ltd., The Wright Dust Feed Mechanism, G.A. 180 -
Instructions of Use, Publication DF 170, Issue No. 3, 22 Minerva Road,
London, NW10GHS, England, November 1975.
4. Ahlberg, N. S., and J. W. Winchester, Atmospheric Environment, 12:1631,
1978.
5. Mercer, T. T., Aerosol Technology in Hazard Evaluation, Academic Press,
New York, 1973.
6. Cooper, C., G. Langer, and J. Rosinski, Air Pollution Control Assn. J.
18:57, 1979.
7. Katz, M., ed. , Methods of Air Sampling and Analysis (2nd edition),
American Public Health Association, 1977.
8. Federal Register, August 13, 1975, p. 34024.
9. A Technical Assistance Document—Use of the Flame Photometric Detector
Method for Measurement of Sulfur Dioxide in Ambient Air, EPA-600/4-78-
024, Research Triangle Park, NC, May 1978.
10. Von Lehmden, D. J. , Suppression Effect of C02 on FPD Total Sulfur Air
Analyzers and Recommended Corrective Action, Proceedings, Fourth Joint
Conference on Sensing of Environmental Pollutants. New Orleans, La.,
November 6-11, 1977.
11. Federal Register, December 14, 1977, p. 62971.
12. Matthews, R. D., R. F. Sawyer, and R. W. Schefer, Interferences In Chemi-
luminescent Measurement of NO and N02 Emissions from Other Nitrogen-
Containing Compounds, Environmental Science and Technology, 11 (12):1092-5,
November 1977.
13. Winer, A. M., J. W. Peters, J. P. Smith, and J. N. Pitts, Jr., Response
of Commercial Chemiluminescent N0-N02 Analyzers to Other Nitrogen Contain-
ing Compounds, Environmental Science and Technology. 8:1118, 1974.
14. Ellis, E. C., Technical Assistance Document for the Chemiluminescence
Measurement of Nitrogen Dioxide, EPA Environmental Monitoring Series,
EPA-600/4-75-003, Environmental Protection Agency, Research Triangle
Park, NC, December 1975.
15. Paur, R. J., and F. F. McElroy, Technical Assistance Document for the
Calibration of Ambient Ozone Monitors. EPA Environmental Monitoring
Siries. EPA-600/4-79-Q57, Environmental Protection Agency, Research
Triangle Park, NC 27711, September 1979.
16. McElroy, F. F., Transfer Standards for the Calibration of Ambient Air
Monitoring Analyzers for Ozone, EPA Environmental Monitoring Series,
EPA-600/4-79-056, Environmental Protection Agency, Research Triangle
Park, NC 27711, September 1979.
113
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17. Federal Register, October 5, 1978, p. 46258.
18. Koirtyohann, S. R., and J. W. Wen, Analytical Chemistry, 45:1986, 1973.
19. Darley, E. F., K. A. Dettner, and E. R. Stephens, Analysis of Peroxyacetyl
Nitrates by Gas Chromatography with Electron Capture Detection, Analy-
tical Chemistry, 35 (4):589-91, April 1963.
20. Holdren, M. W., and R. A. Rasmussen, Moisture Anomaly in Analysis of
Peroxyacetyl Nitrate (PAN), Environmental Science and Technology, 10
(2):185-7, February 1976.
21. Watanabe, I., and E. R. Stephens, Reexamination of Moisture Anomaly in
Analysis of Peroxyacetyl Nitrate, Environmental Science and Technology,
12 (2): February 1978.
22. Stephens, E. R., in: Advances in Environmental Sciences and Technology,
Pitts and Metcalf, eds., Volume I, Wiley, New York, 1969.
23. Bergman, F. J., and M. C. Sharp, Measurement of Atmospheric Sulfates:
Evaluation of the Methyl thymol Blue Method, Environmental Monitoring
Series, EPA-600/4-76-Q15, Environmental Protection Agency, Research
Triangle Park, NC, March 1976.
24. Monitoring System for Collection and Analyses of Ambient Benzene Levels
in Urban Atmosphere, report to the EPA EMSL by PEDCo Environmental. Inc.
under Contract No. 68-02-2722, Assignment No. 7. Appendix. January 1979.
114
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SECTION 7
QUALITY CONTROL/QUALITY ASSURANCE GUIDELINES
FOR RESEARCH INVOLVING ANIMALS
7.1 INTRODUCTION
Research involving animals presents a source of significant data variabil-
ity—the complex biochemical system of the animal itself. This variability is
affected by the animal's genetic, physiological, psychological, and environ-
mental condition. The purpose of this section is to outline procedures to
control these variables and thereby assist the investigator in attaining high
quality results that are reproducible, accurate, and therefore scientifically
valid.
Quality control (QC) begins with experimental design and extends through
completion of the final report. The purpose of these QC guidelines is to
highlight areas in the research protocol where variance can become significant;
it is not the purpose of these QC guidelines to dictate or to inhibit the
investigator's experimental design. These guidelines address proper QC proce-
dures for the animal staff (Section 7.2) and the research laboratory (Sec-
tion 7.3). General guidelines for administration of test agents are also
included (Section 7.3.2). More specific and detailed procedures are available
in the references (see Section 7.5).
These guidelines (Sections 7.2 and 7.3) represent health care standards
that are to be met or exceeded by all intramural and extramural HERL-supported
animal research activities. They are consistent with the Environmental Protec-
tion Agency (EPA) and the Food and Drug Administration (FDA) Good Laboratory
Practice (GLP) regulations.1 2 Additional sources were also used,3"12 with
extensive reference being made to HERL-RTP's Laboratory Animal Staff's standard
operating procedures, which are revised annually, and contain information on
animal support facilities available to the Health Effects Research Laboratory
(HERL). Copies can be obtained from HERL-RTP's, Animal Care Coordinator
(MD-70).13 Figure 7-1 is an example of a quality assurance systems audit
115
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ANIMAL FACILITY CHECKLIST
Laboratory Animal Management
( ) Caging conditions
( ) Environmental control _
( ) Caging size
( ) Sanitary maintenance
( ) Building maintenance
( ) Storage of feed
( ) Identification of animals
( ) Overcrowding of animals _
( ) Illumination
( ) Feeding and watering of animals
( ) Vermin control
( ) General storage conditions
( ) Waste disposal
( ) Physical plant conditions
( ) Husbandry practices
( ) Hazardous materials handling and facilities
( ) Recordkeeping
Laboratory Animal Quality and Health
( ) Quarantine and isolation of animals
( ) Adequate veterinary care
( ) Diagnosis, treatment and control of animal diseases
( ) Separation by species
Personnel
( ) Personnel health program
( ) Adequacy of personnel (training and numbers)
( ) Protective clothing
Use of Laboratory Animals
( ) Surgical and postsurgical care
( ) Euthanasia procedures
(S) = Satisfactory
(U) = Unsatisfactory
(NA) = Not applicable
Inspection by
Inspection date Location
Figure 7-1. Sample quality assurance inspection checklist for
an animal care facility (from AAALAC certification
checklist). Section 7.2 should be consulted for
details.
