PB32-228966
IERL-RTP Procedures Manual: Level 1
Environmental Assessment Biological Tests
Litton Bionetics, Inc.
Kensington, MD
Prepared for
Industrial Environmental Research Lab
Research Triangle Park, NC
Oct
U.S. DEPARTMENT OF COMMERCE
National Technical Information Service
-------
United States EPA - 600/8 - 81 - 024
Environmental Protection , , «m
Agency October 1981
&EPA Research and
Development
IERL-RTP PROCEDURES MANUAL:
LEVEL 1 ENVIRONMENTAL ASSESSMENT
BIOLOGICAL TESTS
Prepared for
Office of Environmental Engineering and Technology
Office of Environmental Processes and Effects Research
Office of Solid Waste
Office of Toxic Substances
Prepared by
Industrial Environmental Research
Laboratory
Research Triangle Park, NC 27711
-------
TECHNICAL REPORT DATA
(Pleau read Juaruerioru on the rtvtru be fan completing)
1. REPORT NO.
. .
EPA-600/8-81-024
3. RECIPIENT'S ACCESSION NO.
PRfl7 228966
* T1TtEAN09U8TITLEIERL-RTP Procedures Manual:
1 Environmental Assessment, Biological Tests
5. REPORT DATE
October 1981
6. PERFORMING ORGANIZATION CODE
7. AUTHORIS)
D. J. Brusick and R. R. Young
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Litton Bionetics, Inc.
5516 Nicholson Lane
Kensington, Maryland 20795
10. PROGRAM ELEMENT NO.
ONRACT/GRANT NO.
68-02-2681, Task 501
12. SPONSORING AGENCY NAME AND ADDRESS
13. TYPE OF REPORT AND PERIOD COVERED
EPA, Office of Research and Development
Industrial Environmental Research Laboratory
Research Triangle Park, NC 27711
13. TYPE OF REPORT AND PERIOD
Task Final; 3/78-9/81
14. SPONSORING AGENCY CODE
EPA/600/13
is. SUPPLEMENTARY NOTES
pr04ect officer ^Ra
919/541-2558. The report supersedes EPA-600/7-
mond G. Merrill, Mail Drop 62,
-043.
i6. ABSTRACT
manuai gives revised procedures for Level 1 environmental assess-
ment biological tests, and supersedes the first edition, EPA-600/7-77-043 (NTL3
No. PB 268484), published in April 1977. The revised biological procedures comple-
ment the Level 1 chemical and physical procedures published in October 1978 as
TERL-RTP Procedures Manual: Level 1 Environmental Assessment (Second Edition),
EPA-600/7-78-201 (NTB No. PB 293795). Level 1 is a screening phase that identi-
fies , categorizes , and ranks the pollutant potential of influent and effluent streams
from industrial and energy-producing processes. The manual is a guide to sampling
and analysis professionals in planning and executing the bioass ay portion of a phased
environmental source assessment program. The manual gives the goals , strategies ,
and philosophy of a phased approach to environmental assessment. It introduces col-
lection and pretest handling procedures for environmental samples and the recom-
mended Level 1 biological test protocols used to analyze the samples. Basic quality
control procedures are discussed, as are possible bioassay procedures for Levels 2
and 3.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b. IDENTIFIERS/OPEN ENDED TERMS
c. COSATI Field/Group
Pollution
Bioassay
Quality Control
Sampling
Analyzing
Pollution Control
Stationary Sources
Environmental Assess-
ment
Biological Tests
13JB
06A
13H.14D
14B
is. DISTRIBUTION STATEMENT
Release to Public
19. SECURITY CLASS (Thii Report)
Unclassified
21. NO. OP PAGES
149
20. SECURITY CLASS (This page)
Unclassified
22. PRICE
CPA Form 2220-1 (»-7J|
-------
NOTICE
THIS DOCUMENT HAS BEEN REPRODUCED
FROM THE BEST COPY FURNISHED US BY
THE SPONSORING AGENCY. ALTHOUGH IT
IS RECOGNIZED THAT CERTAIN PORTIONS
ARE ILLEGIBLE, IT IS BEING RELEASED
.IN THE INTEREST OF MAKING AVAILABLE
AS MUCH INFORMATION AS POSSIBLE.
-------
EPA - 600/8 - 81 - 024
OCTOBER 1981
IERL-RTP PROCEDURES MANUAL:
LEVEL 1 ENVIRONMENTAL
ASSESSMENT
BIOLOGICAL TESTS
SUBMITTED BY:
D, j. BRUSICK AND R. R. YOUNG
LITTON BIONETICS, INC.
5516 NICHOLSON LANE
KENSINGTON, MARYLAND 20895
EPA CONTRACT NO. 68-02-2681
PROJECT OFFICER: " RAYMOND G. MERRILL
PROCESS MEASUREMENTS BRANCH
STRSAL ENVIRONMENTAL RESEARCH LABORATORY
U.S. ENVIRONMENTAL PROTECTION AGENCY
RESEARCH TRIANGLE PARK, NC 27711
PREPARED FOR:
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, DC 20545
-------
DISCLAIMER
This report has been reviewed by the Industrial Environmental Research
Laboratory, U.S. Environmental Protection Agency, and approved for publica-
tion. Approval does not signify that the contents necessarily reflect
the views and policies of the U.S. Environmental Protection Agency, nor
does mention of trade names or commercial products constitute endorsement
or recommendation for use.
-------
FOREWORD
This bioassay procedures manual has been prepared as a guide for studies
to be conducted by the Industrial Environmental Research Laboratory of
the Environmental Protection Agency (EPA), Research Triangle Park, North
Carolina. To assist in its preparation, a subgroup of the Environmental
Assessment Steering Committee was formed. The subcommittee, composed of
EPA experts in health and ecological effects, was given the responsibility
of recommending specific bioassays. This subcommittee recommended an
initial series of tests which were reviewed by the committee as a whole,
various other bioassay experts within EPA and others in industry and
universities. This manual is a revision of and supersedes the "IERL-RTP
Procedures Manual: Level 1 Environmental Assessment, Biological Tests
For Pilot Studies," published in April 1977 (EPA-600/7-77-043, NTIS
PB 268484).
The bioassay procedures in this manual are designed to complement the
chemical and physical procedures of the Level 1 environmental assessment
program and to be an integral part of a comprehensive source assessment
strategy. The purpose of Level 1 is to obtain preliminary information,
identify problem areas, and provide the basis for the ranking of streams
for further consideration in the overall environmental assessment.
This manual is written to guide sampling and analysis professionals in
planning and executing the bioassay portion of an environmental source
assessment program. The recommended biotests for testing the toxicity
and mutagenicity of feed and waste streams of industrial processes are
described with a brief summary of procedures for collecting and preparing
the samples to be tasted. A more detailed discussion of the sampling
program and the procedures for chemical and physical testing of industrial
process feed and waste streams is provided in the companion publication:
"IERL-RTP Procedures Manual: Level 1 Environmental Assessment, Second
Edition," published in October 1978 (EPA-600/7-78-201, NTIS PB 293795).
A more detailed discussion of each bioassay procedure is included in the
references cited in each chapter of this manual.
Chapter 1 of this manual defines the goals and strategy employed in Level 1
testing and gives the background and philosophy of the phased approach
to environmental assessment.
Chapter 2 discusses the Level 1 sampling activities and pretest-handling
procedures that can be used for most industrial complexes. For each
sample type, the discussion focuses on the general problem as well as
specific problems of preparation needed for sampling, the actual sampling
procedures and packaging of samples for shipment.
Chapters 3 through 5 specify the Level 1 health effects, aquatic and
terrestrial bioassay schemes. The schemes identify the methods of
analyses, anticipated output and level of effort required for implementa-
tion and the basic format for presenting the results of the tests.
m
-------
Chapter 6 describes the data management, including data collection forms
and an approach to consolidated toxicity assessment for multitest data
for health and ecological effects.
Chapter 7 outlines the recommended quality control and documentation
procedures necessary to verify the quality of the assays performed. ...
Chapter 8 provides a brief discussion of testing beyond that defined as
Level 1.
-------
ABSTRACT
This manual presents the revised procedures for Level 1 environmental
assessment biological tests. The manual supersedes the first edition
published in April 1977 (EPA-600/7-77-043, NTIS PB 268484). The biological
procedures in this manual are designed to complement the Level 1 chemical
and physical procedures published in IERL-RTP Procedures Manual: Level 1
Environmental Assessment (2 ed.), October 1978, (EPA-600/7-78-201, NTIS
PB 293795). Level 1 is a screening phase that identifies, categorizes,
and ranks the pollutant potential of influent and effluent streams from
industrial and energy-producing processes. The manual is written to
guide sampling and analysis professionals in planning anq executing the ,.
bioassay portion of a phased environmental source assessment program.
This manual presents the goals, strategies and philosphy.of a phased
approach to environmental assessment. It introduces collection and pre-
test handling procedures for environmental samples and the recommended
Level 1 biological test protocols used to analyze the samples. Basic
quality control procedures are discussed, as are possible bioassay proce-
dures for Level 2 and Level 3.
-------
CONTENTS
Page No.
DISCLAIMER i i
FOREWORD i i i
ABSTRACT - - v
FIGURES ix
TABLES -- — x
ACKNOWLEDGEMENTS — xi
CHAPTER 1. BACKGROUND AND GENERAL INFORMATION 1
1.1 Introduction: Definition of Level 1 1
1.2 Application of Level 1 Testing and Its Interpretation 1
1.2.1 Strategy of a Phased Approach.for Level 1
Testing 2
1.2.2 Definition of Level 1 Sampling and Analysis 3
1.2.3 Goals of Level 2 Sampling and Analysis - 3
1.2.4 Goals of Level 3 Sampling and Analysis -- 3
1.3 Multimedia Sampling Procedures 4
1.3.1 Classification of Streams for Sampling
Purposes .4
1.3.2 Phased Approach Sampling Point Selection
Criteria 5
1.4 Data Requirements and Pretest Planning 5
1.4.1 Process Data Needs 5
1.4.2 Pretest Site Survey 5
CHAPTER 2. SAMPLE COLLECTION, STORAGE, IDENTIFICATION, AND
PRETEST HANDLING 9
2.1 Introduction 9
2.2 Procedures for Sample Identification, Packaging
and Transport . 13
2.3 Sample Preparation Prior to Bioassay Testing 16
2.3.1 Solid Material Grinding and Particle
Sizing 16
2.3.2 Organic Extraction of Particulates 16
2.3.3 Preparation of Sorbent Resin Extracts —- 18
2.3.4 Concentration of Organic Material in
Aqueous Environmental Samples 18
2.3.5 Analysis of Extracts and Organic Liquids
for Total Organic Content . 22
2.3.6 Concentration of Extracts and Solvent
Exchange Procedure 22
2.3.7 Particulate Removal from Glass Mat
Filters - 23
2.3.8 Concentration of Aqueous Samples by
Lyophilization 24
2.3.9 Leachate Preparation 25
2.4 Special Problems 25
2.5 Sample Amounts Required for Level 1 Bioassays 26
-------
Page No.
CHAPTER 3. LEVEL 1 HEALTH EFFECTS BIOASSAYS 29
3.1 Introduction and Rationale 29
3.2 Ames Salmonel1 a/Microsome Mutagenesis Assay 33
3.2.1 Introduction and Rationale 33
3.2.2 Materials and Methods 33
3.2.3 Experimental Design 35
3.2.4 Results and Data Interpretation 38
3.3 Rabbit Alveolar Macrophage (RAM) Cytotoxicity
Assay 40
3.3.1 Introduction and Rationale 40
3.3.2 Materials and Methods - 41
3.3.3 Experimental Design .--_-- 41
3.3.4 Results and Data Interpretation 42
3.4 Rodent Cell Clonal Toxicity Assay 45
3.4.1 Introduction and Rationale 45
3.4.2 Materials and Methods 46
3.4.3 Experimental Design 46
3.4.4 Results and Data Interpretation 47
3.5 Acute Iji Vivo Toxicity Test in Rodents 48
3.5.1 Introduction and Rationale : 48
3.5.2 Materials and Methods 49
3.5.3 Experimental Design 49
3.5.4 Results and Data Interpretation 50
CHAPTER 4. LEVEL 1 AQUATIC ECOLOGICAL ASSAYS - 53
4.1 Introduction and Rationale 53
4.1.1 General Materials and Methods for Aquatic
Ecological Assays 54
4.2 Static Acute Toxicity Tests with Freshwater Fish
and Daphnla • 60
4.2.1 Introduction and Rationale 60
4.2.2 Materials and Methods 60
4.2.3 Test Organisms 61
4.2.4 Experimental Design 62
4.2.5 Results and Data Interpretation 64 .
4.3 Freshwater Algae 120-Hour Screening Test 66
4.3.1 Introduction and Rationale 66
4.3.2 Materials and Methods 66
4.3.3 Test Organisms :--——- 67
4.3.4 Experimental Design . 69
4.3.5 Results and Data Interpretation — r 70
4.4 Static Acute Toxicity Tests with Marine Fish and
Mysids 71
4.4.1 Introduction and Rationale 71
4.4.2 Materials and Methods — 71
4."4.3 Test Organisms 72
4.4.4 Experimental Design 73
4.4.5 Results and Data Interpretation 75
4.5 Marine Algae 96-Hour Screening Test 76
4.5.1 Introduction and Rationale. 76
4.5.2 Materials and Methods 76
VII
-------
Page No.
4.5.3 Test Organisms 78
4.5.4 Experimental Design 78
4.5.5 Results and Data Interpretation 79
4.6 Bioaccumulation Procedure for Industrial and Energy
Source Samples 81
4.6.1 Introduction and Rationale 81
4.6.2 Materials and Methods 81
4.6.3 Experimental Design 83
4.6.4 Results and Data Interpretation 83
CHAPTER 5. LEVEL 1 TERRESTRIAL ECOLOGICAL TESTS - 85
5.1 Introduction and Rationale 85
5.2 Plant Stress Ethylene Test 86
5.2.1 Introduction and Rationale 86
5.2.2 Materials and Methods 87
5.2.3 Experimental Design 89
5.2.4 Results and Data Interpretation 92
5.3 Root Elongation Test -- 93
5.3.1 Introduction and Rationale 93
5.3.2 Materials and Methods 93 .
5.3.3 Experimental Design 96
5.3.4 Results and Data Interpretation 99
5.4 Insect Toxicity Assay 102
5.4.1 Introduction and Rationale 102
5.4.2 Materials and Methods — 103
5.4.3 Experimental Design 103
5.4.4 Results and Data Interpretation 105
CHAPTER 6. LEVEL 1 DATA FORMATTING AND ANALYSIS - 107
6.1 Introduction 107
6.2 Degree of Sensitivity of Level 1 Bioassays 110
CHAPTER 7. LEVEL 1 QUALITY CONTROL AND QUALITY ASSURANCE
REQUIREMENTS — —- 113
7.1 Introduction - - 113
7.1.1 General Quality Control Required for
Bioassay Performance • 113
7.2 Requirements for Quality Assurance 113
7.2.1 Quality Assurance Samples . 113
7.3 Requirements for Quality Control 115
CHAPTER 8. ENVIRONMENTAL ASSESSMENT BEYOND LEVEL 1 117
REFERENCES 119
APPENDIX A - SISTER CHROMATID EXCHANGE IN CHINESE HAMSTER
OVARY CELLS - 125
GLOSSARY - -. - 130
VTM
-------
FIGURES
Number Page No.
1.1 Biological Analysis Overview 6
2.1 Level 1 Bioassay Sample Collection Form 14
2.2 Level 1 Bioassay Sample Processing Form 15
2.3 Field Portable Sampling Cartridge (to scale) 20
3.1 Ames Salmonella/Microsome Mutagenesis Assay 36
5.1 Complete Stress Ethylene Exposure Set-Up Showing
the Various Components and Their Intercon-
nections 88
5.2 Plant Enclosure for Plant Stress Ethyl-ens Test — 91
5.3 Glass Tank With Glass Pegs Cemented in Place 95
5.4 Glass Tank With Glass Plates in Position Between
Pegs 98
5.5 Seedlings Showing Methods of Measuring Roots 100
5.6 Preparation of Treatment Chambers for
Drosophila 104
6.1 Health Effects Critical Data Summary Form 108
6.2 Aquatic Ecological Effects Critical Data Summary
Form 109
6.3 Bioassay Summary Form 111
8.1 Proposed Scheme for a Second Stage Evaluation of
Level 1 Results - 118
A.I Sister Chromatid Exchange (SCE) 126
IX
-------
TABLES
Number Page No.
2.1 Summary of Sampling Methods 10
2.2 Level 1 Bioassay Sample Characteristics 11
2.3 Recommended Test Sample/Bioassay Compatabilities — 12
2.4 Pretest Sample Preparation 17
2.5 Minimum Requirements for Sampling Cartridges 21
2.6 Anticipated Sample Amounts Required to Conduct
Level 1 Tests - - 27
3:1 Characteristics of Level 1 Health Effects Bioassays-- 30
3.2 Level 1 Data Presentation 31
3.3 Advantages and Limitations of Level 1 Health Effects
Screening Tests 32
3.4 Salmonella typhimurium Strain Characteristics 34
3.5 Positive Control Mutagens • 37
3.6 Acceptable Spontaneous Revertants Per Plate 38
3.7 Ames Assay Evaluation Criteria * 39
3.8 RAM Assay Evaluation Criteria 45
3.9 CHO Assay Evaluation Criteria 48
3.10 Definition of Pharmacological Toxic Signs — 51 ...
3.11 Acute In Vivo Rodent Assay Evaluation Criteria 52
4.1 Characteristics of Level 1 Aquatic Ecological Effects
Bioassays 55
4.2 Recommended Prophylactic and Therapeutic Treatments
for Freshwater Fish 58
4.3 Sample Size Requirements for Aquatic Ecological Assays 59
4.4 Summary of Test Conditions, Freshwater Fish or
. Macroinvertebrate Test --.-- . 63
4.5 Definition of Fish Behavior Terms • '--.- 65
4.6 Composition of Algal Assay Medium (AAM) 68
4.7 Incubation Conditions for Freshwater Algal Assay
Organisms 69
4.8 Summary of Test Conditions, Marine Fish or Macro-
invertebrate Test 74
4.9 Composition of Marine Algal Assay Medium (MAAM) 77
4.10 Incubation Conditions for Marine Algal Assay Organisms 79
4.11 Definition of Toxicity Categories for Aquatic
Ecological Assays 80
4.12 Partition Coefficients of Chemicals Used for
Calibration 82
4.13 Sample Quantitites for Bioaccumulation 83
4.14 Approximate Concentrations for Calibration Samples -.- 84
5.1 Characteristics of Level 1 Terrestrial Ecological
Effects Bioassays 86
5.2 Plant Stress Ethylene Test Evaluation Criteria 92
5.3 Hand Screens for Sizing Seeds 96
5.4 Root Elongation Test Evaluation Criteria 102
5.5 Drosophila Insect Toxicity Assay Evaluation
Criteria 106
7.1 Ames Salmonella/Microsome Mutagenesis Assay Quality
Assurance Audit 114
7.2 CHO Clonal Toxicity Assay Quality Assurance Audit — 115
-------
ACKNOWLEDGEMENTS
This manual was prepared under the direction of Raymond G. Merrill,
Industrial Environmental Research Laboratory, Research Triangle Park,
North Carolina (IERL-RTP), who was the project officer. This task was
part of EPA's Environmental Assessment, contract number 68-02-2681 with
Litton Bionetics, Inc.
Special acknowledgement is given to Kenneth M. Duke, Battelle-Columbus
Laboratories, for his valuable contributions to this project; for his
involvement with the first edition and for the contributions he provided
to this edition of the Level 1 manual.
Acknowledgement is given to members of the Environmental Assessment Bio-
assay Subcommittee. This committee was composed of EPA personnel from
the Office of Environmental Engineering and Technology (OEET), the Office
of Health Research (OHR), the Office of Environmental Processes and Effects
Research (OEPER) and the Office of Health and Environmental Assessment
(OHEA). These individuals had the responsibility of selecting the proce-
dures to be implemented in the phased approach to environmental assessment.•
Members of this committee are listed on the following page.
Acknowledgement is also given to D. Curt Hutchirrson, Algirdas G. Vilkas and
Thomas A. Gezo of Union Carbide Corporation, Environmental Services,
Tarrytown, NY for their extensive contributions to the Aquatic and Terres-
trial Ecological Effects Sections.
Special acknowledgement is given to the many helpful discussions with
Dr. W.W. McFee, Purdue University.
-------
ENVIRONMENTAL ASSESSMENT STEERING COMMITTEE:
BIOASSAY SUBCOMMITTEE MEMBERS
James A. Dorsey
Industrial Processes Division, MD-62
Industrial Environmental Research
Laboratory
Environmental Protection Agency
Research Triangle Park, NC 27711
William B. Horning
Newtown Fish Toxicology Station
3411 Church Street
Cincinnati, OH 45244
Joellen Lewtas (Huisingh)
Health Effects Research Laboratory
MD-68
Environmental Protection Agency
Research Triangle Park, NC 27711
Raymond G. Merrill
Industrial Processes Division, MD-62
Industrial Environmental Research
Laboratory
Environmental Protection Agency
Research Triangle Park, NC 27711
Michael A. Pereira
Health Effects Research Laboratory
Environmental Protection Agency
Cincinnati, OH 45268
Shahbeg Sandhu
Health Effects Research Laboratory,
MD-68
Environmental Protection Agency
Research Triangle Park, NC 27711
Gerald F. Stara
Environmental Criteria Assessment Office
Environmental Protection Agency
Cincinnati, OH 45268
David T. Tingey
Environmental Research Laboratory
Environmental Protection Agency
200 SW 35th Street
Corvallis, OR 97330
Gerald E. Walsh
Environmental Research Laboratory
Environmental Protection Agency
Sabine Island
Gulf Breeze, FL 32561
Michael 0. Waters
Health Effects Research Laboratory,
MD-68
Environmental Protection Agency
Research Triangle Park, NC 27711
OTHER MAJOR CONTRIBUTORS
Gary Chapman
Western Fish Toxicology Station
1350 S.E. Goodnight Avenue
Corvallis, OR 97330
Lawrence Claxton
Health Effects Research Laboratory,
MD-68
Environmental Protection Agency
Research Triangle Park, NC 27711
Kenneth M. Duke
Battelle-Columbus Laboratories
505 King Avenue
Columbus, OH 43201 '
John W. Laskey
Reproductive Physiology Section
Environmental Protection Agency
Research Triangle Park, NC 27711
xn
-------
CHAPTER 1
BACKGROUND AND GENERAL INFORMATION
1.1 INTRODUCTION: DEFINITION OF LEVEL 1
The Industrial Environmental Research Laboratory of the Environmental
Protection Agency, Research Triangle Park, North Carolina (IERL-RTP),
has developed a three-phased approach to performing an environmental
assessment—the testing of feed and waste streams associated with indus-
trial and energy processes in order to define control technology needs.
Each phase or level involves distinct sampling and analytical activities.
While Level 2 and 3 assessments are briefly described in Chapter 8., this
biological procedures manual focuses on Level 1 sampling and bioassays.
A companion manual describes the chemical and physical analysis proce-
dures for Level 1 and the details of sampling procedures (1).
Physical and chemical characterization of environmental emissions is
critical to the definition of, need for and design of control technology.
However, the final objective of the IERL-RTP's environment assessment is
the control of industrial emissions to meet environmental goals that
limit the release of substances that cause harmful human health or eco-
logical effects. Consequently, the testing of industrial feed and waste
streams for biological effects is needed as a complement to the physical
and chemical data to ensure that the assessment is comprehensive. Biolog-
ical testing can provide a direct measure of the toxicity and/or mutageni-
city of substances to organisms that chemical analysis cannot. This is
especially important when dealing with substances for which there is
little available data on toxicity or when assessing complex mixtures
where synergisms and antagonisms may alter the toxicity of the.individual
chemical constituents.
It should be stressed that the results of Level 1 tests are not intended
for regulatory actions or recommendations, nor are they to be used as
tests of acceptability or non-acceptability of emission release. The
three-phased sampling and testing strategy was developed to focus avail-
able resources (both manpower and dollars) on industrial emissions which
have a high potential for causing measurable health or ecological effects
and for providing chemical and biological information on all sources of
industrial emissions.
1.2 APPLICATION OF LEVEL 1 TESTING AND ITS INTERPRETATION
The phased approach as it applies to Environmental Assessment requires
three separate levels of sampling and analytical effort. The first level
provides (1) preliminary environmental assessment data, (2) identification
of problem areas and (3) the data needed for the ranking of energy and
industrial processes, streams within a process and components within a
stream, for further consideration in the overall assessment. Level 2
sampling and analysis is designed to provide additional information that
-------
will confirm and expand the data base developed in Level 1. The Level 2
results provide a more detailed characterization of biological effects
of the toxic streams, define control technology needs and may define the
probable cause of a given problem. Level,3 utilizes appropriate sampling
and analysis methodology to monitor the specific problems identified in
Level 2 so that the toxic or inhibitory components in a stream can be
determined exactly as a function of time and process variation for control
device development. Sublethal chronic effects are also monitored in
Level 3.
1.2.1 Strategy of a Phased Approach for Level 1 Testing
A phased approach offers potential benefits both in terms of the quality
of information that is obtained for a given level of effort and in terms
of the costs per unit of information. This approach has been investigated
and compared to the more traditional approaches (2) and has been found
efficient in both time and funds required for assessment.
Implementation of a phased approach recognizes that it is impossible to
accommodate every conceivable condition on the first sampling or analysis
effort. There is a possibility that many streams or even the entire
installation may not be emitting hazardous substances in quantities of
environmental significance. Conversely, certain streams or sites may
have such problems that a control technology development program can be
initiated in parallel with a Level 2 effort. If either of these situa-
tions can be determined by a simplified set of sampling and analysis
techniques (Level 1), considerable savings in both time and money will
result. When budgetary limitations require sampling only those instal-
lations most in need of control technology, a simplified sampling and
analysis methodology can usually establish which installations should be
given priority.
The three levels are closely linked to the overall environmental assessment
effort. Level 1 identifies the questions that must be answered by Level 2,
and Level 3 monitors the problems identified in Level 2 to provide infor-
mation on chronic effects and for control device design and development.
The following situation is an example of the relationship of the levels
to each other.
Level 1 biological testing results indicated that a small quantity of an
effluent has inhibitory effects on algal growth, adverse effects on a
specified percentage (EC™) of the population in the Daphnia bioassay
and gives a positive microbial mutagenicity test. Level 1 chemical test-
ing indicated that polycyclic organic materials (POM) were present in
significant amounts. Considering these results, Level 2 biological
sampling and analysis would determine such factors as toxic effect over
a long time period, bioaccumulation at low trophic levels (primary
producers and consumers) and persistence of toxicity in the receiving
waters. Level 2 chemical testing is used to identify and quantify the
POM compounds and any other pollutant classes identified at Level 1 as
accurately as possible. This combination of biological and chemical
-------
testing can identify the exact nature of the toxic substance(s) and deter-
mine if a complex biological effect such as synergism, antagonism or
bioaccumulation is occurring. Level 3 testing will be used for long-term
continuous monitoring. Chemical testing will provide information on
seasonal or feedstock variations of the previously identified toxic
substance(s). Long-term biological testing will serve as an integrator
of such variations. In addition, Leve.l 3 biological testing will identify
possible chronic health and ecological effects. The entire data package
can then be used in designing the control technology development program
for the stream.
