United States
Environmental Protection
Agency
Office of Research and
Development
Washington, DC 20460
EPA/600/4-90/031
January 1991
Manual for the Evaluation of
Laboratories Performing
Aquatic Toxicity Tests
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EPA/600/4-90/031
MANUAL FOR THE EVALUATION OF LABORATORIES
PERFORMING AQUATIC TOXICITY TESTS
by
Donald J. Klemml, Larry B. Lobring^,
and William H. Horning 11^
^Aquatic Biology Branch, Quality Assurance Research Division
^Inorganic Chemistry Branch, Chemistry Research Division
3 Aquatic Biology Branch, Quality Assurance Research Division
Environmental Monitoring Systems Laboratory - Cincinnati
ENVIRONMENTAL MONITORING SYSTEMS LABORATORY - CINCINNATI
OFFICE OF MODELING, MONITORING SYSTEMS, AND QUALITY ASSURANCE
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
Printed on Recycled Paper
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NOTICE
This document has been reviewed in accordance with-U. S. Environmental
Protection Agency Policy and approved for publication. Mention of trade
names or commercial products does not constitute endorsement or
recommendation for use.
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FOREWORD
Environmental measurements are required to determine the chemical
and biological quality of drinking water, surface waters, groundwaters,
wastewaters, sediments, sludges, and solid waste. The Environmental
Monitoring Systems Laboratory - Cincinnati (EMSL-Cincinnati) conducts
research to:
o Develop and evaluate methods to identify and measure the
concentration of chemical pollutants.
0 Identify and quantitate the occurrence of viruses, bacteria,
and other human pathogens and indicator organisms.
0 Measure the toxicity of pollutants to representative species
of aquatic organisms and determine the effects of pollution on
communities of indigenous freshwater, estuarine, and marine
organisms, including the phytoplankton, zooplankton, periphyton,
macrophyton, macroinvertebrates, and fish.
0 Develop and operate a quality assurance program to support
achievement of data quality objectives for environmental
measurements.
The Federal Water Pollution Control Act Amendments of 1972
(PL 92-500), the Clean Water Act (CWA) of 1977 (PL 95-217), and the Water
Quality Act (WQA) of 1987 (PL 100-4) explicitly state that it is national
policy that the discharge of toxic substances in toxic amounts be
prohibited. Determination of the toxicity of effluents, therefore, plays
an important role in identifying and controlling toxic discharges into
surface waters. The guidelines in this manual were developed for use by
the U.S. Environmental Protection Agency (USEPA) regional and state
programs, and the National Pollutant Discharge Elimination System (NPDES)
compliance monitoring program to provide standardized procedures for
conducting on-site audits and evaluations of laboratories performing
toxicity tests of effluents and surface waters.
Thomas A. Clark
Director
Environmental Monitoring Systems
Laboratory - Cincinnati
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PREFACE
This document provides guidelines for evaluation of biological
laboratories involved in toxicity testing and in the culturing of
freshwater and marine fish, invertebrates, and plants for use in effluent
and surface water toxicity tests. The subjects covered include:
evaluation criteria, preparation for the audit and evaluation,
organizational history, laboratory personnel, facilities, equipment and
supplies, methodology, sample collection, handling, and preservation,
quality assurance, records and data reporting, safety, and report
preparation. The evaluator performing on-site audits and evaluations of
aquatic biology laboratories must have working knowledge of the NPDES
program and sufficient knowledge and experience with biomonitoring and
toxicity testing methodology. The manual was developed to aid the
National Pollutant Discharge Elimination System (NPDES)
evaluator/inspector in performing the Compliance Evaluation Inspections
(CEI) and the Performance Audit Inspection (PAI) specified in the USEPA
(1988a), NPDES Compliance Inspection Manual.
While no formal national USEPA certification program is in place for
aquatic biology laboratories performing toxicity tests as part of the
NPDES program, guidelines are needed that describe an overall laboratory
evaluation program capable of producing valid toxicity data for use in a
NPDES permit. Guidelines have been used for several years to assess the
capability of the Regional EPA laboratories to provide biological data of
acceptable quality.
An overall laboratory evaluation program consisting of four phases is
described. Phase I includes preliminary contact between the laboratory
and the regulating authority to determine mutually agreeable dates for
the on-site inspection, submission ef completed pre-survey information
forms, and submission of available data on the use of reference toxicants
or performance evaluation samples by the laboratory prior to the on-site
visit. Phase II is the on-site visit by a qualified evaluator,
consisting of a meeting with the senior laboratory staff to explain the
audit or evaluation process, a tour of the laboratory facility,
one-on-one discussions with the technical staff, examination of documents
and records, completion of the on-site evaluation forms and checklists,
and an exit debriefing by the evaluator providing a verbal laboratory
performance rating of acceptable, minimally acceptable, or unacceptable.
Phase III is submission of a final report by the evaluator to the
laboratory with a rating indicating reconciliation of any differences and
corrective actions required by the laboratory. The final report must
clearly state the capabilities of the laboratory to provide acceptable
biological data. Phase IV consists of follow-up activities such as
technical assistance, resolving major deficiencies, and revisiting the
laboratory, if required, to inspect corrected deficiencies and major
changes in the laboratory.
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ABSTRACT
This manual describes guidelines and standardized procedures for
conducting on-site evaluations of laboratories performing toxicity
tests. Included are pre-survey information activities, on-site
evaluation activities, evaluation criteria, organizational history and
laboratory staff, facilities, equipment, instruments, supplies, culturing
and testing methodology, sample collection, handling, preservation,
preparation, quality assurance and data handling, safety and general
practices, evaluation report and performance rating. Supplementary
information on chain-of-custody guidelines, quality control checklist for
self-biomonitoring toxicity tests, standard operating procedures (SOPs)
format, culturing criteria SOP format, pre-survey information forms,
on-site laboratory evaluation forms and checklists, and on-site toxicity
test conditions and test acceptability criteria checklists is provided in
the Appendices.
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TABLE OF CONTENTS
Foreword . . iii
Preface iv
Abstract v
Acknowledgments viii
1. Introduction 1
2. Evaluation Criteria for Toxicity Testing
Laboratories 3
3. Preparation for the Evaluation 6
4. Organizational History and Laboratory Staff 8
5. Laboratory Facilities 10
6. Equipment, Instruments, and Supplies 13
7. Methodology 16
8. Sample Collection, Handling, Preservation,
and Preparation . . 18
9. Quality Assurance and Data Handling 22
10. Safety and General Practices 25
11. Evaluation Report 27
Selected References 29
Appendices 33
A. Chain-of-Custody Guidelines 34
B. Quality Control CheckList for Self-Biomonitoring
Toxicity Tests 40
C. Example of Standard Operating Procedures (SOPs) Format ... 42
D. Example of Culturing Criteria SOP Format 45
E. Pre-Survey Forms: Toxicity Test Laboratory Evaluation ... 46
F. On-Site Laboratory Evaluation Forms and Checklists 63
G. Recommended Toxicity Test Conditions and Test Acceptability
Criteria: On-Site Checklists 72
VII
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ACKNOWLEDGMENTS
The manual was prepared in part using the.following sources:
toxicity test manuals, "Methods for measuring the acute toxicity of
effluents to freshwater and marine organisms," "Short-term methods for
estimating the chronic toxicity of effluents and receiving waters to
freshwater organisms," "Short-term methods for estimating the chronic
toxicity of effluents and receiving waters to marine and estuarine .
organisms," by EMSL-Cincinnati; the draft outlines "Performance audit
inspection guidance document for the NPDES permit program," by
EMSL-Cincinnati; the draft outline "Aquatic toxicity test laboratory
evaluation criteria" by EMSL-Cincinnati; the draft document "Evaluation
of aquatic biology programs" by EMSL-Cincinnati; "Manual for the
certification of laboratories analyzing drinking water. Criteria and
procedures quality assurance," 1990a, by the Office of Drinking Water,
Washington, D.C.; previous inspections of toxicity testing programs, by
USEPA, Regions I, III, and IV; and manuals and checklists for performance
audit inspection, evaluation, or certification for aquatic toxicity
testing used by USEPA Regions I, III, IV, and state regulatory agencies,
i.e., North Carolina, South Carolina, Massachusetts, New Jersey, and New
York.
This manual was reviewed by the following members of the
EMSL-Cincinnati Bioassay Subcommittee and its parent committee, the
Biological Advisory Committee. The reviewers represented individuals
from EMSL-Cincinnati;, USEPA regional and headquarters programs, and other
research and development (ORD) laboratories.
Peter Nolan, Environmental Services Division, Region 1
William Peltier, Environmental Services Division, Region 4
Michael Bastian, Environmental Services Division, Region 6
Charles Faultry, Environmental Services Division, Region 6
Terry Hollister, Environmental Services Division, Region 6
Delia Oteiza, Environmental Services Division, Region 6
James F. Pendergast, Water Management Division, Region 6
Bob Reeves, Environmental Services Division, Region 6
Loys Parrish, Environmental Services Division, Region 8
Margarete A. Heber, Office of Water Enforcement and Permits,
Headquarters, Washington, D.C.
Jerry Smrchek, Office of Toxic Substances, Headquarters, Washington,
D.C.
Gary B. Collins, Environmental Monitoring Systems Laboratory -
Cincinnati
James Lazorchak, Environmental Monitoring Systems Laboratory -
Cincinnati
Philip A. Lewis, Environmental, Monitoring Systems Laboratory -
Cincinnati
Timothy W. Neiheisel, Environmental Monitoring Systems Laboratory -
Cincinnati
Quentin H. Pickering, Environmental Monitoring Systems Labratory -
Cincinnati
vn
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Cornelius I. Weber, Environmental Monitoring Systems Laboratory -
Cincinnati
John Winter, Environmental Monitoring Systems Laboratory -
Cincinnati
Mary M. Sullivan, Quality Assurance Research Division, provided
valuable secretarial assistance, and Betty J. Thomas, Publication
Assistance, EMSL-Cincinnati, provided a thorough editorial review.
IX
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SECTION 1
INTRODUCTION
1.1 The Federal Water Pollution Control Act (FWPCA) Amendments of 1972
(PL 92-500), the Clean Water Act (CWA) of 1977 (PL 95-217), and the Water
Quality Act of 1987 (PL 100-4) were enacted to restore and maintain the
chemical, physical, 'and biological integrity of the Nation's waters
(Section 101[a]). The legislation contains other specific or implied
requirements for the collection of biomonitoring data in at least 15
sections. The Declaration of Goals and Policy, Section 101(a)(3) of
these laws, states that "it is the national policy that the discharge of
toxic pollutants in toxic amounts be prohibited." The principal
mechanism for reducing and eliminating the discharge of toxic substances
is through implementation of the National Pollutant Discharge Elimination
System (NPDES) program established by Section 402(a)(l) of the FWPCA.
1.2 During the 1970's and early 1980's, acute toxicity tests were used by
the U.S. Environmental Protection Agency (USEPA) regional programs and
states to estimate the safe concentration of toxic effluents in receiving
waters (USEPA, 1973; 1975; 1978c; 1985a). These methods were
supplemented later with short-term (sub-chronic or chronic) toxicity
tests (nine days or less) to estimate the chronic toxicity of effluents
(USEPA, I988c; 1989c).