116
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checklist for an animal care facility, Figure 7-2 is a QC checklist for animal
care procedures and Figure 7-3 is a principal investigator's QC checklist for
animal testing.
7.2 ANIMAL RESEARCH PROGRAM QUALITY CONTROL
7.2.1 Animal Facility Design Quality Control
Quality control guidelines for animal care facilities include but are not
limited to:
1. Service areas should include: Animal housing rooms of identical
design (dimensions should vary only by suites); an office; a surgery
room with adjacent preoperative, scrub, and postoperative areas; a
cage wash area and clean cage storage area; feed and bedding storage
rooms (separate); a diet kitchen; a diagnostic laboratory; shower
and locker rooms; a necropsy room; strategically located receiving,
quarantine, and isolation areas; a biohazard suite; a supply storage
room; a lounge or lunch area; a waste holding area; and experimental
procedure rooms.
2. A clean-dirty floor and work flow pattern that includes air locks
and ultraviolet lights.
3. A specially designed, separate biohazardous^study area including:
shower-out capability, HEPA-filtered isolation cubicles, an autoclave
an ultrasound cleaner, a vertical laminar flow biological safety
hood (Class II Type B) that is exhausted outside the work area
through charcoal filters, a sink with bench surface, and a waste
storage area.
4. Environmentally controlled features per room should include:
a. Ventilation in animal rooms consisting of 100 percent fresh air
with 10-15 changes per hour.
b. Humidity controlled at 55 ± 5 percent (relative) with indicators
displayed. Humidity should range between 40 to 70 percent.
c. Temperature controlled at 72° ± 2° F (range between 64° and
80° F) with indicators displayed and temperature recorded
by a high-low thermometer.
d. Lighting controlled automatically (timers recessed into walls
outside of room) at strengths of 75 to 100 footcandles. Light
fixtures should be sealed against water and vermin.
e. Dog kennels should be soundproofed, especially ceilings, ducts,
and doors.
117
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ANIMAL HUSBANDRY CHECKLIST
( ) Proper quarantine, cage identification
and space
( ) Sanitation
( ) Food, water, bedding contamination
( ) Environmental conditions
( ) Health status
( ) Separation of species
( ) Vermin control
( ) Surgical conditions
( ) Animal care committee and management
( ) AAALAC accreditation
( ) Standard operating procedures (SOP)
( ) Personnel protection
(S) = Satisfactory
(U) = Unsatisfactory
(NA) = Not applicable
Inspection by
Inspection date Location
Figure 7-2. Sample QC checklist for animal care. (Section 7.2.2 should
be consulted for details.)
118
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INVESTIGATOR CHECKLIST FOR ANIMAL TESTING
( ) Background genetic information
( ) Transportation
( ) Environmental laboratory conditions
( ) Health status
( ) Sacrifice, surgery, quarantine
( ) Age
( ) Caging materials and environment
( ) Restraints, handling
( ) Nourishment
( ) Safety
( ) Drug route: Injection
Feed
( ) Toxic compounds
( ) Drug purity _^
( ) Drug stability
( ) Controls
( ) Drug interactions
( ) Recordkeeping
( ) Standard operating procedures (SOP)
( ) Biostatisties
( ) D.V.M./Pathologist
( ) Standard reporting forms
(S) = Satisfactory
(U) = Unsatisfactory
(NA) = Not applicable
Inspection by
Inspection date Location
Figure 7-3. Sample QC checklist for investigator using animals.
(Section 7.3 should be consulted for details.)
119
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5.
6.
f. Air pressure should be properly balanced and slightly negative
to clean corridor but positive to soiled corridors.
Strategically located, separate quarantine and conditioning areas.
All selected materials utilized in the
tate efficient and hygienic operation.
conform to all pertinent regulations.
features are defined as follows:
animal quarters should facili-
Structural design should
Structural materials and
a. Corridors should be 7 ft wide with coved floor-wall junctions
to facilitate cleaning. Curbs, guardrails, and bumpers should
be installed for the protection of corners, doors, and wall-
mounted devices.
b. Animal room doors should open into rooms and measure 42 in.
wide by 84 in. high. Each should feature a lock, window, inset
handles, and an automatic closing device with a floor- or
wall-mounted stop. Doors should seal when closed to prevent
vermin and/or insect entrance. All doors should be metal and
should feature kickplates.
c. Floor surfacing should be barrier-sealed, smooth, nonabsorbant,
nonslip, and wear-, acid-, and solvent-resistant. It should be
monolithic with a minimal number of joints and should be able
to withstand the wear and tear of racks, cages, and other heavy
equipment.
d. Walls should be well sealed, have smooth surfaces, and be
resistant to detergents and other cleaning agents. They should
be free of cracks, holes, and imperfect junctures.
e. Ceilings should be smooth, waterproof, and free of penetrations
and imperfect junctures.
f. Floor drains may not be necessary, especially in rodent housing
areas where wet/dry vacuum cleaning suffices. Floor drains
(>4 in. diameter) should be provided in some rooms to offer
flexibility. In areas of
>6 in. diameter with flush
be designed at 0.25 in./yd.
drains should be included.
rooms.
7.2.2 Animal Husbandry Quality Control13
heavy usage, drains should measure
rims provided. Floor slope should
The capability to cap and seal
There should be no drains in surgery
Appropriate animal husbandry quality control procedures should include
but are not limited to:
1. Animal identification methods should be especially designed for the
particular species.
120
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2- Quarantine of animals should be consistently carried out in a separate
area designed for this purpose. The procedures governing operations
in this area should be strictly adhered to at all times.
3. Proper cage population densities should be maintained at all times.
Refer to Table 7-1 for specific space recommendations.
4. Sanitization should be accomplished through the use of carefully
selected products, schedules, and application patterns.
5. Animal caging should be sanitized by following a specific schedule
for each species and project. The cage washers should be properly
functioning at all times and records of internal temperature monitor-
ing should be maintained.
6. Animal feeds should be appropriately selected, properly stored, and
monitored for contamination and deterioration. See Table 7-2 for
suggested maximum contamination levels. Table 7-3 illustrates QC
tests done for HERL-RTP.
7. Fresh water should be available at all times (unless specifically
denied); it should be potable and consistently monitored for contami-
nation. Automatic systems should be carefully monitored for appro-
priate function and contamination.