1.2.2 Definition of Level 1 Sampling and Analysis
At the initiation of an environmental assessment, little is known about
the specific sampling requirements of a source, hence the emphasis is on
survey tests. Sampling and analysis at this level are designed to show,
within broad general limits, the presence or absence and, where possible,
the approximate levels of toxicity associated with a source. The results
of this phase are used to establish priorities for additional testing
among a series of energy and industrial sources, streams within a given
source and components within streams. Level 1 has as its most important
function the ranking of specific streams and components for the Level 2
effort.
1.2.3 Goals of Level 2 Sampling and Analysis
The Level 2 sampling and analysis goal is to provide definitive data
required in the environmental assessment of a source. Consequently, the
goal of Level 2 sampling and analysis is obtaining statistically repre-
sentative samples, expanding information on the nature of the biological
response and finally, where possible and when necessary, identifying and
quantifying the toxic substance(s). Level 2 analyses are the most critical
of all three levels because they must provide confirmation of the results
obtained in Level 1 and give an accurate characterization of the potential
of the source to cause adverse environmental effects.
Level 2 must provide sufficient detailed information on the problems
delineated by Level 1 so that control stream priorities, total environ-
mental damage and an initial estimate of process/control system regions
of overlap can be established.
1.2.4 Goals of Level 3 Sampling and Analysis
Level 3 testing involves long-term monitoring of components specific to
the stream of concern. The sampling and analysis are directed towards
the integration of effects over time to account for seasonal or feedstock
variations. These efforts are also geared to assess the chronic health
and ecological effects of the stream components.
-------
1.3 MULTIMEDIA SAMPLING PROCEDURES
The Level 1 procedures described in the IERL-RTP sampling and chemical
analysis manual (1) can be utilized to acquire process effluent and feed-
stock samples. The Level 1 environmental assessment program, at a minimum,
must acquire a sample of each process feedstock and effluent stream, and
of fugitive air/water emissions. The feed stream data are necessary to
establish a baseline for comparison. The effluent stream sampling program
is required to determine the mass emissions rate and the environmental
impact which will result.
1.3.1 Classification of Streams for Sampling Purposes
Comprehensive assessments are organized around the five general types of
samples found in industrial and energy-producing processes rather than
around the analytical procedures that are required to collect the samples.
This facilitates the complex task of organizing the manpower and equipment
necessary for successful field sampling. The Level 1 chemical and physical
manual (1) should be consulted before sampling is undertaken to avoid
confusion in classifying streams or stream samples.
The five sample types are:
(1) Gas/Vapor (Non-particulate laden) - These include samples from
process streams, vents and effluents. Samples contain inor-
ganic and organic gaseous components.
(2) Gaseous Streams (Particulate or aerosol laden) - This involves
sampling contained air or gas streams such as in ducts or stacks.
Samples include particulates and higher molecular weight organics
with boiling points greater than 100°C.
(3) Liquid/Slurry Streams - Liquid streams are defined as those
containing less than 5 percent solids. Slurry streams are
defined as those containing greater than 5 percent solids.
Liquid or slurry streams are classified as aqueous or nonaque-
ous. A stream sample that contains more than 0.2 percent
organics is considered nonaqueous.
(4) Solids - These include a broad range of material sizes from
large lumps to powders and dusts, as well as nonflowing wet
pastes. Nonflowing wet pastes may be formed either by wetting
solids with aqueous or nonaqueous liquids or may be highly
viscous liquids such as some tars or oils. The distinction
between solids and slurries can become blurred.
(5) Fugitive Emissions - The characteristic of this general sample
type is that the emissions are transmitted to the environment
without first passing through some stack, duct, pipe or channel
designed to direct or control their flow. The sample may be
in any of the above physical forms and may result from non-ducted
gaseous, participate or liquid emissions from the overall plant
or process units.
-------
A flow diagram which shows the overall relationship of the samples to
the Level 1 analysis scheme is presented in Figure 1.1.
1.3.2 Phased Approach Sampling Point Selection Criteria
The selection of sampling points, where phased sampling techniques are
employed, relies on the concept previously stated: that Level 1 sampling
is oriented towards obtaining data with relaxed accuracy requirements
for determination of the pollution potential of a source, whereas Level 2
sampling is intended to acquire more accurate data necessary for a defini-
tive environmental assessment. The recommendations in this manual are
restricted to Level 1 sampling and analysis. Stream parameters such as
flow rates, temperature, pressure and other physical characteristics
will be obtained on both levels within the objectives of a given level
of sampling-.;- ., .
1.4 DATA REQUIREMENTS AND PRETEST PLANNING
Prior to the actual sampling and analysis, the data needs must be estab-
lished and used to identify analysis requirements and sampling problems.
The following paragraphs present a summary of data requirements and plan-
ning which must be applied (1-3). Specific recommendations associated
with each of the process streams are discussed in the appropriate chapters
of this manual.
1.4.1 Process Data Needs
Before traveling to a plant for a pretest site survey, it is necessary
to become familiar with the chemistry and operational characteristics of
the operations as well as any pollution control processes. The detailed
process data necessary for the sampling and analysis effort, as well as
the overall environmental assessment, are described in the sampling and
chemical companion to this manual (1):
The data collected must be consistent with the overall Level 1 objectives.
Thus, the minimum amount of data for a given stream is flow rate per
unit time at a given temperature and pressure. Additional data that may
be necessary are average flow per unit time, the effect of process varia-
tions on stream flow and composition, and normal variations in flow and
compositions with variations in process cycling. It is expected that
professional sampling and analysis personnel, in conjunction with the ..
EPA Project Officer, Industrial Environmental Research Laboratory, Research
Triangle Park (PMB-IERL-RTP), will select the appropriate data.requirements
for a given industry.
1.4.2 Pretest Site Survey
After establishing the necessary process data needs and selecting a tenta-
tive set of sampling points, a pretest site survey should be performed.
The pretest site survey must include provision for identification of
streams for bioassay sample collection. Since the sample requirements
-------
SAMPLE FOR BIOLOGICAL ANALYSIS8
Gases and Suspended
Particulate Matter
Gaseous
Grab Samples
Partlculates
Sorbent
Resins
[ <5umJ
Liquids
•
*T5sr «**
Extract
Concentrate
and Solvent
Exchange
y
• Plant Stress
Ethylene
• Microbial
Mutagenesis
•RAM
Cytotoxlcity
• CHO
Cytotoxlcity
•Insect
Toxicity
Concentrate
For Microhial
and Rodent Solvent
Tests Exchange
Extracts
• Microblal
Mutagenesis
• CHO
Cytotoxlcity
• Insect
Toxicity
With Suspended
Solids
Solids
Filter|2mm
sieve]
Grind
• Microbial Mutagenesls
• CHO Cytotoxicity
• Rodent Acute Toxicity
• Aquatic Toxicity b
• Root Elongation
• Insect Toxicity
S?
3
•o
ID
Preparation
•Microbial Mutagenesls
•RAM Cytotoxicity
• CHO Cytotoxicity
•Rodent Acute Toxklty
•Aquatic Toxicityb
•Insect Toxicity
Analysis
a Consult Table 2.3 for .Recommended Test Sample/Bloassay Capabilities and Table 2.4 (or Test Sample Preparation.
kAquatic Tests Include Freshwater or Marine Fish, Invertebrate and Algal Tests.
Figure 1.1 BIOLOGICAL ANALYSIS OVERVIEW
-------
for bioassay testing are larger than requirements for chemical analysis,
the presite survey must be sufficiently detailed to allow definition of
the correct process stream, the proper location and sampling methodology
prior to the arrival of the field sampling team. This presite survey
may or may not recommend the same sampling methods for chemical and bio-
logical analysis, however, both types of samples should represent the
same process stream over approximately the same time period.
-------
CHAPTER 2
SAMPLE COLLECTION, STORAGE, IDENTIFICATION, AND PRETEST HANDLING
2.1 INTRODUCTION (1,3,4)
Level 1 sampling strategy presumes that all streams leaving the process
will be sampled unless empirical data equivalent to Level 1 already exist.
Further, Level 1 sampling is not based on a priori judgements as to the
composition of streams. It is presumed that prior knowledge about the
source is, at best, incomplete. Predictions and extrapolations of existing
data should be used only as a check on Level 1 assessments and not es a
replacement for it (3).
Level 1 sampling programs make maximum use of existing sampling and stream
access sites. While care must be exercised to limit sample bias, the
commonly applied concepts of multiple point, isokinetic or flow propor-
tional sampling are not rigidly adhered to. A series of discrete samples
are taken, when appropriate, and are combined proportional to stream
flow to produce a single "average" for analysis. Alternatively, a single
sample of each stream is collected under average process operating condi-
tions.
This chapter briefly discusses the general methodology for obtaining
gaseous, particulate, liquid and solid feedstock and waste-stream samples
for the biological analyses. An overview of the sampling procedures is
presented in Table 2.1. A more detailed description is included in the
IERL-RTP Procedures Manual: Level 1 Environmental Assessment (Second
Edition) (1).
The types of samples obtained from the procedures outlined in Table 2.1
are usually mixtures and present problems for biological test systems
that have been developed and validated primarily with pure chemicals.
Table 2.2 summarizes the characteristics .of samples collected from various
sources.
All Level 1 bioassays are not suitable for the entire range of sample
types obtained from industrial sources. Certain tests, for example,
provide reliable results with solid samples that are soluble in organic
solvent carriers, but the same test is not reliable if used to evaluate
a gas or slurry. In other situations, the amount of sample required for
applicable bioassays is too large to permit the tests to be performed on
the available sample (such as with SASS samples).
Suitability of test systems for specific samples must be judged on an
individual basis but, as shown in Table 2.3, some generalizations and
recommendations can be made with respect to bioassay/test sample compat-
ability.
-------
TABLE 2.1 SUMMARY OF SAMPLING METHODS
Source Type
Number
of Samples
General Method
General Follow-Up
Gas and Vapor
(Non-particulate Laden)
Gaseous Streams
Containing Particulates
1 process
cycle or
5 hours
continuous
sample
Liquid/Slurry
Solid
Fugitive Emissions
(Air or Water)
1
1
High pressure line
Grab purge
Evacuated grab
SASSe
• Heat exchange
• Tap
• Dipper
• Automatic
• Manual grab
• Boring or auger techniques
Air: high volume sampler equipment
with a 3.5 pm filter and an
XAD-2 cartridge, evacuated
grab sampler, or SASS.
Water: plug collectors
A glass-bulb sample
container is used for chemical
and some bioassay analysis sub-
samples. Teflon or Tedlar
bags are used to hold and transport
larger samples for bioassays.
Particulates (10, 10-3, 3-1 urn) are
collected in cyclones and on filters
(
-------
TABLE 2.2 LEVEL 1 BIOASSAY SAMPLE CHARACTERISTICS
Source
Sample
Description
Characteristics
Air
Gas/Vapor (Non- Grab Gas
particulate laden)
Gaseous Streams SASS Cyclone Solids > 3 urn
(particulate/aerosol (10 urn + 3 urn)
laden) SASS - Cyclone Solids 1-3 urn
Process Fugitive
Emissions
Fugitive Gases
Liquids
All Sources
Solids
Piles, Conveyors,
Bins, etc.
SASS - Filter Solids < 1 urn
XAD-2 extract
SASS
XAD-2 Resin
High-Volume
Sampler
High-Volume
Sampler
Grab
Grab or
Composite
Grab or
Composite
Solids
XAD-2 extract
Gas
Untreated
Untreated solids
Organic, inorganic or both.
Sample limited by storage capacity.
May be inorganic, organic or both.
SASS samples may have limited size.
Same as above.
On fiberglass mat. Combine with
SASS 1-3 Mm if possible.
Organics in dichloromethane.
Requires solvent exchange
before bioassay.
Organic, inorganic or both.
Same as SASS XAD-2 Resin
Same as Grab above
Aqueous, nonaqueous or organic.
Solution, suspension or slurry.
Unlimited sample except for fugitive
run-off.
Coal, ash, residues, products; organic
and inorganic; unlimited sample.
-------
TABLE 2.3 RECOMMENDED TEST SAMPLE/BIOASSAY COMPATABILITIES
Health Effects Bioassays3
1.
Sample Type
Gas/Vapor (Non-
Ames
BC
RAM
NC
CHO
NC
WAT
NC
Ecological Effects Bioassays3
Aquatic
TestsD
NC
Terrestrial Tests
PSE
R
RE .
NC .
. II
B
particulate)
2. Liquids (<5% Solids)
A. Aqueous R A R R
B. Nonaqueous R A R A
3. Solids and Slurries
(>5% Solids)
A. Soluble R A R R
B. Insoluble R R A R
C. SASS par-
ticulates R R A NC
R
A
R
R
NC
B
B
B
B
R
A
R
R
NC NC
Standard test abbreviations are as follows:
Ames: Ames Salmonella/microsome mutagenesis assay.
RAM: Rabbit alveolar macrophage cytotoxicity assay.
CHO: Rodent cell clonal toxicity assay.
WAT: Acute i_n vivo test in rodents (whole animal test).
PSE: Plant stress ethylene test.
RE: Root elongation test.
IT: Insect toxicity assay.
Aquatic tests include marine or freshwater fish, invertebrate and algal bioassays.
Identification of compatibility abbreviations:
R: Recommended for Level 1 environmental assessment testing.
NC: Sample not compatible with test methodology.
A: Compatible with bioassay with no modifications to protocol. Not recommended
for routine Level 1 testing, but may provide additional information.
B: Compatible"with bioassay with modification to protocol. Not recommended for
Level 1 testing.
Nonaqueous liquids include samples with greater than 0.2 percent organics,
solvent exchange samples and sorbent resin extracts. Extracts must be solvent
exchanged to dimethylsulfoxide (DMSO) for testing.
12
-------
2.2
PROCEDURES FOR SAMPLE IDENTIFICATION, PACKAGING, AND TRANSPORT
In order to evaluate samples properly, information regarding the collec-
tion, processing and shipping of these samples must be transmitted by
the sampling contractor with the samples to the bioassay contractor.
The forms provided here are proposed for use by the field sampling Manager
and the EPA Project Officer to transmit the necessary information. The
first form, "Level 1 Bioassay Sample Collection Form," (Figure 2.1) details
the manner in which the samples are collected and processed before shipment
to the biological testing laboratory. The second form, "Level 1 Sample
Processing Form" (Figure 2.2), defines the types of bioassays to be per-
formed on the sample. It is completed by the Project Officer responsible
for the environmental assessment. Copies of the sample collection forms
should be: attached to all final reports.
Upon receipt of each test substance in the laboratory performing the
bioassays, the following information should be recorded in a permanent
copy and maintained on file for reference purposes:
(a) Date of Sample Receipt
(b) Sponsor's Name
(c) Sample Identification
(d) Quantity and Condition of Sample Received
(e) Physical Description of the Sample
(f) Storage Conditions and Location
(g) Sample Disposition and Disposal
These records are for internal quality control purposes and need not be
attached to the report.
An acceptable system of sample coding is listed below. This system will
permit more rapid identification of samples and yet maintain a high degree
of uniformity between chemical and bioassay sample codes.
1C l-3)jm cyclone catch HMB
3C 3-10 |jm cyclone catch HI
IOC . . > 10 urn cyclone catch
PF-a Particulate filter(s) HIB
PR CH2Cl2/Methanol probe and
cyclone rinse AI
MR CH2C12 organic module
rinse AI-1B
XR XAD-2 resin
XRB XAD-2 resin blank AI-2B
CD-0 Neat condensate MCB
CD-LE CH2C12 extract of MMB
condensate FF
CD-AE Acidified, extracted CF
condensate FA
HM HN03 module rinse BA
HN03 blank
First (H202) impinger -
Special handling
First (H202) impinger
blank - Special handling
2nd and 3rd (APS) impinger
composite
2nd (first APS) impinger
blank
3rd (secon'd APS) impinger
CH2Cl2/blank
CH2Cl2/Methanol blank
Liquid (oil) fuel feed
Solid (coal) fuel feed
Fly ash
Bottom ash
13
-------
FIGURE 2.1 LEVEL 1 BIOASSAY SAMPLE COLLECTION FORM
I. SAMPLE INFORMATION
1. Sample No. Collection Date
2. Sampling Site
3. Field Sampling Manager (on-site)
4. Contractor Contract No.
5. EPA Project Officer Program Name
6. Source Sampled
7. Discharge Rate of Source (Volume/Time)
8. Quantity Sampled/Units
9. Sample Description (liquid, slurry, solid, extract, appearance, etc)
10. Other Information as Applicable
Collection temp. Sampling location
pH Sampling technique
Other __
II. HANDLING & SHIPPING
1. Describe Sample Treatment Prior to Shipping (e.g., transfers,
extractants, stored undiluted, grinding, solvents used)
2. Field Storage and Shipping Conditions
Container Temperature Light
D Amber Glass a Ambient a Shield from light
a Polyethylene Bottle a Refrigerate (0 to 4°C)
a Coated Bag or Bottle a Freeze (-20°C)
a Teflon or Tedlar Bags D Dry Ice
D Other
3. Approximate Time in Storage and Time in Shipping
4. Sample Shipped to -
5. Mode and Carrier for Shipping
6. Comments
(This form should be completed by the on-site sampling manager and accompany
each sample. . A copy should be forwarded to EPA project officer and should
be attached to the Final Report).(Information necessary for bioassay
contractor to perform tests and additional information on source can be
found in engineering report.)
14
-------
FIGURE 2.2 LEVEL 1 8IOASSAY SAMPLE PROCESSING FORM
I. SAMPLE IDENTIFICATION
Sample No.
Bioassay Contractor
EPA Project Officer
Brief Sample Name
Collection Date
Program Manager
II. SAMPLE TYPE AND LAB PROCESSING
Basic Type
Solid
Subtype
Liquid
Gas
a Solid Granular
a Slurry, > 5% Solids
n Particulates from Filter
a Filter and Particulates
D Suspensions, < 5% Solids
a Aqueous
a Nonaqueous
n Extract
a Condensate
a Other ____i^
a Pressure Collection
a Vacuum Collection
Processing
D Grind, a <5 |jm or a <3/8 in.
a Extract Particulates with
. Organic Solvent
D Remove Particulate from
Filter
a Prepare Water Leachate
a Concentrate
D Solvent Exchange
a Evaporate to Dryness
a Filter
D Test as is
D Other
III. BIOASSAYS REQUESTED AND QUANTITY REQUIRED
Health Effects
Solid
a Mutagenesis (Ames)
a Macrophage toxicity (RAM)
D Rodent Cell Clonal toxicity (CHO)
n Mice iji vivo toxicity (WAT)
Aquatic Ecological Effects
a Freshwater fish toxicity
D Freshwater invertebrate
a Freshwater algae
a Marine fish toxicity
D Marine invertebrate
a Marine algae
Terrestrial Ecological Effects
D Plant stress ethylene (PSE)
(1,365 liters gas)
D Plant root elongation (RE)
D Insect toxicity (IT)
D Other tests (explain on back)
0.1 g
0.1 g
10.0 g
10 kg
0.5 kg
0.25 kg
10 kg
2 kg
0.25 kg
2.5 kg
0.5 g
Aqueous
Liquid
5 ml
45 ml
45 ml
50 ml
40 L
2 L
1 L
40 L
8 L
1 L
10 L
20 ml
Nonaqueous
Liquid
5 ml
2 ml
2 ml
20 ml
1
0.2
0.1
• 1
0.8
0.1
10 ml
IV. RECEIPT OF SAMPLE
Date of Receipt
Received by
This form is completed by the EPA project officer and transmitted to the
bioassay contractor. The amount of nonaqueous .liquid required for aquatic
testing is dependent upon the water solubility of the liquid sample.
15
-------
2.3 SAMPLE PREPARATION PRIOR TO BIOASSAY TESTING
Level 1 environmental sampling procedures provide samples which represent
the "average" composition of solid, liquid and gaseous streams of indivi-
dual processes. Biological testing of these Level 1 samples is generally
limited to testing of the whole sample which is consistent with the survey
nature of this environmental assessment program. The testing of frac-
tionated samples or specific components of a given sample involves a
degree of specificity more appropriate to Levels 2 and 3 testing.
Pretest sample preparation should be limited only to those procedures
required to process the sample to a form compatible with biological
testing. Pretest processing for specific sample types should be. stand-
ardized and applied as uniformly as possible across all Level 1.tests as
shown in Table 2.4. Data evaluation may be skewed if test materials are
not subjected to identical pretest processing in all bioassays. The
final ranking of a process stream for potential toxicity is based on a
composite of bioassay results. If some of the bioassay results are
derived from extracted or concentrated samples and other test results
are based on whole, unprocessed samples, the composite ranking may be
skewed toward greater toxicity. When samples are processed prior to
testing, the results of the tests should be adjusted to reflect the changes
in concentration introduced during the processing procedure. This chapter
includes a brief description of how samples should be handled in preparation
for biological analysis.
2.3.1 Solid Material Grinding and Particle Sizing
Insoluble solid samples should be ground to particles of 5 urn or less in
size. Grinding should be accomplished in a way which does not heat the
sample. Manual methods such as mortar and pestle or automatic methods
such as cryoscopic impact grinding* may be used. Cryoscopic impact
grinding is accomplished at liquid nitrogen temperatures. Approximately
100 to 250 mg of the sample are placed in the impaction unit dry or along
with a small amount of a suitable vehicle, such as absolute ethanol.
The sample is recovered from the grinder in a larger volume of vehicle.
The suspension is then run through a filter system utilizing 25 urn nylon
prefilter and a 5 urn nylon final filter.t The 5 urn particle criterion
in the resulting filtrate can then be verified by light microscopy.
Samples can then be evaporated to dryness, weighed and resuspended in
the appropriate medium for the biological tests.
2.3.2 Organic Extraction of Particulates
In addition to testing solids and particulates directly, individual pro-
jects or certain bioassays such as the Ames test may require the prepara-
tion and testing of organic extracts of these sample types. Solid
materials including SASS cyclone probe and filter particulate, ash, and
other samples collected from high-temperature process streams may be
extracted for 24 hours with dichloromethane in a Soxhlet apparatus (1).
*For example, Spex Freezer/Mill, Spex Industries, Inc., Metuchen, NJ 08840.
tFor example, Tetko, Inc., 422 Saw Mill River Road, Elmsford, NY 10523.
16
-------
TABLE 2.4 PRETEST SAMPLE PREPARATION
Ecological Effects Tests3
Health Effects Tests Aquatic Terrestrial Tests
Sample Type AmesRAMCHOWAT Tests PSE RE IT
1. Gas/Vapor (Non- —c — — — — WS
particulate)
2. Liquids
(<5% Solids)
A. Aqueous CX (WS) WS CL WS — WS WS
B. Nonaqueousd SE (SE) SE (SE) (SE) — (SE) SE
3. Solids and Slurries
(>5% Solids)
A. Soluble WS (WS) WS WS WS — (WS) WS
B. Insoluble EX GR (GR) WS (LE) LE — (LE) EX
C. SASS
particulates FE.EX FE (FE) — — — ~ (FE.EX)
aStandard test abbreviations are explained in Table 2.3.
Aquatic tests include marine or freshwater fish, invertebrate and algal bioassays.
cldentification of sample preparation abbreviations:
CL: Concentrate 4- to 10-fold by lyophilization (Section 2.3.8)
CX: Test neat sample in minimum Ames test. If negative, concentrate up to
1000-fold and solvent exchange to DMSO (Sections 2.3.4 and 2.3.6).
EX: Test whole sample in standard test. If negative, at the direction of the
Project Officer, extract organics from particulates, solvent exchange to
DMSO (Section 2.3.2) and retest.
FE: Test SASS particulates as supplied, for 1 urn or less fraction, remove
particulate from filter (Secton 2.3.7).
GR: Grind to 5 urn or less (Section 2.3.1).
LE: Test aqueous leachate of insoluble solids (Section 2.3.9).
SE: Test nonaqueous liquids without preparation, extract sorbent resins and
solvent exchange to DMSO (Sections 2.3.3 and 2.3.6).
WS: Test gas or aqueous liquid samples as supplied and soluble solids as solutions.
( ): Sample compatible with test, but not required for Level 1 testing (see Table 2.3).
—: Sample not compatible with test or test requires modification, not required for
d Level 1 (see Table 2.3).
Nonaqueous liquids include samples with greater than 0.2 percent organics, solvent
exchange samples and sorbent resin extracts.
17
-------
After cooling, the nonvolatile organic content of the extract is deter-
mined by gravimetric (GRAV) analysis. An aliquot, not to exceed 10 percent
of the sample, is processed by the GRAV method as described in Section 2.3.5
of this manual. The remainder of the sample is evaporated to dryness
under nitrogen at <40°C. The sample is then treated as a nonvolatile
organic solid, and redissolved or suspended in DMSO. The objective is
to achieve a final concentration of 15.mg (minimum) to 100 mg (preferred)
of organics per milliliter of dimethylsulfoxide (DMSO) in a sufficient
volume of sample for testing.
Extracts of solid or particulate samples collected in a manner that
prevents volatilization of moderately boiling organics (bp 100° to 300°C)
are analyzed for total organics, concentrated and solvent exchanged by
the procedures used to process SASS sorbent extracts (Sections 2.3.5 and
2.3.6).
2.3.3 Preparation of Sorbent Resin Extracts
The XAD-2 sorbent is extracted for 24 hours in a large Soxhlet extrac-
tion apparatus with dichloromethane as described in IERL-RTP Procedures
Manual: Level 1 Environmental Assessment (Second Edition) (1). The
boiling solvent level is maintained throughout extraction. An aliquot
of the sample is assayed for total organic content by the methods discussed
in Section 2.3.5. The extract must then undergo concentration and solvent
exchange with, preferably, DMSO before incorporation into the microbial
mutagenesis, cellular toxicity or insect toxicity tests (see Section 2.3.6).
Care must be taken not to bring the resin extract to dryness at any time
during concentration or solvent exchange.
2.3.4 Concentration of Organic Material in Aqueous Environmental Samples
Introduction. Organic separation and concentration of water samples for
mutagenesis testing for Level 1 is accomplished using XAD-2 and XE-347
porous resins. Both resins are available from Rohm and Haas Co., Phila-
delphia, PA. After extensive review and experimental work on sorbents (5),
it is apparent that no single resin can be used to sample adequately the
wide range of chemical classes present in aqueous environmental samples.
XAD-2 has high affinity for non-polar species but fairly low affinity
for polar compounds. The best resins for adsorption of polar species
are the Amber-sorb XE-340 series, with XE-347 exceeding the volumetric
capacity of the others. Using this information, a two-stage sampling
cartridge is described in this section. Samples entering the cartridge
would encounter the XAD-2 first where polar organic material is adsorbed.