1.3 Based on the growing use Of effluent toxicity tests for the control
of toxic discharges, USEPA issued a national statement: "Policy for the
Development of Water Quality Based Permit Limitations for Toxic
Pollutants," in the Federal Register Vol. 49, No. 48, Friday, March 9,
1984. The policy proposed the use of toxicity data to assess and control
the discharge of toxic substances through the NPDES permits program. The
policy also states that "biological testing of effluents is an important
aspect of the water quality-based approach for controlling toxic
pollutants. Effluent toxicity data, in conjunction with other data, can
be used to establish control priorities, assess compliance with state
water quality standards and set permit limitations to achieve those
standards." A technical support document and permit writer's guide on
the use of effluent and receiving water toxicity data were prepared by
the Office of Water Enforcement and Permits (OWEP) and the Office of
Water Regulations and Standards (OWRS) to provide additional guidance on
the implementation of the biomonitoring policy (USEPA, 1985b; 1988a;
1990a).
1.4 Dischargers of pollutants are issued permits under Section 402 of
the Act which set specific limits and operating conditions to be met by
the permittee. Section 308 of the Act authorizes inspections and
monitoring to determine whether NPDES permit conditions are being met.
The Section provides for self-monitoring and USEPA monitoring. USEPA
monitoring consists of either evaluating self-monitoring data or
performing on-site monitoring. Also, Section 308 and 402 of the Act
provide for the delegation of Federal NPDES program authority to States
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to issue permits and conduct permit compliance monitoring. Section
122.44 (d)(1)(IV)(V), in addition, specifies that there are some
conditions that require the permit to contain effluent limits for whole
effluent toxicity.
1.5 USEPA Regional Laboratories are currently audited by the
EMSL-Cincinnati. USEPA Regional Laboratories are in turn responsible for
auditing and evaluating state laboratories, and USEPA Regional
Laboratories and/or some state laboratories are responsible for auditing
or evaluating permittee laboratories, such as county, municipal,
industrial, utility, and contract service laboratories.
1.6 Presently there is no formal USEPA certification program for
laboratories performing aquatic toxicity tests as part of the NPDES
permits program. This manual is to provide uniform guidelines for the
evaluation of biological laboratories to ensure high quality data. USEPA
Regions and several states have requested common guidelines that can be
used to evaluate the capability of federal, state' university, private,
and local laboratories to produce data on the toxicity of effluents and
receiving waters.
1.7 Proposed operation of the certification program is as follows:
EMSL-Cincinnati is to certify USEPA Regional laboratories for biological
analyses. Regional certification officers are responsible for
certification of state laboratories and in turn the state laboratories
are responsible for certifying local laboratories. Local laboratories
include any state, county, industrial, municipal, utility, federal or §•
private consulting laboratory excluding USEPA Regional and principal ^
state laboratories. EMSL-Cincinnati would also offer an annual training
program for all evaluators.
1.8 Regional laboratories and principal state laboratories should
annually provide toxicity data using reference toxicants supplied by
EMSL-Cincinnati and pass an on-site evaluation every three years.
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SECTION 2
EVALUATION CRITERIA FOR TOXICITY TESTING LABORATORIES
2.1 To comply with the criteria set forth in this manual, laboratories
performing toxicity tests must:
2.1.1 Maintain a qualified staff (see Section 4).
2.1.2 Develop, implement, and maintain a document describing the quality
assurance and quality control program, including a laboratory quality
assurance plan to ensure that precision, accuracy, completeness,
comparability, and representativeness of data are known and documented.
2.1.3 Develop data quality objectives (DQOs) so that determination of
data quality is accomplished and met (USEPA, 1984b; 1989a; 1989b). DQOs
are qualitative and quantitative statements developed by data users to
specify the quality and precision of data needed to support specific
decisions or regulatory actions. Establishment of DQOs involves
interaction of decision-makers and the technical staff.
2.1.4 Develop and maintain detailed written standard operating
procedures (SOPs) for all toxicity tests, culture methods, equipment and
instruments, glassware cleaning procedures, sample collection, sample
preservation, preparation, chain-of-custody procedures, chemical
analyses, quality control, and data analyses. Quality control procedures
and techniques must also be included in the SOPs.
2.1.5 Use approved or recommended methods (see Section 7). The
culturing, toxicity testing conditions, physical and chemical analyses,
test acceptability criteria, and statistical methods for data analyses
are found in the following toxicity test manuals (unless otherwise noted):
*EPA/600/4-85/013 - Methods for Measuring the Acute Toxicity of
Effluents to Freshwater and Marine Organisms,
Third Edition or latest edition. NTIS (#PB
85-205383) $31.00
*EPA/600/4-87/028 - Short-term Methods for Estimating the Chronic
Toxicity of Effluents and Receiving Waters to
Marine and Estuarine Organisms, First Edition
or latest edition. NTIS (fPB 89-220503) $45.00
*EPA/600/4-89/001 - Short-term Methods for Estimating the Chronic
Toxicity of Effluents and Receiving Waters to
Freshwater Organisms, Second Edition or latest
edition. NTIS (#PB 89-207013) $31.00
* Avail able from the National Technical Information Service (NTIS),
5285 Port Royal Road, Springfield, VA 22165.
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2.1.6 Calculate the LC50 (or EC50) for acute toxicity tests using the
statistical methods described in the appropriate Agency toxicity test
manual.
2.1.7 Calculate the No-Observed-Effect Concentration (NOEC) and
Lowest-Observed-Effect Concentration (LOEC) and the linear interpolation
method for chronic toxicity tests as described in the appropriate Agency
toxicity test manual.
2.1.8 Conform to the specified toxicity test methods or the approved
NPDES methodology for analytical methods, sample collection,
preservation, and preparation, sample containers, sample holding times,
and chain-of-custody of samples.
2.1.9 Provide at least 150-200 square feet of laboratory space and 15-20
linear feet of laboratory bench space (see Section 5). Laboratory space
must be appropriate to the types and numbers of tests performed. The
building must provide adequate lighting, cooling, and heating to maintain
appropriate environmental conditions for culturing organisms and toxicity
tests. Hot and cold running water must be available for equipment
cleaning.
2.1.10 Provide and maintain separate, compartmentized areas in the
laboratory for culturing, toxicity testing, and other chemical analyses
(e.g. extractions, ammonia analyses).
2.1.11 Maintain the facilities, instruments, equipment, and supplies so
that environmental controls of test conditions meets the criteria for the
tests (see Sections 5 and 6).
2.1.12 Report to the regulatory authority, equipment changes or other
changes that would affect the ability of the testing laboratory to meet
culturing or toxicity test criteria.
2.1.13 Provide appropriate glassware, chemicals, apparatus, .disposable
supplies, and equipment necessary for culturing and toxicity testing
conditions.
2.1.14 Have available the instrumentation for measurements of dissolved
oxygen, temperature, conductivity, salinity, and pH. The capability to
determine alkalinity and total hardness and to detect total residual
chlorine may also be required.
2.1.15 Maintain daily records for physical, chemical, and biological
data including all culturing and toxicity testing. Records and data
reporting must be kept by the laboratory for not less than three years
(USEPA, 1982) or as required by the regulating authority.
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2.1.16 Conduct a standard reference toxicant testing program on
organisms cultured by the laboratory in order to verify the health and
sensitivity of the offspring. The health and sensitivity of the
offspring must be determined at least once each month or as recommended
in the acute or chronic toxicity test methods or as specified in the
NPDES permit. If the laboratory is performing acute and chronic toxicity
tests, a reference toxicant(s) must be used for both tests. Each testing
program must be described in a separate section of the SOP.
2.1.17 Develop and maintain control charts for each
reference-toxicant-organism combination, and successive toxicity values
should be plotted and examined to determine if results are within
prescribed limits.
2 1.18 Maintain reproducing cultures of test organisms in the
laboratory. Use of test organisms for regulatory purposes that are not
maintained as a viable laboratory culture may be accepted on a
case-by-case basis upon receipt of written permission from regulatory
authority. Organisms purchased must be acclimated. A reference toxicant
must be used to determine and document health and sensitivity of the
purchased organisms.
2.1.19 Demonstrate satisfactory performance on evaluation samples
submitted by the regulatory authority requiring the toxicity test. Each
laboratory must maintain a written record and report analyses of
performance evaluation samples to the proper authority.
2.1.20 Comply with local, state, and federal regulations for handling
and disposing of toxic and hazardous waste.
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SECTION 3
PREPARATION FOR THE EVALUATION
3.1 The evaluator conducting on-site audits and evaluations of aquatic
biology laboratories performing toxicity tests must have working
knowledge of the NPDES compliance monitoring program and sufficient
knowledge and experience with biomonitoring and toxicity testing
methodology.
3.2 The laboratory audit or evaluation program consists of four phases.
3.2.1 Phase I includes preliminary contact between the laboratory and
the regulating authority to determine mutually agreeable dates,
submission of completed pre-survey information forms (Appendix E) by the
laboratory prior to the on-site visit and submission of results of
performance evaluation samples.
3.2.1.1 Prior to the on-site visit, the laboratory is required to submit
(1) all the "Laboratory History" pre-survey information forms provided in
Appendix E, (2) an organizational chart, and (3) a copy of its Quality
Assurance Plan.
3.2.1.2 The pre-survey information forms (Appendix E) must be completed
and returned to the evaluating authority 30 days prior to the on-site
visit.
3.2.1.3. The laboratory staff is available during the on-site visit.
3.2.2 Phase II is the on-site visit by an evaluator consisting of an
introduction and meeting with the senior laboratory staff involved in the
toxicity program to explain the evaluation process. During the on-site
visit the evaluator must:
3.2.2.1 Insure that the laboratory has a QA plan in effect by
determining if the laboratory has written procedures (QA plan or
equivalent) for conducting its quality assurance program.
3.2.2.2 Tour the laboratory facility.
3.2.2.3 Review and verify the items completed on the pre-survey forms
during the on-site visit.
3.2.2.4 Have one-on-one discussions with the technical staff.
3.2.2.5 Review the records and written standard operating procedures for
compliance with the required toxicity test methods, using the completed
pre-survey forms.
3.2.2.6 Examine the quality assurance data to determine if the quality
assurance program is being implemented.
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3.2.2.7 Evaluate the procedures and equipment used for those specific
analyses for which the laboratory has requested the evaluation, using the
criteria in this manual.
3.2.2.8 Use the pre-survey forms (Appendix E) and the on-site evaluation
forms and checklists (Appendix F and Appendix G) as verification
checklists during the on-site visit.
3.2.2.9 Review the results of the evaluation at a debriefing conference
with the laboratory director, manager, or supervisor and appropriate
staff members.
3.2.2.10 Discuss deviations in the observed culturing and toxicity test
precedures and records.
3.2.2.11 Recommend changes in equipment and supply needs, staffing
requirements, and facility improvements.
3.2.2.12 Give a verbal laboratory performance rating of acceptable,
minimally acceptable, or unacceptable.
3.2.2.13 Agree on a schedule for review of the draft report by the
laboratory personnel and submission of the final evaluation report.
3.2.3 Phase III is submission of the final report to the laboratory with
a rating by the evaluator indicating reconciliation of any evaluation
criteria differences (see Section 2) and corrective actions required by
the laboratory (see Section 11).
3.2.3.1 The report must clearly state the capabilities of the laboratory
to provide acceptable biological and toxicological data.
3.2.4 Phase IV should consist of follow-up activities.
3.2.4.1 Technical assistance to help resolve deficiencies found during
the audit or evaluation process.