8. Animal bedding should be appropriately selected, stored, and moni-
tored for contamination.
9. Vermin control program should be carefully designed, applied, and
monitored.Consistent records should be kept and descriptive infor-
mation concerning the agents used should be available at all times.
10. Surgical procedures should be performed by qualified personnel only,
humane methods should be strictly adhered to, thorough records
maintained, appropriate preoperative protocols filed, and related
equipment properly maintained.
11. Personnel should have appropriate training and experience and their
performance should be routinely evaluated.
12. Standard operating procedures should be thoroughly spelled out,
understood, and consistently applied in all areas.13
13. The Animal Care Advisory Committee should meet regularly, the meet-
ing minutes should be carefully recorded and promulgated, and the
specific functions of the committee should be well understood by
each member.
14. Isolation of diseased animals (if so indicated) should be done
promptly and in an area strategically separated from the main hous-
ing area.
121
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TABLE 7-1. SPACE RECOMMENDATIONS FOR LABORATORY ANIMALS
Animals
Mice
Rats
Hamsters
Guinea pigs
Rabbits
Cats
Dogs"
Primates"
Group 1
Group 2
Group 3
Group 4
Group 5
Pigeons
Coturnix quail
Chickens
Sheep and
goats
Type of Floor Area/
Weight Housing Animal
< 10 g Cage
10-15 g Cage
16-25g Cage
> 25 g Cage
< lOOg Cage
1 00-200 g Cage
20 1-300 g Cage
> 300 g Cage
< 60 g Cage
60-80 g Cage
81-100g Cage
> lOOg Cage
£ 350 g Cage
> 350 g Cage
< 2 kg Cage
2-4 kg Cage
4-6 kg Cage
> 6 kg Cage
s 4 kg Cage
> 4 kg Cage
< 15 kg Pen or run
15-30 kg Pen or run
> 30 kg Pen or run
< 15kg Cage
15-30 kg Cage
> 30 kg Cage
s 1 kg Cage
s 3 kg Cage
s 1 5 kg Cage
>15kg Cage
> 25 kg Cage
— Cage
— Cage
< 0.5 kg Cage
0.5-2 kg Cage
2-4 kg Cage
> 4 kg Cage
< 25 kg Pen
25-50 kg Pen
> 50 kg Pen
39 cm2 (6 in.2)
52 cm2 (8 in.2)
77 cm2 (12 in.2)
97 cm2 (15 in.2)
110cm2(17in.2)
148 cm2 (23 in.2)
187 cm2 (29 in.2)
258 cm2 (40 in.2)
64. 5 cm2 (10.0 in.2)
83. 9 cm2 (13.0 in.2)
103.2 cin*(16.0 in.2)
122.6 cm2 (19.0 in.2)
277 cm2 (43 in.2)
652cm2(10l in.2)
0.1 4m2 (1.5 ft2)
0.28m2 (3.0 ft2)
0.37m2 (4.0 ft2)
0.46 m2 (5.0 fl2)
-------
TABLE 7-1 (continued)
Animals
Swine
Cat lie
Horses
WeiKhl
< 50 kg
50- 100 kg
> 100kg
< 350 kg
350-450 kg
45 1-550 kg
55 1-650 kg
> 650 kg
< 75 kg
75-200 kg
20 1-500 kg
50 1-600 kg
60 1-700 kg
> 700 kg
__
Type of
Housing
Pen
Pen
Pen
Stanchion
Stanchion
Stanchion
Stanchion
Stanchion
Pen
Pen
Pen
Pen
Pen
Pen
Tie stall
Pen
Floor Arc;i/
Animal HriRht'
0.5fJIH2(6 (I2) —
Ml m2(l2h
2.70 m2 (30 ft2) —
1.49 m2 (16 It2) —
I.77m!(l8l't2) —
1.95 m2 (22 ll2) —
2. 23m2 (24 ft2) —
2.51 m2(27 ft2) —
2. 23m2 (24 ft2) —
4. 64m2 (51 ft2) —
9.29m2 (100 ft2) —
11. 15m2 (121 ft2) —
13.01 in2 (140 ft2) —
13. 94m2 (151 ft2) —
4. 09m2 (44 ft2) —
13. 38m2 (144 ft2) —
" From the resting floor to the cage top.
b These recommendations may require modifications according lo the body
conformation of particular breeds. As a further general guide, the height of a dog
cage should be equal to the height of the dog over the shouldcis (at the withers) plus
at least 6 in. (15.2 cm), and the width and depth of the cage should each be equal to
the length of the dog from the tip of the nose to the base of the* tail plus at least 6 in.
(15.2cm).
'The primates are grouped according to approximate size. Hxamplcs of species
that may be included in the various groups are:
Group I—marmosets, tupaias, and infants of various species
Croup 2—cebus and similar species
Group 3—macaques and large African species
Group 4—baboons, and nonbrachialing monkeys larger than 15 kg
Group 5—great apes and brachiating species
If primates are housed in groups in pens, only compatible animals should be kept. The
minimal height of pens should be 6 ft (1.83 m). Resting perches and appropriate
shelter should also be provided. In all cages, the minimal cage height for chimpanzees
and brachiating species (orangutans, gibbons, and spider and woolly nionkc>s) should
be such that the animal can swing from the cage ceiling without having its feet touch
the floor of the cage when fully extended.
d Sufficient headroom must be provided for birds to stand erect.
SOURCE: Guide for the Care and Use of Laboratory Animals,
U.S. DHEW, Publication No. (NIH)78-23, 1978.
123
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TABLE 7-2. MAXIMUM CONCENTRATIONS OF FEED CONTAMINANTS CONSIDERED
ACCEPTABLE FOR NATURAL INGREDIENT RATIONS MANUFACTURED FOR USE AT THE
NATIONAL CENTER FOR TOXICOLOGICAL RESEARCH AT JEFFERSON, ARKANSAS
Agents Maximum Concentration
Cadmium 0.05 ug/g
Selenium 0.50 ug/g
Polychlorinated biphenyls 0.50 ug/g
Total DDT (DDE, DDT, TDE) 0.05 ug/g
Mercury 0.05 ug/g
Arsenic 0.25 ug/g
Lead 1.00 ug/g
Dieldrin 0.01 ug/g
Lindane 0.01 ug/g
Heptachlor 0.01 ug/g
Malathion 0.50 ug/g
Estrogenic activity 2.00 ug/kg
Total aflatoxins (B^ B2, Gl5 G2) 1.00 ug/kg
1976, ILAR News, used by permission of the Institute of Laboratory Animal
Resources, National Research Council.