Then, any polar materials breaking through the XAD-2 would be adsorbed
by the XE-347 stage.
The sequential XAD-2/XE-347 sorbent cartridge is recommended for general
and compound-specific sampling of organics from water at levels of 10-100
ppm and below. If an aqueous stream of unknown organic loading, contains
more than 100 parts-per-million of organics, breakthrough may occur before
a 10 liter total sample is collected. Also, if more than 1 g of organic
material is recovered from the sampling cartridge recommended here, break-
through should be suspected.
18
-------
Column Construction. A plan for a full-scale field portable sampling
cartridge appears in Figure 2.3. Any system composed of glass, Teflon
and/or stainless steel which meets the minimum size requirement may be
used. The rationale for the choice of parameters listed here is presented
in reference 5.
It has been demonstrated that linear velocities of eluent greater than
4.0 cm/min produce undesirable reduction in the volumetric capacity of
the column.
The minimum length of the resin bed was determined by the retention
characteristics of the resins used. The column design described here
has been shown capable of collecting compounds of interest from water
at 95 percent efficiency.
It is generally accepted that an adsorbent bed should have a length-to-
diameter ratio considerably greater than one to avoid channeling and
back-eddy effects. The cartridge in Figure 2.3 was thus somewhat "over
designed" and would be sufficient for collection of maximum of 30 liters,
at the specified 42 ml/min flow rate. However, recoveries from the
cartridge should not be significantly reduced by using the larger quantity
of resin so long as the desorption solvent flow is in a counter direction
to the original sample flow, or the resins are separated and extracted
separately.
Alternate column sizes and corresponding flow rates are listed in Table 2.5.
These sizes meet the minimum requirements for diameter, flow rate and
volumetric capacity described above. Table 2.5 is provided to facilitate
construction of the proper sampling cartridge from units available from
commercial suppliers.*
Preparation of Resins. Resins are cleaned in batches. To remove fines
and preservatives, the resins are first washed with deionized water.
The remaining material is transferred to a Soxhlet apparatus, and consecu-
tively extracted with three solvents: distilled, deionized water (eight
hours), methanol (24 hours) and methylene chloride (24 hours). After
extraction, the resins are removed from the Soxhlet apparatus and stored
in acid-washed, amber glass bottles under methanol.
Preparation. Packing the trap with the resin beads is easiest when vacuum
is applied to the bottom (by aspirator). The cleaned resin beads are
loaded with a metal spoon and packed by drawing organic free water through
the bed. The XAD-2 resin is loaded first into the bottom stage; then a
separating ring is dropped in, followed by the XE-347 resin, finally,
the top is secured and both end caps tightened.
Field and Laboratory Sampling. Aqueous samples are to be concentrated
1000-fold by this procedure. The final requested volume for Ames testing
is 5 ml, so 5 liters of aqueous sample are passed through the column by
"For example, Bio-Rad Laboratories, 2200 Wright Avenue, Richmond, CA 94804.
19
-------
Teflon Disc
1.74" x 1/16"
Teflon gasket
O.D. - 1.75"
1.0." 1.50"
x 1/16"
L.
6.
*•
'
I
JO"
1J50"-
-J
i ,
— ^
•MMH
rs \
ii™
&
•
»
\
L OD. h
Screen
OD.-
I.D. -
Screen
(not at
(solder
I
Stainless Insert to
hold screen
c
Stainless screen 1.325 mesh
(not attached) ID. - 1.375" x 1/8"
Stainless screen -f 325 mesh
O.D. - 1.75" x 1/16" Stainless
All holes 1/32" dia.
Figure 2.3 FIELD PORTABLE SAMPLING CARTRIDGE [to scale]-
20
-------
TABLE 2.5 MINIMUM REQUIREMENTS FOR SAMPLING CARTRIDGES
Column ID
cm
1.5
2.0
2.5
5
Length3
cm
40
20
13.
10b
Maximum
Flow Rate
ml/min
7
12
20
42
Time in
Sample 10
20
13
8.
4
hrs.
liters
5
Column length includes provision for both XAD^2 and XE-347. Half-length
separate columns of each resin may be connected in series to satisfy this
length. Longer column lengths are acceptable, however, an unused cartridge
containing XAD-2 and XE-347 should be extracted and subject to bioassay as
.a solvent-method blank.
Ten (10) cm is the minimum length of the cartridge based on a 2:1 length-
to-width ratio.
gravity feed or by using a laboratory metering pump*. All delivery lines
should be Teflon or stainless steel connected by swagelock fittings.
The first component the sample encounters as it enters the cartridge is
a cavity which is filled with glass wool. The purpose of this is to
remove any particulate material from the sample stream before it reaches
the resin.
Note that the direction of flow is up (against gravity). This is done
to minimize problems caused by bubbles or channeling as a result of the
action of gravity on the system.
Sample Recovery. The cartridge is loaded in a manner that keeps the two
sequential resin beds separate, so that the extraction and analyses of
the adsorbed compounds can be made separately. The contents of each
half of the cartridge are placed in separate Pyrex Soxhlet thimbles which
have been previously cleaned. To remove as much water as possible, these
thimbles are attached to suction flasks and aspiration vacuum is applied,
drawing most of the water through. After 15-30 seconds of air-drying,
the thimbles are placed in Soxhlet extractors. Samples are extracted
for 24 hours. The resultant organic samples from the XAD-2 and the XE-347
are combined and are dried using anhydrous sodium sulfate which was cleaned
and prepared as described in reference (1). Alternatively the resins
may be extracted in situ using a continuous extraction apparatus (5).
IH S1'tu extraction must be done in a way that back-flushes the methylene
chloride solvent through the XE-347 first, through the XAD-2 second and
finally out into the receiving flask. The total organic content of the
Tor example, Fluid Metering, Inc., Oyster Bay, NY 11771.
21
-------
extract is analyzed according to the methods in Section 2.3.5. Concen-
tration of the organic sample to 5 ml and solvent exchange to DMSO is
performed as described in Section 2.3.6 of this manual.
2.3.5 Analysis of Extracts and Organic Liquids for Total Organic
Content
Extracts and organic liquid samples are analyzed for total organic content
using analytical methods recommended in the IERL-RTP Procedures Manual (1).
Samples can contain both moderately volatile organic compounds (bp 100°
to 300°C) and nonvolatile organics (bp >300°C). Organic content of :
extracts must be determined by the appropriate methods (below) before,
and possibly after, any procedure which requires solvent evaporation.
Total Chromatographable Organics (TCP) Analysis. Quantitative anal-ysis
of moderately volatile materials is achieved by a gas chromatographic .
procedure called total Chromatographable organics (TCO) analysis (Section
9.4.1,. Reference 1). TCO is used only with SASS and aqueous-sample sorbent-
resin extracts. TCO analysis is not appropriate for samples that have
been collected or stored under conditions which permit volatilization of
low to moderate boiling organics.
Because materials in the TCO volatility range may be lost to varying
degrees during solvent evaporation, it is important that this analysis
be performed on extracts and solutions before and after any concentration
step.
Gravimetric (GRAV) Analysis. Nonvolatile organic components in all
extracts are quantitated by gravimetric (GRAV) analysis (Section 9.4.2.,
Reference 1). This simple procedure consists of taking an aliquot of
the sample and evaporating it in a preweighed aluminium weighing pan.
The sample is dried to constant weight (±0.1 mg) and the residue weight
determined. The GRAV nonvolatile organic content is calculated from the
residue weight and reported as one number for the whole sample. At least
10 mg of sample residue should be weighed, but no more than 10 percent
of the sample should be used for GRAV analysis. This procedure is suitable
for all extract sample types including extracts from SASS particulates
and residues from high-temperature processes. The GRAV analysis method
is also applicable for solid-weight determination of slurries. Highly
viscous liquids and pastes are weighed directly, placed in a suitable
solvent and dosed on a weight-per-volume basis.
2.3.6 Concentration Of Extracts and Solvent Exchange Procedure*
It will usually be necessary to concentrate the organic material from
sorbent-resin extracts to a volume of 10 ml for subsequent analysis.
Total organic content of the extract is determined by GRAV and TCO analysis
(Section 2.3.5) before and after solvent evaporation. It is recommended
that concentration to slightly less than 10 ml volume (i.e., 8 or 9 ml)
be accomplished using a Kuderna-Danish (K-D) apparatus with a three-ball
"For all extracts except SASS particulate fractions and residues from
high temperature process.
22
-------
Snyder column for volumes less than 1 liter. For volumes greater than 1
liter, a rotary evaporator should be used to reduce the initial volume
to approximately 100 ml. The resulting sample may be concentrated further
by K-D. It is essential that the extract not be reduced to dryness at
this point in the scheme to prevent loss of volatile material. The con-
centrated extract should then be transferred to a graduated container
(e.g., Kuderna-Danish receiver or centrifuge tube) and the volume restored
to 10 ml.
The concentration process should be stopped if material begins to drop
out of solution. In that case, the extract should be restored to a con-
venient volume in which the material is redissolved.
Bioassay testing requires that the dichloromethane solvent be eliminated
before the sample extract is applied to the test system. The appropriate
volume of extract is carefully reduced to 1 ml at £40°C under a gentle
stream of nitrogen (tapped from a liquid-nitrogen cylinder, if possible,
to minimize impurities). The solvent evaporates rapidly, so it is impor-
tant that this operation be done under constant surveillance to ensure
that the volume is not reduced below 1 ml. It is also necessary to warm
the samples slightly, either by hand or water bath at <40°C, to prevent
condensation of atmospheric moisture in the sample caused by evaporative
cooling.
One milliliter of DMSO is added and mixed by gentle agitation. The volume
is reduced to a total of 1.5 ml. Another 1 ml of DMSO is added, mixed
and the volume is reduced to 2.25 ml. The exchange is repeated with
another 1 ml of DMSO and the volume is reduced to. a final volume of
3 ml. Other DMSO volumes may be used if 3 ml of DMSO does not give a
suitable sample preparation.
2.3.7 Particulate Removal From Glass Mat Filters
The 1 urn or less fraction of SASS train particulate samples is often
supplied for biological testing still embedded on the surface of a glass-
mat filter. The purpose of this procedure is to optimally remove parti-
culate on glass mat filters while minimizing glass-shard removal.
Filters should be cleaned before sampling to remove shards from the filters.
This is done by sonicating blank filters in an ultrasonic water bath*
for two hours in cyclohexane. Filters should be placed in sterile glass
dishes, large enough so the filter is not bent, and covered by at least
3 cm of solvent. Standard 142-mm filters (such as Reeve Angel 934 AH
filters) can be cleaned individually in 150-mm x 75-mm crystallizing dishes-
using approximately 400 ml of cyclohexane. After cleaning, th'e filters
are removed to a clean, lint-free surface to dry. Once dry, the filters
are dessicated for at least 12 hours, filters are weighed then stored in
labeled petri dishes. Care should be taken in handling cleaned and loaded
filters to mimimize the release of glass shards to particulate samples.
"For example, Sonicor, Sonicor Instrument Corporation, Copiague, NY 11726.
23
-------
Each filter is placed in a sterile glass dish (such as that used in condi-
tioning) and cyclohexane added to a depth of approximately 1 cm (200 ml
in the 150-mm x 75-mm crystallizing dish). The dish is placed in the
ultrasonic cleaner (with at least 4 cm of bathwater or as recommended by
the manufacturer) and sonicated for five minutes. The filter is removed
to a clean, lint-free surface loaded side-up, and covered with a clean
paper towel.
Solvent is transferred to a 500-ml, round-bottom flask using a 25-ml
solvent rinse of the dish. The filter is sonicated in cyclohexane for a
second five minute period. The solvent and rinse from the second sonica-
tion is combined with the solvent obtained from the first wash. Mul-
tiple filters are occasionally supplied as one test sample. Particulate
suspensions from multiple-filter samples are combined in one common round-
bottom flask. The suspension is evaporated with a rotary evaporation
apparatus between additions so that no more than 300 ml are in the flask
at any one time.
The particulate suspension, whether from one filter or combined from
several, is evaporated to a small volume with a rotary evaporator. The
concentrated particulate suspension is then transferred to a tared, amber-
glass vial. In transferring the concentrated suspension to the vial,
several sonications using fresh cyclohexane may be necessary to clean
the round-bottom flasks. The solvent is evaporated to dryness under a
stream of nitrogen in a warm-water bath. The dried residue in the vial
is then dessicated for at least 12 hours and then weighed. The weight
gain of the vial is the weight of particulate material available for
testing.
The residue is resuspended in a volume of DMSO to give the desired concen-
tration of particulate. Vial caps should be lined with sterile Teflon
rounds. Samples suspended in DMSO are stored, tightly capped at +4°C
until used.
2.3.8 Concentration of Aqueous Samples By Lyophilization
Aqueous samples supplied for biological testing are often collected from
receiving bodies of water or other sources of water anticipated to have
low toxicant levels. Testing aqueous samples for toxicity in the acute
i_n vivo rodent toxicity assay often requires samples to be concentrated
to bring toxicants above the threshold of assay sensitivity. Corrections
are made in evaluating sample toxicity to compensate for the effects of
concentration.
Aqueous samples for rodent toxicity testing are concentrated 4- to 10-fold
by lyophilization. An aliquot of the sample (commonly 500 ml) is taken
and frozen in a chemically clean, bulk-freeze-drying flask. The sample
is reduced to a little less than one tenth the initial volume using a
large-sized lyophilyzing apparatus* at -40°C with a vacuum of 100 microns
"For example, Virtus Company, Inc., Gardiner, NY 12525.
24
-------
Hg. The sample is then thawed and transferred with deionized or glass-dis-
til led-water rinses to a clean, graduated cylinder. The volume is
increased to the desired volume (usually 50 ml) with deionized or glass- .
distilled water. The sample is transferred to an amber-glass, screw-top
bottle and stored at +4°C. The container label should provide full infor-
mation about the original sample as well as the concentration process.
2.3.9 Leachate Preparation
This procedure is used to leach water-soluble components from solid
samples. Aqueous leachates are prepared by shaking a known weight of
solid with distilled water and separation of the aqueous phase by filtra-
tion.
Solid test samples are ground, if necessary, so as to pass through a
9.5 mm (3/8 in.) standard sieve. Drying of the sample is not recommended
as volatile components may be driven off. A representative portion of
the test material is weighed and placed into the container to be used
for leaching. A volume of dilution water is added to the container equal
(in milliliters) to four times the weight (in grams) of sample. Large
volumes of leachate maybe prepared in a 30-gallon, linear polyethylene
drum. The drum is placed on a drum roller and agitated for 48 hours at
20 ± 2°C. Smaller containers may be used if less leachate is required.
Agitation equipment should produce constant movement of the aqueous phase
equivalent to that of a reciprocating platform shaker operated at 60 to
70 (25-mm: 1-in.) strokes per minute. The suspension is allowed to settle
after 48 hours of agitation. The aqueous phase is separated from any
solid or nonaqueous phases by decantation, centrifugation or filtration
through filter paper, as appropriate. The aqueous phase is vacuum- or
pressure-filtered through a 0.45-um-membrane filter and stored in a sterile
container of a size such that the entire bottle is filled.
2.4 SPECIAL PROBLEMS
Certain types of samples present testing problems. Slurry samples present
unique problems since they tend to precipitate in DMSO. This is parti-
cularly troublesome at high concentrations in the i_n vitro clonal cell
assay. A layer of precipitate covering the cells may result in death
caused by physical problems; solids may resist normal washing procedures
or may interefere with scoring by obliterating the colony.
Many particulate samples derived from the combustion process contain
adhered toxic chemicals which are only slowly extracted from the particles
by most bioassay systems. Preliminary extraction using organic solvents
(e.g., dichloromethane) can be used to acquire concentrated volumes of
the adhering chemicals. However, this approach may have little relevance
to normal i_n vivo toxicity; it may skew the bioassay responses and identify
most combustion process streams as highly toxic and candidates for higher
•level testing. Bioassay of extract from particulate samples is not
generally required for Level 1 assessment but may be done i_n addition to
testing the whole particulate for comparative purposes (see Table 2.4).
25
-------
2.5 SAMPLE AMOUNTS REQUIRED FOR LEVEL 1 BIOASSAYS
A frequent problem encountered in environmental assessment is the avail-
ability of sufficient sample to conduct all of the desired tests.
Table 2.6 lists each test conducted in Level 1 and the anticipated amount
of sample needed. It must be kept in mind, however, that the level of
toxicity will determine the final amount required. The amount of test
material, in some cases, is also dependent upon the characteristics of
the sample. For example, the maximum applicable dose (MAD) and the
volume of nonaqueous liquid samples required for aquatic ecological
testing is dependent upon the solubility determined for each sample
before testing is initiated.
The use of weights or volumes in test systems must be uniform. To achieve
this uniformity, the following rules will be used to direct the units of
concentration:
1. A sample with a density approximating that of water and no suspended
particles will be tested as a liquid, using microliters (pi) as the
unit of volume.
2. A sample which is a solid or a slurry consisting of > 5 percent of
the total as solids will be tested as a solid material using micro-
grams (ug) as the unit of weight.
3. A suspension which contains less than 5 percent of its total as a
solid will be tested as a liquid.
4. SASS sorbent resin extracts in DMSO will be tested as a nonaqueous
liquid using microliters as the unit of volume. However, the dose
will also be calculated based on the concentration of organics using
ug as the unit of weight. An attempt will also be made to calculate
the volume of original gas sampled per unit weight of organics.
26
-------
TABLE 2.6 ANTICIPATED SAMPLE AMOUNTS REQUIRED TO CONDUCT LEVEL 1 TESTS
Bioassay
HEALTH EFFECTS
Ames Salmonella
RAM Toxicity
CHO Clonal Toxicity
Rodent Toxicity
AQUATIC ECOLOGICAL EFFECTS
Freshwater Fish
Freshwater Invertebrate
Freshwater Algae
Marine Fish
Marine Invertebrate
Marine Algae
Solid
(grams)
2.0 (.5)b
0.1 (.025)
0.1 (.025)
10.0 (5)
10 kg
(7.5 kg)
0.5 kg
(0.3 kg)
0.25 kg
(0.13) kg
10 kg
(7.5 kg)
2 kg
(1-2 kg)
0.25 kg
(0.13 kg)
Liquid
(mi 1 1 i
. Aqueous
5 (1.5)c
45 (15)
45 (15)
50 (25)e
40L (30L)
2L (1.5L)
1L (0.6L)
40L (30L)
8L (6L)
1L (.61)
liters)
Nonaqueous
5 (1.5)
2 (0.6)
2 (0.6)
20 (10.0)
1L (750)f
200 (150)
100 (60)
1L (750)
800 (600)
100 (60)
Gas
(liters)
d
—
—
_ «~
—
—
—
—
—
—
TERRESTRIAL ECOLOGICAL EFFECTS
Plant Stress Ethyl ene
Root Elongation
Insect Toxicity
d
2.5 kg
.5 (.1)
d
10L (5L)
20 (10)
d
d
10 (5)
1,365
—
d
Nonaqueous liquid include samples with greater than 0.2% organics, solvent
exchange samples, and extracts.
The first value is the requested sample size for Level 1 testing. The value
in parentheses is the minimum feasible sample size to conduct the test.
cWhen concentrated (Section 2.3.4), up to 5 liters (1.5 liters minimum)
are required.
Sample form is compatible to bioassay but modifications are beyond the scope
of this edition of the Level 1 manual.
eWhen samples are concentrated (Section 2.3.8) up to 0.5 liters (0.25 liters
,minimum) are required.
The maximum applicable dose (MAD) and the volume of nonaqueous liquid samples
required for aquatic ecological testing is dependent upon the solubility deter-
mined for each sample before testing is initiated. For additional information,
contact the Technical Support Staff, Process Measurements Branch, IERL-RTP, U.S.
EPA, Research Triangle Park, NC 27711.
27
-------
CHAPTER 3
LEVEL 1 HEALTH EFFECTS BIOASSAYS
3.1 INTRODUCTION AND RATIONALE
The Level 1 health effects tests include assays for determining toxicity
and mutagenicity at several levels in organisms ranging in complexity
from bacteria to mammalian cells in culture (both permanent cell lines
and primary cells) to intact animals. Table 3.1 describes the biological
characteristics of the target organisms in this group of tests. The
tests are able to detect molecular changes such as DNA mutation (Ames
test), acute cell toxicity (RAM and CHO tests) and complex toxicological
responses in intact animals (WAT test).
Table 3.2 summarizes the types of data obtained, the nature of the observed
response and the need for statistical analysis in the Level 1 assays.
This group of tests offers broad coverage of toxicity with the concomitant
advantages of low cost, reproducibility, rapid performance period and
small sample sizes (Table 3.3). These features are consistent with the
goals of Level 1 environmental assessment.
Health effects bioassays are used to determine the concentration of test
material that produces either a defined mutagenic or toxic effect on the
test organisms in a short period of time. The Ames Salmonella/ microsome
mutagenesis assay (Ames) identifies the minimum effective concentration
(MEC) of a test sample that produces significant mutagenesis in any of
four tester strains of Salmonella typhimurium used.
The rabbit alveolar macrophage assay (RAM) measures four endpoints relating
to cell death and metabolic impairment, following 20 hours of continuous
exposure. The effective concentration of toxicant that reduces each
parameter to 50 percent of the control (EC5Q) is calculated. The ECcg
is also estimated in the rodent cell (CHO) clonal toxicity assay basea
upon the reduction in colony-forming ability of the cells following
24 hours of continuous exposure. Mortality and physiological observations
are recorded in both the quantal and quantitative phases of the acute i_n
vivo test in rodents (whole animal test, WAT). For samples exhibiting
toxicity in the quantal phase, the dose lethal to 50 percent of the animals
(LDt-n) is calculated. Additional health effects endpoints may be measured
in modified versions of the CHO toxicity test.
Results from Level 1 health effects tests are interpreted by using evalua-
tion criteria unique to each test. Test samples are ranked according to
relative mutagenicity or toxicity using guidelines presented in the results
and data interpretation section for each test.
Preceding page blank 29
-------
TABLE 3.1 CHARACTERISTICS OF LEVEL 1 HEALTH EFFECTS BIOASSAYS
Characteristic
Cell Type/Organ System
End Point(s) Measured
Salmonella
Mutagenesis
Prokaryotic Cell-
Enteric Bacteria
Species
Point Mutation
Cytotoxicity Assays
RAM
Eukaryotic-Primary
Rabbit Macrophage
Cells
Lethality and
Metabolic Impairment
CHO
Eukaryotic-
Hamster
Cell Line
Cell Lethality
WAT
Integrated Organ
and Tissue Systems
Lethality-
Toxic Signs
Amenable to Sample Types
Data Expression
Special Features
Solids, Liquids,
Particulates
Solids, Liquids,
Particulates
Positive or Negative EC50 (Viability, ATP)
Requires In Vitro
Activation System
to Detect Active
Metabolites
Especially Effective
for Particulate
Samples Because
Cells Are Phago-
cytic
Solids, Liquids,
Particulates
EC5Q (Clonal)
Detects effects
on Reproductive
Capacity of Cells.
Same Cells May Be
Used For SCE
Assay
Solids, Liquids,
Particulates
LD5Q or Toxic Signs
Can Detect Complex
Toxicological
Phenomena that
Are Dependent on
Interactions of
Several Organ
Systems
-------
TABLE 3.2 LEVEL 1 DATA PRESENTATION
Assay
Type of Data Obtained
Response Summarized
Statistical
Analysis
Salmonella Assay
Mutagenic responses in one or more bacterial Positive or negative
strains compared against established
criteria for a positive effect
Determination of the lowest tested
concentration giving a response
(Minimum Effective Concentration, MEC)
Dose-response effect can
be graphed
MEC for positive responses
Toxicity can be estimated
Not normally performed.
However, if each
dose is assayed
in replicate, a
mean ± SD can be
calculated
RAM Assay
Trypan blue dye exclusion as an estimate
of living and dead cells
ATP measurement using a fluorometer
Replicate cultures per dose evaluated
ECc0 values using percent
inability and viability
index calculated as per-
cent of control (curves
are plotted)
EC,-n value using ATP/106
Cglls and ATP/flask
calculated as percent
of control (curves
are plotted)
Mean ± SD calculated
for replicate samples
EC5Q and 95% confi-
dence limits can be
calculated.
CHO Assay
Number of surviving colonies at each
concentration
Survival = colonies of treated cells
colonies of control cells
Replicate cloning per dose
x 100
Survival curve is graphed
ECrn value derived from
50% reduction in colonies
relative to control
Mean ± SD calculated
for replicate samples
EC5Q and 95% confi-
dence limits can be
calculated.
WAT Assay
Lethality
toxic signs
and descriptions of
LD50 Value derived from 50%
reduction in survival
Li tchfi eld/Wi1coxi n
Analysis for LD5f,
-------
TABLE 3.3 ADVANTAGES AND LIMITATIONS OF LEVEL 1 HEALTH EFFECTS SCREENING TESTS
Advantages
Limitations
Cost Effective
Rapid
Large Data Base for Chemicals Tested
Good Reproducibility
Salmonella Assay Correlates with Mammalian
Carcinogenesis For Many Classes of Chemicals
RAM Assay Amenable to Study of Particulates
Assays Represent Sensitive Targets for Toxic
Agents
Use of S9 Mix in Salmonella Assay Permits
Evaluation of Metabolites
Small Sample Size Required to Conduct Assay
Except for the Rodent Toxicity Assay
Extrapolation of Results to Humans Uncertain
High Level of Technical Ability Required in Some
Assays
Route of Exposure Not Always Relevant to Human
Experience
Prediction of Toxicity Jji Vivo Not Highly
Quantitative
Limited Opportunity To Evaluate Volatile
Components of Industrial Emissions
Indirect Mammalian Toxicity (e.g., Neurotoxicity)
Can Be Detected in the Rodent Assay
-------
3.2 AMES SALMONELLA/MICROSOME MUTAGENESIS ASSAY
3.2.1 Introduction and Rationale
The Ames assay is based on the property of selected Salmonella typhimurium
mutants to revert from an histidine-requiring state (auxotrophy) to an
histidine-synthesizing state (prototrophy) as a result of exposure to
mutagens (6). The test is designed to mimic mammalian metabolic proces-
ses by the incorporation of a mammalian liver 9,000 X g microsomal fraction
(S-9) and the cofactors necessary to generate enzymatic activity. The
test is used as a screen for mutagenic activity of pure compounds, complex
mixtures or component fractions. It has recently been demonstrated that
most initiating carcinogens have mutagenic activity. In several compara-
tive studies the Ames assay has demonstrated approximately 80 to 85 percent
accuracy in detecting known animal carcinogens as mutagens (7). However,
some known carcinogens are negative in the test (e.g., diethylstilbestrol,
natulan, asbestos and some carcinogenic metals) or only very weakly posi-
tive. This may be because many of the negative agents are not believed
to be initiating carcinogens (8). Chemicals which are mutagenic in the
Ames system but have not been shown to be carcinogenic in mammals are
also known. These mutagens may represent a unique class of chemicals,
or the animal tests may not be sufficiently sensitive to detect their
carcinogenic effect. Continued improvement of the present bacterial
strains, addition of new strains, standardization of the Salmonella assay
and re-evaluation of the conventional animal carcinogenesis data may
reduce this level of error; however, a perfect correlation between muta-
tion and carcinogenesis is unlikely. The following discussion is intended
as a general description of the test; a detailed study design is presented
in the published method (6).