3.2.4.2 The evaluating authority should be notified of any major changes
in personnel, equipment, and laboratory facilities.
3.2.4.3 If a follow-up visit to the laboratory by the evaluator is
necessary to determine whether the deficiencies have been resolved and-
found acceptable, a time period should be scheduled by the evaluator and
proper authority of the laboratory.
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SECTION 4
ORGANIZATIONAL HISTORY AND LABORATORY STAFF
4.1 History ;
4.1.1 Prior to the on-site visit, the evaluator should become familiar
with the staff, facilities, and biological testing experience of the
laboratory. The information, gathered during the preliminary contact
phase (Phase I) and by use of pre-survey information forms and check
lists, may be expanded upon and clarified during the on-site senior staff
meeting (Phase II). If the evaluator is not familiar with the
laboratory, a tour of the facility must be given during the initial
on-site visit.
4.1.2 Laboratories should demonstrate an active biological testing
program sufficient to insure that the technical staff maintains expertise
in their respective fields.
4.1.3 This information includes the client served, client satisfaction,
materials tested, species (organisms) used, test methods, and quality
assurance program.
4.1.4 Past performance data on reference toxicants, quality control and
performance evaluation samples, compliance monitoring data previously
submitted, and previous evaluation reports should be reviewed to identify
potential problem areas to concentrate on during the on-site visit. This
review will allow the evaluator to provide assistance in areas of
greatest need and more efficiently utilize the time allotted to the
on-site visit.
4.1.5 The laboratory is requested to complete and return the pre-survey
information forms and submit an organizational chart, a resume for the
supervisor, each professional biologist/analyst, and technican involved
in the culturing and toxicity testing (Appendix E).
4.2 Laboratory Staff
4.2.1 The importance of a competent supervisor/manager and professional
staff with relevant training and experience is necessary in order to
generate valid toxicity testing data.
4.3 Manager or Supervisor
4.3.1 Qualification and Responsibilities
4.3.1.1 Is responsible for the overall performance of the laboratory in
its execution and reporting of analyses.
4.3.1.2 Must have sufficient academic training and experience to
properly implement testing and a quality assurance program.
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4.3.1.3 Minimum of a bachelor of science degree in biological sciences
or closely related science curricula and three years laboratory
experience in aquatic toxicity testing or a master of science degree in
biological or closely related science and at least one year laboratory
experience in culturing and aquatic toxicity testing.
4.4 Professional Biologist/Analyst
4.4.1 Staff Qualifications
4.4.1.1 The biologist/analyst performs toxicological tests with no or
minimal supervision.
4.4.1.2 Academic Training: Minimum of a bachelor's degree in areas of
biology, zoology, fisheries, chemistry, environmental science, or related
fields. •
4.4.1.3 Job Training: Minimum of two weeks formal or on-the-job
training each from a federal agency, state agency, or academic
institution in culturing and toxicity testing of effluents and surface
waters. The amount of training would depend on academic background.
4 4.1.4 Experience: At least one year of bench experience with no or
minimal supervision in culturing and toxicity testing aquatic organisms
used in the NPDES program.
4.5 Biological Technician
4.5.1 The technician performs toxicity tests and culturing of aquatic
organisms with supervision from a professional biologist/analyst.
4.5.2 Academic training: Minimum of high school education. Two years
of college in biology, zoology, chemistry, or related fields is
recommended.
4.5.3 Job Training: One week of training each in toxicological testing
and the culturing of aquatic organisms. Personnel should take advantage
of courses available from federal and state agencies, or academic
institutions.
4.5.4 Experience: At least one year of culturing aquatic organisms and
bench experience in toxicity testing.
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SECTION 5
LABORATORY FACILITIES
5.1 General Requirements
5.1.1 Minimum standards for the instrumentation, equipment (see Section
6), and culture units (see USEPA, 1985a; 1988c; 1989c) to perform the
toxicity tests are inherent to the production of valid data from fixed or
mobile laboratories.
5.1.2 The laboratory facilities should be clean, air conditioned (20°C
to 25°C), well ventilated, adequately lighted at the bench top (100 +
ft-c), and have adequate workspace. It is recommended that 150 to 200
square feet/person be available. The laboratory should contain at least
10 to 15 linear feet of usable bench space per biologist/analyst to
accommodate periods of peak work load. The laboratory must have hot and
cold running water.
5.1.3 The laboratory should be secure and be maintained in a clean,
organized manner. The laboratory should provide safeguards (see Section
10) to avoid electric shock, prevent accidental chemical spills,
equipment failures, and prevent fires.
5.1.4 The laboratory should have policies, procedures, and provisions
for the disposal of chemical and toxic wastes. Exhaust hoods are
required for the handling of toxic chemicals and samples. This includes
venting for sample preparation, extractions, and toxicity testing.
5.1.5 Contamination-free work areas should be provided for handling of
test materials.
5.1.6 Adequate facilities should be provided for storage of samples and
test materials, including cold storage.
5.1.7 Adequate space should be available for culturing test organisms
and preforming the toxicity test. Organisms should be shielded from
external disturbances.
5.1.8 Culturing, toxicity testing, and chemical analyses should be done
in separate areas.
5.1.9 Volatile compounds or toxic samples should not be used or stored
near culture units.
5.1.10 Temperature control of the toxicity tests, culture units, and
holding tanks should be achieved using circulating water baths, heat
exchangers, or environmental chambers.
5.1.11 Air used for aeration must be free of oil and fumes.
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5.2 Laboratory Pure Water
5.2.1 Only satisfactorily tested reagent grade water from deionization
or distillation units is used to prepare media, reagents, and
dilution/rinse water for culture and toxicity test methods (ASTM
Standards and Standard Methods, latest editions).
5.2.2 Reconstituted or synthetic water made with laboratory pure water
for culturing and toxicity testing should be tested monthly to assure it
meets the following requirements and demonstrates no toxicity to the test
organisms:
5.2.2.1 Conductivity ( <_ 0.1 umhos conductivity or > 17 megohm
resistivity at 25°C).
5.2.2.2 pH (5.5-7.5) .
5.2.2.3 Total Chlorine residual (non-detectable).
5.2.2.4 The dilution water made with laboratory pure water should
demonstrate no toxicity to the test organisms.
5.2.3 If laboratory pure water does not meet the above requirements,
supplementary analyses must be performed to determine the cause.
5.2.4 Toxic metals and organics must not be present in the laboratory
pure water.
5.2.4.1 The "USEPA Ambient Water Quality Criteria Documents" and USEPA
(1987d) Quality Criteria for Water 1986, Office of Water Regulations and
Standards, U.S. Environmental Protection Agency, Washington, D.C., EPA
440/4/86-001 provide data and guidance on acceptability and toxicity of
individual metals and organics to aquatic organisms and should be
consulted.
5.3 Dilution Water
5.3.1 The choice of the dilution water (reconstituted laboratory pure
water or surface water) used in the tests will depend largely on the
objectives of the study, and what is required in the NPDES permit. The
dilution water should demonstrate no toxicity to the test organisms (see
USEPA, 1985a; 1988c; 1989c).
5.3.2 To prepare a synthetic (reconstituted) freshwater or a synthetic
(artificial) seawater, use only reagent grade chemicals or recommended
commercial sea salts (see USEPA, 1985a; 1988c; 1989c).
5.3.3 To prepare a synthetic seawater from manufactured synthetic sea
salts, follow the directions of the manufacturer and USEPA (1988c) in
making the dilution water. To prepare hypersaline brine derived from
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natural seawater, see USEPA (1988c). Artificial seawater should be used
only if specified in the culturing and toxicity testing methods.
5.4 Glassware Washing
5.4.1 The guidance provided below is intended to eliminate toxicity
associated with glassware. If controls show toxicity additional cleaning
steps may be required.
5.4.1.1 All glassware, sample containers, test vessels, pumps, tanks,
and other equipment that need cleaning or come in contact with effluent
must be washed and rinsed with laboratory pure water to remove surface
contaminants (see USEPA, 1985a; 1988c; and 1989c).
5.4.1.2 New plastic ware, used for sample collection or organism exposure
vessels, may or may not require soaking or rigorous cleaning. It may be
sufficient to rinse the new containers once with deionized water and
sample water before use, but control tests with the new containers may be
required to see if toxicity occurs before using them in toxicity
testing. If toxicity is found during the control tests, more rigorous
cleaning will be required.
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SECTION 6 ,
EQUIPMENT, INSTRUMENTS, AND SUPPLIES
6.1 Necessary and appropriate equipment, instruments, and supplies must
be available in adequate quantities for culturing test organisms and for
performing the toxicity tests being conducted. The evaluator should
limit the review to those tests performed routinely.
6.2 All materials in contact with a sample or the test organisms should
be nontoxic. Materials used should not reduce or add to the sample
toxicity. Adequate survival and growth in the cultures and test control
solutions and acceptable performance on reference toxicants, would imply
appropriate materials are being used. The date of receipt and lot number
of disposable supplies being used should be recorded. Any changes in
cleaning procedures should be noted so that changes in performance might
be correlated with these items.
6.3 Manufacturers' operating manuals, standard operating procedures
(SOPs) and equipment maintenance log books should be evident to the
evaluator, and available and used by the operator/analyst. Balances and
other major equipment should be serviced regularly. Lists of reagents
and consumable materials are specified in the latest editions of the
USEPA acute and chronic toxicity test methods.
6.4 Design, performance or use specifications for selected equipment and
supplies for toxicity test methods are provided below:
6.4.1 pH Meter: Scale of 0-14 pH units with accuracy and scale
readability to at least j^O.l units. Laboratories are encouraged to
purchase meters capable of functioning with specific ion or other
electrodes. Units may be line or battery powered.
6.4.2 Dissolved Oxygen Meter: Capable of measurements at 0-100%
saturation. Field or laboratory units with accuracy specifications of at
least 0.1 mg/L are acceptable.
6.4.3 Analytical Balance: Capable of accurately weighing to 0.01 mg
(0.00001 g). -The balance must be seated on a steady base to prevent
vibration and protected from air currents. Class S certified weights
should be available, and the balance must be checked with the weights
each time it is used to document acceptable performance of the balance.
6.4.4 Conductivity Meter: Suitable for checking reagent water quality
and saline water. Should be readable in ohms or megohms and should have
a range from 2 ohms to 20 megohms. Unit may be line or battery operated.
6.4.5 Thermometer: Mercury-filled, centigrade thermometer or digital
thermometer with 1°C or finer subdivisions. Continuous recording
electronic-chart thermometer or bulb thermographs capable of documenting
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a 1°C or less temperature change are acceptable. A certified or
National Institute of Standard Technology (NIST) traceable thermometer
should be available for calibration checks.
6.4.6 Drying Oven: Gravity or mechanical convection unit with
selectable temperature control from room temperature to 180°C.
6.4.7 Refractometer: Hand held, automatic temperature compensated
refractometer calibrated for salinity measurements from 0-160 parts per •
thousand (ppt).
6.4.8 Compound Microscope, Dissecting Microscope, and Magnifying Lens:
Hand held or supported, with appropriate light source, for examining
small organisms in the test chambers or for examining cells on microscope
slides.
6.4.9 Radiometer (light meter): Capable of measuring the intensity of
ambient room light over a range of at least 0-200 uE/m2/s (0-1000
ft-candles).