124
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TABLE 7-3. MONTHLY QC/QA TESTS PERFORMED FOR HERL/LAS
Nasopharyngeal cultures for Mycoplasma
Pathogenic respiratory bacteria (e.g., Staphylococcus aureus, E. coli,
Streptococcus viridans, Proteus mirabilis, and £. morgani).
Pseudomonas (detection) from animal drinking water, feces, lung wash-
ings, and feeder surfaces, utilizing Pseudosal agar.
Murine virus antibody determination in serum.
Endoparasite detection for Syphacia muris and S. obvelata, Eimeria sp.,
Aspicularis tetraptera. Heterakis spumosa. Hymenolepsis nana and
H. diminuta (utilizing zinc sulfate flotation and anal tape methods).
Ectoparasites: Myocoptes musculinus, Myobi a ratti and M. musculi,
Demodex sp. Polyplax spinulosa and £. serrata, and Notoedres sp.
Feed (Wayne Rabbit Diet, Purina Monkey Chow, Wayne Guinea Pig Diet,
Purina Laboratory Chow, Wayne Lab Blox) and bedding (heat-treated white
pine shavings) analysis for:
3.
9.
Aflatoxins
Metals
Arsenic
Bismuth
Cadmi urn
Lead
Magnesium
Manganese
Mercury
Bacterial Contamination
Standard Plate Count for:
Coliforms (total and fecal)
E. Coli
M.B.A.S.
Yeast and Mold
Pseudomonas
Salmonella
Shige11a~
Pesticides
Alpha BHC
Beta BHC
Gamma BHC - Lindane
Delta BHC
Heptachlor
Aldrin
Heptachlor Epoxide
ODE
DDD
DDT
Mi rex
Methoxychlor
Dieldrin
Endrin
Telodrin
Estimated PCB's
Ronnel
Ethion
Trithion
Diazinon
Methyl Parathion
Ethyl Parathion
Malathion
Bacterial colony counts of animal rooms sampled immediately upon comple-
tion of room sanitization.
Temperature and humidity are checked twice daily (a.m. and p.m.) in all
rooms.
SOURCE: Linko, R. Standard Operating Procedures, HERL/LAS ACC, Room P302
EPA, RTP NC, 27711, 1980.
Note: Monthly summary reports can be obtained from the
EPA Animal Care Coordinator.
125
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15. Adequate personnel protection against inhalation of dust particles
and exposure to carcinogens/mutagens must be provided.
16. Animal colony facilities should be accredited by the American Associa-
tion for Accreditation of Laboratory Animal Care (AAALAC), as proof
of high quality animal care.
7.3 INVESTIGATOR QUALITY CONTROL
High quality data are obtained more consistently when the entire research
team consists of knowledgeable and dedicated individuals. Listed below are
important parameters that investigators should be familiar with and should
incorporate into the planning of their experimental protocols.
7.3.1 Experimental Laboratory Environment
1. The genetic background of the test species should be thoroughly
assessed before selection.
2. The health status of the test species should be thoroughly evaluated.
3. The investigator should be fully aware of the effects of improper
transportation on the research animal between laboratories or between
the original supplier and the animal facility.
4. The laboratory environment should be as similar as possible to the
animal housing environment with respect to temperature, humidity,
ventilation, lighting, noise, and other important features (see
Table 7-4).
5. The particular cage environment should be as similar as possible to
the animal housing cage environment with respect to population dens-
ity and bedding materials.
TABLE 7-4. RECOMMENDED TEMPERATURE AND RELATIVE
HUMIDITY FOR COMMON RODENTS
Temperature n , . .
c Relative
Rodent °C °F humidity (%)
Mouse
Hamster
Rat
Guinea pig
20-24
20-24
18-24
18-24
68-75
68-75
65-75
65-75
50-60
40-55
45-55
45-55
1976, ILAR News, used by permission of the Institute of Laboratory Animal
Resources, National Research Council.
126
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6. Proper methods should be selected when sacrificing animals.
7. Proper methods of restraint should be uniformly used to minimize
stress.
8. Cage population density should be carefully monitored and maintained
using standard guidelines (Table 7-1).
9. Animal feed and water quality should be the same as that used in the
animal housing area (see Tables 7-5, 7-6, and 7-7). These parameters
should be monitored continuously and the findings documented and
recorded in a bound notebook.
10. Proper procedures to avoid cross-contamination between species
should be carefully adhered to in the research laboratory.
7.3.2 Experimental Compound Testing
1. Experimental compound testing procedures should be thoroughly under-
stood and uniformly applied, using the following guidelines:
a. Injectable routes of administration should be used whenever
possible, as injection affords the most reliable method of test
compound absorption.
b. The investigator should be thoroughly aware of the positive
and/or negative effects of administering test compounds in the
feed or drinking water.
c. The investigator should be thoroughly aware of the safety
aspects of adding carcinogenic/mutagenic type compounds to the
feed or drinking water.
d. The investigator should be thoroughly aware of the safety
aspects of using highly toxic and/or carcinogenic injectable
compounds with respect to the disposition of all contaminated
and/or waste products.
e. The investigator should be thoroughly aware of the chemical and
biological purity as well as impurity of the test compound
before initiating the study.
f. The investigator should be thoroughly aware of the chemical
stability and concentration of the test compound before study
initiation. Samples should be collected and properly stored
for later reference.
g. The investigator should thoroughly characterize the chemical
and biological properties of all positive controls, including
vehicle compounds, before initiating the study.
127
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TABLE 7-5. AVERAGE DAILY NUTRIENT REQUIREMENT IN PERCENTAGE OF WHOLE DIET8
KJ
oo
Species (Adult)
Nutrients
Protein
Fats
Carbohydrates
Calcium
Phosphorous
Magnesium
Sodium chloride
Potassium
Iron
Copper
Cobalt
Iodine
Manganese
Zinc
Vitamin A
Thia^iine (Ba)
Riboflavin (B2)
Pantothenic
acid (B3)
Niacin (B5)
Pyridoxine (B6)
Choi ine
Cyanocobalamin
(Bi2)
Vitamin C
Vitamin D
Vitamin E
Vitamin K
Inositol
Folic acid
Biotin
Calcium panto-
thenate (Ba)
Para- ami nobenzoic
acid (PABA)
Monkey
(Rhesus)
15-20%
3-5%
45-55%
0.86%
0.47%
0.11%
0.5%
0.56%
144 ppm
12 ppm
0.54 ppm
84 ppm
47 ppm
20 ppm
Required
0. 75 mg/kg-D
0.75 mg/kg-D
Required
38 mg/kg-D
1.3 mg/kg-D
?