3.2.2 Materials and Methods
Indicator organisms. The indicator organisms to be used are the Salmonella
typhimurium tester strains developed by Dr. Bruce Ames (TA-1535, TA-1537,
TA-98, and TA-100). They are histidine-deficient variants and are used
to detect reverse mutations, which are either frameshift (TA-1537 and
TA-98) or base-pair substitutions (TA-1535 and TA-100), as indicated by
reversion to histidine prototrophy (Table 3.4).
Liver microsome preparations. The activation system for mutagenesis
screening consists of Aroclor 1254, induced S-9 fraction derived from
rat livers. Male, Sprague-Dawley rats weighing approximately 200 g each
.are used. Induction is accomplished by a single intraperitoneal injection-
of Aroclor 1254 (diluted in corn oil to 200 mg/ml) into each rat five
days before sacrifice at a dosage of 0.5 mg/g of body weight. All rats
are deprived of food (not water) 12 hours before sacrifice.
The following steps are carried out at 4°C using co.ld sterile solutions
and glassware. The livers (10 to 15 g) are aseptically removed from the
rats and placed into a cold, preweighed beaker containing 10 to 15 ml
of 0.15 M KC1.
33
-------
TABLE 3.4 SALMONELLA TYPHIMURIUM STRAIN CHARACTERISTICS
Strain
Designation
TA-1535
TA-1537
TA-98
TA-100
Gene
Affected
his G
his C
Ms D
Ms G
Additional Mutations3
Repai r
A uvr B
A uvr B
A uvr B
A uvr B
LPS R Factor
rfa
rfa
rfa pKMlOl
rfa pKMlOl
Mutation Type
Detected
Base-pair
substitution
Frames hi ft
Frameshift
Base- pair
substitution
Reference 6 presents a discussion of the additional mutations and
description of the abbreviations.
After the livers are washed and weighed in this beaker, they are removed
with forceps to a second beaker containing 3 ml of the KC1 solution per
gram of wet-liver weight. The livers are then minced with sterile scis-
sors, transferred to a chilled glass homogenizing tube, and homogenized
in.an ice bath by passing a low-speed motor-driven pestle through the
livers a maximum of three times. The chilled homogenates are then placed
into centrifuge tubes and centrifuged for 10 minutes at 9,000 X g at 4°C.
The resulting supernatant is decanted, transferred in 2-ml amounts to
small storage tubes, quickly frozen in dry ice and stored at -75° to
-80°C in a low-temperature freezer. This supernatant is known as the
S-9 fraction. Sufficient S-9 for use each day is thawed at room tempera-
ture and kept on ice before and during use. The extent of bacterial
contamination of the S-9 fraction should be determined. The S-9 mix may
be filter sterilized (0.45 urn porosity filter) if required.
The quality of each S-9 lot is determined before the lot is released for
general use. The enzymatic activity of S-9 is measured by testing refer-
ence mutagens, such as benzo-a-pyrene or 2-anthramine, in the Ames assay.
Metabolic activation is required for these chemicals to be detected as
mutagens. The protein content of the S-9 is also determined. The normal
range of values is 28 to 45 mg of protein per milliliter of S-9.
Metabolic activation mixture. The S-9 microsomal mix is prepared according
to the recommendations of Ames described earlier. The mix contains per
ml: S-9 (0.1 ml), MgCl2 (8 umoles), KC1 (33 umoles), D-glucose-6-phosphate
(5 umoles), nicotinamide adenine dinucleotide phosphate (NADP) (4 umoles)
and sodium phosphate, pH 7.4 (100 umoles). The S-9 mix is prepared fresh
each day and is maintained on ice before and during use. Use of the S-9
mix should not exceed 6 hours at 0°C.
34
-------
Bacteriological media. The minimal-glucose agar medium for histidine-
requinng strains used in mutagenesis assays is a 1.5 percent Difco-Bacto
agar in Vogel-Bonner Medium E with 2 percent glucose. Top agar (0.6 per-
cent purified agar, 0.1 M NaCl) contains 0.05 mM histidine and 0.05 mM
biotin to permit the bacteria to undergo several divisions.
3.2.3 Experimental Design
General test procedures. The plate-incorporation version of the Ames
assay is recommended for routine use in Level 1 mutagenicity assessment.
In the plate-incorporation assay, the sample is added directly to molten
top agar (45°C) which is then poured onto the plates along with the test
organism and the liver S-9 activation system (Figure 3.1). Test samples
'are diluted in water, dimethylsulfoxide, ethanol or acetone so that
constant volume aliquots are added to each plate. The solvents are listed
in order of preference. Maximum liquid volume of test sample and vehicle
should not exceed 0.2 ml per plate. Once the overlay has solidified,
the plates are incubated at 37°C for 48 to 72 hours. Typically, -48 hours
is specified unless there is evidence of growth inhibition. Plates are
then scored for the number of revertants per plate.
It is recommended that the standard pi ate-incorporation assay be performed
in duplicate for each test concentration and control. Controls for each
test include solvent or vehicle controls to measure the spontaneous rever-
sion frequency for each tester strain. A sterility check of the test-
material solution or suspension is made. Positive controls consisting
of compounds which both do and do not require metabolic activation are
conducted concurrently with each assay. Quality control procedures are
summarized in Chapter 7 and detailed in Reference 9.
Testing strategy and dose selection are dependent upon sample type and
availability. If test material is limited, it is advisable to test test
agents with the Salmonella strains TA-98 and TA-100 only prior to con-
ducting complete mutagenic assays. This step may reduce the amount of
chemical used if the test material proves to be highly toxic or mutagenic.
Solid samples are tested for mutagenicity at the maximum applicable
dose (MAD) of 5 mg per plate and at five lower concentrations of 2.5,
1, 0.5, 0.1 and 0.05 mg per plate. Additional sample types that are also
tested on a weight-per-volume basis are slurries and sample extracts
of known organic content. If the initial test is negative, it should be
repeated after one week for confirmation with strains TA-98 and TA-100.
If the results are positive, repeat studies should be performed over a
narrower concentration range with strains showing positive results in
the initial test to identify the minimum effective concentration (MEC).
Solid samples may be tested up to 10 mg per plate if there is evidence
of a weak-positive effect at 5 mg/plate. The requirement for retesting
is dependent upon the availability of sufficient quantities of test
samples.
All nonaquebus liquids are tested in a minimum assay starting at the MAD
concentration of 200 ul per plate, and at lower concentrations of 100,
50, 10, 5 and 1 ul/plate. Samples are retested after one week if the
35
-------
AMES ASSAY [PLATE INCORPORATION METHOD]
Molten [43°C to 45°C] overlay agar
appropriately supplemented*
0.05 ml to 0.2 ml
Test, positive or solvent
control chemical
0.1 ml
Aliquot of an overnight culture
of bacteria [109 cells/ml]
Aliquot of
buffer
0.5 ml
-S-9
0.5 ml S-9 mix [hepatic
+ S-9 ' homogenate from PCB
pretreated rat plus
necessary cofactors]
Overlay poured on selective
bottom agar medium
Plates incubated at 37°C
for approximately 2 days
The numbers of revertants/plate counted
.Data analyzed
Interpretation/Conclusion
*A modification of this test called the "Preincubation Modification" consists of a 15-20 minute
preincubation of the cells, chemical and S-9 at 37'C before the overlay is added. Certain agents
not active in the standard method will be positive in this modification.
'Figure 3.1 AMES SALMONELLA/MICROSOME MUTAGENESIS ASSAY
36
-------
test is negative with strains TA-98 and TA-100. Mutagenic samples are
retested over a narrower range of concentrations with those strains
showing positive results initially to identify the MEC.
A modification to the Ames mutagenesis assay is used to test receiving-
body-water samples and other dilute aqueous samples. Frequently mutagens
occur in environmental samples below the threshold of sensitivity for
many classes of chemicals in the Ames test. Aqueous samples often need
to be concentrated to detect mutagenic activity. Testing concentrated
aqueous samples may appear to contradict the screening nature of Level 1
assessment. However, pretest processing is acceptable to enhance the
sensitivity of the Ames test because of the need for ranking of process
streams, for confirming investigations and/or for control technology
application.
Testing strategy requires aqueous samples to be tested at the MAD of
200 ul per plate. If the neat sample at 200 ul per plate is mutagenic,
a repeat test is performed over a narrower dose range with strains showing
a mutagenic response to identify the MEC. Aqueous samples that are nega-
tive in the minimum assay are concentrated 1000-fold on sorbent resins
as discussed in Sections 2.3.4, 2.3.5, and 2.3.6. The sample extract in
DMSO is tested over the range of 200, 100, 50, 10, 5, and 1 ul per plate.
Samples that are negative are retested one week later with strains TA-98
and TA-100; those that are positive are retested with a narrower range
of concentrations with strains giving positive results if the MEC is not
identified in the initial assay.
Positive controls. Positive-control mutagens are run with each strain.
Both direct-acting compounds and compounds requiring activation are used.
Each positive-control chemical has a preferred solvent. Untreated controls
using the appropriate solvent are run for each indicator strain. Table 3.5
lists the chemicals, solvents and concentrations which may be routinely
used as positive controls for each strain.
TABLE 3.5 POSITIVE CONTROL MUTAGENS
Assay
Nonactivation
Chemical
Sodium azide
2-nitrofluorene
(NF)
9-aminoacridine
(9AA)
Solvent
Water
Dimethyl-
sulf oxide
Ethanol
Concentration
per Plate (ug)
10
10
50
Salmonella
Strains
TA-1535,
TA-98
TA-1537
TA-100
Activation
2-anthramine
(ANTH)
Oimethyl-
sulfoxide
2.5 TA-1535, TA-1537
TA-98, TA-100
37
-------
Negative controls. Both a negative (untreated cells) and a solvent (test
sample vehicle) control are conducted concurrently with each assay. The
concentration of solvent used in the solvent control is equal to the
maximum concentration of solvent used in dosing the test material. If
no solvent is used, only the untreated control is conducted.
Modifications of the assay. In addition to the plate-incorporation method
of analysis, other procedures may be of value in chemical assessment.
Certain types of materials such as dialkylnitrosamines and hydrazines
are not active in the standard method but require a preincubation of the
test agent, S-9 mix and indicator organisms for approximately 30 minutes
at 37°C prior to the addition of overlay agar (10). Selection of the
preincubation modification should be made after discussions with the
source contact regarding the chemistry of the sample:or under direction
from the EPA Project Officer.
3.2.4 Results and Data Interpretation
Acceptance of test data is based primarily on control test results.
Negative or solvent controls for each strain should have a background
mutant frequency similar to the ranges presented in Table 3.6. Positive
controls should give a positive mutagenic response when analyzed by the
evaluation criteria that follow.
TABLE 3.6 ACCEPTABLE SPONTANEOUS REVERTANTS PER PLATE
Strain Revertants/Plate
TA-1535
TA-1537
TA-98
TA-100
20 ± 10
15 ± 10
50 ± 25
150 ± 75
Because the procedures to be used to evaluate the mutagenicity of the
test article are semiquantitative, the criteria to be used to determine
positive effects are inherently subjective and are based primarily on a
historical data base.
Plate-test procedures do not permit exact quantisation of the number of
cells surviving chemical treatment. At low concentrations of the test
article, the surviving population on the treatment plates is essentially
the same as that on the negative-control plate. At high concentrations,
the surviving population is usually reduced by some fraction. One require-
ment in evaluating Ames mutagenesis data is that the selected doses range
over at least two log concentrations, the highest of these doses being
selected possibly to show slight toxicity as determined by subjective
criteria.
38
-------
Mutagenicity is evidenced by reversion to prototrophy and colony formation
on the selective culture medium. A sample may be considered mutagenic
if the number of induced revertants is more than three times the solvent-
control value for strains TA-1535 and TA-1537 or more than two times the
solvent-control value for strains TA-98 and TA-100.
The demonstration of dose-related increases in mutant counts is an impor-
tant criterion in establishing mutagenicity. Generally, a positive dose-
response over three test concentrations should be observed in conjunc-
tion with the relative fold increases identified above. A factor that
might modify dose-response results for a mutagen would be the selection
of doses that are too low (usually mutagenicity and toxicity are related).
If the highest dose is far lower than a toxic concentration, no increases
may be observed over the dose range selected. Conversely, if the lowest.
dose employed is highly cytotoxic, the test article may kill any mutants
that are induced, and the test article will not appear to be mutagenic.
The goal of Level 1 Ames testing is to rank source streams by relative
degree of genetic toxicity (mutagenicity). Samples identified as muta-
genic by the criteria above are then ranked by the evaluation criteria
presented in Table 3.7. The lowest concentration giving a positive
response in any strain is identified as the minimum effective concentra-
tion (MEC) for that sample. The final report should identify the MEC
and mutagenicity category for each sample. Samples with no detectable
activity at the maximum applicable dose (MAD) are ranked not detectable
(ND). A convenient method to derive the MEC is to graphically represent
the dose-response data corrected for the spontaneous background. The
MEC is the concentration required to induce a two- (TA-98 and TA-100
strains) or three-fold (TA-1535 and TA-1537) increase over the spontaneous
value. The MEC value should fall on the linear part of the dose response
curve.
TABLE 3.7 AMES ASSAY EVALUATION CRITERIA
Mutagenic
Activity
High
Moderate
Low
Not dectable
Solids
(MEC in mg/plate)
<0.05
0.05-0.5
0.5-5
>5
Liquids
(MEC in ul/plate)
<2
2-20
20-200
>200
Organic Extracts3
(MEC in ul/plate)
<2
2-20
20-200
>200
aConsult Section 3.2.4 for explanation of evaluation criteria for aqueous
sample extracts.
39
-------
Aqueous samples are evaluated in a slightly different manner from solids
and nonaqueous liquids. Aqueous samples are evaluated initially as uncon-
centrated liquids. Those samples ranked as nondetectable are then concen-
trated 1000-fold and tested as an organic extract. The processed sample
is then ranked by the criteria in Table 3.7 under organic extract. Reports
will include the data and final evaluation for both the unconcentrated
and the organic extract. The ranking of organic extracts of aqueous
samples is meaningful only to similar sample types; comparison of data
to other sample types is not recommended.
3.3 RABBIT ALVEOLAR MACROPHAGE (RAM) CYTOTOXICITY ASSAY (11-15)
3.3.1 Introduction and Rationale
Primary cell cultures of rabbit alveolar macrophages (RAM) are used to
measure cell death and metabolic impairment resulting from i_n vitro expo-
sure to particulate and soluble toxicants. The RAM cells constitute a
first line of pulmonary defense because of their ability to engulf and
remove particulate materials which are deposited in the lung. Primary
RAM cell cultures exhibit many of the metabolic and functional attributes
of the original tissues. Therefore, it is appropriate that this cell
type be used to define the acute cellular toxicity of airborn particulates
and associated chemicals as part of the Level 1 health effects environ-
mental assessment.
The RAM cytotoxicity assay has been employed effectively in cytotoxicity
screening of a wide variety of pure chemicals, mixtures and environmental
contaminants. Recently, this system has been applied in evaluating the
relative cellular toxicities of hazardous metallic salts (11) and parti-
culate effluents from coal gasification, fluidized bed combustion and
conventional coal-combustion processes (12). This cytotoxicity screening
system has also been used to "rank" the toxicities of industrial particulates
collected from SASS cyclone sampling trains. Compared to conventional,
whole-animal tests for acute toxicity, this assay is more rapid, less
costly and requires significantly less sample. However, the assay employs
isolated cells rather than intact animals, so the information may be
difficult to use to predict health hazards of toxic chemicals.
The standard RAM assay is used to develop cytotoxicity data following
20 hours of continuous exposure to test materials. The observed endpoints
are cell viability as measured by trypan blue dye exclusion and metabolic
impairment as measured by cellular adenosine triphosphate (ATP) levels.
Results are expressed in terms of an EC5Q concentration, which, is the
estimated test concentration causing a 50 percent reduction in each
measured parameter relative to the control. Statistical methods are
recommended for the calculation of the EC5Q values and the 95 percent
confidence limits for each EC™ determination.
The RAM assay is used primarily for solid or particulate samples for
Level 1 assessment. Aqueous and nonaqueous liquids are compatable with
this test but are normally tested in the rodent cell (CHO) clonal toxicity
assay discussed in Section 3.4.
40
-------
3.3.2 Materials and Methods
Indicator cells. This assay employs short-term primary cultures of
alveolar macrophages obtained by lung lavage of male and female New
Zealand white rabbits. The rabbits are individually housed and fed
antibiotic-free laboratory rabbit chow and water ad libitum. The animals
are examined for the absence of respiratory illness and for over-all
general health prior to collection of macrophage.
Media. The cells are maintained and treated in Eagle's Minimum Essential
Medium (EMEM) with Earle's salts supplemented with 10 percent fetal bovine
serum (heat-inactivated), 100 units/ml penicillin, 100 ug/ml streptomycin
and 0.5 ng/ml amphotericin B (Fungizone). Kanamycin (35 ug/ml) may also
be added as extra protection against bacterial contamination. Intermediate
steps require EMEM with 0 and 20 percent fetal bovine serum (FBS).
An alternate medium, Medium 199, may be substituted for EMEM and supple-
mented in the same manner. This medium is also available commercially
as a lOx concentrate.
Test material. Solid samples are tested as supplied or are finely ground
to 5 urn or less (see Section 2.3.1) and tested as a suspension in culture
medium. Dry particulate material, aqueous liquids, suspensions and slur-
ries are added directly to the culture medium and tested as a suspension
or solution. Liquids containing less than 0.2 percent organic solvent
can generally be tested directly. Samples dissolved in organic solvents
are solvent-exchanged into dimethylsulfoxide (DMSO) according to Section
2.3.6 before testing.
3.3.3 Experimental Design
Dosage selection. Test materials are pre-screened in triplicate at the
maximum applicable dose (MAD) of 1000 ug/ml for solids and particulates,
600 ul/ml for aqueous samples or 20 ul/ml for nonaqueous liquids and
sorbent extracts in DMSO. Testing will be terminated and the sample
categorized as having toxicity that is not detectable (ND) if no parameter
is depressed 50 percent or more relative to the control. If any parameter
is depressed 50 percent or more, a definitive test is undertaken with
another population of macrophage. Solid and particulate samples are
tested in triplicate using a dose range which includes 1000, 500, 100,
50 and 10 ug/ml. Aqueous samples are tested unfiltered (or filtered-
through a 0.45 urn membrane filter if necessary) at concentrations of
600, 200, 60, 20 and 6 ul/ml. Nonaqueous samples solvent exchanged into
DMSO are tested in triplicate at 20, 10, 5, 1 and 0.5 ul/ml. Dosage and
evaluation of organic extracts are also based upon ug/ml of organics
when organic content of a sample is known.
Routine pre-screen testing at MAD concentrations is efficient for the
testing of multiple samples. The pre-screen test may be eliminated in
situations where the sample size is limiting or where only a single sample
is to be tested. The dose range may be modified to accomodate sample
characteristics or previously gathered toxicity data. Inclusion of addi-
tional dose-levels or replicas per dose may be desirable.
41
-------
Controls. In all cases, triplicate cultures of untreated cells are estab-
lished and analyzed as the control. If the test material is dissolved
in an organic solvent (normally DMSO), cultures exposed to the solvent
will constitute the control values. The final concentration of solvent
in the growth medium shall be one percent or less, with the exception of
the highest dose which shall have no more than two percent solvent.
Procurement of cells. Macrophages are collected from healthy rabbits
weighing 1.5 to 2.5 kg. Animals are sacrificed by injection of sodium
pentoborbital (60 mg/kg) into the marginal ear vein. The neck and chest
area are irrigated with 70 percent ethanol and a tracheostomy is performed
using sterile operating procedures. Thirty ml of 0.85 percent saline
(23°C) are instilled into the lungs via a catheter and allowed to remain
for 15 minutes. The lavage fluid is removed with a sterile 50-ml syringe
and placed into a sterile 50-ml centrifuge tube on ice. Five to nine
subsequent lavages are performed and fluid is collected, except the saline
is removed shortly after its introduction into the lungs. Any tubes of
lavage fluid found to contain blood or mucous are discarded.
The cells are centrifuged at 450 x g for 15 minutes, preferably at 4°C.
The supernatant is aspirated, the cells resuspended in fresh cold saline,
and the cells centrifuged again. After the second centrifugation the
cells are resuspended in EMEM containing 20 percent heat-inactivated
fetal bovine serum (FBS) and are pooled. Differential cell counts are
performed using Wright stain; a minimum of 200 cells is counted. Cells
are also counted by hemocytometer and viability determined using trypan
blue dye exclusion.
Macrophage populations are discarded if specific parameters are not in
the normal ranges. The macrophage fraction of the pooled lavage fluid
should be greater than 90 percent of the nucleated cells. Cells are
discarded if viability, as determined by trypan blue dye exclusion, is
substantially less than 95 percent. The normal yield per rabbit is approx-
imately 30 x 106 cells; occasionally up to 50 or 60 x 106 cells are
obtained (12). If animals yield more than 60 x 106 or less than 20 x 106
cells, the collection is discarded.
Treatment of cells. Cells are diluted with EMEM (20 percent FBS) to
between 5 x 105 and 1 x 106 cells per ml. Aliquots of cell suspension
(2 ml) are then added to each pregassed (5 percent C02) test flask.
Three flasks are prepared for each concentration and control.
Test samples, whether for the initial prescreen at the MAD concentration
or the definitive test, are processed by appropriate pretest procedures
discussed in Section 2.3. In addition, particulate samples are leached
before testing. Particulate samples are weighed, vortexed dry for two
minutes to break up aggregates and are suspended in EMEM (0 percent FBS)
at 2000 ug/ml. The particulate suspension is preincubated for 20 hours
on a rocker platform at 37°C in a 5 percent C02-humidified atmosphere.
For all test samples, dilutions are performed with EMEM (0 percent FBS)
to twice the desired final concentration for each dose level. Two ml of
42
-------
sample are added per culture flask. Each flask therefore contains between
1 and 2 x 106 cells in 4 ml of EMEM containing 10 percent serum and the
test agent at the desired final concentration.
A different procedure is used for preparing the high dose (600 ul/ml) of
aqueous test samples. To achieve this high concentration, lOx EMEM (or
lOx Medium 199, if this alternate medium is used for the entire assay) is
diluted with the test sample, the cell suspension and the medium supple-
ments.
If the test substance causes a color change in the culture medium, the
pH is determined in additional treated flasks. After the exposure period,
the pH of the medium in the experimental flasks is again recorded. If
the sample causes a drop in the pH below 6.8 or an increase above 7.6,
the sample is retested under pH-adjusted conditions. The pH is adjusted
to pH 7.2 using ultra-pure HC1 or NaOH.
The dosed flasks are incubated at 37°C with loosened caps in a 5-percent
C02-humidified atmosphere. After setting for 30 minutes to permit attach-
ment, the flasks are rocked on a platform rocker (12 oscillations per
minute) for the remainder of a 20-hour exposure period.
Cell viability assay. At the end of the treatment period, the medium
containing unattached cells is decanted into a centrifuge tube on ice.
The attached cells are rinsed with 1 ml of 0.1 percent trypsin with 0.01
percent EDTA and then incubated with 2 ml of the trypsin/EDTA solution
for approximately 5 minutes at 37°C. The trypsinate rinse and decanted
medium are combined for each culture to yield a 7-ml cell suspension for
subsequent analyses.
A 1.0-ml aliquot is removed for cell count and viability determination.
The aliquot is combined with 0.5 ml of 0.4 percent trypan blue and counted
by hemocytometer after five minutes. A minimum of 200 cells is counted.
The percent viability is determined for each dose level and compared to
the control. The number of viable cells at each dose level is also
compared to the number of viable cells in the control to yield the vi-
ability index.
ATP assay. ATP is determined using a fluorometer (such as Dupont Model
760 Luminescence Biometer*) according to procedures supplied with the
instrument. Aliquots of the cell suspensions are removed and analyzed
immediately for ATP content. A 0.1-ml aliquot of cell suspension is
added to a 0..9-ml mixture of 90 percent DMSO and 10 percent 0.01 M
morpholinopropanesulfonic acid (MOPS) and vortexed for ten minutes.
After two minutes at room temperature, 5.0 ml of cold 0.01 M MOPS buffer
at pH 7.4 is added and the extract is mixed thoroughly and placed on
ice. Aliquots of 10 ul are injected into cuvettes containing 0.7 mM
crystalline luciferin, 100 units luciferase, 0.01 M magnesium sulfate,
and 0.01 M MOPS buffer (pH 7.4) at 25°C in a total volume of 100 ul.
The emitted, light is measured photometrically in the luminescence biometer.
"E.I. du Pont de Nemours and Co., Inc., Wilmington, DE 19898.
43
-------
The biometer is calibrated daily with standard ATP solutions to provide
a direct read-out of the ATP content. Each test sample is assayed at
least two times to demonstrate consistent readings.
ATP values are reported as average ATP per flask and ATP per 106 total
cells for each dose level and both parameters are also expressed as a
percent of the control. The dimension used for ATP concentration is
femtogram (fg) which is equivalent to 10-1S gram.
3.3.4 Results and Data Interpretation
Report. The reports will include the experimental protocol, screened
doses, pH values (if appropriate),-determination of the EC™ values and
95 percent confidence limits for four different parameters and the toxicity
ranking of the sample. The EC5Q value is defined as the test concentration
causing a reduction in a measured parameter by 50 percent relative to
the control. The four assay parameters are the percent viable cells in
the treated cultures, viability index, ATP per culture flask, and ATP
per 106 cells.
Assay acceptance criteria. The assay will be considered acceptable for
evaluation of the test results if the following criteria are met. The
macrophage population is 90 percent or greater of the total nucleated
cells collected by lung lavage. The percent viability of the macropahges
used to initiate the assay is 95 percent or greater. The survival of
viable macrophages in the negative control cultures over the 20-hour
treatment period is 70 percent or greater. A sufficient number of data
points (for five test concentrations or less) is available to clearly
locate the EC™ of the most sensitive test parameter within a toxicity
region as denned under Data Evaluation. The data points critical to
the location of the EC™ for the most sensitive parameter are the averages
of at least two treatea cultures. If all the test parameters yield EC™
values greater than 1000 ug/ml for solids, 600 ul/ml for aqueous solutions,
or 20 ul/ml for organic solutions, the curves for ATP content and viability
index parameters do not exceed 120 percent of the appropriate control.
Data evaluation. A substantial quantity of raw toxicity data is produced
from the RAM assay. Standard forms should be used to record both raw
and analyzed data as recommended in the companion volume giving proposed
quality control and quality assurance procedures (9).