6.4.10 Water Purification System: Consisting of any combination of
distillation, reverse osmosis, ion exchange, activated carbon and
particle filtration capable of producing nontoxic deionized water of 18
megohms (0.05 umho conductivity) resistivity. Commercially available
cartridge systems are preferred (See ASTM, Volume 11.01, D 1193,
"Standard Specification for Reagent Mater," Type I Reagent Water).
6.4.11 Mechanical Shaker: Variable speed capable of providing orbital
motion at a rate of 100 cycles per minute.
6.4.12 Fluorometer or UV-VIS Spectrophotometer: Suitable for
measurements of chlorophyll a_ and performing colorimetric analyses.
6.4.13 Electronic Particle Counter: Coulter counter or equivalent
capable of mean cell volume computation (MCV).
6.4.14 Environmental Chamber/Incubator: Capable of maintaining
temperatures of 200C + 2QC, 240C +2°C, and/or 250 +_ loc
6.4.15 Autoclave: Capable of producing 1.1 Kg. cm? (15 psi) pressure
at 1210C (2500F).
6.4.16 Refrigerator: Explosion proof and capable of maintaining a
temperature of 4°C for sample storage; lockable, if for
chain-of-custody requirements.
6,4.17 Freezer: Capable of maintaining a temperature of -2QOC for
storage.
6.4.18 Air Compressor, Air Pumps: capable of producing oil free air.
6.4.19 Standby GeneratordlO VAC): For electrical backup in emergency.
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6.4.20 Light Box: For illuminating embryos, larvae, and organisms
during examination.
6.4.21 Desiccator: For keeping specimens free of moisture.
6.4.22 Amperometric Titrator: For measurement of free, total, and
combined chlorine.
6.4.23 Vacuum Pump: Electric powered, capable of providing vacuum in
the range of 1-25 Hg.
6.4.24 Counting Chamber: Hemacytometer, Palmer-Maloney,
Sedgwick-Rafter,. for counting sea urchin gametes and algal cells
(Selenastrum capricornutum).
6.4.25 Centrifuge: General purpose bench top or floor model; producing
lOOOxg; capable of accepting bottles or tubes appropriate for the sample
volumes used.
6.4.26 Exhaust Hood: For handling reagents, potentially-toxic samples,
and controlling toxic fumes.
6.4.27 Water Bath: For controlling test temperatures of 20-25°C +
1°C.
6.4.28 Personal Computer (PC): for data analyses.
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SECTION 7
METHODOLOGY
7.1 One of the most important aspects of the evaluation process is to
determine if approved methods are being used by the laboratory. All
activities including organism culturing, sample collection, handling,
preservation, and preparation, toxicity testing, physical and chemical
analyses, and toxicity test data analysis must be covered by written standard
operating procedures, protocols or analytical procedures. These written SOP
documents must be available to, and understood and used by, the laboratory
staff. The evaluator must also determine if these written procedures are
consistent with any required (approved) methods and if any significant
deviations occur in either the written procedures or in the implementation of
the procedures. Any deviations from standard test methods should be
documented.
7.2 The evaluator should review the methods used by the laboratory,, as
stated on the completed pre-survey forms, prior to the on-site visit. The
following sources of methods should be available to the laboratory staff:
7.2.1 Standard methods for the examination of water and wastewater,. American
Public Health Association, 1015 Fifteenth Street NW, Washington, D.C 20005
(latest edition).
7.2.2 Annual Book of ASTM Standards, Vols. 11.01, 11.02, and 11.04.
American Society for Testing and Materials, 1916 Race Street, Philadelphia,
PA 19103 (latest editions).
7.2.3 Methods for measuring the acute toxicity of effluents to freshwater
and marine organisms, EPA-600/4-85-013 or latest edition. U.S. Environmental
Protection Agency, EMSL-Cincinnati, Cincinnati, OH 45268.
7.2.4 Short-term methods for estimating the chronic toxicity of effluents
and receiving waters to freshwater organisms, EPA/600/4-89-001 or latest
edition. U.S. Environmental Protection Agency, EMSL-Cincinnati, Cincinnati,
OH 45268.
7.2.5 Short-term methods for estimating chronic toxicity of effluents and
receiving waters to marine and estuarine organisms, EPA/600/4-87-028 or
latest edition. U.S. Environmental Protection Agency, EMSL-Cincinnati,
Cincinnati, OH 45268
7.2.6 Methods for chemical analysis of water and wastes, EPA/600/4-79-020 or
latest edition. U.S. Environmental Protection Agency, EMSL-Cincinnati,
Cincinnati, OH 45268.
7.2.7 Handbook for analytical quality control in water and wastewater
laboratories, EPA/600/4-79-019 or latest edition. U.S. Environmental
Protection Agency, EMSL-Cincinnati, Cincinnati, OH 45268.
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7.2.8 Methods for organic chemical analysis of municipal and industrial
wastewater, EPA/600/4-82/057 or latest edition. U.S. Environmental
Protection Agency, EMSL-Cincinnati, Cincinnati, OH 45268.
7.2.9 Technical support document for water quality-based toxic control.
Office of Water Enforcement and Permits, Office of Water.Regulations and
Standards, EPA/440/4-85-032 or latest edition.. U.S. Environmental
Protection Agency, Washington, D.C. 20460.
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SECTION 8
SAMPLE COLLECTION, HANDLING, PRESERVATION, AND PREPARATION1
8.1 Introduction
8.1.1 Specific requirements for sample collection and handling should be
specified in NPDES permits and/or compliance monitoring documents. The
evaluator should become familar with the requirements contained in these
documents.
8.1.2 The following items are only applicable to laboratories delegated
responsibility for sample collection. All laboratories are responsible for
items 8.1.5 and 8.1.6.
8.1.3 Sampling frequency must conform to that specified by permits or
regulations. Collectors should be trained in sampling procedures and approved
by the appropriate regulatory authority or its designated representative.
8.1.4 Applicable state regulations pertaining to chain-of-custody must be
followed. An example of chain-of-custody procedures is presented in Appendix
A.
8.1.5 The report form must include the date and time of sample arrival at the
laboratory and the date and time analysis begins.
8.1.6 Holding/transit time between sampling and analysis must not exceed 36
hours. If the laboratory is required by permits and regulations to examine
samples within 36 hours, the laboratory is to indicate that the data may be
invalid because of excessive delay before sample processing (see 8.4.1).
8.2 Effluent Sampling
8.2.1 The effluent sampling point should be the same as that specified in the
NPDES discharge permit (USEPA, 1988a).
8.2.2 Conditions for exception would be the following, but they must be
approved by permitting authority:
8.2.2.1 Better access to a sampling point between the final treatment and the
discharge outfall.
8.2.2.2 If the processed waste is chlorinated prior to discharge to the
receiving waters, it may also be desirable to take samples prior to contact
with the" chlorine to determine toxicity of the unchlorinated effluent.
Adapted from: USEPA (1985a), (1988c), and (1989c).
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8.2.2.3 In the event there is a desire to evaluate the toxicity of the
influent of municipal waste treatment plants or separate wastewater streams in
industrial facilities prior to their being combined with other wastewater
streams or non-contact cooling water, additional sampling points may be chosen.
8.2.3 The decision on whether to collect grab or composite samples should be
specified in the NPDES permit and is based on the objectives of the test and
an understanding of the short and long-term operations and schedules of the
discharger. If the effluent quality varies considerably with time, which can
occur where holding times are short, grab samples may be preferable because of
the ease of collection and the potential of observing peaks (spikes) in
toxicity. However, the sampling duration of a grab sample is so short that
full characterization of an effluent over a 24-h period would require a
prohibitive number of separate samples and tests. Collection of a 24-h
composite sample, however, may dilute toxicity spiking and averages the
quality of the effluent over the sampling period. A lengthy discussion of the
advantages and disadvantages of sample types (grab or composite samples) is
found in USEPA (1985a) or (1989c). The regulatory program or NPDES permit
should dictate sample collection objectives.
8.2.4 Aeration during collection and transfer of effluents should be
minimized to reduce the loss of volatile chemicals.
8.2.5 Definitive tests performed for NPDES permit purposes, unless otherwise
specified in permit, require daily effluent sample collection and daily
renewal of test solutions.
8.3 Receiving Water Sampling
8.3.1 It is common practice to collect grab samples for receiving water
toxicity studies.
8.3.2 When non-toxic receiving water is required for a test, it may be
collected upstream from the outfall or from other surface water known to be
uncontaminated and which has properties similar to the receiving water (see
USEPA, 1989c). If the objective of the test is to determine the additive
effects of the discharge on receiving water which may be contaminated, the
test is performed using dilution water consisting of receiving water collected
daily upstream from the outfall.
8.3.3 Dilution water to be taken from the receiving water upstream from the
outfall, is collected at a point as close as possible to the outfall, but
upstream from or outside of the zone influenced by the effluent.
8.3.4 To determine the extent of the zone of toxicity in the receiving water
downstream from the outfall, receiving water samples are collected at several
distances downstream from the discharge. The time required for the
effluent-receiving-water mixture to travel to sampling points downstream from
the outfall, and the rate and degree of mixing, may be difficult to
ascertain. It may not be possible to correlate downstream toxicity with
effluent toxicity at the discharge point unless a dye study is performed. The
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toxicity of receiving water samples from five stations downstream from, the
discharge point can be evaluated using the same number of test vessels and
test organisms as used in one effluent toxicity test with five effluent
dilutions.
8.4 Sample Handling, Preservation, and Shipping
8.4.1 If the data from the samples are to be acceptable for use in the NPDES
Program, the elapsed time from collection of a grab or composite sample to its
first use for initiation of the test, or for test solution renewal, should not
exceed 36 h. In no case should a sample be used in a test more than 72 h
after removal from the sampling device. Composite samples must be chilled
during collection and maintained at 4°C until warmed up for use.
8.4.2 Samples Used in On-Site Tests
8.4.2.1 Samples collected for on-site tests should be used within 24 h.
8.4.3 Samples shipped to Off-Site Facilities
8.4.3.1 Samples collected for off-site toxicity testing should be chilled
during collection and maintained at 4°C until used or shipped iced to the
central laboratory, and there transferred to a refrigerator (4°C) until
used. Every effort must be made to initiate the test with an effluent sample
on the day of arrival in the laboratory.
8.4.3.2 Samples may be shipped in 4-L (1-gal) CUBITAINERSR, or new plastic
"milk" jugs. All sample containers should be rinsed with source water before
being filled with sample. CUBITAINERSR and plastic jugs are not to be
reused. CUBITAINERSR and plastic jugs used for effluents or toxic surface
water samples should be punctured after use to prevent reuse.
8.4.3.3 Several sample shipping options are available, including Express
Mail, air express, bus, and courier service. Express Mail is delivered seven
days a week. Shipping and receiving schedules of private carriers on weekends
vary with the carrier.
8.5 Sample Preparation
8.5.1 With Ceriodaphnia dubia or other cladoceran invertebrate and fish
tests, effluents and surface waters must be filtered through a (60 urn)
plankton net to remove indigenous organisms that may attack or be confused
with the test organisms (see Ceriodaphnia dubia test methods, USEPA, 1989c,
for details). Surface waters used in algal toxicity tests must be filtered
through a 0.45 urn pore diameter filter before use. It may be necessary to
first coarse-filter the dilution and/or waste water through a nylon sieve
having 2-4 mm holes to remove debris and/or break up large floating or
suspended solids. Caution: filtration may remove toxicity.