25 ug/kg-o
25 mg/kg-D
Required
Required
?
7
1.3 mg/kg-D
250 ug/kg-D
—
--
Dog
20-24%
4-9%
40-60%
1.1%
0.9%
0.05%
1.5%
0.9%
57 mg/kg-D
7 mg/kg-D
2.5 mg/kg-D
1.5 mg/kg-D
5 mg/kg-D
5 mg/kg-D
1.5 mg/kg-D
7.3 mg/kg-D
2.2 mg/kg-D
2.3 mg/kg-D
10. 5 mg/kg-D
9. 7 mg/kg-D
1.21 g/kg-D
0.03 mg/kg-D
None
6.6 ug/kg-o
48 mg/kg-D
7
7
7
7
--
7
Cat
30-40%
20-30%
25-35%
Required
"
25,000 IU/kg-0
4 mg/kg-D
4 mg/kg-D
5 mg/kg-D
40 mg/kg-D
4 mg/kg-D
3 g/kg-D
Trace
None
1,000 lU/kg-D
136 lU/kg-D
7
7
None
7
--
Required
Rat
16-20%
5.0%
45-55%
0.6%
0.6%
0.05%
0.5%
0.2%
25 mg/kg-D
5 mg/kg-D
Trace
Trace
0.002%
0.005%
12,000 lU/kg-D
4 mg/kg-D
4 mg/kg-D
--
15 mg/kg-D
0.4 mg/kg-D
1 g/kg-D
5 Mg/kg-D
None
300 lU/kg-D
20 mg/kg-D
0.1 mg/kg-D
0.3 mg/kg-D
7
0.3 ppm
10 mg/kg-D
--
Mouse
16-20%
3-12%
45-55%
0.5%
0.5%
0.05%
0.5-1.0%
0.2%
25 mg/kg-D
5 mg/kg-D
Trace
Trace
0.002%
0.005%
500 lU/kg-D
3 mg/kg-D
4 mg/kg-D
--
30 mg/kg-D
1 mg/kg-D
1 g/kg-D
5 ug/kg-D
None
18 lU/kg-D
40 lU/kg-D
1 mg/kg-D
10-100 mg/kg-D
7
0. 34 ppm
8.5 mg/kg-D
--
Rabbit
15%
3-5%
45-55%
Required
"
350 mg/kg-D
0.7%
Required
"
7 mg/kg-D
Required
ii
None
None
200 mg/kg-D
Required
Required
None
None
Required
7 rag/ kg- D
7
7
Required
Required
None
7
Guinea pig
20%
3-5%
45-50%
1.2%
0.6%
0.34%
Required
1.6%
Required
n
11
n
12 mg/kg-D
16 mg/kg-D
16 mg/kg-D
20 mg/kg-D
100 mg/kg-D
16 mg/kg-D
1.5 g/kg-D
None
200 mg/kg-D
None
60 mg/kg-D
10 mg/kg-D
None
10 mg/kg-D
None
—
100 mg/kg-D
Hamster
24%
3.5%
45-65%
0.6%
0.35%
7
7
7
7
7
7
7
7
7
13,000 IU/kg-D
6 mg/kg-D
6 mg/kg-D
40 mg/kg-D
Required
6 mg/kg-D
7
None
None
None
25 mg/kg-D
Required
7
None
None
—
7
NOTE: Fiber or roughage in standard commercial diets is about 10 percent for the guinea pig, 15 percent for the rabbit, 2.5 to 5.5 percent
for the other common laboratory animals listed here.
©1979, Manual for Laboratory Animal Technicians, used by permission of
American Association for Laboratory Animal Science.
-------
TABLE 7-6. BIOLOGICALLY EFFECTIVE CONCENTRATIONS OF SELECTED
HEAVY METALS
Element Species
As Mouse
Rat
Cd Mouse
Rat
Pb Mouse
Rat
Ag Mouse
Rat
Se Mouse
Rat
End point
Reproduction
Growth and mortality
Mortality
Mammary tumors
Growth
Reproduction
Mortality
Renal vasculature
Hypertension
Mortality
Growth
Antibody formation
6-Aminolevulinate
dehydratase
Mortality
Mortality and growth
Hypertension
Behavior and learning
Kidney ultrastructure
Reproduction
Growth and mortality
Mammary tumors
Growth, mortality
and tumors ,
AAF- induced tumors
Hepatitis
Dietary or water
concentration
(ppm)
No
Effect effect
5
5
10
62
10
5
0
1
5
14
10
5
5
5
2
2
3
3
2
2
0
2
5
.5b 31.25b
.2
—
5
™
1
b 2'5
b
•5h 0-lh
.5b 0.5b
Decreased incidence, increased tumor growth rate.
Mixed in the diet, all others were administered in water.
CDecreased tumor incidence.
Increased latent period.
©1980, Journal of Toxicology and Environmental Health, Vol. 6, used by
permission of Hemisphere Publishing Corporation.
129
-------
TABLE 7-7. BIOLOGICALLY EFFECTIVE CONCENTRATIONS OF SELECTED
ORGANIC FEED CONTAMINANTS
Dietary
concentration
(ppm)
Compound Species
Aflatoxin Mouse
(BO
Rat
DDT Mouse
Rat
Dieldrin Mouse
Rat
Heptachlor Mouse
Rat
Lindane Mouse
Rat
Malathion Rat
End point
Liver lipids
Liver tumors
Liver hyperplasia
Liver tumors
Reproduction
Neoplasia
Liver tumors
Liver tumors
Lung tumors
Liver morphology
Microsomal . enzymes
Microsomal enzymes
Reproduction
Mortal ity
Liver tumors
Microsomal enzymes
Microsomal enzymes
Liver weight
Brain lesions
Liver weight
Liver tumors
Microsomal enzymes
Microsomal enzymes
Tumor induction and
liver morphology
Microsomal enzymes
Microsomal enzymes
and liver weight
Liver weight
Cholinesterase
Choi inesterase and
EEC
DMBA- induced tumors
Effect
0.114
1
0.001
0.015
-
3
2
250
10
5
2.5
1
2.5
10
0.1
5
5
1
0.34
1
5
5
2
-
50
20
100
1,000
380a
250
No
effect
0.057
—
—
—
3
-
-
20
-
1
2
0.2
-
1
-
1
2
0.1
0.02
-
1
1
-
50
20
2
-
100
-
-
Estimated equivalent.
©1980, Journal of Toxicology and Environmental Health, Vol. 6, used by
permission of Hemisphere Publishing Corporation.
130
-------
h. The investigator should be thoroughly aware of the solubility
parameters of the compounds involved as well as their possible
interactions in animal systems and their expiration dates.
i. The investigator should monitor the stability of the tested
compounds and maintain thorough records of the results.