Toxicity data can be analyzed using statistical methods developed by
Garrett and Stack (16). Four statistical programs are available for a
programmable calculator with magnetic-card capability. Data generated
by the programs include the mean and standard deviation of replicate
values, probability (P) values, the estimated dose level at which a re-
sponse attains a given magnitude (normally the EC™), and the 95 percent
confidence limits for a given EC5Q determination.
The calculated EC™ value for each parameter is evaluated using Table 3.8.
The toxicity ranking of the test material is determined by the assay
parameter with the lowest EC™ value. Organic extracts are evaluated
44
-------
and ranked as solid samples based upon the organic content of the sample
(ug organics/ml). The EC5n of sorbent extracts of SASS train samples is
also converted to the equivalent volume of gas originally sampled.
TABLE 3.8 RAM ASSAY EVALUATION CRITERIA
Toxicity
High
Moderate
Low
Not Detectable
Solids
(EC5Q in ug/ml)
<10
10 to 100
100 to 1000
>1000
Aqueous Liquids
(EC5Q in ul/ml)
<6
6 to 60 :.
60 to 600
>600
Nonaqueous Liquids3
(EC5Q in ul/ml)
<0.2
0.2-2
2-20
>20
Criteria for nonaqueous liquids are tentative and under evaluation.
3.4 RODENT CELL CLONAL TOXICITY ASSAY
3.4.1
Introduction and Rationale
Permanent cell cultures of Chinese hamster ovary cells (CHO) are used in
this assay for measuring test-material toxicity. The observed end point
is the reduction in colony-forming ability of single cells after an i_n
vitro exposure to test materials. CHO cells have been widely used in
evaluating pure chemicals, mixtures and environmental samples for cyto-
toxicity.
CHO cells have characteristics which make them suitable for Level 1 toxi-
city assessment and ranking. The cell line is well characterized and
cultures are easily maintained. CHO cells are sensitive indicators of
toxicity and provide a good correlation of toxicity with other Level 1
tests. The assay requires only a small amount of sample, is inexpensive
and requires only seven days for completion. CHO cells have been shown
to be capable of engulfing environmental particulate materials (12). In
addition to these characteristics, CHO cells can be easily and inexpen-
sively used to simultaneously measure both genetic toxicity and cytotoxi-
city (17,18); this capability may be highly desirable when more detailed
studies of sample toxicity are required. Suggested procedures to expand
the Level 1 CHO toxicity assay to include a genetic end point are discus-
sed in Appendix A.
The CHO clonal toxicity assay is primarily used for aqueous and non-aqueous
liquids, but is also compatible with solid and particulate samples.
Results are expressed in terms of the EC5Q concentration, which is the
45
-------
estimated test concentration causing a reduction in colony-forming ability
by 50 percent. Statistical methods can be used for the calculation of
the ECcQ value and the 95 percent confidence limits.
3.4.2 Materials and Methods
Indicator Cells. A cell line originally derived from Chinese hamster
ovarian tissue and designated CHO-K1 is used for this assay. The cells
spontaneously transformed to a stable, hypodiploid line of rounded, fibro-
blastic cells with unlimited growth potential. Monolayer cultures have
a fast doubling time of 11 to 14 hours and normally can be cloned at 80
percent or greater efficiency. Permanent stocks are maintained in liquid
nitrogen and laboratory cultures are maintained by serial subculturing.
Laboratory cultures are also periodically checked by culturing methods
for mycoplasma contamination.
Media. The CHO-K1 cell line has an absolute requirement for proline and
therefore must be maintained in a culture medium containing this ami no
acid. Ham's Nutrient Mixture F12, which contains 3 x 10-4 M L-proline is
normally used, supplemented with 10 percent fetal bovine serum and 2 mM
L-glutamine. Experimental cultures also contain 100 units penicillin,
100 ug streptomycin and 0.5 ug amphotericin B (Fungizone) per milliliter.
A lOx formulation of Ham's F10 medium, which also contains proline, is
used for the testing of aqueous samples in order to avoid dilution of
medium components.
Test material. Solid samples are either tested as supplied or ground to
fine particles (Section 2.3.1). Insoluble solids may be leached by
suspending the sample material in Ham's F12 for 20 hours at 37°C on a
rocker platform. . .'.;.-
Aqueous liquids and suspensions containing less than 0.2 percent organic
solvent are directly added by volume to the medium. A lOx concentrate
of Ham's F10 is used to prepare the high dose (600 ul/ml) for aqueous
liquids and suspensions. Samples supplied in organic solvents, such as
sorbent extracts, are usually concentrated and solvent exchanged into
DMSO before testing (Section 2.3.6).
3.4.3 Experimental Design
Dosage selection. Dry particulate material is dissolved or suspended in
growth medium and tested in triplicate using a dose range which includes
1000, 500, 100, 50 and 10 ug/ml. Aqueous samples are tested unfiltered
(or filtered through a 0.45 urn membrane filter if necessary) a.t concentra-
tions of 600, 200, 60, 20 and 6 ul/ml. Nonaqueous samples and samples
that are solvent exchanged into DMSO are tested in triplicate at 20, 10,
5, 1 and 0.5 ul/ml. The dose range may be modified to accommodate sample
characteristics or previously gathered toxicity data. Inclusion of addi-
tional dose-levels or replicas per dose may be desirable.
46
-------
Controls. In all cases, untreated cells will be plated to establish the
control cloning efficiency. If the test material is dissolved in an
organic solvent (usually DMSO), cells exposed to solvent in the medium
are cloned to provide the reference cloning efficiency for the effect of .
the test substance. The final concentration of solvent in the growth.
medium will be one percent or less with the exception of the highest
dose, which will have no more than two percent solvent. All controls
are performed in triplicate.
Clonal toxicity assay. Cells from sub-confluent monolayer stock cultures
in logarithmic growth phase are trypsinized with 0.25 percent trypsin,
counted by hemocytometer, and reseeded into a series of 60-mm culture
dishes at 200 cells per dish. The cultures are incubated for 6 to 16 hours.
at 37°C in a humidified five percent C02 atmosphere to allow attachment
of the cells and recovery of growth rate.
Test material is then applied (three dishes per dose) and the cultures
are returned to the incubator. After a 24-hour exposure period, the
medium is aspirated and the cells are washed three times with Dulbecco's
phosphate buffered saline (PBS) (prewarmed to 37°C). Fresh medium (5 ml)
is placed on each culture and incubation continued for an additional six
days to allow colony development. Medium is drained from the cultures
and the surviving colonies are washed with PBS, fixed in ethanol and
stained with Giemsa. Colony counting is performed manually; colonies
smaller than 50 cells are not counted.
If the test substance causes a color change, the pH of the medium is
recorded for the doses that produced a color change. At the end of the
exposure period, the pH of the discarded medium for which initial pH
measurements were made is again recorded. If the sample causes a drop
in the pH below 6.8 or an increase above 7.6, the sample is retested
under pH-adjusted conditions. The pH is adjusted to pH 7.2 using ultra-
pure HC1 or NaOH.
3.4.4 Results and Data Interpretation
Report. The reports will include the experimental protocol, screened
doses, pH values (if appropriate), colony counts, percent survivals (colony
counts relative to control colony counts), EC™ values with 95 percent
confidence limits and toxicity ranking of the sample.
Assay acceptance criteria. The assay is considered acceptable for evalua-
tion of the test results if the following criteria are met... .Average .
cloning efficiency of the CHO-K1 cells in the negative controls is
70 percent or greater, but not exceeding 115 percent. Distribution of
colonies in the treated cultures is generally uniform over the surface
of the culture dish. Data points for each test concentration critical
to the location of the EC™ are the averages of at least two treated
cultures. A sufficient number of test concentrations are available to
clearly locate the EC™ with a toxicity region as defined under Data
Evaluation. If the EC™ value is greater than 1000 ug of solid sample/ml,
600 ul of aqueous sample/ml or 20 ul of nonaqueous sample/ml, the plotted
curve does not exceed 110 percent of the negative control.
47
-------
Data evaluation. Toxicity data can be analyzed using statistical methods
developed by Garrett and Stack (16). Four statistical programs are avail-
able for a programmable calculator with magnetic-card capability. Data
generated by the programs include the mean and standard deviation of
replicate values probability (P) values, the estimated dose level at
which a response attains a given magnitude (normally the EC™), anc' ^ne
95 percent confidence limits for a given EC™ determination. An alternate
method to estimate EC™ values is to graphically fit a curve by eye through
relative survival data plotted as a function of the logarithm of the
applied concentration.
TABLE 3.9 CHO ASSAY EVALUATION CRITERIA
Solids
Toxicity ^^50 ^n Ma/1"!)
High <10
Moderate 10 to 100
Low 100 to 1000
Not Detectable >1000
Aqueous Liquids Nonaqueous Liquids3
(EC™ in ul/ml) (EC™ in |jl/ml)
OU OU
<6
6 to 60
60 to 600
>600
<0.2
0.2-2
2-20
>20
Criteria for nonaqueous liquids are tentative and under evaluation.
The toxicity ranking of the sample is determined by the EC™ value and
the evaluation criteria in Table 3.9. Organic extracts are evaluated
and ranked as solid samples based upon the organic content of the sample
(ug organics/ml). The EC™ of sorbent extracts of SASS train samples is
also converted to the equivalent volume of gas originally sampled.
3.5 ACUTE IN VIVO TOXICITY TEST IN RODENTS
3.5.1 Introduction and Rationale
Because of the complex mixture of chemical compounds in environmental
samples and the potential for additive or synergistic action, the rodent
HI vivo screen is considered to be a valuable test method.
The advantages of the ui vivo toxicity assays are embodied mainly in the
fact that the toxicologTcal assessment is performed in whole animals.
There is a significant background of test data on a wide range of toxi-
cants for the rodent systems, thus supplying needed information for reli-
able interpretation of results with complex effluents (19).
48
-------
The main disadvantage of an acute rodent toxicity study is a possibly
unsatisfactory prediction of toxicity induced by long-term/low-level
exposures. An additional consideration is the need for multi-gram quan-
tities of test material which may prohibit testing where small amounts
of sample are available, such as from source streams containing gaseous
and particulate material.
Mice and rats are usually the two animals of choice for the measurement
of acute toxicity. These choices are made because of the availability
of uniform strains, ease of housing, their small size, relatively low
cost and a large volume of published toxicologic data.
3.5.2 Materials and Methods
Test organisms. Random-bred weanling mice (21 to 28 days old) are used
for Level 1 acute In vivo rodent tests. Weanlings are used because they
are likely to be more sensitive to toxic effects of test materials than
adult mice. In addition, significantly less test material is required
for dosing.
Weanling mice may be purchased directly or females with timed pregnancies
may be obtained from laboratory breeding colonies or from commercial
suppliers. Litters are adjusted to five males and five females shortly
after birth to help standardize and enhance pup growth.
Weanlings from random-bred laboratory rats may be used if difficulties
are encountered in gavaging weanling mice because of their small size.
Rats have been found to be equally as sensitive as mice for this assay.
Test material. Solid materials are generally tested without processing.
Samples that are not finely divided may be ground with a mortar and pestle.
There are no particle size requirements for this test but material should
be fine enough to pass through a 24-gauge gavage needle when in suspension.
Solids are solubilized or suspended in deionized water. Primary dosing
suspensions are prepared 24 hours in advance to permit water soluble
materials to leach into the water at room temperatre. Other vehicles
such as corn oil, olive oil or carboxymethyl cellulose may be used if
water is not suitable.
Aqueous-liquid samples are concentrated 4- to 10-fold (Section 2.3.8).
Nonaqueous liquids are used without preparation. Organic extracts, such
as SASS sorbent resin extracts, should be concentrated and solvent ex-
changed according to Section 2.3.6.
3.5.3 Experimental Design
Since the major objective of the Level 1 biological testing procedure is
to identify toxicological problems at minimal cost, it is recommended
that a two-step approach be taken to the initial acute ui vivo toxico-
logical evaluation of unknown compounds. The first is based on the quanta!
(all-or-none) response and the second on the quantitative (graded) res-
ponse. Normally, the quantal test is used to determine the necessity
49
-------
for carrying out the quantitative assay. If no animals die following
exposure to the in the quantal test, further i_n vivo testing is not
initiated and the sample toxicity is categorized as not detectable (NO).
Quantal. Five male and five female weanling random-bred mice (21 to
28 days old) are used for dosing at the maximum applicable dose (MAD)
(5 ml/kg for liquids and 5 gm/kg for solids). The weight of each animal
is determined before dosing. The test material is administered by gavage
to this population of animals in one, or up to three fractionated doses
within eight hours, to give a total dose of 5 ml/kg for liquids or 5 gm/kg
for solids (solubilized or suspended in a suitable vehicle). The sample
is administered using 1-ml plastic syringes and 24-G gavage needles.
The test material should be well mixed when aliquots are removed for
dosing. The maximum volume administered to weanling mice should not
exceed 0.3 ml at each administration.
Immediately following administration of the test substance and at frequent
intervals during the first day, observations of the frequency and severity
of all toxic signs or pharmacological effects (Table 3.10) are recorded
on an observation checklist (9). Particular attention is paid to time
of onset and disappearance of signs. Daily observations are made and
recorded on all animals for a 14-day period. At termination of the
observation period, all surviving animals are weighed and killed; gross
necropsies are then performed. Necropsies are also performed on all
animals that die during the course of this study.
Quantitative. If a single animal in the quantal study dies during the
14-day observation period, a quantitative study is performed. Fifty
weanling mice or rats, equally divided by sex, are used for this study.
After determining good health in the study population following seven
days of quarantine, the animals are randomly divided into five groups of
ten animals (five of each sex). The test substance is administered in
graded dosages according to the following schedule: 1.0, 0.5, 0.1, and
0.05 g/kg or ml/kg. A different dosage schedule may be selected depending
on the results of the quantal study. A control group receives an amount
of the vehicle equal to the maximum amount of vehicle used in dosing the
test material. The observations, study duration, and necropsy procedures
are carried out as described for the quantal test.
3.5.4 Results and Data Interpretation
Quantal. If no mortality occurs in the quantal study, no further studies
will be performed with the test substance and the LD5Q should be reported
as greater than 5 ml/kg or 5 g/kg. The test material is ranked as having
nondetectable toxicity (NO) at the maximum applicable dose (MAD). Effluent
samples which produce harmful effects i_n vivo and do not result in deaths
should be noted in the results summary. Such observations are difficult
to quantitate but provide insight into the sublethal effects of a sample
on rodents. Further investigations may be recommended from observation
of nonlethal toxic effects. Initial and final average body weights of
the dosed groups are determined and recorded. Gross observations of
the necropsies are made and summarized in the report.
50
-------
TABLE 3.10 DEFINITION OF PHARMACOLOGICAL TOXIC SIGNS
Organ System
Observation and
Examination
Common Signs of Toxicity
CNS and
somatomotor
Autonomic
nervous system
Respiratory
Cardiovascular
Gastroi ntesti nal
Skin and fur
Mucous membranes
Eye
Others
Behavior
Movements
Reactivity to various
stimuli
Cerebral and spinal
Muscle tone
Pupil size
Secretion
Nostrils
Character and rate
of breathing
Palpation of cardiac
region
Events
Abdominal shape
Feces consistency
and color
Peri anal region
Color, turgor,
integrity
Conjunctiva, mouth
Eyeball
Transparency
Rectal or paw skin
General Condition
Change in attitude to observer,
unusual vocalization, restless-
ness, sedation
Twitch, tremor, ataxia, cata-
tonia, paralysis, convulsion,
forced movements
Irritability, passivity,
anaesthesis, hyperaesthesis
Sluggishness, absence of reflexes
Rigidity, flaccidity
Myosis, mydriasis
Salivation, lacrimation
Discharge
Bradypnoea, dyspnoea, Cheyne-
Stokes respirations, Kussmaul
breathing
Thrill, bradycardia, arrhy-
thmia, stronger or weaker
beat
Diarrhea, constipation,
Flatulence, contraction
Unformed, black or clay colored
Soiled
Reddening, flaccid skinfold,
eruptions, piloerection
Discharge, congestion,
hemorrhage, cyanosis, jaundice
Exophthalmus, nystagmus
Opacities
Subnormal, increased temperature
Abnormal posture, emaciation
51
-------
Quantitative. The LD™ will be calculated by the method of Litchfield
and Wilcoxin (20) or Other suitable method (e.g. PROSIT) for ID™ deter-
mination. If the data are not suitable for calculation of a precise
LDcQ, i.e., total mortality occurs for the lowest dose, an estimate of
the LDcQ could be made or the LD5Q could be expressed as less than
0.05 mi/kg or 0.05 g/kg. Occasionally, it may be necessary to use higher
dosages, lower dosages or another series of intermediate dosages depending
on the initial results.
The calculated LD5Q value is used to rank the test sample. Evaluation
criteria for toxicity categorization are presented in Table 3.11.
Observations are made and recorded daily on all animals for the 14-day
period. As in the quantal phase, no attempt is made to quantitate or
rank the observations. The average animal body weight of each group is
determined initially and at the termination of the experiment. The average
weights, and the weights as fractions of the control are reported for
each dose level. Necropsy observations are recorded and reported.
TABLE 3.11 ACUTE IN VIVO RODENT ASSAY EVALUATION CRITERIA
Toxicity
High
Moderate
Low
Not Detectable
Solids
(LD5Q in g/kg)
<0.05
0.05 to 0.5
0.5 to 5
>5
Liquids
(LD5Q in ml /kg)
<0.05
0.05 to 0.5
0.05 to 5
>5
52
-------
CHAPTER 4
LEVEL 1 AQUATIC ECOLOGICAL ASSAYS
4.1. INTRODUCTION AND RATIONALE
Biological responses and bioaccumulation must be considered, as well as
chemical and physical parameters, when assessing the potential impact of
complex wastes on the aquatic environment. Biological testing for aquatic
ecological effects usually consists of static acute toxicity tests on
selected organisms representative of the various trophic levels. Evalua-
tion of the bioaccumulation of components in complex mixtures is accomp-
lished using a laboratory technique for simulating bioaccumulation pheno-
mena.
Acute toxicity tests are used to determine the concentration of test
material that produces an adverse effect on a specified percentage of
the test organisms in a short period of time. Because mortality is nor-
mally an easily detected and an obviously important adverse effect, the
most common acute toxicity test is the acute lethality test. The index
most often used with fish is the 96-hour median lethal concentration
(96-hour LC5Q) and for macroinvertebrates the 48-hour effective concen-
tration (48-Hour ECj:0). The LC5Q is a statistically derived estimate of
the concentration of toxicant in dilution water that is lethal to 50
percent of the test organisms during continuous exposure for a specified
period of time, based on data from one experiment. This may be supple-
mented, in fish tests with effects on behavior. The EC5Q for macroinver-
tebrates is an estimate of the concentration of test material that results
in the immobilization of 50 percent of the test organisms during continuous
exposure for a specified period of time in one experiment. Immobilization
is defined as lack of movement except for minor activity of appendages.
This measured effect is used because death is not always easily determined
with some invertebrates.
In algal tests the principle criterion of toxicity is the effect on growth
during continuous exposure for a specified period of time. The exposure
period for the freshwater algal bioassay is 120 hours, while that for
the marine algal bioassay is 96 hours. The 96- or 120-hour effective
concentration (ECcn), the concentration in which algal growth is inhibited
by 50 percent as compared with growth in the control, is statistically
estimated. For samples which stimulate algal growth, the stimulatory
concentration (SC20) is calculated. The 96- or 120-hour SC2Q is defined
as the concentration causing a stimulation in growth of 20 per.cent rela-
tive to the control after 96 or 120 hours of exposure. Other related
criteria which may be useful are the effects on rates of growth, on maxi-
mum standing crops, and on algal biomass at the end of the assay.
It may also be possible to establish the approximate concentration of
test material which produces no observable deleterious effect by any of
the criteria under study, which is the No Observed Effect Concentration
(NOEC).
53
-------
Since the reporting for each test is unique, guidelines are given for
individual tests in each section and summarized in Table 4.11.
The recommended test organisms in freshwater tests are the algae Selenas-
trum capricornutum, juvenile fathead minnow Pimephales promelas, and
early instars of Daphm'a magna. The recommended test period is 120 hours
for the algal test, 96 hours for the fish study, and 48 hours for the
daphnid study. Thus, the principal finding obtained from an algal study
is the 120-hour EC™ or SC2Q, from the fish study the 96-hour LC5Q and
from the daphnid study the 48-hour EC5Q. Table 4.1 describes the biologi-
cal characteristics of the aquatic bioassays.
The suggested test organisms in marine tests are the algae Skeletonema,
costatum, the juvenile sheepshead minnow Cyprinodon variegatus, and the
adult mysid Mysidopsis bahia. The primary parameters of toxicity obtained
from a marine algal study are the 96-hour EC5Q, or SC20, from the marine
fish study the 96-hour LCrQ and from the mysTa study the 96-hour EC,,,.
The characteristics of the marine aquatic tests are also described Tn
Table 4.1.
The aquatic tests described in this section are well suited for Level 1
environmental assessment testing because they develop useful information
quickly and at low cost. The six recommended aquatic bioassays have
been routinely used by EPA and others to monitor the biological impact
of effluents on the environment. There already exists a body of published
material and technical expertise which can assist in the application and
interpretation of Level 1 aquatic testing. These tests measure the effect
of a test material on organisms that represent three successively higher
trophic levels characteristic of either fresh or marine waters. Selection
of the freshwater or marine battery of tests is made based on the type
of receiving water into which the effluent is discharged. Their principal
limitations are (1) that they usually do not closely simulate the char-
acteristics of the receiving waters into which the test effluent is
actually being discharged, and (2) that the species tested may not be
representative of the most sensitive species native to those waters.
They do, however, make it possible to rank municipal and/or industrial
effluents in order of relative toxicity.
The procedures for the aquatic ecological assays have been developed
largely from References 21 and 22. Modifications to the original proto-
cols have been made where necessary to adapt tests to the requirements
of Level 1 environmental assessment.
4.1.1. General Materials and Methods For Aquatic Ecological.Assays
Materials and methods that are common to all, or nearly all, Level 1
aquatic ecological tests are presented in this section. The section for
each specific test discusses materials and methods unique for that test
and identifies which of the general materials and methods are applicable.
54
-------
TABLE 4.1
CHARACTERISTICS OF LEVEL 1 AQUATIC ECOLOGICAL EFFECTS BIOASSAYS
Characteristic
Static Acute
Aquatic Bioassay
Algal Bioassay
Freshwater Species
Marine Species
End Point(s) Measured
Amenable to Sample Types
Data Expression
Special Features
Fish - Fathead Minnow,
Invertebrate - Daphnia
Fish - Sheepshead Minnow,
Invertebrate - Mysidopsis
Lethality
Liquids, Solids (leachates)
LC50
Can detect whole-animal
effects on key aquatic
ecological consumers
Selenastrum
Skeletonema
Cell Population Growth
Liquids, Solids (Leachates)
EC50' SC20
Effective measure of
toxicity to aquatic
producers
Facilities. The facilities should include tanks equipped for temperature
control and aeration for holding and acclimating test organisms, as well
as a constant temperature area or recirculating water bath for the test
vessels. If the use of reconstituted dilution water is necessary, there
should be a tank for its preparation. If air is used for aeration, it
must be free of oil and fumes. The test facility must be well ventilated
and free of fumes. Illumination should be provided of an intensity and
duration that is specified in the Materials and Methods section for each
test.
Construction materials. Materials that come in contact with effluent
samples, stock solutions or test solutions should minimize sorption of
any constituents of the test material and not contain .any substances -
that can be leached or dissolved by the water. Glass, #316 stainless
steel, and perfluorocarbon plastics must be used whenever possible to
minimize leaching, dissolution and sorption. Unplasticized plastics may
be used for holding and acclimation tanks and in the water supply system.
Rubber, copper, brass, and lead should be avoided. If stainless steel
is used it must be welded, never soldered. Silicone adhesive used to
cement glass containers sorbs some organochlorine and organophosphorus
55
-------
compounds which are difficult to remove; therefore, as little adhesive
as possible should be in contact with test-material solutions and extra
beads of adhesive should be on the outside, not the inside, of the con-
tainers.
Test containers. Fish tests should be conducted in 19.6-liter wide-mouth
soft-glass jars or in all-glass containers 30 cm wide, 60 cm long and
30 cm high. Oaphnids should be exposed in 3.9-liter wide-mouth soft-glass
bottles, in 3.3-liter battery jars or in 250-milliliter beakers. Mysid
tests are conducted in 2-liter culture dishes containing 1 liter of test
medium. Freshwater and marine algal tests should be conducted in Erlen-
meyer culture flasks of Pyrex or Kimax type of glass. The flask size is
not critical, but because of C02 limitations, the volume-to-volume ratio
is. The recommended contents-to-fTask-volume ratios for hand-shaken
flasks are:
25 ml in 125 ml flask
50 ml in 250 ml flask
100 ml in 500 ml flask
Maximum permissible contents-to-volume ratios in continously shaken flasks
should not exceed 50 percent.
Cleaning and preparation of glassware.• Each container for fish or macro-
invertebrate testing must be cleaned before use. A new container must
be (1) washed with non-phosphate detergent, (2) rinsed with 100 percent
acetone, (3) rinsed with water, (4) rinsed with 5 percent nitric acid
and (5) rinsed thoroughly with tap or other clean water. After testing,
each container should be (1) emptied, (2) rinsed with water, (3) cleaned
with a material suitable for removing the toxicant tested (such as acid
to remove metals and bases and solvent to remove organic compounds) and
(4) rinsed thoroughly with water. Dilute acid is also used to remove
mineral deposits. Containers should be disinfected for one hour with an
iodophor, 200 mg hypochlorite per liter or a quaternary ammonium salt
such as 800 ppm Roccal II* with at least one thorough scrubbing during
the hour, then rinsed thoroughly. For safety, acid and hypochlorite
should not be used together.
All glassware used in freshwater or marine algal testing is prepared as
above with the following exception. The final rinse should be of deionized
water filtered through a 0.22 urn membrane filter if the Coulter Counter
is to be used in freshwater algal assays. Flasks are dried in an oven
at 50° to 70°C. Foam plugs are inserted and the glassware is autoclaved
for 20 minutes at 1.1 kg/cm2 (15 psi) and 121°C. Cooled flasks are stored
in closed cabinets.
Receipt and quarantine for fish. Stock fish shipped from outside sources
may have been subjected to changes in temperature, dissolved oxygen and
pH, handling disturbances and other stresses, and should be examined
carefully for health and vigor. Holding water should be introduced gradu-
ally into the shipping bags, and fish observed for abnormal behavior.
"National Laboratories, Montvale, NJ 07645
56
-------
When the difference in water temperature between the bag and holding
tank is 2°C or less, fish from one bag should be introduced into the
tank and observed for acute stress. If acute stress is not seen, the
remaining fish may be introduced into the tank in a similar manner.