&.5.2 The dissolved oxygen (DO) concentration in the dilution water should be
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near saturation prior to use. Aeration will bring the DO and other gases into
equilibrium with air, minimize oxygen demand, and stabilize the pH.
8.5.3 If the dilution water and effluent must be warmed to bring them to the
prescribed test temperature, supersaturation of the dissolved gases may become
a problem. To prevent this problem, the effluent and dilution'water are
checked for dissolved oxygen (DO) with a probe after heating to 25°C. If
the DO is greater than 100% saturation (8.5 mg/L) or lower than 40%
saturation, the solutions are aerated moderately with a pipet tip for a few
minutes until the DO is within the prescribed range.
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SECTION 9
QUALITY ASSURANCE1 AND DATA HANDLING
9.1 Quality Assurance/Quality Control Definitions2
9.1.1 There is often confusion about the specific definitions of the terms
"Quality Assurance" and "Quality Control". While many people recognize a
distinction, the terms are often used interchangeably.
9.1.2 Quality Assurance is the total program for assuring the reliability of
monitoring data.
9.1.3 Quality Control is the routine application of procedures for
controlling the measurement process.
9.2 Each laboratory shall have a written quality assurance (QA) plan. A
routine, on-going, quality assurance (QA) program and quality assurance plans
are necessary to insure and document the quality of the data produced.
Quality assurance/quality control (QA/QC) should be a continous process
implemented throughout the entire culturing and toxicity testing program.
9.2.1 Three guidance documents are available from USEPA to assist in
preparation of the QA plan. USEPA (1980b) describes management policies,
organization, objectives, principles, and general procedures to establish how
data of known and acceptable quality should be produced. USEPA (1980c) is a
general guidance document, and USEPA (1984a) is a more detailed guidance
document that combines a work plan with the QA project plan.
9.2.3 The QA program should cover all aspects of the biological testing
activity, including sampling and sample handling, test conditions, equipment,
methodology, record keeping and data evaluation, which are subject to QA/QC
procedures. The data quality indicators (e.g, the desired precision in
controls and in reference toxicants) and objectives developed with this QA
program including overall precision and accuracy should accompany all data
produced by the laboratory.
9.2.4 The determination of data quality is -accomplished through the
development of data quality objectives (DQOs) by the interaction of
decision-makers and the technical staff (see USEPA, 1984b; 1989a; 1989b).
9.2.4.1 DQOs are qualitative and quantitative statements developed by data
users to specify the quality of data needed to support specific decisions or
regulatory actions.
Adapted from: USEPA (1978c), (1985a), (1988c), (1989c), and (1979c).
2USEPA (1978a). Newsletter Quality Assurance. Environmental Monitoring and
Support Laboratory - Cincinnati.
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9.2.5 For general guidance on good laboratory practices related to toxicity
testing, see: FDA (1978), USEPA (1979a, 1980a, 1980e, 1988b), and DeWoskin
(1984).
9.3 Appropriate sampling, sample handling and preservation should include
considerations for selection of. the sampling locations and number of samples
necessary to adequately represent the source. Additional considerations
concern the use of composite or grab samplers, sample containers, volume of
sample required, collection of appropriate dilution waters, preservation at
4°C if samples are held for more than 24 hours. Location, date, and time of
sampling must be recorded as well as the date and time that analysis was
initiated. If necessary, chain-of-custody procedures should be used.
9.4 The test organisms must be in good health, be disease-free, show minimum
mortality in holding tanks, and demonstrate "acceptable" performance in
control test solutions. Additionally, the health of the organism should be
documented by periodic use of reference toxicant testing.
9.5 Documentation of instrument and equipment performance, calibration, and
maintenance must be available for review. Balances and other major equipment
should be serviced annually.
9.6 Methods and procedures used should be well documented and available to
the staff and the evaluator. Any deviations from the methods as written, or
options used, should be noted.
9.7 Field and laboratory/bench data should be kept in bound notebooks.
Additional data such as bench sheets, sample receipt forms or chain-of-custody
records should be referenced and noted. Use of electronic (computer) data
bases is acceptable if adequate security and backup are maintained.
9.8 Data evaluation procedures should be reviewed to determine if adequate
statistical support is available and used. Data from toxicity tests should be
plotted as a preliminary step to aid in interpretation of results and to help
detect problems and expected trends or patterns in the responses. Recommended
statistical programs should be used to calculate results.
9.9 Reference toxicants and control charts should be used to document (1) the
health of the organisms used, (2) data quality, and (3) the overall laboratory
performance. Control charts or acceptance ranges should be constructed or
calculated and routine QC data plotted or compared to these pre-established
criteria. The laboratory should demonstrate that the QC data generated is
used to document data quality.
9.10 Proper documentation of the entire biological testing process is
critical in supporting the validity of the data produced. Bound notebooks or
secure computer data bases should be used to maintain detailed records of all
data required by the methods and covered in this manual. Annotations to the
record should be made as soon as possible (at least daily) to minimize or
prevent any loss of information. These records should be made available to
the evaluator during the on-site visit. The record should be complete enough
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to follow a sample chronologically through the entire collection, measurement,
data collection and sample disposal process. Data reports should include the
DQOs and data quality indicators developed by the laboratory's quality
assurance program as an aid to the ultimate data user.
9.11 Procedures for confirming data validity (e.g., checking final reports to
laboratory bench sheets) should be performed.
9.12 All bound notebooks should have consecutively numbered pages and no
pages should be missing. Pages with errors should be corrected near where the
error occurs and initialed and dated by the person making the correction.
Pages with multiple errors or with major problems such as spills or illegible
ink marking should not be discarded, but should remain in the bound notebook.
The next page or a page nearby should be used to re-enter information.
Waterproof plastic-coated paper notebooks are available.
9.13 All information reported on the pre-survey information forms and
checklists should be confirmed and possibly expanded upon during the on-site
visit.
9.14 Data validation may include a comparison of final reports with lab bench
sheet data.
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SECTION 10
SAFETY AND GENERAL PRACTICES
10.1 Introduction
10.1.1 While safety is not an integral part of laboratory evaluation, the
evaluator should be aware of unsafe conditions and lack of, or weaknesses in
a, formal safety program. This program should begin with management support
and include the assignment of health and safety responsibility, maintenance of
safe working conditions, establishment of safety training, accident reporting,
medical and first aid treatment and acceptance of the program by the staff.
Written safety policies should be available to the laboratory staff and to the
evaluator for review.
10.1.2 Basic good housekeeping practice should also be evident in a neat and
orderly laboratory and office environment. These practices must be adequate
to protect the staff from physical injury and exposure to hazardous or toxic
substances, to avoid interference with laboratory operations, and to assure
the production of valid data of known quality (see USEPA, 1985a; 1988c; 1989c).
10.1.3 Training on the use of safety equipment, first aid and medical
emergency treatment should be documented.
10.2 Safety Equipment and Supplies
10.2.1 Necessary and appropriate safety apparel such as aprons, lab coats,
respirators, gloves, safety glasses and shoes, and hard hats should be
available.
10.2.2 First aid kits, fire extinguishers and blankets, safety showers and
emergency spill kits should be available as well as a record of their
maintenance and inspection.
10.2.3 Mobile and remote locations,should be equipped with a communication
system to summon help in case of an emergency. It is recommended that
personnel not work alone in the field.
10.2.4 Facilities should be available for soap and water cleaning of exposed
body parts that may be contaminated by effluent samples.
10.3 Safety Practices
10.3.1 AIT personnel hand!ing.environmental samples known or suspected to
contain human waste should be immunized against disease causing agents.
10.3.2 The exterior of all sample containers should be protected from
contamination during sample collection and handling. If the outside of the
container is contaminated, it should be decontaminated to prevent human
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exposure. Sample spills must be cleaned up and/or neutralized (all acids), as
required, to prevent human exposure.
10.3.3 Sample labels and identification should be adequate to alert personnel
of potential or known hazards.
10.3.4 Material safety data sheets should be available for all chemical
preservatives and reagents used.
10.3.5 Work with and disposal of effluent samples should be performed in
compliance with current federal, state, and local rules pertaining to
hazardous materials.
10.3.6 All electrical equipment should be properly grounded. Ground fault
interrupters should be used in all wet lab areas.
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SECTION 11
EVALUATION REPORT
11.1 Prior to termination of the on-site visit, the evaluator should
have an exit debriefing meeting with the laboratory senior staff. At
this debriefing the following findings should be discussed:
11.1.1 Any deviations or inadequacies in procedures, documentation,
and/or records.
11.1.2 Recommendations concerning equipment and supply needs, staffing,
and facility improvements.
11.1.3 Areas in which the evaluating authority can provide technical
assistance.
11.1.4 Performance rating of the laboratory: acceptable, minimally
acceptable, or unacceptable.
11.1.5 During this open discussion it is important to provide the
laboratory with all of the findings, to rate the performance of the
laboratory, and to provide recommendations for correction and
improvement. In some instances all the findings that are discussed will
be incorporated into a draft report. This will avoid any
misunderstandings and allow time for the laboratory to take appropriate
corrective actions prior to preparation and distribution of the final
report. It should be stressed that the report is intended to provide
positive information on how the laboratory may improve performance and
not intended to be a negative or punitive device. It should be
determined and agreed upon at this meeting what the distribution of the
final report shall be.
11.2 After completion of the on-site visit, the evaluator prepares a
draft narrative report that contains all of the information pertinent to
the evaluation that was discussed at the debriefing and actions taken as
a result of the evaluation. The draft report should be sent to the
laboratory for review, comment and action prior to any outside
distribution. Additional follow-up visits may be required to verify
corrections of serious problems. From this draft report and review
comments the final report is prepared that includes the general headings
and information listed below:
11.3 Title Page
11.3.1 This page should include the following: Name of laboratory,
address, date of the on-site visit, name, title, address, and signature
of the evaluator.
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11.4 Commendations
11.4.1 Point out those areas where the laboratory exceeds the minimum
standards of performance.
11.5 Deviations
11.5.1 List each deviation and describe it in detail. Provide
recommendations or appropriate corrective actions. Point out
specifically those areas where deviations occurred previously and where
recommended corrective actions were not taken.
11.6 Remarks
11.6.1 Recommend improvements which, while not affecting the evaluation
status, would improve laboratory operations.
11.7 List of Personnel
11.7.1 List names and title of all technical personnel along with the
functions that each one normally performs. Also identify the critical
laboratory personnel that could influence the performance rating of the
laboratory if their job functions were changed. Note in this section
that the evaluating authority must be notified if any of the critical
personnel are involved in changes of job function and how the critical
functions will be performed in the future.
11.8 Signatures
11.8.1 The evaluation report should be signed by the evaluator and any
accompanying letter or action metoorandum should state the performance
rating granted and be signed by the evaluating authority.
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SELECTED REFERENCES
Annual Book of ASTM Standards, Vols. 11.01, 11.02, and 11.04. 1989.
American Society for Testing and Materials, 1916 Race Street,
Philadelphia, PA 19103 (latest editions).
APHA. 1989. Standard Methods for the Examination of Water and Wasterwater.
17th Ed. American Public Health Association, Washington, D.C. 20005.
Berner, D.B. 1985. ,The taxonomy of Ceriodaphnia (Crustacea: Cladocera) in
U.S. Environmental Agency cultures. Environmental Monitoring and Support
Laboratory, U.S. Environmental Protection Agency, Cincinnati, Ohio
45268. EPA/600/4-86-032.
Biesinger, K.E., L.R. Williams, and W.H. van der Schalie. 1987. Procedures
for conducting Daphnia magna toxicity bioassays. Environmental Monitoring
Systems Laboratory, U.S. Environmental Protection Agency, Las Vegas,
Nevada 89114. EPA/600/8-87/001.