7.3.3 Experimental Design and Data Reporting
1. All data, including experimental design, observations, and informa-
tion on test animal physiological parameters, should be fully docu-
mented on standard forms or in a bound notebook, whichever is more
appropriate.
2. Any changes or deviations from standard operating procedures (SOP)
should be documented in writing after approval from the principal
investigator.
3. A qualified biostatistician should review all research protocols to
ensure proper implementation of statistical procedures.
4. A Doctor of Veterinary Medicine should be consulted for recommenda-
tions on experimental procedures and test animal health verification.
7.4 SUMMARY
The purpose of this section was to provide the staff and scientific
investigators of HERL-RTP with basic information necessary to provide proper
animal care. Following these quality control and protocol guidelines will
increase sensitivity, quality, and reproducibility of results and the longevity
of test animals.
A properly designed animal research program should address total data
quality, including quality control and quality assurance activities. The
major effort in quality control is made by EPA/HERL-RTP, the operating organiza-
tion. Consequently, the principal investigator and the laboratory animal
staff are responsible for quality control. Internal quality assurance is the
responsibility of the animal care coordinator and the animal care committee.
External quality assurance is provided by outside audits conducted by organiza-
tions such as AAALAC ensure that proper animal handling techniques are being
followed. These external audits are encouraged because they will assist in
the objective definition of problem areas and reward and document HERL-RTP1s
ability and commitment to obtaining animal data of the highest quality.
Recent provisional accreditation of the HERL-RTP animal facilities by AAALAC
has verified that animal care is acceptable.
131
-------
In summary, quality control is consistent with the implementation of good
scientific methodology and with the systematic attention to eliminating the
causes of poor data quality.
7.5 REFERENCES
1. Environmental Protection Agency (EPA) Good Laboratory Practice Standards
for Health, Federal Register, Vol. 44, No. 91, Wednesday, May 9, 1979,
pp. 27362-27375.
2. Food and Drug Administration (FDA) Nonclinical Laboratory Studies Good
Laboratory Practice Standards, Federal Register Vol. 43, No. 247, Friday,
December 22, 1978, Part II, pp. 59986-60025.
3. Greenman, D. L., W. L. Oiler, N. A. Littlefield, and C. J. Nelson, Com-
mercial Laboratory Animal Diets: Toxicant and Nutrient Variability,
Journal of Toxicology and Environmental Health, 6:235-246, 1980.
4. Guide for the Care and Use of Laboratory Animals, U.S. DHEW, Publication
No. (NIH)78-23, 1978.
5. Long-term Holding of Laboratory Rodents, Institute of Laboratory Animal
Resources, ILAR News, XIX(4), 1976.
6. Sontag, H., N. Page, and U. Saffiotti, Guidelines for Carcinogen Bioassay
in Small Rodents, NCI Technical Report Series 1, DHEW Publication Number
(NIH)76-801-NC1-CG-TR-1, 1976.
7. Manual for Laboratory Animal Care, Ralston Purina Company, Checkerboard
Square, St. Louis, Missouri 63188, 1978.
8. Manual for Laboratory Animal Technicians, American Association for Labor-
atory Animal Science (AALAS), 210 N. Hammes Avenue, Suite 205, Joliet, IL
60435, Publication 67-3, December 1, 1979.
9. Guide to the Care and Use of Experimental Animals, Volume 1, Canadian
Council on Animal Care, 1105-151 Slaten Street, Ottawa, Ontario K1P5H3,
1980.
10. Green, E.L. (ed.), Biology of the Laboratory Mouse, McGraw-Hill Book
Company, 1966, pp. 1-7067
11. Baker, J., J. R. Lindsey, and S. H. Weisbroth, The Laboratory Rat, Volume I,
Biology and Diseases, Academic Press, 1980, pp. 1-435.
12. Baker, J., J. R. Lindsey, and S. H. Weisbroth, The Laboratory Rat, Volume II
Research Applications, Academic Press, 1980, pp. 1-276.
13. Linko, R., Standard Operating Procedures, HERL/LAS ACC, Room P302, Environ-
mental Protection Agency, Research Triangle Park, NC, 27711, 1980.
132
-------
APPENDIX A
CHEMICAL PRODUCTS USED IN THE HERL-RTP
ANIMAL CARE FACILITIES
CHEMICAL PRODUCTS USED IN THE EPA ANIMAL CARE FACILITIES*
I. Approved Disinfectants
A. Nolvasan-S, Chlorhexidine based virucide, Fort Dodge Laboratories,
Inc.
Active ingredient:
l,l'-Hexamethyl(exebis)-[5-(p-chlorophenyl) biguanide] diacetate--2%
Concentration: 3 ounces Nolvasan-S to 1 gallon water.
B. T.B.Q. Germicidal Detergent, quaternary ammonium-based germicide,
Vestal Laboratories.
Active ingredients:
N,N-bis [2-(omega-hydroxpoly(oxyethylene)) ethyl] alkylamine--12%
Alkyl (50% C14, 40% C12, 10% Ci6) dimethyl benzyl ammonium
chloride--(trade name: Zephiran chloride) 8%
Concentration: 2 ounces T.B.Q. to 1 gallon water.
C. BGC-3 broad-spectrum germicidal cleaner, multiphenolic based germi-
cide, DuBois Chemicals.
Active ingredients:
Sodium lauryl ether sulfate--99%
Sodium ortho-phenylphenate--8.85%
Sodium ortho-benzyl-para-chlorophenate--7.92%
Isopropyl alcohol--?.39%
Sodium para-tertiary-amylphenate--4.10%
Tetrasodium ethylenediamine tetracetate--0.95%
Concentration: 0.5 (1/2) ounce BGC-3 to 1 gallon water.
II. Detergents
A. Life Sci 100, alkaline ware washing agent, DuBois Chemicals.
Ready-mixed concentration provided.
B. Life Sci 200, concentrated liquid phosphoric acid cleaner, DuBois
Chemicals.
Ready-mixed concentration provided.
*Source: R. Linko, Standard Operating Procedures, HERL/LAS ACC, Room P302,
No. 247, Environmental Protection Agency, Research Triangle Park, NC, 1980.
133
-------
C. Septisol Solution (Hexachlorophene), health care personnel hand
wash.
Ready-mixed concentration provided.
D. Du-DRI, concentrated, acidic, liquid rinse additive, DuBois Chemi-
cals.
Ready-mixed concentration provided.
III. Odor Counteractants
A. Arrest, water-based maskant, DuBois Chemicals.
Concentration: 2 ounces Arrest to 1 gallon water.