To prevent spread of disease, incoming fish for stock should be quaran-
tined for at least two weeks and observed for abnormal behavior and para-
sites. The quarantine tanks should be prepared in advance by thorough
scrubbing and cleaning with an industrial cleaner, rinsing with water,
sterilizing with a quaternary ammonium salt such as 800 ppm Roccal II,
and rinsing with at least three changes of water before filling with
dilution water. If after two-weeks' quarantine they show no signs of
infection or abnormal behavior they are transferred to stock-holding
tanks, otherwise, they are either discarded or treated as described in
Disease treatment for fish.
To prevent initiation and spread of disease, nets, buckets, fish graders
and hands should be routinely disinfected with 200 ppm Roccal II before
being placed in the water.
Disease treatment for fish. Freshwater fish may be chemically treated
to cure or prevent diseases by using the treatments recommended in
Table 4.2. Some of the treatments (formalin and potassium permanganate)
listed in Table 4.2 may also be used to cure or prevent diseases in marine
fishes. However, if a group of either type of fish is severely diseased,
the entire lot should be destroyed. Generally, the fish should not be
treated during the first 16 hours after arrival at the facility because
they may be stressed because of collection or transporation and some may
have been treated just prior to transit. Tests must not begin with treated
fish for at least four days after treatment. Tanks and test chambers
which'may be contaminated with undesirable microorganisms should be dis-
infected following the procedures outlined in Cleaning, in this section.
Test material. The test material may be a solid, aqueous liquid, or
nonaqueous liquid. Quantity of sample required, is listed in Table 4.3.
Samples are usually tested directly without preparation, however, some
test materials require pretest preparation. Table 2.4 lists pretest
preparation requirements for individual sample types and Section 2.3
details specific procedures. Aqueous effluents and aqueous leachates of
solid samples should be run directly and must not be aerated or altered
in any way, except that aqueous effluents may be filtered through a sieve
or screen with holes 2 mm or larger to remove large particles. Nonaqueous
samples are diluted in the appropriate solvent with uniform aliqupts :.,
added to each test container or are added directly by volume and diluted
with dilution water. If possible, samples should be solvent Exchanged
to a solvent compatible with the test organisms. The maximum applicable
dose is determined uniquely for each nonaqueous liquid sample based upon
the solubility of the sample in water and the toxicity of solvents used
to test organisms. Samples must be covered at all times and violent
agitation must be avoided. Undissolved materials must be uniformly dis-
persed by gentle agitation immediately before a portion of the sample is
removed for use. In handling samples containing highly volatile sub-
stances, it may be desirable to add the test sample below the surface of
the dilution water.
57
-------
TABLE 4.2 RECOMMENDED PROPHYLACTIC AND
THERAPEUTIC TREATMENTS FOR FRESHWATER FISHC
Disease
Chemical
Cone. , mg/1
Application
External
bacteria
Monogenetic
trematodes ,
fungi and
external .
protozoa
Parasitic
copepods
Benzalkonium chloride
(Hyamine 1622®)
Nitrofurazone (water mix)
Neomycin sulfate
Oxytetracycline hydrochloride
(water soluble)
Formalin plus zinc-frse
malachite green oxalate
Formalin
Potassium permanganate
Sodium chloride
Dexon® (35% AI)
Trichlorfon
(Masolen®)
1-2 AIb
3-5 AI
25
25 AI
25
0.1
150-250
2-6
15,000-30,000
2000-4000
20
0.25 AI
30-60 minc
£
30-60 min^
30-60 min
30-60 minc
1-2 hours0
30-60 min°
30-60 min
5-10 min dip
c,e
30-60 min
f
These recommendations do not imply that these treatments have been cleared
or registered for these uses. Appropriate State and Federal regulatory
agencies should be consulted to determine if the treatment in question can
be used and under what conditions the uses are permitted. These treatments
should be used only on fish intended for research. They have been found
dependable, but efficacy against diseases and toxicity to fish may be altered
by temperature or water quality. Researchers are cautioned to test treatments
on small lots of fish before making large-scale applications. Prevention
of disease is preferred, and newly acquired fish should be treated with the
formalin-malachite green combination on three alternate days if possible.
However, in general, fish should not be treated on the first day they are
in the facility. This table is merely an attempt to indicate the order of
preference of treatments that have been reported to be effective. Before a
treatment is used, additional information should be obtained from such sources
as Davis (23), Hoffman and Meyer (24), Reichenbach-Klinke and Elkan (25),
. Snieszko (26) and van Duijn (27).
AI - active ingredient.
Treatment may be accomplished by (1) transferring the fish to a static
treatment tank and back to a holding tank; (2) temporarily stopping the
flow in a flow-through system, treating the fish in a static manner, and
resuming the flow to flush out the chemical or (3) continuously adding a
stock solution of the chemical to a flow-through system by means of a metered
.flow or the technique of Mount and Brungs (28).
One treatment is usually sufficient except for "Ich", which must be treated
daily or every other day until no sign of the protozoan remains. This may
take 4 to 5 weeks at 5 to 10°C and 11 to 13 days at 15 to 21°C. A tempera-
gture of 32°C is lethal to Ich in 1 week.
^Minimum of 24 hours, but may be continued indefinitely.
Continuous treatment should be employed in static or flow-through systems
until no copepods remain, except that treatment should not be continued for
over 4 weeks and should not be used above 27°C.
58
-------
TABLE 4.3 SAMPLE SIZE REQUIREMENTS
FOR AQUATIC ECOLOGICAL ASSAYS
Liquid
Type of Test
Freshwater Fish
Freshwater Invertebrate
Freshwater Algae
Marine Fish
Marine Invertebrate
Marine Algae
Solid
(kilograms)
10 (7.5)b
0.5 (0.3)
0.25 (0.13) '<
10 (7.5)
2 (1.2)
0.25 (0.13)
Aqueous
(liters)
40 (30)
2 (1.5)
1 (0.6)
40 (30)
8 (6)
1 (0.6)
Nonaqueous"
(milliliters)
1000 (750)c
200 (150)
100 (60)
1000 (750)
800 (600)
100 (60)
Nonaqueous liquids include aqueous samples with greater than 0.2% organics,
nonaqueous liquids, solvent exchange samples, and extracts or leachates in
a nonaqueous (organic) vehicle.
The first value given is the requested sample size for routine Level 1
testing. The value in parentheses is the minimum feasable sample size
• to conduct the test. . .
The maximum applicable dose (MAD) and tha volume of nonaqueous liquid
samples required for aquatic ecological testing is dependent upon the
solubility of the sample in water. The MAD is determined for each sample
before testing is initiated. For additional information, contact the
Technical Support Staff, Process Measurements Branch, IERL-RTP, U.S. EPA,
Research Triangle Park, NC 27711.
If testing is to be done on-site, the tests should begin within eight
hours of collection. If testing is to be done at a laboratory, the samples
should be placed on ice for preservation during the transportation and
testing performed as soon as possible after laboratory receipt of the
samples. Samples should be stored at 4°C if testing is not initiated
upon sample receipt. The temperature of the sample should be adjusted
to that of the test (± 2°C) before portions are added to the dilution
water. Solid materials may be added directly to dilution water.
Dissolved oxygen concentration. Aeration of test solutions during the
test should be avoided to minimize loss of highly volatile materials.
It should be noted in the final report if the dissolved oxygen concentra-
tion is less than 40 percent saturation in any test chamber for freshwater
fish or Daphnia tests, or less than 60 percent for marine fish or mysid
tests. Neither freshwater nor marine algal tests have defined dissolved
oxygen concentration standards.
59
-------
4.2 STATIC ACUTE TOXICITY TESTS WITH FRESHWATER FISH AND DAPHNIA
4.2.1 Introduction and Rationale
The static toxicity tests with freshwater fish and Daphnia provide a
large amount of data in a short period of time. The recommended test
organisms are the juvenile fathead minnow, Pimephales promelas, and early
instars of Daphnia magna. The static acute exposure period for the fat-
head minnow is 96 hours and 48 hours for the daphnid study. The 96-hour
mean lethal concentration (96-hour LCc0) is calculated for the fathead
minnow. Because death is not always easily determined in Daphnia, the
48-hour effective concentration (48-hour EC5Q) is calculated for Daphnia.
4.2.2 Materials and Methods j
General procedures listed for all aquatic tests in section 4.1.1 are
applicable to the static acute toxicity tests with freshwater fish and
Daphnia. Specific areas discussed in Section 4.1.1 that should receive
careful attention are: facilities, construction materials, test con-
tainers, cleaning and preparation of glassware, receipt and quarantine
for fish, disease treatment for fish, test material and dissolved oxygen
concentration. Materials and methods unique to freshwater fish and
Daphnia tests are included below.
Dilution water. A minimal criterion for acceptable dilution water is
that healthy organisms will survive in it for the duration of acclima-
tion and testing without showing signs of stress such as discoloration
or unusual behavior. Water in which daphnids will survive and reproduce
satisfactorily should be an acceptable dilution water for tests with
freshwater organisms.
The dilution water should be of constant quality and analyzed by the
methods given in References 29, 30, 31, and 32 to ascertain that none of
the following substances exceeds the maximum allowable concentration
shown:
Maximum
Pollutants Concentration
Suspended solids 20 mg/1
Total organic carbon 10 mg/1
Un-ionized ammonia 20 ug/1
Residual chlorine 3 ug/1
Total organophosphorus pesticides 50 ng/1
Total organochlorine pesticides plus PCB's 50 ng/1
The dilution water is considered to be of constant quality if the monthly
ranges of hardness, alkalinity and conductivity are within 10 percent of
their respective means and if the monthly range of pH is less than
0.4 units. Reconstituted dilution water may be prepared according to
the method of Marking and Dawson (33). For comparability of results
between tests, hardness should be as close as possible to 100 mg/1 as
CaC03.
60
-------
4.2.3 Test Organisms
Species. The juvenile fathead minnow, Pimephales promelas, and early
instars of Daphm'a magna are the species to be used in Level 1 freshwater
static acute toxicity tests. The fathead minnow is a warm-water fish of
ponds, lakes and sluggish streams. Daphnids occur in nearly all types
of freshwater habitats. Both species,, because of their wide geographic
distribution, important places in the aquatic food web, temperature
requirements, wide pH tolerance, ready availability and ease of culture,
have been recommended as bioassay organisms by the Committee on Methods
for Toxicity Tests with Aquatic Organisms (21).
Source. Fathead minnows may be obtained from private, State, or Federal
fish hatcheries, or captured from wild populations in relatively unpolluted
areas. However, collecting permits may be required by local and State
agencies. Fish collected by electroshocking should not be used. Daphm'a
should be reared in the testing facility from laboratory cultures.
Sizes, life stages. Fathead minnows used in testing should weigh between
0.5 and 1.0 g each. All fish in each test should be from the same year
class, and the standard length (tip of snout to end of caudal peduncle)
of the longest fish should be no more than twice that of the shortest
fish. Weights and lengths should be determined by measuring representative
specimens before the test or control fish after the test. Very young
fish (not yet actively feeding), spawning fish and spent fish should not
be used.
Daphm'a magna used in testing should be in the early instar stages of
their life cycle. All organisms in a test must be from the same source
and as healthy and uniform in size and age as possible.
Culturing, care and handling. Fathead minnows are maintained at 20-22°C
in a flow-through system with a turnover of at least two volumes daily,
or in a recirculating system in which the water is passed through a carbon
filter and an untraviolet sterilizer.
Daphm'a magna are maintained in a static system at 19-22°C. Tanks must
be siphoned periodically to remove debris and water added as necessary
to maintain volume. Cultures must be maintained under optimum conditions
at all times to prevent formation of ephippial eggs; daphnids from cultures
in which ephippia are being produced must not be used in testing.
Both species should be fed at least once a day, at which time careful
observations should also be made for mortality and for signs of disease,
stress and injury. Fish are fed a commercial fish food such as Purina
Trout Chow*. Daphnia are fed 1.25 mg (dry weight) of a mixed freshwater
algal culture per liter of water daily. Dead and abnormal fish or Daphnia
should be removed as soon as they are observed.
"Ralson Purina Co., St. Louis, MO 63188.
61
-------
Water quality should be held constant as described earlier and tempera-
ture changes should not exceed 3°C in any 12-hour period. Fish tanks
should be scrubbed at least twice a week.
The organisms should be handled as little as possible. When handling is
necessary, it should be done as gently, carefully and quickly as possible
so that the organisms are not needlessly stressed. Small dip nets are
best for handling fish and wide-bore pipettes for Daphm'a. Organisms
that touch dry surfaces or are dropped or injured during handling should
be discarded.
From the time test organisms are first cultured or received they should
be shielded from disturbances; overcrowding should be avoided.
Holding and acclimation. After collection or transportation, the fish
or Daphnia should be held in and acclimated to the dilution water for at
least two days before beginning a test under the same holding conditions
as described earlier in Care and handling.
A group of animals must not be used for a test if individuals appear to
be diseased or otherwise stressed or if more than 5 percent die within
48 hours prior to beginning the test. If a group fails to meet these
criteria, it must be discarded or treated and held an additional 4 days.
Fathead minnows should not be fed for 48 hours prior to the beginning of
a test. However, the Daphnia may be fed up to the beginning of the test.
4.2.4 Experimental Design
Test procedure. Unless the approximate toxicity of the sample is-already
known, at least six concentrations of test material should be prepared.
The highest dose should be at the maximum applicable dose (MAD) for that
sample type (Table 4.11) unless physical characteristics of the sample
or other previously gathered toxicity data contravene this. The concen-
trations should be in a geometric series; each one should be at least
50 percent of the next higher one.
In fathead minnow tests, at least 10 fish must be exposed to each test
concentration per replicate with two replicates per concentration used
in the test. For Daphnia magna tests, 10 organisms per replicate with
three replicates per concentration should be used. The use of more organ-
isms and replicate test containers and random assignments of test organisms
to containers is desirable.
The fathead minnow tests should be conducted at 22 ± 2°C, and those with
Daphnia at 19 ± 2°C. A photo period of 16 hours light and 8 hours dark
is used for both tests. Neither type of test animal should be fed during
exposure. The test conditions are summarized in Table 4.4.
62
-------
TABLE 4.4 SUMMARY OF TEST CONDITIONS,
FRESHWATER FISH OR MACROINVERTEBRATE TEST
Fathead Minnow,
Pimephales promelas
Daphnia magna
Temperature, °C
Photoperiod, hours
light:dark
Water quality, hardness
mg/1 as CaC03
Container size
Test volume
Organisms per container
Replicates
Feed
Duration, hours
Measurements of D.O.
and pH, hours
22 ± 2
16:8
100
19.6 liters
15 liters
10
2
No
96
0, 24, 48, 72, 96
19 ± 2
16:8
100
250 ml
200 ml
10
3
No
48
0, 48
Each test requires a control which consists of the same dilution water,
conditions, procedures and organisms as used in the test concentrations.
If any solvent other than water is present in the test concentration, a
solvent control is also required. This solvent control is treated the
same as the control except that the amount of solvent used in dosing the
test containers is added to this solvent control.
In the fathead minnow test there should be 15 liters of test solution or
control water in each 19.6-liter jar. If 30 x 30 x 60 centimeter con-
tainers are used, the solution should be between 15 and 20 centimeters
deep.
In the daphnid test there should be 2 to 3 liters of solution or control
water in each 3.9-liter wide-mouth bottle or 3.3-liter battery jar, or
200 milliliters in each 250-milliliter beaker.
Test organisms should be placed in the test and control vessels not more
than 30 minutes after the test solutions are prepared. Ten fish in each
vessel and 10 daphnids in each replicate are recommended. Chemical,
physical and biological data are taken and recorded as described in
63
-------
Section 4.2.5 for the duration of the test (96 hours for the fathead
minnow, 48 hours for the daphnid test.
If no toxicity is detected at any concentration and the MAD dose was
tested, then no further testing is required. The test material may be
reported as having no detectable toxicity. Test materials that kill or
immobilize all or nearly all the test organisms should be retested at a
lower dose range.
Biological loading. The biological loading in each test and control
vessel should not exceed 0.8 g of test organism per liter or be so high
as to (1) reduce dissolved oxygen concentration below 40 percent saturation,
(2) raise the concentration of metabolic products above acceptable levels
or (3) stress the organisms by overcrowding, any of which may invalidate
the test results.
4.2.5 Results and Data Interpretation
Chemical and physical data. In the fathead minnow test, dissolved oxygen
concentration, and pH should be measured at the beginning of the test
and every 24 hours thereafter in the controls and in the high, medium
and low concentrations. Conductivity should be measured at the beginning
of the test in the control and each test concentration. Temperature of
the water bath or controlled-temperature area should be recorded continu-
ously or every 24 hours.
In the Daphnia test, dissolved oxygen, pH, and conductivity (when required)
on the high, medium and low concentrations, and temperature should be
recorded initially and at 48 hours.
Concentration of un-ionized ammonia, if required, can be obtained by
measuring total ammonia and consulting Reference 30.
Biological data. Mortality is the effect most often used to define acute
toxicity to aquatic organisms. Criteria for death are usually lack of
movement, especially of gill movement in fish, and lack of reaction to
gentle prodding.
Because death is not always easily determined with some invertebrates,
an ECcQ may be calculated rather than an LC5Q. The principal criterion
for effect on Daphnia is immobilization, defined as lack of movement
except for minor activity of appendages.
Mortality or immobilization and abnormal behavior should be recorded
every 24 hours for the duration of the test. Observations of test
materials which produce harmful effects i_n vivo, but do not result in
deaths, are difficult to quantitate. Such observations provide insight
into the sublethal effects of a sample on aquatic organisms and may be
used to recommend further investigation of the test material. Dead or
immobilized organisms should be removed as soon as they are observed.
Table 4.5 lists definitions of fish behavior terms, and suggested code
for recording and reporting. If more than 10 percent of test organisms
in any control die or are immobilized, the entire test is unacceptable.
64
-------
TABLE 4.5 DEFINITION OF FISH BEHAVIOR TERMS
Code Term Definition
]~General Behavior!Observable responses, of the test fish, individually or in
groups, to the range of factors constituting their environment.
a. Quiescent: marked by a state of inactivity or abnormally low activity; motion-
less or nearly so.
b. Hyperexcitable: reacting to stimuli with substantially greater intensity than
control fish.
c. Irritated: exhibiting more or less continuous hyperactivity.
d. Surfacing: rising and remaining unusually long at the surface.
e. Sounding: diving suddenly straight to the bottom; remaining unusually long at
the bottom.
f. Twitching: moving the body or parts of the body with sudden jerky movements.
g. Tetanous: in a state of tetany; marked by intermittent tonic spasms of the
voluntary muscles.
h. Flaccid: lacking tone, resilience or firmness; weak and enfeebled; flabby.
i. Normal: unaffected by or not exposed to a particular experimental treatment;
conforming to the usual behavioral characteristics of the species.
2. Swimming. Progressive self-propulsion in water by coordinated movement of
tail, body, fins.
a. Ceased: Broken off or tapered off to a stop.
b. Erratic: Characterized by lack of consistency, regularity or uniformity;
fluctuating, uneven; eccentric.
c. Gyrating: Revolving around a central point; moving spirally about an axis.
d. Skittering: skimming hurriedly along the surface with rapid body movements.
e. Inverted: turned upside down, or approximately so.
f. On side: turned 90° laterally, more or less, from the normal body orientation.
3. Pigmentation. Color .of skin due to deposition or distribution of pigment.
a. Light discolored: color appearance lighter than usual for the species.
b. Dark discolored: color appearance darker than usual for the species.
c. Varidiscolored: color appearance abnormally varied; mottled.
4. Integument. The skin.
a. Mucus shedding: observably losing mucous skin coating to an abnormal degree.
b. Mucus coagulation: showing observable clumping or clotting of the mucous skin
coating, especially at the gills.
c. Hemorrhagic: visibly bleeding as from gills, eyes, anal opening.
5. Respiration. Physical action of pumping water into mouth and out through gills,
so as to absorb oxygen.
a. Rapid: observably faster than normal to a significant degree..
b. Slow: observably slower than normal to a significant degree. .
c. Irregular: failing to occur at regular or normal intervals.
d. Ceased: broken off or tapered off to a stop.
e. Gulping air: swimming at surface with mouth open and laboriously pumping
surface water and air through gills.
f. Labored: performed with apparently abnormally great difficulty and effort.
No Observed Effect Concentration: The highest test concentration in which fish expe-
rience no mortality and exhibit no observable behavioral abnormalities at any time
during a specified period of exposure to the test material. Ordinarily determined for
periods from the start of testing to the end of each successive 24 hours.
65
-------
Laboratory data forms. Forms for recording chemical, physical and bio-
logical data in both fathead minnow and Daphm'a tests are illustrated in
Quality Control documents (9).
Calculations. The concentration of test material lethal to 50 percent
of the population (LC,-n) and 95 percent confidence limits should be deter-
mined at 24-, 48-, 72-, and 96-hour exposures for fish tests, and the
EC50 and 95 percent confidence limits at 24- and 48-hour exposures for
Dapnnia magna tests. Any of several methods including moving average,
Spearman Karber, Litchfield-Wilcoxin, probit or binomial may be used.
For a discussion these methods, refer to the review article by Stephan
(34). The results (96 hours for fish and 48 hours for Daphnia) are
evaluated according to Table 4.11 which defines the toxicity categories.
4.3 FRESHWATER ALGAE 120-HOUR SCREENING TEST
4.3.1 Introduction and Rationale
Unicellular algae are important producers of oxygen and form the basis
of the food web in aquatic ecosystems. Since algal species and communities
are sensitive to environmental changes, growth may be inhibited or stimu-
lated by the presence of pollutants. Therefore, the response of algae
must be considered when assessing the potential ecological effects of
.industrial or municipal discharges on aquatic ecosystems.
A simple screening test for toxicity to algae can be conducted in
120 hours. Algae are exposed to various concentrations of the test
material; growth is measured at 120 hours. Results are expressed in
terms of the EC™ (the lowest test concentration causing inhibition of
growth by 50 percent relative to the control) and the no observed effect
concentration (NOEC, the highest test concentration in which growth is
not significantly different from that in the control). Stimulatory
effects, if any, should be noted and expressed mathematically in terms
of SC-n and used for estimation of bioactivity of effluent.
4.3.2 Materials and Methods
General procedures listed for all aquatic tests in Section 4.1.1 are
applicable to the static acute toxicity test with freshwater algae.
Specific areas discussed in Section 4.1.1. that should receive careful
attention are facilities, construction materials, test containers, cleaning
and preparation of glassware, and test material.
Materials and methods unique to freshwater algal tests follow.
Equipment. Equipment should include a constant-temperature room or
incubator capable of providing temperature control of 24 ± 2°C. A gyro-
tary shaking apparatus capable of 100 oscillations per minute should be
available for test culture flasks. Continuous illumination of 4300 ±
650 lumens/ m2 (400 ft-c) is required for freshwater green algae. Over-
head cool-white fluorescent bulbs should be used. Light intensity is
66
-------
measured adjacent to the flask at liquid level using a light meter capable
of being calibrated against National Bureau of Standards lamps.
Culture containers for this and other aquatic tests are discussed in
Section 4.1.1. Erlenmeyer culture flasks of-Pyrex or Kimax glass are
used in either 125-, 250-, or 500-ml sizes. The recommended contents-to-
volume ratio in hand-shaken flasks is 1 to 5 and should not exceed 50
percent for continuously shaken flasks (35). Flask closures must permit
gas exchange and prevent contamination. Foam plugs* are suggested.
Since some brands may be toxic, each laboratory must determine for each
type of closures purchased whether or not there are any significant effects
on algal growth.
-';
Support equipment includes an autoclave or pressure cooker capable of
producing 1.1 kg/cm2 (15 psi) at 121°C and a dry-heat oven capable of a
temperature of 120 ± 1°C. A Coulter Counter with a mean-cell-volume
computer (MCV/MHR) or high-quality microscope is needed for biomass
measurements.
Freshwater Algal Nutrient Medium. Algal Assay Medium (AAM) is prepared
in the order listed in Table 4.6 by adding 1.0 ml of each of the macro-
nutrient stock solutions, to 900-ml deionized water, with mixing after
each addition. Then 1.0 ml of the micronutrient stock is added and the
final volume brought to 1 liter with deionized water. The mixture is
filter-sterilized by passing through a 0.22 |jm porosity membrane filter
(pre-rinsed with 100-ml deionized water) into a sterile container.
Medium is stored in the dark at 4°C to reduce possible photochemical
changes and bacterial growth. <•
4.3.3. Test Organisms
For freshwater algal assays, the recommended test organism is Selenastrum
capricornutum, a unicellular non-motile chlorophyte that is easily main-
tained in laboratory cultures.t
Stock cultures of algae should be maintained at 24°C ± 2°C under continuous
illumination in AAM. It is recommended that several cultures be maintained
on agar under axenic conditions. Transfers of stock cultures should be
made every six to eight days to provide cultures with a sufficient number
of cells growing exponentially for test inoculations. All transfers
must follow standard microbiological techniques to ensure a minimum of
contamination. Prior to establishing an algal culture, the culture techni-
ques of Miller et aj., should be reviewed (35).
xFor example, Gaymar Industries, Inc., Orchard Park, NY 14127.
tCultures can be obtained from Joseph C. Green, Environmental Reseach
Laboratory, U.S. EPA, Corvallis, Oregon 97330.
67
-------
TABLE 4.6 COMPOSITION OF ALGAL ASSAY MEDIUM (AAM)
Macronutrients
Stock Solutions
Nutrient Composition
Prepared Medium
Compound
NaN03
NaHC03
,
K2HP04
MgS04-7H20
MgCl2-6H20
CaC12-2H20
Concentration
(g/D
25.500
15.000
1.044
14.700
12.164
4.410
Element
N
Na
C
K
P
S
Mg
Ca
Concentration
(mg/D
4.200
11.001
2.143
0.469
0.186
1.911
2.904
1.202
Micronutrients
Stock
Compound
H2B03
MnCl2-4H20
ZnCl2
CoC12-6H20
CuCl2-2H20
Na2Mo04-2H20
FeCl3-6H20
Na2EDTA-2H20
Solutions
Concentration
(mg/D
185.520
415.610
3.271
1.428
0.012
7.260
160.000
222.000
Nutrient
Composition
Prepared Medium
Element
B
Mn
Zn
Co
Cu
Mo
Fe
"
Concentration
(ug/1)
32.460
115.374
1.570
0.354
0.004
2.878
33.051
• «
Other forms of the salts may be used as long as the resulting
concentrations of elements are the same.
68
-------
4.3.4.
Experimental Design
Preparation of Toxicant. Depending on its nature, the test material is
prepared by one of two methods. In the first method, solids or non-aqueous
liquid materials may be added directly by weight or volume respectively
to the algal medium or a concentrated stock solution may be prepared in
deionized water (or a solvent such as ethanol, acetone, dimethylforamide
or triethyleneglycol) and equal volume aliquots of a small size are added
to each treatment. If it is not possible to prepare a homogenous solution
of the toxicant, it must be added directly into each replicate flask.