DeWoskin, R.S. 1984. Good laboratory practice regulations: a comparison.
Research Triangle Institute, Research Triangle Park, North Carolina 27711.
FDA. 1978. Good laboratory practices for nonclinical laboratory studies.
Part 58, Fed. Reg. 43(247):6001-60020, December 22, 1978.
Pennak, R.W. 1989. Fresh-Water Invertebrates of the United States. Protozoa
to Mollusca, John Wiley & Sons, Inc., New York.
USEPA. 1971. Algal assay procedures: bottle test. National Eutrophican
Program. U.S. Environmental Protection Agency, Environmental Research
Laboratory, Corvallis, Oregon 97333.
USEPA. 1973. Biological field and laboratory methods for measuring the
quality of surface waters and wastes. C.I. Weber (ed.). Office of
Research and Development, U.S. Environmental Protection Agency,
Cincinnati, Ohio 45268. EPA-670/4-73-001.
USEPA. 1975. Methods for acute toxicity tests with fish, macroinvertebrates,
and amphibians. Environmental Research Laboratory, U.S. Environmental
Protection Agency, Duluth, Minnesota. 55804. EPA-660/3-75-009.
USEPA. 1977a. Qua!ity assurance research plan FY-1978-82. Office of
Research -and Development, U.S. Environmental Protection Agency,
Washington, D.C. 20460. EPA-600/8-77-008.
USEPA. 1977b. Occupational health and safety manual. Office of Planning and
Management, U.S. Environmental Protection Agency, Washington, D.C. 20460.
29
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USEPA. 1978a. Quality Assurance Newsletter. Volume 1, No. 1, Environmental
Monitoring and Support Laboratory, Cincinnati, Ohio 45268
USEPA. 1978b. Procedure for the evaluation of environmental monitoring
laboratories. Environmental Monitoring and Support Laboratory, U.S.
Environmental Protection Agency, Cincinnati, Ohio 45268.
EPA-600/4-78-007.
USEPA. 1978c. Methods for measuring the acute toxicity of effluents to
aquatic organisms. W. Peltier, Chairman, Bioassay Advisory Committee.
Second edition. Environmental Monitoring and Support Laboratory -
Cincinnati, U.S. Environmental Protection Agency, Cincinnati, Ohio.
EPA-600/4-78-012.
USEPA. 1979a. Good laboratory practice standards for health effects.
Paragraph 772.110-1 - Standards for development of test data. Fed. Reg.
44:227362-27375, May 9, 1979.
USEPA. 1979b. Interim NPDES compliance biomonitoring inspection manual.
Office of Water Enforcement and Permits (EN-338), U.S. Environmental
Protection Agency, Washington, D.C. 20460.
USEPA. 1979c. Handbook for analytical quality control in water and
wastewater laboratories. Environmental Monitoring and Support Laboratory,
U.S. Environmental Protection Agency, Cincinnati, Ohio. 45268.
EPA-600/4-79-019.
USEPA. 1979d. Methods for chemical analysis of water and wastes.
Environmental Monitoring and Support Laboratory, U.S. Environmental
Protection Agency, Cincinnati, Ohio. 45268. EPA-600/4-79-020.
USEPA. 1980a. Proposed good laboratory practice guidelines for toxicity
testing. Paragraph 163.60-6. Fed. Reg. 45:26377-26382, April 18, 1980.
USEPA. 1980b. Guidelines and specifications for preparing quality assurance
program plans. Report No. QAMS-004180. U.S. Environmental Protection
Agency, Washington, D.C. 20460.
USEPA. 1980c. Interim guidelines and specifications for preparing quality
assurance project plans. Report No. QAMS-005180. U.S. Environmental
Protection Agency, Washington, D.C. 20460.
USEPA. 1980d. Appendix B - Guidelines for deriving water quality criteria
for the protection of aquatic life and its uses. Fed. Reg., Vol. 45, No.
231, Friday, November 28, 1980.
USEPA. 1980e. Physical, chemical, persistence, and ecological effects
testing; good laboratory practice standards (proposed rule). 40 CFR 772,
Fed. Reg. 45: 77353-77365, November 21, 1980.
30
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USEPA. 1982a. Manual for the certification of laboratories analyzing drinking
water. Criteria and procedures quality assurance. Office of Drinking
Water (WH-550). U.S. Environmental Protection Agency, Washington D C
20460. EPA-570/9-82-002.
USEPA. 1982b. Methods for organic chemical analysis of municipal and
industrial wastewater. Environmental Monitoring and Support Laboratory
U.S. Environmental Protection Agency, Cincinnati, Ohio. 45268.
EPA/600/4-82/057.
USEPA. 1982c. Handbook for sampling and sample preservation of water and
wastewater. Environmental Monitoring and Support Laboratory, U.S.
Environmental Protection Agency, Cincinnati, Ohio. EPA-600/4-82-029.
USEPA. J984a. Guidance for preparation of combined work/quality assurance
project plans for environmental monitoring. Report No. OWRS QA-1. U S
Environmental Protection Agency, Washington, D.C. 20460.
USEPA. 1984b. The development of data quality objectives. Prepared by the
EPA Quality Assurance Management Staff and the DQO Workgroup. U.S
Environmental Protection Agency, Washington, D.C. 20460.
USEPA. 1985a. Methods for measuring the acute toxicity of effluents to
freshwater and marine organisms. W.H. Peltier and C. I. Weber (eds.).
Third edition. Environmental Monitoring and Support Laboratory, U.S.
Environmental Protection Agency, Cincinnati, Ohio. EPA-600/4-85-013.
USEPA. 1985b. Technical support document for water quality-based toxic
control. Office of Water Enforcement and Permits and Office of Water
Regulations and Standards, U.S. Environmental Protection Aqency
Washington, D.C. 20460. EPA-440/4-85-032.
USEPA. 1987a. Permit writer's guide to water quality-based permitting for
toxic pollutants. Office of Water, U.S. Environmental Protection Aqency
Washington, D.C. 20460. EPA/440/4-87-005.
USEPA. 1987b. Availability, adequacy, and comparability of testing
procedures for the analysis of pollutants established under Section 304(h)
of the Federal Water Pollution Control Act. Report to Congress.
Environmental Monitoring Systems Laboratory, U.S. Environmental Protection
Agency, Cincinnati, Ohio. 45268. EPA/600-9-87-030.
USEPA. 1987c. The risk assessment guidelines of 1986. Office of Health and
Environmental Assessment, U'.S. Environmental Protection Agency
Washington, D.C. 20460. EPA/600/8-87/045.
USEPA. 1987d. USEPA Quality Criteria for Water 1986. Office of Water
Regulations and Standards, U.S. Environmental Protection Aqency
Washington, D.C. 20460. EPA/440/4-86/001.
31 '
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USEPA. 1988a. NPDES compliance inspection manual. Office of Water
Enforcement and Permits (EN-338), U.S. Environmental Protection Agency,
Washington, D.C. 20460.
USEPA. 1988b. 40 CFR Part 160 - Good laboratory practice standards.
pages 153-164.
USEPA. 1988c. Short-term methods for estimating the chronic toxicity of
effluents and receiving waters to marine and estuarine organisms. C.I.
Weber, W.B. Horning, D.J. Klemm, T.W. Neiheisel, P.A. Lewis, E.L.
Robinson, J. Menkedick, and F. Kessler (eds.). Environmental Monitoring
and Support Laboratory, U.S. Environmental Protection Agency, Cincinnati,
Ohio. 45268. EPA-600/4-87/028.
USEPA. 1989a. Ecological assessment of hazardous waste sites: A field and
laboratory reference. Environmental Research Laboratory, U.S.
Environmental Protection Agency, Corvallis, Oregon. 97333.
EPA/600/3-89-013.
USEPA. 1989b. Proceedings of the Second Annual Ecological Quality Assurance
Workshop. Exposure Assessment Division, Environmental Monitoring Systems
Laboratory, U.S. Environmental Protection Agency, Las Vegas, Nevada.
89114.
USEPA. 1989c. Short-term methods for estimating the chronic toxicity of
efluents and receiving waters to freshwater organisms. C.I. Weber, W.H.
Peltier, T.J. Norberg-King, W.B. Horning, II, F.A. Kessler, J.R.
Menkedick, T.W. Neiheisel, P.A. Lewis, D.J. Klemm, Q.H. Pickering, E.L.
Robinson, J.M. Lazorchak, L.J. Wymer, and R.W. Freyberg (eds.). Second
edition. Environmental Monitoring Systems Laboratory - Cincinnati, U.S.
Environmental Protection Agency, Cincinnati, Ohio. 45268.
EPA-600/4/89/001.
USEPA. 1990a. Manual for the certification of laboratories analyzing
drinking water. Criteria and procedures quality assurance. Office of
Water, U.S. Environmental Protection Agency, Washington, D.C. 20460.
EPA/570/9-90-008.
USEPA. 1990b. Technical support document for water quality-based toxic
controls. Office of Water Enforcement and Permits and Office of Water
Regulations and Standards, U.S. Environmental Protection Agency,
Washington, D.C. 20460. In Press.
32
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APPENDICES
A. Chain-of-Custody Guidelines 34
B. Quality Control Checklist For
Self-Biomonitoring Toxicity Tests .... 40
C. Example of Standard Operating Procedures
(SOPs) Format ...... 42
D. Example of Culturing Criteria SOP Format . 45
E. Pre-Survey Forms: Toxicity Test Laboratory Evaluation . . 46
F. On-Site Laboratory Evaluation Forms and
Checklists 53
G. Recommended Toxicity Test Conditions and Test
Acceptability Criteria: On-Site Checklists 72
33
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APPENDIX A
CHAIN-OF-CUSTODY GUIDELINES^
1. Introduction
1.1 Written procedures for sample handling should be available and followed
whenever samples are collected, transferred, stored, analyzed, or destroyed.
For the purposes of litigation, it is necessary to have accurate records which
can be used to trace the possession and handling of samples from the moment of
collection through analysis. Secure, computer based data management systems
may be appropriate. The procedures defined here represent a means to satisfy
this requirement.
1.1.1 A sample is in someone's "custody" if:
it is in one's actual physical possession;
it is in one's view, after being in one's physical possession;
it is in one's physical possession and then locked up so that no one can
tamper with it, and it is kept in a secured area, restricted to authorized
personnel only.
2. Sampling Collection, Handling, and Identification
2.1 It is important that a minimum number of persons be involved in sample
collection and handling. Guidelines established in standard manuals for
sample collection, preservation, and handling should be used (e.g., USEPA,
NPDES Compliance Inspection Manual EN-338 (1988a); Methods for Measuring the
Acute Toxicity of Effluents to Freshwater and Marine Organisms (1985a) or
latest edition, Environmental Monitoring and Support Laboratory, U. S.
Environmental Protection Agency, Cincinnati, Ohio. EPA/600-4-85-013,,
2.2 Field records should be completed at the time the sample is collected and
should be signed or initialed, including the date and time, by the sample
collector(s)"-
2.3 Field records should contain the following information:
a. unique sample number;
b. date and time;
c. type of sampler;
Adapted from: USEPA (1982a) and (1990a),
34
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d. source of sample (facility name, location, and sample type);
e. preservative .used (if preserved);
f. analyses required;
g. name of collector(s);
h. pertinent field data (pH, DO, Cl residual, etc.);
i. serial number on seals and transportation cases.