IV. Insecticides
A. Baygon 1.5, emulsifiable insecticide, Mobay Chemical Corporation.
Active ingredients:
2-(l-Methylethoxy) phenol methylcarbamate--13.9%
Concentration: 8 ounces Baygon 1.5 to 1 gallon water.
B. Diazinon-4E, insecticide, Stephenson Chemical Company, Inc.
Active ingredients:
0,0-diethyl 0-(2-isopropyl-6-methyl-4-pryimidinyl)
Phosphorothioate--47.55%
Tetrachloroethylene--8.70%
Aliphatic petroleum distillates—26.15%
C. Drione, insecticide, FMC Corporation.
Active ingredients:
Pyrethrins—1%
Piperonyl butoxide, technical —10%
Amorphous silica gel--40%
Petroleum hydrocarbons--49%
Concentration: This insecticide is applied as a powder as packaged.
V. Miscellaneous.
A. Chlorox
B. Comet
C. Lysol
134
-------
APPENDIX B
ZOONOSES—EXPERIMENTAL ANIMALS TO MAN
A: BACTERIAL DISEASES: —
Disease in Man
9. Pscudotuberculosis
10. Rat Bite Fever
11. Salmonellosis
12. Shigellosis
Baciltary Dysentry
13. Tetanus
14. Tuberculosis
15. Tularemia
16. Vibriosis
Causative Agent
P.pseudotuberculosis
and some Fungi
S.monlliformis
Spirillam minus
Salmonella spp.
Shigella spp.
Cl.tetanl
M. tuberculosis
M.bovis
M. avium
F. tularensis
Vibrio fetus
Common
Vertebrate Hosts'
Rodents, Pigeons, Turkeys
Canaries & misc. wild birds
Rodents
Rodents
Farm animals, Rodents
Reptiles, Amphibia
Non-human primates
Horse and other
Equidae
Primates, Cattlo, Dogs
Cattle. Dogs
Poultry, Swine. Sheep
Rabbits, Wild rodents
Sheep
Domestic ruminants
Means ol Spread
Contact
Ingestion
Rodent bites, Ingestion
Rodent bites
Ingestion; Inhalation
Contact
Contact, fecal
contamination
Contaminated
puncture wounds
Contact, Ingestion
Inhalation, Needles —
hypodermic & tattoo
Contact
Ingestion
Unknown (contact?)
Vectors and
Notes on Cycle
—
—
—
—
Soil, many
mammalian spp.
Anthropozoonotic"
Biting Insects
and ticks
—
• Only more common host species are listed.
" Man is the primary vertebrate host.
©1980, Guide to the Care and Use of Experimental Animals, used by permission
of the Canadian Council on Animal Care.
-------
APPENDIX B
ZOONOSES—EXPERIMENTAL ANIMALS TO MAN (cont'd)
B. RICKETTSIAL - DISEASES:
Causative Agent
R. akari
R. rickettsia
R.australis
R.sibiricus
R. mosseri
Coxiella
burnetti
Diseases In Man
Rickettsial Pox
Rocky Mountain
Spotted Fever
Queensland, North
Asian Tick Typhus etc.
of Eastern/Asiatic
Countries
Murine Typhus
Q (ever
Common
Vertebrate Hosts
Mice
Rodents. Rabbits
Young sheep & dogs
Various Mammalian
Tick-host species
Rats; Mice
Cattle; Sheep
Means of Spread; Vectors, Cycle Notes
Mite bites; Altodermanyssus sanguineus
Tick bites; Dermacentor spp;
American dog tick
Tick bites; ticks themselves
may act as reservoirs with
tick to tick passage
Flea bites from rat (leas; Rat to rat spread by lice also
Inhalation; No overt disease in natural hosts;
virus concentrated in placenta
C. ARBOVIRUS DISEASES:
E.E.E. virus
W.E.E. virus
V.E.E.
S.L.E
Powassan virus
Colorado tick —
borne virus
Various Asian
Arboviruses
Eastern Equine
Encephalomyelltis
Western Equine Enceph
Venezuelan Equine
Encephalitis
St. Louis Encephalitis
Powassan Encephalitis
Colorado tick
(ever
Various tick-borne
Hemorrhagic Fevers
Birds; Horses
Birds; Horses
Horses
Birds
Rodents (?)
Ground' squirrels;
Peromyscus spp.
Rodents; Hares; Monkeys
Mosquito bites; Bird/Mosquito/
Horse natural cycle
...,„.
Natural cycle Horse/Mosquito only
Natural cycle Bird/Mosquito only
Tick bites;
Tick bite; Tick/small Rodent
natural cycle
Tick bites: Sub tropical climate conditions lavor
cycle.
©1980, Guide to the Care and Use of Experimental Animals, used by permission
of the Canadian Council on Animal Care.
-------
APPENDIX B
ZOONOSES—EXPERIMENTAL ANIMALS TO MAN (cont'd)
D. OTHER VIRUS DISEASES:
Causative Agent
Hemorhagic lever virses
L.C.M. virus
Herpes simiae
Hepatitis virus
Rabies virus
Diseases In Man
S. American and Korean
Hemorhagic fever. Lassa
(ever
Lymphocytic Chprio —
Meningitis
Herpes B.Encephalitia
Hepatitis A.
Rabies
Common
Vertebrate Hosts
Wild rodents
Mastomys ratalensis
Rodents; numerous other
mammals
Rhesus; Other Macaca
Chimpanzees;
Dogs, Bats & many others
Means of Spread, Vectors, Cycle Notes
Contact; Contamination of food etc. with rodent
excreta. Direct contact
Contact; Inhalation; Congenital transmission;
Tissue culture transmission.
Contact; bite wounds; Old World monkeys.
Contact; Anthropozoonotic diseases'.
Bites: Saliva contact; virus cone. In salivia.
• Man is primary host. Measles (Rubeola) is another anthropozoonotlc virus to non-human primates.
E. FUNGAL and PROTOZOAN DISEASES:
Trychophyton spp.
Microsporum spp.
Other dermatophytes
Histoplasma
capsulatum
Coccidioides
immitis
Toxoplasma gondil
Trypanasoma spp.
Plasmodiuma spp.
Leishmania spp.
Entamoeba
histolytica
Ringworm
dermatomycoses
Hlstoplasmosis
Coccidioidomycosls
Toxoplasmosls
Blood protozoan
diseases
Amebiasis
Amebic dysentery
Dog. Cal. G. Pig
Rodents and Farm animals
Dogs, other domestic
and wild species
Cattle, Dogs and
occassionally other spp.
Cats; occassionally other
other domestic & lab spp.
Non human Primates,
Rodents Domestic and
wild spp.