The second method, for aqueous effluents or aqueous leachates of solids,
allows testing by percent volume (volume/volume). Nutrients are added
to one liter of effluent in the same quantities as in the control algal
assay medium.. The effluent is used up to 80 percent volume-per-volume
in the.test. Additional test concentrations are prepared on a volume-
percent basis by mixing appropriate volumes of effluent with control
medium.
Test Procedure. Six test concentrations and a negative control are
normally tested, with four replicates of each. Three replicates are
inoculated with algae while the fourth serves as a blank. Three repli-
cates are necessary for statistical analyses and the blank is necessary
to correct biomass measurements for particulates which may be present in
the test treatments.
If a solvent is necessary for the preparation of the test material, a
solvent control must be included. All flasks and the solvent control
must contain the same amount of solvent. The toxicity of commonly used
solvents should be determined in each laboratory using this bioassay to
help select suitable solvent levels.
1. Inoculum. A 6- to 8-day-old stock culture is used as the inoculum
source. Population density in the stock culture is determined by direct
counting or spectrophotometry. A volume of inoculum calculated to yield
an initial concentration of 10,000 cells/ml is added aseptically to each
flask. The volume of inoculum added should be between 0.1 and 1.0 ml.
2. Incubation. Incubation conditions for the test alga are given in
Table 4.7. Test flasks are incubated for 120 hours.
TABLE 4.7 INCUBATION CONDITIONS FOR FRESHWATER ALGAL ASSAY ORGANISMS . .
Species
Light
Intensity,
lumens/nr
Photoperiod
Hours of
Light: Dark
Shaking Speed,
Oscillations
Per Minute
Temperature, °C
Selenastrum
capricornutum 4300 ± 650
24:0
100
24 ± 2
69
-------
3. Response monitoring. After 120 hours of exposure, algal growth is
measured by either of the following methods: (a) electronic particle
counting or (b) direct counting. Cursory microscopic observation is
desirable to reveal any abnormalities in cell shape or condition.
a) Electronic particle counting. A Model ZBI Coulter electronic particle
counter with mean cell volume computer (MCV/MHR) is used. A C-1000 Chan-
nelyzer may be used rather than the MCV/MHR, but it is neither preferred
nor recommended. The MHR Computer must be calibrated with the Organic
Calibration material; biomass may be determined indirectly by the following
equation:
S. capricornutum mg dry weight I-1 = [(A) - (P)] x MCV (urn3) x F
where A is the algal cell counts ml-1, P is the particulate counts of
the blank ml-1, MCV is the mean cell volume and F is the correlation co-
efficient to be determined by each laboratory.
If there are particles in the test material, it is usually possible to
eliminate counts contributed by the particles from the total counts. If
the particles are in the same size range as the algal cells, the blank
flasks are counted and these counts subtracted from the total counts.
The advantage of this method is that it allows for determination of bio-
mass produced in addition to cell numbers.
b) Direct counting. A hemacytometer counting chamber and a microscope
are used.Two samples are taken from each flask, and two counts are
made of each sample. Whenever feasible, 400 cells per replicate are
counted in order to obtain ± 10 percent accuracy at the 95 percent confi-
dence level. This method permits visual inspection of the condition of
algal cells and discrimination between algal cells and debris or parti-
culates in the test material.
4.3.5 Results and Data Interpretation
Calculations. Percent inhibition (I), or stimulation (S), is calculated
for each concentration according to the following formula:
Percent Response = T - C ,, ,g0 Positive response - Stimulation
C - IN Negative response = Inhibition
where C is the mean growth in the control (mg I-1), T is the mean growth
in the treated culture (mg I-1) and IN is the dry weight of inoculum used
(mg I-1).
Different endpoints may be calculated from the percent response vs. con-
centration data. For samples which are inhibitory, an an EC™ (defined
as the lowest test concentration causing growth inhibition of 50 percent
relative control) is calculated. For samples which are stimulatory, an
SCpQ (defined as the lowest concentration causing growth stimulation of
20 percent relative to control) is calculated.
70
-------
A linear regression analysis is performed on the test results. The X-axis
should be percent effluent and the y-axis should be percent effect on
growth. Results of linear regression analysis are used to calculate the
concentration of effluent causing any effect on growth inhibition or
stimulation) by the following formula.
T = ±E - t c
s
T = Concentration of test material.
s = Slope of regression analysis
c = Constant from regression analysis.
E = Endpoint tested (i.e., EC5Q, SC2Q).
A" predicted NOEC, defined as the highest test concentration in which
growth was not significantly different from that in the control, may
also be calculated. The methods of Dunnett (36,37) or Williams (38,39)
are recommended for determining the NOEC.
The 120-hour EC™ results are evaluated according to criteria defined in
Table 4.11 which will permit the test material to be ranked by toxicity
category.
4.4 STATIC ACUTE TOXICITY TESTS WITH MARINE FISH AND MYSIDS
4.4.1 Introduction and Rationale
The methods recommended for static acute toxicity tests on marine
fish and mysids provide a large volume of data in a short period of time.
Principles of Level 1 testing with marine organisms are similar to testing
with freshwater organisms, as described in Section 4.2.
The recommended test animals in marine tests are the juvenile sheepshead
minnow, Cyprinodon variegatus, and the adult mysid, Mysidopsis bahia.
The recommended tests for both species are static acute exposures that
allow calculation of the 96-hour LC50 for fish and the 96-hour ECcg for
mysids. Because death is not always easily determined with some inverte-
brates, an ECj-Q is calculated rather than LCcn for the mysid.
The following procedures have been adapted largely from References 21
and 40.
4.4.2 Materials and Methods
General procedures listed for all aquatic tests in Section 4.1.1 are
applicable to the static acute toxicity tests with marine fish and mysid.
Specific areas discussed in Section 4.1.1 that should receive careful
attention are: facilities, construction materials, test containers,
cleaning and preparation of glassware, receipt and quarantine for fish,
disease treatment for fish, test material and dissolved oxygen concentra-
tion. Materials and methods unique to marine fish and mysid tests are
as follows.
71
-------
Dilution water. Artificial sea salts* are used for preparation of marine
dilution water. Salts are added to glass-distilled or deionized water
to attain the appropriate salinity which is confirmed with a salinometer.
Mysid tests are conducted at a salinity of 22-26 parts-per-thousand (ppt)
and sheepshead minnow tests are conducted at a salinity of 10 parts-per-
thousand.
The dissolved oxygen concentration of dilution water should be between
90 and 100 percent saturation. Water that may be contaminated with un-
desirable microorganisms should be passed through an ultraviolet steril-
izer.
4.4.3. Test Organisms
Species. The species to be used for Level 1 marine tests are the juvenile
sheepshead minnow (Cyprinodon variegatus) and adult bay mysid (Mysidopsis
bahia).
Source. Mysids may be collected from wild populations in relatively
unpolluted areas, purchased from commercial suppliers, or cultured in
the laboratory according to the method of Nimmo et a]_. (41).
Juvenile sheepshead minnows may be cultured according to the method of ..
Schimmel et al_. (42) or purchased from commercial suppliers.
Culturing, care and handling. Methods for handling, rearing and static
testing of the mysid are given in Nimmo et al_. (41) and Borthwick (43).
Schimmel et a_L (42) describes a method for culturing sheepshead minnows.
These references should be consulted prior to establishing laboratory
culture systems for both mysid and sheepshead minnows.
During holding, acclimation and testing, the organisms must not be dis-
turbed unnecessarily, either by excessive handling or excessive movement
around the tanks. Handling should be done as gently, carefully and quickly
as possible.
Stock fish should be held at 20 ± 2°C. If they are collected at another
temperature, they should not be subjected to more than 2°C change in
temperature in any one-hour period or to more than a 5 ppt change in
salinity (the fishes will be raised at 10 ppt) in any 24-hour period.
Crowding during acclimation must be avoided. Commercial flake foodt
should be fed to the fish once a day. The fish should show no signs
of stress such as discoloration, altered behavior or disease and must
be kept for at least two days in acclimation tanks.
Holding temperature for mysids is between 22 and 25°C with a salinity
range of 22 to 26 ppt; they should be cultured and maintained within
these limits.
*For example, Rila Products, Teaneck, NJ 07666
tFor example, Longlife Aquarium Products, Harrison, NJ 07029.
72
-------
Mysids must be fed approximately two live 48-hour-old Artemia nauplii
per mysid each day. It is imperative to maintain a sufficient quantity
of food in the culture system at all times to prevent cannibalism.
A group of fish or .mysids must not be used for a test if individuals
appear to be diseased or otherwise stressed, or if more than five percent
die within the 48 hours immediately prior to the beginning of the test.
If a group fails to meet these criteria, all individuals must be discarded
or treated and held an additional four days. It may be more practical to
discard the entire group.
4.4.4 Experimental Design
Test Procedure. Marine aquatic ecological assays with fish and macro-
invertebrates are parallel in design to the corresponding freshwater
tests. Ten fish and 10 mysid per replicate are exposed to six concen-
trations of test material for 96 hours. There should be a minimum of
two replicates for fish and three replicates for mysid for each test
concentration or control. Use of more test organisms and replicate test
containers as well as random assignment of test organisms to containers
is desirable.
The highest dose should be at the maximum applicable dose (MAD) for that
sample type (see Table 4.11) unless physical characteristics of the sample,
sample size or previously gathered toxicity data contravenes this. The
concentrations should be in a geometric series, each one at least 50 per-
cent of the next higher.
The sheepshead minnow tests should be conducted at 20 ± 2°C and those
with mysid at 22 to 25°C. A phote period of 16 hours light and 3 hours
dark is used for the sheepshead minnow and continuous light for the mysid.
The fish should not be fed for 48 hours before the beginning of a test,
or during the test. However, mysids require food and must be fed during
acclimation and testing. They should be given approximately 20 48-hour
old Artemia nauplii (per 10 mysids) daily. The test conditions are sum- •-
marized in Table 4.8.
Each test requires a negative control with the same dilution water, condi-
tions, procedures and organisms as used in the test concentrations. If
any solvent other than water is present in the test concentration, a
solvent control is also required. This solvent control is treated the
same as the negative control except that the same amount of solvent used
to dose the other test containers is added to the control containers.
In the sheepshead minnow test there should be 15 liters of test solution
or control water in each 19.6-liter jar. If 30 x 30 x 60-cm containers
are used, the solution should be between 15 and 20 cm deep.
73
-------
TABLE 4.8 SUMMARY OF TEST CONDITIONS,
MARINE FISH OR MACROINVERTEBRATE TEST
Sheepshead minnow
Cypn'nodon variegatus
Mysidopsis bahia
Temperature, °C
Photoperiod,
hours light:dark
Water quality,
salinity, ppt
Container size
Test volume
Organisms per
container
Replicates
Feed
Duration, hours
Measurements, hours
D.O., pH
Salinity, hours
20 ± 2
16:8
10
19.6 liters
15 liters
10
2
No
96
0, 24, 48, 72, 96
0, 96
22 - 25
Continuous/ light
22 - 26
2 liters
1 liter
10
3
Yes
26
0, 24, 48, 72, 96
0, 96
In the mysid test there should be 1 liter of solution or control water
in each 2-liter beaker or culture dish.
Organisms should be placed in the test and control vessels not more than
30 minutes after the test solutions are prepared. Chemical, physical
and biological data are taken and recorded as described below for the
duration of the test.
If no toxicity is detected at any concentration and the MAD dose was
tested, then no further testing is required. The test material may be
reported as having no detectable toxicity. Test materials that kill or
immobilize all or nearly all the test organisms should be retested with
a lower dose range.
74
-------
Biological loading. The biological loading in each test and control
vessel should not exceed 0.8 g of test organism per liter or be so high
as to (1) reduce dissolved oxygen concentration below 60 percent saturation
(2) raise the concentration of metabolic products above acceptable levels
or (3) stress the organisms by overcrowding, any of which may invalidate
the test results.
4.4.5 Results and Data Interpretation
Chemical and physical data. Dissolved oxygen concentration and pH should
be measured at the beginning of the test and every 24 hours thereafter
in the controls and in the high, medium and low test concentrations.
Temperature of the water bath or controlled-temperature area should be
recorded continuously or every 24 hours.
Concentration of un-ionized ammonia, if required, can be obtained by
measuring total ammonia and consulting Reference 30.
Methods for the foregoing tests are described in References 21 and 43.
Biological data. Mortality is the effect most often used to define acute
toxicity to aquatic organisms. Criteria for death are usually lack of
movement, especially of gill movement in fish, and lack of reaction to
gentle prodding.
Because death is not always easily determined with some invertebrates,
an ECcQ may be calculated rather than an LC5Q. The principal criterion
for effect on mysids is immobilization, defined as lack of movement except
for strongly diminished activity of appendages. ~'
Death or immobilization and abnormal behavior should be recorded every:
24 hours for the duration of the test. Dead organisms should be removed
as soon as they are observed. If more than 10 percent of test organisms
in any control die or are immobilized, the entire test is unacceptable.
Observations of test materials, which produce harmful effects j_n vivo,
but do not result in deaths, are difficult to quantitate. Such observa-
tions provide insight into the sublethal effects of a sample on aquatic..
organisms and may be used to recommend further investigation of the test
material.
Table 4.5 lists definitions of fish behavior terms and a code for recording
and reporting.
Laboratory data forms. Forms for recording chemical, physical-and biolo-
gical data in both sheepshead minnow and mysid tests are illustrated in
Quality Control documents (9).
Calculations. The concentration of test material lethal to 50 percent
of the population (LC5Q) and 95 percent confidence limits should be deter-
mined (when possible) at 24-, 48-, 72-, and 96-hour exposure for fish
tests and the EC,-0 and 95 percent confidence limits at the same time
points for the mysid tests. Any of several methods which have been
75
-------
reviewed by Stephan (34) including moving average, Spearman-Karber, Litch-
field-Wilcoxin, probit or binomial may be used. The results are evaluated
according to criteria defined in Table 4.11 which permits the test mate-
rial to be ranked by toxicity category.
4.5 MARINE ALGAE 96-HOUR SCREENING TEST
4.5.1 Introduction and Rationale
The fundamental principles of the marine algae screening test are identical
to those described for the freshwater algae screening test (Section 4.3.1).
4.5.2 Methods and Methods
General procedures listed for all aquatic test in Sections 4.1.1. are
applicable to the static acute toxicity test with marine algae. Specific
areas discussed in Section 4.1.1 that should receive careful attention
are: facilities, construction materials, test containers, cleaning and
preparation of glassware and test material. Materials and methods unique
to marine algal tests are as follows:
Equipment. Equipment should include a constant-temperature room or incu-
bator capable of providing temperature control of 20 ± 2°C. A gyrotary
shaking apparatus capable of 60 oscillations per minute should be available
for test culture flasks. Illumination of 4300 ± 650 lumens/m2 (400 ft-C)
is required for marine algae with a photoperiod of 14 hours light and
10 hours dark. Overhead cool-white fluorescent bulbs should be used.
Light intensity is measured adjacent to the flask at liquid level using
a light meter capable of being calibrated against National Bureau of
Standards lamps.
Culture containers for this and other aquatic tests are discussed in
Section 4.1.1. Erlenmeyer culture flasks of Pyrex or Kimax glass are
used in either 125-, 250- or 500-ml sizes. The recommended contents-to-
volume ratio for hand-shaken flasks is 1 to 5 and should not exceed
50 percent for continuously shaken flasks. Flask closures must permit
gas exchange and prevent contamination. Foam plugs* are suggested.
Since some brands may be toxic, each laboratory should determine for
each type of closures purchased whether or not there are any significant
effects on algal growth.
Support equipment includes an autoclave or pressure cooker capable of
producing 1.1 kg/cm2 (15 psi) at 121°C and a dry-heat oven capable of a
temperature of 120 ± 1°C. A high-quality microscope or spectrophotometer
is needed for biomass counting.
"For example, Gaymar Industries, Inc., Orchard Park, NY 14127.
76
-------
Marine Algal Nutrient Medium. Synthetic sea water is prepared by adding
approximately 30 g of a commercial salt mix* to 1 liter of deionized
water. Salt mix is added with continuous stirring until the salinity is
30 parts-per-thousand when all the salt is dissolved. Salinity may be
measured with a refractometer, salinity-conductivity meter or salinometer.
Nutrients are added to the synthetic sea water. For stock culture medium;
30-ml metal mix, 2.0-ml minor salt mix and 1.0 ml vitamin mix are added
to 1 liter (final volume) of synthetic sea water. Composition of these
mixes is given in Table 4.9. Metal mix used for stock culture medium
contains EDTA whereas that used for toxicity tests does not. To prepare
medium for toxicity tests and control, 15-ml metal mix (without EDTA),
1.0 ml minor salt mix and 0.5 ml vitamin mix are added to 1 liter (final
volume) of synthetic sea water.
Media are sterilized by autoclaving at 1.1 kg/cm2 (15 psi) at 121°C for
15 minutes. Culture and test media are cooled and held at least 12 hours
to allow the pH to stabilize.
TABLE 4.9 COMPOSITION OF MARINE ALGAL ASSAY MEDIUM (MAAM)
Compound Concentration
Metal mix
FeCl2-6H20 0.048 g/1
MnCl2-4H20 0.144 g/1
ZnS04-7H20 0.045 g/1
CuS04-5H20 0.157 mg/1
CoCl2-6H20 0.404 mg/1
H3B03 1.140 g/1
Na2EDTAa 1.000 g/1
Minor salt mix
K3P05 3.0 g/1
NaN03 50.0 g/1
Na2Si03-9H20 20.0 g/1
Vitamin mix
Thiamine hydrochloride 50.00 mg/100-ml
Biotin 0.01 mg/100 ml
B12 0.10 mg/100 ml
aEDTA added only to metal mix used for stock culture medium.
"For example, Rila Products, Teaneck, NJ 07666.
77
-------
4.5.3 Test Organisms
The recommended test organism for marine algal assays is Skeletonema
costatum (22). Algal cultures are available from the Culture Collection
of Algae, Department of Botany, University of Texas, Austin, TX 78712.
Stock cultures of algae should be maintained at 20 ± 2°C under a 14-hour
light, 10-hour dark photoperiod in marine algae culture medium. It is
recommended that several cultures be maintained on agar under axenic
conditions. Transfers of stock cultures should be made every six to
eight days to provide cultures with a sufficient number of cells growing
exponentially for test inoculations.
4.5.4 Experimental Design
Preparation of Toxicant. Depending on its nature, the test material is
prepared by one of two methods. The first method is for solids or non-
aqueous liquids. These materials may be added directly by weight to the
algal medium or a concentrated stock solution may be prepared in deionized
water (or other solvent); equal-volume aliquots of a small size are added
to each treatment. If it is not possible to prepare a homogeneous solution
of the test material, it must be added directly into each replicate flask.
The second method of test material preparation is for aqueous effluents
and aqueous leachates of solids. Nutrients are added in the same quanti-
ties as in the control medium to 1 liter of the effluent. The effluent
is used as the 100 percent test material treatment. Additional test
concentrations are prepared on a volume-percent basis by mixing appropriate
volumes of effluent medium mixture with control medium. Thus, all test
treatments and controls will contain the same amount of nutrients unless
the effluent contained nutrients.
Test Procedure. Six concentrations of test material should be prepared,
as well as negative controls and solvent control if needed, all with
three replicates. A fourth replicate for each treatment contains test
material and nutrient medium, but is not inoculated with algae. This
blank is necessary to correct biomass measurements for particulates which
may be present in the test material.
If a solvent is necessary for the preparation of the test mcterial, a
solvent control must be included. All test flasks and the solvent control
must contain the same amount of solvent. Solvent levels should be as
low as possible so as not to interfere with test results. The toxicity
of commonly used solvents should be determined in each laboratory using
this bioassay to help select suitable solvent levels.
1. Inoculum. A six- to eight-day-old stock culture is used as the
inoculum source. Population density in the stock culture is determined
by direct counting or spectrophotometry. A volume of inoculum calculated
to yield an'initial concentration of 30,000 cells/ml is aseptically added
to each flask. The volume of inoculum added should be between 0.1 and
1.0 ml.
78
-------
2. Incubation. Incubation conditions for marine algal assays are given
in Table 4.10 below.
TABLE 4.10 INCUBATION CONDITIONS FOR MARINE ALGAL ASSAY ORGANISMS
Light Photoperiod, Shaking Speed,
Intensity, Hours of Oscillations Temperature,
Species Lumens/m2 Light:Dark Per Min °C
Skeletonema 4300 ± 650 14:10
costatum
60
20 ± 2
3. Response monitoring. After 120 hours of exposure, algal growth is
measured by one of the following methods: (a) direct counting, or
(b) spectrophotometry. Microscopic observation is desirable to reveal
any abnormalities in cell shape or condition.
a) Direct counting. A hemacytometer counting chamber and a microscope
are used.Two samples are taken from each flask, and two counts are
made of each sample. Whenever feasible, 400 cells per replicate are
counted in order to obtain ± 10 percent accuracy at the 95-percent confi-
dence level. This method has the advantage of allowing for the discrimi-
nation between algal cells and debris or particulates in the test material.
b) Spectrophotometry. A rapid technique for biomass monitoring is
absorbance. This is the preferred method for Level 1 estuarine algal
studies. A double-beam spectrophotometer with high-quality cuvettes
should be used. A sample is withdrawn from each flask and placed in the
cuvette. Absorbance is read against the proper blank for each concentra-
tion. Because absorbance is a complex function of the volume, shape and
pigmentation of the algae, a calibration curve should be constructed to
establish the relationship between absorbance and concentration (absor-
bance is determined on known dilutions of the control culture). The
effect of particulates in the test material, if any, on the absorbance
readings should also be determined.
4.5.5 Results and Data Interpretation
Calculations - Percent inhibition (I), or stimulation (S), is calculated
for each concentration according to the following formula:
Percent Response = T - C w ,QQ Positive Response = Stimulation
C - IN Negative Response = Inhibition
where C is the absorbance or cell numbers in the control (mg I-1) T is
the absorbance or cell numbers in the treated culture, (mg I-1) and IN
is the dry weight of inoculum used (mg I-1). Different endpoints
79
-------
TABLE 4.11 DEFINITION OF TOXICITY CATEGORIES FOR AQUATIC ECOLOGICAL ASSAYS
00
o
Assay
Freshwater
Fish
Freshwater
Invertebrate
Freshwater
AlgaeT
Marine
Fish
Marine
Invertebrate
Marine
Algae
Activity Measured
96-hr LC,nc
(lethality)
48-hr EC5Qe
(immobilization)
120-hr EC5Q
(growth inhibition)
96-hr LC,n
(lethality?)
96-hr EC5Q
(immobilization)
96-hr EC5Q
(growth inhibition)
Sample
Type
S
AL
S
AL
S
AL
S
AL .
S
AL
S
AL
MADb
1
100
1
100
1
80
1
100
1
100
1
100
Range of LC^
Units
g/L
percent
g/L
percent
g/L
percent
g/L
percent
g/L
percent
g/L
percent
High
<0.01
<20
<0.01
<20
<0.01
<20
<0.01
<20
<0.01
<20
<0.01
<20
Moderate"
0.01-0.1
20-75
0.01-0.1
20-75
0.1-0.1
20-75
0.01-0.1
20-75
0.01-0.1
20-75
0.1-0.1
20-75
or ECcnConcentrations
Low0
0.1-1
75-100
0.1-1
75-100
0.1-1
75-80
0.1-1
75-100
0.1-1
75-100
0.1-1
75-100
Not Detectable
NDd at >1
ND at >100
ND at >1
ND at >100
ND at >1
ND at >80
ND at >1
ND at >100
ND at >1
ND at >100
ND at >1
ND at >100
S = solid, AL = aqueous liquid. Nonaqueous liquids are evaluated on an individual basis because of
variations in samples such as solubility in water, vehicle, percent organic vehicle and percent solids.
The maximum applicable dose and range of doses able to be tested for nonaqueous liquids is dependent
upon the solubility of such samples in water. Because of this variability, evaluation criteria
. have not yet been developed.
MAD = Maximum applicable dose.
.LCrn = Calculated'concentration expected to kill 50 percent of population.
aNDjy Not detectable.
f:EC,-Q = Calculated concentration expected to produce effect in 50 percent of population.
The MAD for routine freshwater algae testing of liquids is 80 percent rather than 100 percent as in the
other tests. Evaluation of low (L) or not detectable (ND) results for this test should take this into ac-
count. Samples can be retested at higher concentrations.
-------
may be calculated from the percent response vs. concentration data. For
samples which are inhibitory, an ECc0 (defined as the calculated test
concentration causing growth inhibition of 50 percent relative to control)
is calculated. For samples which are stimulatory, an SC^Q (defined as
the calculated concentration causing growth stimulation or 20 percent
relative to control) is calculated. For all samples the no observed
effect concentration (NOEC) is established or the EC™ or SC-g are calcu-
lated using the method previously noted in Section 4.3.5. Tne results
are evaluated according to criteria defined in Table 4.11 which permits
the test material to be ranked by toxicity category.
4.6. BIOACCUMULATION PROCEDURE FOR INDUSTRIAL AMD ENERGY SOURCE
SAMPLES
4.6.1. Introduction and Rationale
The need for inclusion of a simple screening procedure in Level 1 environ-
mental assessment to evaluate the bioaccumulation of components in complex
mixtures that would accumulate in aquatic organisms has been established.
Such a procedure will establish the need to perform subchronic or chronic
biological tests at Level 2.
A high-performance liquid chromatographic (HPLC) procedure is available
that fills the basic requirements of a screening bioaccumulation test.
The HPLC procedure is based on known correlations between octanol/ water
partition coefficients (expressed as Log P) and bioconcentration (44).
This section describes the procedure to be used on complex mixtures from
energy and industrial sources.
4.6.2
Materials and Methods
A high-performance liquid chromatograph equipped with a solvent gradient
accessory and a 254 nm ultraviolet detector is employed. The column
recommended is a Varian* preparative reverse phase Micropack CH column
(part number 03-912152-72) which consists of a 30 cm x 8 mm id stainless-
steel cylinder filled with 10 micron lichrasorb to which octadecylsilane
is permanently bound. Equivalent columns of the same dimensions are
acceptable. A reverse phase guard column (Whatman or equivalent) prior
to the preparative reverse phase column is recommended.
The Varian preparative column, operated at room temperature will give
the proper separation efficiency when an isocratic mobile phase of 85/15
methanol water at a flow rate of 2.0 ml/min is used. The column should
be allowed to equilibrate for at least two void volumes (approximately
10 min) before injection of any sample. After each analysis, the column
is cleaned by programming the mobile phase to 100 percent methanol.
Organic solvents should be distilled in glass or of equivalent quality.
Water should be free of organic contaminants.
"Varian Associates, Inc., Palo Alto, CA 94303.
81
-------
Calibration. Seven standards (Table 4.12) are used to calibrate the
retention and sensitivity characteristics of individual instruments.
These seven standards represent a range of retention times and P values
within which the majority of bioaccumulation components of environmental
mixtures will elute.