2.4 Each sample is identified by affixing a pressure sensitive gummed
label or standardized tag on the container(s) (Figure 1). This label
should contain the sample number, source of sample, preservative used,
and the collector(s) initials. Analysis required should be identified.
Where a label is not available, the same information should be written on
the sample container with an indelible marking pen.
2.5 The sample container should then be placed in a transportation case
along with the chain-of-custody record form (Figure 2.), pertinent field
records, and analysis request form. The transportation case should then
be sealed and labeled. All records should be filled out legibly in pen.
The use of locked or sealed chests will eliminate the need for close
control of individual sample containers. However, there will undoubtedly
be occasions when the use of a chest will be inconvenient. On these
occasions, the collector should place a seal around the cap of the
individual sample container which would indicate tampering if removed.
3. Transfer of Custody and Shipment
3.1 When transferring the possession of the samples, the transferee must
sign and record the date and time on the chain-of-custody record (Figure
2). Custody transfers, if made to a sample custodian in the field,
should account for each individual sample, although samples may be
transferred as a group. Every person who takes custody must fill in the
appropriate section of the chain-of-custody record.
3.2 The field custodian (or field collector if a custodian has not been
assigned) is responsible for properly packaging and dispatching samples
to the appropriate laboratory for analysis. This responsibility includes
filling out, dating, and signing the appropriate portion of the
chain-of-custody record.
3.3 All packages sent to the laboratory should be accompanied by the
chain-of-custody record and other pertinent forms. A copy of these forms
should be retained by the field custodian (either carbon or photocopy).
3.4 Mailed packages can be registered with return receipt requested. If
packages are sent by common carrier, receipts should be retained as part
of the permanent chain-of-custody documentation.
3.5 Samples to be transported must be packed to prevent breakage. If
samples are shipped by mail or by other common carrier, the shipper must
comply with any applicable Department of Transportation regulations.
The package must be sealed or locked to prevent tampering. Any evidence
of tampering should be readily detected if adequate sealing devices are
used.
35
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o
EPA
Station No. Date Tin
Station Location
Ron Metslff
$niiris QH $nd (5r®a$$
mn DO
NntriontR Bact,
Oth^r
Samplers:
le Sequence No.
Grab
CJomp-
Remarks/Preservative:
Figure 1. Example of Sample Identification Tag.
36
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|urv.y
Station
Number
|
w
Station Location
Date
Relinquished by: Signature
Relinquished by: Signature
Relinquished by: Signature
Relinquished by: Signature
Dispatched by: Signature
Time
Collectors: Signature
Sample Ty
Water
Comp. Grab
P«
Air
Soq.No.
No. of
Containers
Received by: Signature
Received by: Signature
Received by: Signature
Received by Mobile Laboratory for Field analysis:
Signature
Date/Time
Received for Laboratory by:
Analysis
Required
Date/Time
Date/Time
Date/Time
Date/Time
Date/Time
Metnod of Shipment:
Distribution: Orig.—Accompany Shipment
1 Copy—Survey Coordinator Field Files
Figure 2. Example of Chain-of-Custody Record.
37
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3.6 If the field collector delivers samples to the laboratory, custody
may be relinquished to laboratory personnel. If appropriate personnel
are not present to receive the samples, the samples shall be lockcjd in a
designated area of the laboratory to prevent tampering. Tampering with
field samples is prohibited. The person delivering the samples should
make a log entry stating where and how the samples were delivered and
secured. Laboratory personnel may then receive custody by noting in a
log the absence of evidence of tampering, unlocking the secured area, and
signing the custody sheet.
4. Laboratory Sample Control Procedures
4.1 Sample control procedures are necessary in the laboratory from the
time of sample receipt to the time the sample is discarded. The
following procedures are recommended for the laboratory:
4.1.1 There must be a designated custodian and an alternate person to
act in his or her absence. All incoming samples must be received by the
custodian, who must indicate receipt by signing the accompanying
custody/control forms and who must retain the signed forms as permanent
records.
4.1.2 The custodian must maintain a permanent log book to record,, for
each sample, the person delivering the sample, the person receiving the
sample, date and time received, source of sample, date the sample was
taken, sample identification or log number, how transmitted to the
laboratory, and condition received (sealed, unsealed, broken container,
or other pertinent remarks). This log should also show the movement of
each sample within the laboratory; i.e., who removed the sample from the
custody area, when it was removed, when it was returned, and when it was
destroyed. A standardized format should be established for log entries.
4.1.3 A clean, dry, isolated room, building, and/or refrigerated space
that can be securely locked from the outside must be designated as a
"custody room."
4.1.4 The custodian must ensure that heat-sensitive samples,
light-sensitive samples, radioactive samples, or other sample materials
having unusual physical characteristics, or requiring special handling,
are properly stored and maintained prior to analysis.
4.1.5 Distribution of samples to the analyst performing the analysis
must be made by the custodian.
4.1.6 The laboratory area must be maintained as a secured area,
restricted to authorized personnel only.
38
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4.1.7 Laboratory personnel are responsible for the care and custody of
the sample once it is received by them and must 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 time the analyses are completed.
4.1.8 Once the sample analyses are completed, the unused portion of the
sample, together with all identifying labels, must be returned to the
custodian. The returned tagged sample must be retained in the custody
room until permission to destroy the sample is received by the custodian.
4.1.9 Samples will be destroyed only upon the order of the responsible
laboratory official when it is certain that the information is no longer
required or the samples have deteriorated. The same procedure is true
for sample tags. The log should show when each sample was discarded or
if any sample tag was'destroyed.
4.1.10 Procedures must be established for audits of sample control
information. Records should be examined to determine traceability,
completeness, and accuracy.
39
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APPENDIX B
Quality Control Checklist for Self-Biomonitoring Toxicity Tests1
Permit No.
Facility Name ^
Facility Location
Test Date
Reviewer
Date Reviewed
Permit Requirement
ITEM (CHECK ONE) YES NO
Report Format meets EPA Methods requirement?
(see Weber, et al., 1988, 1989.)
Were EPA test Methods followed?
Test Evaluation:
A. Effluent Sampling
1. Grab or Composite {compare to permit)
2. Was holding time met? (36 hrs. or 72 hrs.)
B. Dilution Water
1. Source
2. Meets EPA requirements
C. Test Type
1. Acute Chronic
2. Test duration
3. Static , Static renewal , Flow-through
4. Chronic renewal , Chronic Flow-through
D. Test Organisms
1. Permit requirement (List species)
2. Age of Test Organisms
3. Source of Test Organisms
^Adapted from: William Peltier, USEPA, Environmental Services Division,
Region IV.
40
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Quality Control Checklist for Self-Biomonitoring Toxicity Tests (Continued)
ITEM(CHECK ONE)YESN0~
E. Test Temperature: (for each day of test)
1. Acute ±20C
2. Chronic ±1°C
F. Dissolved Oxygen > 40% Saturation
G. Total Residual Chlorine (TRC) Measurements
1. Before chlorination
2. After dechlorination
H. Test Results*
1. Controls
a. Acute Tests <_10% mortality after 48 hours
b. Chronic Tests < 20% mortality after 7 days
2. Test acceptability Tor Ceriodaphnia chronic test
a. >15 young/$ and 60% of controls have three broods
3. Effluent Effect
a. Species Species
b. % mortality in __^^
c. 100% effluent ~~~~~
d. LC50/EC50 ~~~~~~~~ "
e. NOEC ___
f- LOEC — ZHZZZIZZ
4. Raw data included for check on errors/inconsistencies
I. Quality Assurance Test Results
1. Type Reference Toxicants '
2. 24 hrs. LC50/EC50 48 hrs. LC50/EC50 ~
3. Acceptable EPA range '
4. Test conducted on species within 30 days of reference
toxicant test
J. Is Self-Biomonitoring Test acceptable to Regulatory Agency?
K. Does Test have to be repeated?
*Note:. For other test species use same format but substitute test
acceptability criteria of organisms in Appendix G.
41
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•APPENDIX C
Example of Standard Operating Procedures (SOPs) Format
This outline should be used as a guideline in the preparation of
the written Standard Operating Procedures (SOPs). Each SOP should be a
separate document Governing each toxicity test organism or group of similar
test organisms. All quality assurance (QA) and quality control (QC)
procedures must be included in the SOPs. SOPs must be prepared for each of
the following activities preformed by the laboratory:
SOP Document:
I. Sample Collection, Handling, Preservation, and Preparation
II. Chain-of-Custody Procedures
III. Equipment/Glassware Cleaning
IV. Culturing and Holding Methods (Each test species is a separate SOP.)
A. Ceriodaphnia dubia
B. Daphnia pulex, D. magna
C. Fathead minnow TPiniephales promelas)
D. Rainbow trout (Oncorhynchus mykiss)
E. Selenastrum capricornutunT
F. Lemna sp.
G. Sheepshead minnow (Cyprinodon variegatus)
H. Inland silverside (Memdia beryl! in a) "
I. Mysidopsis bahia
J. Arbacia punctulata
K. Champia parvula
L. Other
V. Culture Health
A. Ceriodaphnia dubia
B. Daphnia pulex, D. magna
C. Fathead minnow TPimephales promelas)
D. Rainbow Trout (Oncorhynchun mykiss)
E. Selenastrum capricornutum
F. Lemna sp.
G. Sheepshead minnow (Cyprinodon variegatus)
H. Inland silverside (Menidia Beryl!ina)
I. Mysidopsis bahia
J. Arbacia punctulata
K. Champia parvula
L. Other
42
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APPENDIX C
Example of Standard Operating Procedures (SOPs) Format (Continued)
IV. Testing Methods
A. Freshwater Acute Toxicity Tests
1. Ceriodaphnia dubia
2. Daphnia pulex, D magna
3. Fathead minnow TPircephales promelas)
4. Rainbow trout (Oncorhynchus mykiss)
5. Other
B. Estuarine and Marine Acute Toxicity Tests
1. Sheepshead minnow (Cyprinodon variegatus)
2. Inland silverside (Menid ia"bery11ina)
3. Mysid (Mysidopsis bahia)
4. Other
C. Freshwater Short-Term Toxicity Tests
1. Fathead minnow (Pimephales promelas) larval survival
and growth test
2. Fathead minnow (Pimephales promelas) embryo/larval
survival and teratogenicity test
3. Ceriodaphnia dubia survival and reproduction test
4. Selenastrum capricornutum growth test
5. Other
D. Estuarine and Marine Short-Term Toxicity Tests
1. Sheepshead minnow (Cyprinodon variegatus larval
survival and growth test
2. Sheepshead minnow (Cyprinodon variegatus embryo/larval
survival and teratogenicity test.