Dogs. Non human Primates
Direct contact, Ringworm of man can be
transmitted to animals and visa-veras. Soil may
be reservoir.
Inhalation of fungi. May also grow in soil.
Inhalation of air-borne spores
Fungus present In dc.ert soil.
Ingestion of oocyots from cats, Inhalation
Infected meat; Fetal transmission may occur.
Insect vectors — saliva transmission. Some
few species direct transmission.
Contamination of food, usually by man (natural
host) to dogs.
®1980, Guide to the Care and Use of Experimental Animals, used by permission
of the Canadian Council on Animal Care.
-------
Ul
oo
APPENDIX C
PHYSIOLOGICAL AND NUTRITIONAL PARAMETERS
Species
(age & weight)
Monkey
(M. fascicularis)
Mouse
Guinea pig
Hamster
Rabbit
Rat
Baboon
Cat
Rectal
temp.
(° C ± 0.5)
39.0
37.5
39.5
39.0
39.5
37.5
39.0
38.5
Resp. rate
{minimum
X & range)
40
30-54
138
90-180
86
60-110
77
40-120
40
35-56
92
80-150
25
22-35
26
20-30
Heart rate
{minimum
X & range)
220
165-243
470
300-650
280
250-300
332
286-400
260
205-308
350
260-450
115
105-150
150
110-226
Water
required
(daily)
350-950 ml_
3-7 ml
12-15 ml/
100 g body wt.
8-12 mL
80-100 ml/
kg body wt.
20-45 mL
400-600 mL
150 mL
100-200
Urine
excreted
(daily)
150-550 ml
1-3 mL
15-75 mL
6-12 ml
50-90 mL/
kg body wt.
10-15 ml
150-400 mL
50-120 mL
Food
required
(daily)
350-550 g
3-6 g
20-35 g
+ Vit.C. supp.
7-15 g
75-100 g
10-20 g
1-1.5 kg
110-225 g
Digestible
protein
%
17
12
25-30
16
14
12
17
30
©1980, Guide to the Care and Use of Experimental Animals, used by permission
of the Canadian Council on Animal Care.
-------
OJ
SO
APPENDIX D
CLINICAL CHEMISTRY REFERENCE VALUES
Species
Monkey
(M. fascicularis)
Mouse
Rabbit
Rat
Guinea pig
Hamster
Sodium
(mEg/L)
146-152
136
128-186
144
138-160
147
140-156
123
120-149
131
106-146
Potassium
(mEg/L)
4-5
5.3
4.9-5.9
6.0
3.7-6.8
6.2
5.4-7.0
5.0
3.8-7.9
5.0
4.0-5.9
Chloride
(mEg/L)
101-108
108
105-110
103
92-112
102
100-110
94
90-115
95
86-112
Bicarbonate
(mgEg/L)
30
19-35
25.5
20-32
24
16-32
22
13-3.2
21.5
13-30
38.2
33-44
Inorganic
phosphorus
(mg/dL)
5-5.4
6.0
2.3-9.2
4.9
2.3-6.9
7.9
3-11
5.3
3-7.6
5.7
3.4-8.2
Calcium
(mg/dL)
9.5-10.7
6.4
3.2-8.5
9.9
5.6-12.7
11.5
5-14
10.2
5.3-12
9.9
5-12
Magnesium
(mg/dL)
2.3
0.8-3.9
2.8
2-5.4
2.9
1.6-4.4
2.4
1.8-3.0
2.4
1.9-3.5
©1980, Guide to the Care and Use of Experimental Animals, used by permission
of the Canadian Council on Animal Care.
-------
APPENDIX D (continued)
CLINICAL CHEMISTRY REFERENCE VALUES
Species
X range
Monkey
(M. fascicularis)
Mouse
Rabbit
Rat
Guinea pig
Hamster
Glucose
(Md/dL)
60-90
89
63-176
132
78-155
75
50-135
92
82-107
69
33-118
B.U.N.3
(mg/dL)
18-28
19.5
14-28
18.5
9-32
14.5
5-29
23.5
9-32
22
12-26
Cholesterol
(mg/dL)
100-150
64
26-82
26
20-83
27
10-54
30
16-43
53
10-80
Total
protein
(g/dL)
7.5-8.7
6.2
4-8.6
6.8
5-8
7.6
4.7-8.2
5.2
5-6.8
7.1
4-8
Albumin
(g/dL)
2.4-3.4
3
2.5-4.8
3.3
2.5-4
3.7
2.7-5.1
2.6
2.1-3.9
3.3
2.5-4
S.G.O.T.b
(I.V./L)
34-56
36
23-48
71
42-98
63
46-81
47
27-68
100
38-168
S.G.P.T.C
(I.V./L)
21-39
13
2-24
65
49-79
24
18-30
42
25-59
24
12-36
Alkaline
phosph.
(I.V./L)
15-35
19
10-28
130
90-170
87
57-128
70
55-108
17
3-31
Blood urea nitrogen.
3Serum glutamic oxalacetic transaminase.
"Serum glutamic pyruvic transaminase.
-------
APPENDIX E
HEMATOLOGY
Mean Values and Ranges
^
Species
(age & weight)
Monkey
(M. fascicularis)
Mouse
Baboon
Cat
Rabbit
Rat
Guinea pig
Hamster
RBC Mb.
(x 106/mm3) (g/100 ml)
5
4-6
9.2
7-13
5
4-6
7.3
5-10
6.5
5-8
8.5
6-10
5.2
3-7
7.2
4-10
10-12
11.1
10-14
12
8-16
10.5
8-15
13.5
8-17
14.2
11-17
14.3
11-17
16.4
13-19
e ©1980, Guide to the Care and Use of Experimental Animals
e of the Canadian Council on Animal Care.
i
i
PCV
(mL %)
35-43
41.8
33-50
35.6
30-43
40.5
24-45
40.8
31-50
45.9
40-50
43.6
37-50
50.8
39-59
, used by
Platelets
(x 103/mm3)
300-500
240
150-400
100-450
228
100-700
468
250-750
330
150-460
477
250-750
386
300-570
permission
WBC
(x 103/mm3)
5-10
13.6
6-17
5-17
17.0
5-20
8.6
3.0-12.5
9.8
5-13
11.2
6-17
8.1
5-11
Neotrophils
50
30-65
17.2
12-25
27-73
57.1
35-75
45.0
30-65
25.5
5-49
37.0
20-56
25.5
15-35
Lymphocytes
45
25-70
72.3
65-85
26-59
32.2
20-55
40.1
28-85
74.0
43-85
55.7
40-80
70.8
55-92
Blood vol.
(mL/kg)
55-75
70-80
50-70
45-75
45-70
50-65
65-90
65-80
------- |