3. Inland silverside (Menidia beryllina) larval survival
and growth test
4. Mysid '(Mysidopsis bahia) survival, growth, and
fecundity test
5. Sea urchin (Arbicia punctulata) fertilization test .
6. Champia parvula reproduction test
7. Other
VII. Equipment/Chemical Analyses and Calibration of Instruments
A. Dissolved oxygen meters
B. pH meter
C. Conductivity meter
D. Total residual chlorine analysis
E. Hardness analysis
F. Refractometer
43
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APPENDIX C
Example of Standard Operating Procedures (SOP) Format (Continued)
VII. Data Analysis
A. LCso calculation
B. Ceriodaphnia dubia Pass/Fail Results Analysis
C. Dunnetts Procedures
D. Probit Analysis
E. Linear Interpolation Method
F. Other
IX. References
44
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APPENDIX D
Example of Culturing Criteria SOP Format^
Ceriodaphnia dubia
I. Mandatory Requirements
1. Fifteen - twenty young/female.in first three broods.
2. Eighty percent survival of brood animals.
3. Testing is delayed if 1 and 2 above are not met.
4. Brood animals of known age.
5. Brood animals replaced on a specified schedule.
6. Culture water replaced on a specified schedule.
7. Equipment (droppers, reservoir, food containers) sterilized on a
schedule.
8. Chronic reference toxicant tests completed at least quarterly.
9. Coefficient of variation of reference toxicant tests should be <. 50%
for endpoints LC50 and IC50. (NaCl suggested Reference Toxicant).
II. Important Practices
1. Feeding on weekends and holidays.
2. New food prepared at least biweekly.
3. Broods of eight or more used for testing.
4. Age of test animals not more than 24 hours old;
all released within an 8 hour period.
5. Brood animals for a test cultured in water of same hardness,
pH, and conductivity as dilution water for testing.
III. Recommended Practices of High Quality Labs
1. Suspended solids of YCT food measured and feeding level adjusted.
2. Algae included in diet.
3. Algal quantity fed is measured (# cells or optical density).
4. Algal cultures harvested at known age.
5. Standard procedures for cleaning equipment.
6. Males are recognized and counted.
7. Blocking of young from females in test.
Mdapted from: Donald I. Mount and Teresa J. Norberg, USEPA, Environmental
Research Laboratory - Duluth.
45
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APPENDIX E
PRE-SURVEY FORMS: TOXICITY TEST LABORATORY EVALUATION
46
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APPENDIX E
PRE-SURVEY FORM: TOXICITY TEST LABORATORY EVALUATION
Laboratory Name
Address
City . State Zip Code_
Proposed Evaluation Date
Current Federal/State Certification
Laboratory Director
Parent Company/Organization
Toxicity Program Supervisor/Manager
Evaluation Coordinator
Phone
Individual(s) Filling Out Pre-Survey Forms
PLEASE RETURN PRE-SURVEY FORMS BY . TO:
_^
Evaluator(s)
Organization
Address
City State Zip Code
Phone
47
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APPENDIX E
PRE-SURVEY FORM: TOXICITY TEST LABORATORY EVALUATION
CLIENTS SERVED (NPDES PERMITTEES)
Industrial
Municipal
Federal
Interstate
NUMBER OF TOXICITY TESTS PER YEAR
State
Private Sector
Others
Items
MATERIALS TESTED
Tes No~
Comments
Eff1uents
Industrial
Municipal
Sediments
Solid Phase
Suspended Solids
Leachate
Pure Compounds
Criteria Development
Hazard Assessment
Drilling Fluids
Surface Waters
Hazardous Wastes
48
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APPENDIX E
PRE-SURVEY FORM: STAFF RESUME .
Submit a resume for each staff member in the toxicity testing program
which includes the following:
Name:
Address:
Job Title:
Phone Number:
Education: (Degrees, Dates, Institutions Conferring Degree, Major and
Minor Fields)
Toxicity Testing
Formal Training:
On-the-Job Training:
Professional
Experience:
Professional Organizations/
Honor Societies:
Publications:
49
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50
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APPENDIX E
PRE-SURVEY FORM: FACILITY INFORMATION
FIXED LABORATORY
Total space ft2 Culture ft2 Testing ft2
Office ft2
Separate culturing and testing areas Y N
Temperature control system: Manual Automatic
Daily temperature range Recording device available: Yes No
Ventilation: Recirculating Once through
Lighting: Type Photoperiod: h light; h dark
Backup emergency power available: Yes No
Water supply: Source
Lab Treatment
MOBILE LABORATORY
Type Self-propelled Trailer
Length ft Width ft
Total space ft2 Culture ft2 Testl'n9 .ft2
Temperature control system: Manual Automatic
Daily range Recording device available Yes No
Ventilation: Recirculating Once through
Lighting: Type photoperiod: h Light; h dark
Backup emergency power available: Yes No
Water supply: Source _^
Lab Treatment
51
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62
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APPENDIX F
ON-SITE LABORATORY EVALUATION FORMS AND CHECKLISTS
The laboratory must have an informal meeting to introduce the technical
staff and other personnel of the toxicity test program to the evaluator. The
laboratory must present and discuss aspects of the laboratory QA and toxicity
test program. The evaluator must explain the audit or evaluation process if
it is not clear to the laboratory.
The completed pre-survey forms (Appendix E) should be used by the
evaluator as supplemental on-site checklists with Appendix F.
The following items are covered in this appendix:
I. On-Site informal Laboratory Presentation
II. Organization History
III. Laboratory Staff
IV. Facilities and General Equipment
V. Test Equipment, Instruments, and Supplies
VI. Test Organisms
VII. Documentation
VIII. Toxicity Test Methodology
IX. Quality Assurance and Quality Control
X. Data Handling
XI. Summary
63
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APPENDIX F
INFORMAL LABORATORY MEETING AND INTRODUCTION
OF THE TECHNICAL STAFF
I. Organizational Information
Facility Description:
Name/Affi1i ati on:
Address:
Phone Number:
Laboratory Director/Manager:
Type of Evaluation:
Informal meeting and introduction of laboratory staff: Y
Laboratory Personnel Contacted
Name Ti tle
64
-------
APPENDIX F
ON-SITE LABORATORY FORMS AND CHECKLISTS
Note: The completed pre-survey forms (Appendix E) should be used by the audit
or evaluator to supplement the on-site forms and checklists.
II. Organization History
Item
Laboratory On-Site Introduction/
Meeting with staff
Laboratory demonstrates active
toxicity test program
Performance reference toxicant
data available and checked
Organization chart
provided
Yes
No
Comments
III. Laboratory Staff
Item
Pre-survey forms
verification
Appropriate educational background
experience/toxicity testing
Laboratory adequately
staffed
All technical staff available
during evaluation
QA officer report to
senior management
Director/Supervisor/
Manager available during
evaluation
QA officer available
during evaluation
Yes
No
Comments
IV. Facilities and General Equipment
Item
Pre-survey forms
verification
Tour lab
Yes
No
Comments
65
-------
IV. Facilities and General Equipment (Continued)
Item
Tour mobile lab, if
available
Lab work space adequate
Culture space adequate
Toxicity Test space adequate
Lab has distilled/
demineralized water.
Lab has distilled
demineralized water
checked/recorded.
Analytical balance/
calibrated yearly
Balance routinely checked/
class S weights /recorded
logbook
Exhaust hoods provided
Ref ri gerator /freezer
adequate, etc.
Lab maintained in clean/
organized manner
Contamination-free work
areas available for
handling test materials
Culture and test areas
separated
Adequate storage areas
available
Temperature of lab
adequate
Lighting adequate
Air condition/ventilation
adequate
Chemical waste disposal policies/
SOPs available
Lab secure
Yes
No
Comments
V. Test Equipment, Instruments, and Supplies
Item
Pre-survey forms
verification
Yes
No
Comments
66
-------
V. Test Equipment, Instruments, and Supplies (Continued)
Item
SOP(s) verification
Calibration checks/log books
pre-survey forms
Manual available to
operator
Ves
Mo
Comments
VI. Test Organisms
Item
Pre-survey forms
verification
Culture Maintenance
SOP(s) available
Disease control /treatment
protocols documented
Holding/acclimation
facilities adequate
Source of test organisms
documented
Food and feeding program
documented
Freshwater supply /source/quantity
used/quality documented
Estuarine/marine water supply/
source /quantity used/quality/
documented
Yes
No
Comments
VII. Documentation
Item
Hre-survey forms
verification
Yes
No
Comments
67
-------
VII. Documentation (Continued)
Item
Sample custodian designated
Sample procedures/
responsibilities documented
Written SOPs available for
receipt of samples
QA procedures documented/
available to staff
Written SOPs developed for
compiling/maintaining sample
document files
Written SOPs for samples
preservation, storage/
are maintained.
Written SOPs for culture/
test methods
Daily activities/toxicity test
documented
Bound logbooks available/
general chemistry (pH,DO,etc.)
Bound logbooks used, pages
numbered consecutively
Type of work clearly displayed
on logbooks
Logbooks maintained in legible
manner
Are anomalies recorded
routinely
Are inserts permanently affixed
and signed.
Supervisor inspects notebooks/
for appropriate documentation
Yes
No
Comments
VIII. Toxicity Test Methodology (Recommended Toxicity Test Conditions and
Test Acceptability Criteria: On-Site Checklists, see Appendix G.)
Item
Pre-survey forms
verification
Required methods used
Any unauthorized
deviations
Are written SUPS
provided
Yes
No
Comments
68
-------
VIII. Toxicity Test Methodology (Recommended Toxicity Test Conditions
and Test Acceptability Criteria: On-Site Checklists, see
Appendix G.) (Continued)
Item
Biologist/technician
record bench data
in neat accurate manner
Appropriate instrumentation
used with each toxicity test
Yes
No
Comments
IX. Quality Assurance/Quality Control (QA/QC)
Item
Pre-survey forms
verification
Lab maintains QA/QC manual
Manual addresses elements
of QA program, including the
following:
a. Personnel
b. Facilities and equipment
c. Operation of instruments
d. Documentation of SOPs
e. Procurement and inventory
practices
f. Project plans/
'Data quality objectives
g. Reliability of data
h. Data validation
i. Feedback and corrective
action
j. Instrument calibration
Yes
No
Comments
69
-------
IX. Quality Assurance and Quality Control (Continued)
Item
k. Recordkeeping
1. Internal QA/QC audits
m. QC responsibilities/
reporting clearly defined
n. QC charts maintained
for routine analysis
o. QC records show corrective
action to meet QC criteria
p. Supervisory personnel review
data and QC results
Chain-of -custody
maintained
Record keeping
adequate
Instrument Calibration/
logbooks maintained
Reference toxicant evaluations
used
Analytical support/
inorganic analyses
Analytical support/
organic analyses
Yes
No
Comments
X. Data Handling
Item
Recommended statistical
programs used
Data calculations check/
second person
Data calculations
documented
Data analyses capabilities
available
Data and records retained
PC computer (s) available
Yes
~Ro~
Comments
70
-------
XI. Summary.
Item
Evaluation criteria met
Response to evaluation indicates
an awareness to QA
Staff place positive
emphasis on QA/QC?
Responses to QA/QC
open and direct
Cooperative attitude
displayed by staff
Lab places proper emphasis
on QA/QC
QA/QC deficiencies discussed
during debriefing
Overall QA/QC adequate to
accomplish objectives
Corrective actions during
previous evaluations
implemented
Corrective actions result
from this evaluation
Technical assistance
needed
Yes
No
Comments
71
-------
APPENDIX G
RECOMMENDED TOXICITY TEST CONDITIONS AND TEST ACCEPTABILITY CRITERIA:
ON-SITE CHECKLISTS!
Freshwater, Marine, and Estuarine Acute Toxicity Tests
Freshwater Short-Term Tests
Marine and Estuarine Short-Term Tests
lAdapted from: USEPA (1985a), USEPA, (1988c), and USEPA,
(1989c) or latest editions.
72
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