TABLE 4.12 PARTITION COEFFICIENTS. OF CHEMICALS USED FOR CALIBRATION
Compound Log P
Acetone
Benzene
Bromobenzene
Biphenyl
Bi benzyl
pp-DDE
? 4 S ?' 5'
£, t , a, e. , a
- Pentachlorobiphenyl3
0.55 ...,-•:•'
2.13
2.99
3.76
4.81
5.69
6.11
aOther pentachlorobiphenyl isomers with a minimum of 2 and a maximum of
3 chlorine atoms on a ring are acceptable.
Standards are dissolved in a mixture of acetone and cyclohexane (3:1 v/v).
A composite standard is prepared by combining quantities of individual
standards. The concentration of each individual chemical in solution is
adjusted to give a chromatographic peak of 25 to 75 percent full scale
at the highest operating sensitivity of the instrument. (For example,
attenuation 1 at 10 mv full scale).
The six standards are prepared in one solution and used to calibrate the
elution time in units of Log P. Elution times may differ because of
varying void-volume characteristics of HPLC instrumentation.
Sensitivity calibration. For the results from different chromatographic .
systems to be comparable and to insure a minimum detection sensitivity
for specific compounds, the calibration mixture is analyzed prior to
each day's accumulation analyses. Daily calibration eliminates irregu-
larities caused by small changes in flow or solvent characteristics and
acts to monitor column performance during prolonged use as well.
Sensitivity is based on the average quantity of the calibration compounds
which causes a 25-percent scale deflection. The sensitivity is calculated
from the geometric mean of the instrument response to the chemicals listed
in Table 4.12 with the exception of acetone. This value expressed in
ug/25-percent full-scale deflection is defined as Instrument Sensitivity
(IS).
82
-------
4.6.3 Experimental Design
Sample preparation and analysis. Samples should be prepared according
to the methods for organic analysis described in the Level 1 sampling
and chemical analysis manual (1). Since different types of samples are
collected in a source assessment, the amount of sample taken for bioac-
cumulation analysis must be calculated separately for each type. The
quantity of sample used for the analysis is based on the IS. The portions
of Level 1 sample required for analysis for each of the four typical
samples types acquired in Level 1 are shown in Table 4.13.
TABLE 4.13 SAMPLE QUANTITIES FOR BIOACCUMULATION
Sample
Aqueous Liquid
Bulk Solid
Fluegas Particulate
Fluegas Particulate
Sampling
Method
Grab
Grab
SASSa Cyclone
SASSa XAD-2
Quantity for HPLC
Analysis
Extract from IS liters
Extract from IS g
0.03 x (IS) from SASS 30m3
0.03 x (IS) from SASS 30m3
extract
extract
aSASS is the source assessment sampling system described in Reference 44.
Sample analysis must be performed under the same conditions as for the
calibration mixture. Following elution of the sample components, the
column should be flushed with no less than 20 ml of 100 percent methanol.
In some cases, additional components are detected during this phase of
operation. These components should be recorded as having log P > 6.0.
Information acquired from column clean-up must be evaluated carefully
since these post run components can arise from the sample, makeup water
or methanol eluent, or they may be artifacts caused by a sudden change
in eluent.
Following column clean-up, the standard eluent should be passed through
the column for a minimum of 10 minutes to allow column equilibration.
4.6.4 Results and Data Interpretation
Log P for each component is determined from the retention calibration
curve for that day's analysis. The final output of the analysis is a
list of retention times and the corresponding Log P for components which
exceed Log P of 3.5 and the 25 percent full scale sensitivity criterion.
Components eluting in the column clean-up should be reported as Log
P > 6.0.
Interpretation of results from this analysis must be made together with
results from Level 1 bioassays, particularly the ecological effects bio-
assays (Chapters 4 and 5). If the toxicity bioassays show a response,
then further effort at Level 2 first should address the cause and control
83
-------
of the toxic response. If the acute toxicity bioassays show no response,
but bioaccumulation results are positive (i.e., component(s) with Log
P > 3.5), then further effort at Level 2 should deal with the potential
chronic effects of the effluent.
Notes on bioaccumulation analysis. Since the capacity and efficiency of
the reverse phase column are necessarily high, the analysis demands a
precision-packed column. A reverse phase guard column used in series
prior to the bioaccumulation column is recommended to extend the useful
lifetime of the analytical column.
Under normal operating conditions, the concentrations necessary to meet
the 25-percent scale deflection are on the order of those listed in
Table 4.14.
TABLE 4.14 APPROXIMATE CONCENTRATIONS FOR CALIBRATION SAMPLES
Compound Concentration (mg/ml)
Acetone 0.3
Benzene 0.5
Bromobenzene 0.01
Biphenyl 0.2
Bibenzyl 0.2
2, 4, 5, 2', 5' - Pentachlorobiphenyl 0.2
Examples. ..Examples of quantitites of samples required for this analysis
assuming an IS of 1.0 are described here. Aqueous samples are prepared
for analysis by extraction at high and low pH with dichloromethane. For
sufficient sensitivity, the extract from 1.0 liter of sample is required
(i.e., IS liters of sample).
Flue-gas samples are ordinarily taken in quantities sufficient for chemical
and bioassay analyses. The standard Level 1 flue gas sample is taken
with the 5ASS train and represents 30 m3 of flue gas. In order to meet
the sensitivity requirements for bioaccumulation (assuming, for example,
IS = 1.0), a sample representing 1.0 m3 of flue gas is analyzed. For
30 m3, an aliquot of 1.0/30 or .03 of the total SASS sample should be
analyzed.
84
-------
CHAPTER 5
LEVEL 1 TERRESTRIAL ECOLOGICAL TESTS
5.1 INTRODUCTION AND RATIONALE
The Level 1 terrestrial ecological effects tests include assays for deter-
mining toxicity of complex wastes in plant and insect test organisms.
The tests are able to detect sublethal toxic response to stress in plants
(PSE test), sublethal and lethal toxic responses in germinating seeds (RE
test) and acute toxicity and reproductive impairment in insects (IT test).
These tests were selected to provide a range of terrestrial organisms
for assessing the effect of effluent streams on the environment. Test
organisms include maturing plants, germinating seeds and insects. This
group of tests offers testing capabilities for all sample types (including
gases) with the advantages of low cost, reproducibility and relatively
rapid performance time. A future goal for this manual is to include a
test procedure for assessing the impact of effluent samples on soil micro-
organisms (decomposers).
Terrestrial ecological tests are used to determine the concentration of
test material that produces a defined toxic effect on a specified per-
centage of the test organisms in a fixed amount of time. The plant-stress-
ethylene test (PSE test) is designed to assess and rank the toxic effects
of gaseous effluents on plants by measuring the stress ethylene of plant
response and by assessing relative foliar injury in exposed plants. The
root-elongation test (RE test) measures the inhibition of root elongation
and seed germination. Although both parameters are observable toxic :.
responses and are reported, root-elongation inhibition is the preferred
end point. The concentration which inhibits root elongation by 50 percent
of the control (EC™) is estimated and used to rank effluent samples.
The insect-toxicity assay measures the acute toxicity and reproductive
capacity of fruit flies treated with environmental samples. The dose
lethal to 50 percent of the flies (LD5Q) compared to the control is cal-
culated and used to rank test samples. In the optional fertility test,
the effective concentration which reduces the fecundity of surviving
dosed flies to 50 percent of control flies (EC™) is calculated. Char-
acteristics of the Level 1 Terrestrial bioassays are given in Table 5.1.
The Level 1 terrestrial tests represent the state of the art for environ-
mental assessment for terrestrial ecological effects. These tests have ..
not been as thoroughly validated with complex environmental mixtures as
have the health and aquatic ecological effects tests. Because'of the
lack of published procedures and data, a workbook of detailed protocols
for Level 1 terrestrial tests has been published (45). Procedures and
evaluation criteria may be modified as experience is gained in the future.
85
-------
TABLE 5.1 CHARACTERISTICS OF LEVEL 1 TERRESTRIAL ECOLOGICAL EFFECTS BIOASSAYS
Characteristic
Species
Plant Stress
Ethyl ene Test
Bush Bean
Root Elongation
Test
Cucumber, Wheat,
Insect Toxicity
Test
DrosophiTa melanogaster
End Point(s)
Measured
Amenable to
Sample Types
Data Expression
Special Features
Metabolic stress
evidenced by
ethylene production
Gases, Liquids
Only validated
Level 1 Bioassay
for gases; sensitive
Red Clover,
Radish, Lettuce
Root length
Liquids, Solids
(leachates)
Positive or Negative EC
50
Detects toxi-
city to terres-
trial producers;
multiple species
Lethality, Reproductive
capacity
Liquids, Solids
LD50
Detects lethality to
terrestrial consumer
plus can provide data
on fertility
5.2 PLANT STRESS ETHYLENE TEST
5.2.1 ''Introduction and Rationale
This test is designed to assess the toxic effects of gaseous effluents
on plants by measuring the pi ant-stress-ethylene response. Under normal
conditions plants release low levels of ethylene which function hormonally
in the regulation of growth and development. In response to various
stresses, ethylene production increases substantially (46). Critical to
the effectiveness and applicability of the PSE test is that induction of
stress ethylene is proportional to the intensity and duration of the
stress over a wide range of stresses (47). Ethane evolution may also
increase in response to some stresses and is thought to indicate more
severe damage and tissue autolysis (48,49,50). Current evidence suggests
that ethylene is a produce of methionine metabolism and the same biochem-
ical pathways are involved in the production of basal and stress-induced
ethylene (51,52). Ethane may be produced by peroxidation of linolenic
acid upon extensive wounding; this peroxidation may also result in some
ethylene production (52).
86
-------
5.2.2 Materials and Methods
Gas sampling. Gas samples are collected in ten evacuated 150-liter poly-
vinyl fluoride film bags (Tedlar* or equivalent) which are cleaned and
leak tested before use. A minimum of 1365 liters of gas are required
for the PSE test. To facilitate handling and to keep the samples in
darkness, the bags are contained within 55-gallon fiber drums. Access
to the sample bag is through a gate valve attached to the bag and to the
metal drum lid.
Sample bags are filled individually during sampling using an additional
gate valve attached to the outside of the drum and to the stack sample
port with Teflon tubing. During sampling, pressure within the bag is
monitored to follow the filling procedure and to prevent bags from
bursting. ::
The sample drums are promptly shipped to the testing laboratory; samples
should be tested within three days. If a longer storage period is neces-
sary, the sample is stored at 4°C and permitted to warm to the testing
temperature prior to test initiation.
Exposure chamber. Exposure chambers (Figure 5.1) used for this test are
similar to those described by Heck et. a_L (53). A negative-pressure
single-pass flow system is used to draw gas into the chambers which are
then closed to allow static exposure of the plants to the test gas.
Each of the chambers is attached to the test gas intake and exhaust mani-
folds (Figure 5.1). The intake manifold terminates a junction permitting
attachment of the gas-sample-container manifold to which the 10 sample
containers are attached. A high-pressure blower in the exhaust system
is used to draw the sample being tested into the chamber. An internal
air-circulation system provides air circulation during exposure and pre-
vents concentration gradients from developing within the chamber. Inlet
tubing and fittings are made of polypropylene to reduce corrosion and
contamination; exhaust fittings are made of polypropylene, Tygon, and
polyvinylchloride. The operating characteristics of this system are
similar to those reported by Heck et. a_L (53).
Plant culturing. Bush beans, Phaseolus vulgaris L., cultivar Harvester,
are grown from seed for use in the PSE test. A total of eight plants
are required for each test gas concentration and each control. It is
required that 12 be grown for each condition so that uniform plants
may be selected. Two seeds are planted in 10-cm plastic pots using pro-
mix BX or Jiffy Mix as the potting medium. The seedlings are thinned to
one per pot seven to eight days after seeding. Plants are surface-watered
daily with sufficient half-strength Hoagland's nutrient solution (54) to
require the excess solution to drain. They are maintained at average day/
night temperatures of 26°C and 20°C, respectively. A 16-hour photoperiod
is recommended but a minimum 12-hour photoperiod is acceptable. During
the photoperiod, the light intensity must be a minimum of 100 g cal/cnr/day
(200-2500 nra) as measured at plant height. An average light intensity
"E.I. Dupont de Nemours and Co. (Inc.), Wilmington, DE 19898.
87
-------
Sample Container
Manifold
v£**
ajg
jr>
Figure 5.1 COMPLETE STRESS ETHYLENE EXPOSURE SET-UP SHOWING
THE VARIOUS COMPONENTS AND THEIR INTER CONNECTIONS
(only 6 of the 10 sample containers are shown here)
-------
of 200 g cal/cm2/day is preferred. When bush beans are grown in a con-
trol! ed-environment chamber, air is continuously renewed to prevent COj
depletion. The plastochron index (55,56) is used to select plants for
assay to ensure that they are exposed at a standard time of development
and to ensure population uniformity. Bush beans are exposed within
24 hours of the time when the population to be treated exhibits a plasto-
chron index of 1.5 ± 0.3.
5.2.3 Experimental Design
Exposure. Bush beans are exposed to three concentrations of test gas,
with charcoal -filtered air used as a negative control and chlorine gas
used as a posi.tive control. The exposure is carried out sequentially at
room temperature with the control chamber being run first, followed by
the experimental chambers and then positive controls. Exposures to all
test gas concentrations are begun within one hour of the start of controls.
The objective is for each chamber to contain twice as much test gas as
the chamber with the next lower concentration. The mixing curve for the
test gas in the chamber air is logarithamic. Thus, to double the concentra-
tion x which results from a mixing time of y sould require doubling the
mixing time (2y). To double the concentration again (4x) would require
eight times the mixing time (8y). The actual mixing time for any set of
chambers will be a function of the rate of flow of gas through the chamber
and the total quantity of test gas available. The mixing curve for any
chamber design should be empirically determined using an easily detectable
reference gas. Methane at 80 parts-per-million may be used and the change
in chamber concentration measured with a hydrocarbon analyzer.
The flow rate for each chamber is calibrated prior to exposure. A
meter is placed in the exhaust system between the blower and the chamber
and used to calibrate the flowmeter of each chamber. A flow of 5 cfm
(142 1/min) is established using the rotameter for reference; the reading
in centimeters of water on the manometer connected across the orifice is
recorded. The rotameter is subsequently removed and the flow adjusted
with the regulating valve to achieve the previous reading on the manometer,
completing the calibration.
The exposure chambers are prepared by placing eight plants in each chamber
(being sure that adjacent plants do not touch or shade one another) and
activating the internal blowers. The negative control chamber is filled
first. The external blower is turned on, the valves on the control chamber
are opened, and charcoal filtered room air is drawn through the chamber
from the open intake manifold for two. minutes.
Filling of the experimental chambers is conducted analogously. The sample-
container manifold is connected to the ten gas-sample containers and to
the intake manifold. The valves on the first experimental chamber are
opened and the test gas is drawn through the chamber for seven minutes.
The remaining two experimental groups are exposed in the same fashion,
except that the test gas is drawn in for two minutes and one minute, respec-
tively. The concentration of gas doubles from one minute to two minutes
of mixing and again from two to seven minutes. Positive-control chambers
89
-------
are filled with filtered room air as are the negative control chambers.
Sufficient chlorine gas, calculated to give a chamber concentration of
15 parts-per-million, is injected directly into the chamber via a syringe.
Once filled, each chamber is then closed and permitted to incubate for
two hours under a 400-W high-pressure sodium-vapor light at a temperature
of between 22 and 28°C. After two hours of exposure to the test gas, or
control gas mixtures, the contents of each chamber are exhausted through
a charcoal filter and the chambers are opened.
Incubation. Immediately after exposure, plants are carefully removed
from the chambers and are visually inspected for signs of damage. The
plants are then individually sealed with B-620 bags* (Cryovac; 10 x 32 in.;
std. gauge) with a wire support surrounding the plant to prevent the
leaves from touching the bag (Figure 5.2). The bags should be- sealed at
a standard position so as to provide a constant volume for ethylene accu-
mulation. Plants are bagged on a laboratory cart so that further handling
of the enclosed plants is minimized. The enclosed plants are incubated
at 26°C in the dark for four hours while ethylene accumulates within the
bag.
Ethylene and ethane analysis. Ethylene and ethane are quantified by gas
chromatography using a flame ionization detector (57). A Porapak-N column
(2.4 m x 3.2 mm, stainless steel, 80/100 mesh) is used for the separation
and is operated isothermally at 80°C. The injection port and detector
are maintained at 200°C and helium (40 ml/min) is used as carrier gas.
Maximum sensitivity of the instrument is required, as the limit of detec-
tion should be 5 parts-per-billion. The gas chromatograph is calibrated
on each day the samples are to be analyzed using a minimum of five ethylene
standards ranging from 10 to 1000 parts-per-billion to ensure linearity
of response. (The response for ethane is assumed to be equivalent to
the detector response for ethylene;) Peaks are identified by retention
time compared to standards and may be documented further by cochromato-
graphy with standard ethylene and ethane. A 1-ml sample is withdrawn
from the bag surrounding the plant using a 1-ml gastight syringe and is
analyzed by gas chromatography. Ethylene concentrations for negative-
control plants exceeding 50 parts-per-billion indicate improper plant
handling and the test is repeated. For best results, the standard
deviation of the mean ethylene production for each control and experi-
mental exposures should not exceed 1.6.
Assessment of plant injury. At the conclusion of ethylene/ethane analysis,
plants are removed from the bags and are maintained for three days using
the growth conditions previously specified. Plants are then reinspected
for injury. Relative foliar injury as compared to negative controls is
expressed on a scale of 0 to 10 with each increasing unit being equal to
10-percent injury of the total leaf area. The types of injury (e.g.,
leaf lesions, yellowing) should also be noted. In the assessment of
plant injury, any slight foliar injury must be reported as I (10 percent).
Further, only the affected area is to be quantitated. For example,
although an entire leaf may be spotted, the injury is assessed as 2 if
only 20 percent of the leaf surface is necrotic or yellowed.
"Cryovac Division, W.R. Grace and Co., Box 338, Simpsonville, SC 29681.
90
-------
Figure 5.2 Plant Enclosure
91
-------
5.2.4 Results and Data Interpretation
Ethylene concentrations for plants of a given experimental condition are
tabulated and the geometric mean and standard geometric deviation are
determined. A stress-response threshold value is determined which is
1.5 times the mean ethylene concentration of the negative-control plants
(to account for variability) or 50 parts-per-billion, whichever is greater.
A positive response has occurred if the mean for any of the experimental
groups exceeds this threshold. The response evaluated by the criteria
in Table 5.2 is classed as "high" if the threshold is exceeded by the
lowest test gas concentration (one minute); and "moderate" or "low" if
the threshold is exceeded in the two-minute or seven-minute mixing times,
respectively. Ethane data is monitored and recorded for future reference.
Relative plant injury is reported as mean and standard deviation for a
given experimental condition noting the type of injury which was observed.
TABLE 5.2 PLANT STRESS ETHYLENE TEST EVALUATION CRITERIA
Toxicity
High
Moderate
Low
Not Detectable
Lowest Positive
(Mixing Time in
1
2
7
Response
Minutes) Relative
No response at 7 minutes
Foliar Injury
7-10
4-6
1-3
0
Dose levels based upon mixing time in minutes are applicable only if
.flow rates and chamber design given in this chapter are used.
Evaluation based upon foliar injury are tentative and still under evalua-
tion. Each unit is equal to 10-percent injury of total leaf area.
Due to the subjective nature of observation and assessment of plant injury,
ranking of test samples based upon relative foliar injury is difficult
and should not be used for definitive ranking of test material toxicity.
Such information is used by the investigator in the overall evaluation
of a sample or for recommendation for additional Level 1 or 2 testing.
However, in the case of a highly toxic test gas which may damage the
ethylene production mechanism of the plant, foliar damage results are
used to corroborate the highly toxic nature of the sample: little or no
ethylene production with significant foliar damage (50 percent or greater
at the one- or two-minute exposures) confirms a "high" toxicity evaluation.
These observations are analagous to the physiological observations of
mice in acute i_n vivo rodent tests and to behavior observations of fish
in freshwater and marine ecological bioassays.
92
-------
5.3 ROOT ELONGATION TEST
5.3.1 Introduction and Rationale
The development of seed into a mature plant is a series of complex pro-
cesses. Assessment of toxic effects requires selection of a stage in
plant development that is sensitive to a broad range of toxicants and is
important physiologically. Seed germination and root elongation are
critical links in plant development beginning with the dormant embryo
stage and the period of rapid growth when essential plant structures are
formed.
Toxic substances that prevent or reduce germination or root elongation
will decrease plant populations and can reduce crop yields. In natural
systems, those species affected are less able to compete with other
species; thus tolerant species may be selected, resulting in changes in
species diversity, numbers and population dynamics.
Inhibition of seed germination and root elongation has been used in deter-
mining selective toxicities of herbicides (58,59), screening plants for
heavy metal (60,61) and salinity tolerance (62,63) and evaluating toxic
chemicals (64,65) and allelopathic substances (66,67). The root elonga-
tion/seed germination bioassay has several advantages. It is a rapid
test; germination and root elongation can be observed after 115 hours of
incubation. It is a simple test that does not require significant invest-
ments in equipment and facilities or complicated techniques. Personnel
required for performing the bioassay need not be highly skilled.
The same chemical may cause responses at different doses in different
plant species (65). To detect an effect from chemicals of unknown toxi-
city, several plant species should be selected. The species used in
this test—lettuce (butter crunch), Lactuca sativa L.; radish (cherry
belle), Raphanus sativus L.; wheat (Stephens), Triticum aestivum L.;
cucumber (hybrid Spartan valor), Cucumis sativus L.; and red clover
(Kenland), Trifolium pratense L.--are representative of economically
important plants of different plant families. Seed chosen germinates,
grows rapidly, contains no natural .inhibitors and requires no special
pretreatment. All test organisms are grown under identical environmental
conditions (constant temperature, 25°C, constant dark and enclosed to
maintain uniformly high relative humidity).
Although inhibition of root elongation and germination are observable
toxic responses, in this bioassay, root elongation inhibition is the
preferred end point. Usually, elongation is inhibited at lower concen-
trations of toxic substances than is seed germination.
5.3.2 Materials and Methods
Facilities. . Facilities must include work areas for planting seed and
for measurements, preferably isolated from other activities. There should
be a fume hood, distilled water source and refrigeration available at
5°C. The test facility must have a seed germinator and a plant growth
93
-------
chamber or some type of controlled environment chamber capable of main-
taining a uniform temperature at 25°C within ± 1.0°C.
Test containers. One-piece, molded-glass tanks*, with a 6-quart capacity
(approximately 9-% in. (L) x 6-% in. (W) x 7 in. (H)) are used for dosing
seeds. Glass plates (5-1/8 in. x 6 in.) of single-strength window glass
are prepared with polished edges. The glass plates are supported at a
67° angle in the tank with either a glass rack or glass pegs. The use
of glass racks has been found to be superior to the use of glass pegs.
The glass rack is constructed from two glass rods (approximately 9 in.
long) and six half-circles (4-3/4 in. 0.0) of glass tubing connected to
the rods at right angles at 1-3/8-in. intervals. The pegs are 2 to 3 cm
long and 5 mm in diameter. Twenty pegs are cemented with epoxy to the
inside of each glass tank (Figure 5.3).
Equipment. Items specifically needed include a spray bottle with a fog
or mist nozzle, metric ruler, forceps, Soxhlet extraction apparatus,
triple beam balance, pH meter, osmometer, storage bottles and plastic
bags (minimum of 12 in. x 8 in. x 14 in.). An illuminated magnifier may
be helpful for planting, seedling examination and root measurement.
Test organisms. The seeds used in the test are available from commercial
seed companies, state agricultural experiment stations, and laboratories
of the U.S. Department of Agriculture. Seed from one seed lot for each
species should be purchased in amounts adequate for one-year's testing.
Information on seed lot, seed year or growing season collected and germi-
nation percentage should be provided by the source of seed. Only untreated
(not treated with fungicides, repellants) seed is acceptable for Level 1
biological testing.
Size grading of seed. After purchase, size-grading is carried out on
the entire seed lot for each kind of seed. Small samples of 100-150 g
are sized at a time. The seed lot is inspected; trash, empty hulls and
damaged seed are removed. Depending on species, a series of four screens
is selected to separate samples into size classes (see Table 5.3). The
four screens are nested with the screen containing the largest holes on
top and screens with successively smaller holes in sequence below. A
blank or bottom pan collects the fraction that passes through all screens.
Seed is poured onto the top screen and the whole set of nested screens
are shaken (by hand or with a vibrator) until all the seed remains on
one screen or reaches the bottom pan. The separated fractions are col-
lected and the procedure repeated until all the seed in the lot is sized.
That size class which contains the most seed is selected and used exclu-
sively for duration of the tests. The fractions are divided into small
lots, placed in separate envelopes or sacks and stored in moisture-proof
sealed containers in a refrigerator at 5°C.
Preparation of glassware. The glass tanks (fitted with glass pegs or
tanks with glass racks) and glass plates are rinsed in acetone and then ~
thoroughly washed in warm water with a synthetic detergent (e.g. Alconox ).
"For example, Anchor-Hocking Glass Co., Lancaster, OH 43130.
94
-------
Figure 5.3 Glass Tank With Glass Pegs Cemented in Place
95
-------
Following washing, the glassware is rinsed in tap water and in 50 percent
nitric acid. All glassware is rinsed again thoroughly with tap water
and distilled water.
Tissue pager precleam'ng. Eight to ten sheets of single-ply tissue
(Kimwipes ) are placed in a Soxhlet extractor and extracted with distilled
water for a minimum of 24 hours. After extraction, the tissues are
removed, air dried and stored in a dry container or plastic bag.
TABLE 5.3 HAND SCREENS FOR SIZING SEEDS3
Perforated Metal Sheet
Species Round Holes Oblong Holes Wire mesh
Red Clover 1/19, 1/18, 1/17, 1/16
(Fractions of an inch)
Radish 6-1/2, 7, 7-1/2, 8
(64ths of an inch)
Wheat 9, 9-1/2, 10, 10-1/2
(64ths of an inch)
Cucumber 1/13 x 1/2
1/14 x 1/2
1/15 x 1/2
1/16 x 1/2
(fractions of an inch)
Lettuce 6 x 28
6 x 30
6 x 32
6 x 34
(fractions of
an inch; e.g.
1/6" x 1/28")
Supplied by (for example), A.T. Ferrell and Co., Saginaw, MI 48601, or
Seedburo Equipment Co., Chicago, IL 60607.
5.3.3 Experimental Design
Test Medium. The test medium is an effluent sample or aqueous leachate
of a particulate or solid sample. The effluent should be tested as soon
as possible after receipt to minimize changing or altering the sample.
•If it is not possible to test immediately, the sample should be stored
at 0 to 4°C in a closed container. Aqueous leachates of solids are pre-
pared using the procedure in Section 2.3.9. Aqueous leachates of solid
samples should be tested as soon as possible or the solid sample must be
96
-------
stored in closed polyethylene containers until extraction can be made.
Dilutions of the effluent or aqueous extractions should be made
without use of solvents or additives except for distilled water, which
is used as a negative control. Before testing, the pH of the samples
and controls is adjusted to pH 6.5. The highest concentration that
can be used is one for which the pH can be adjusted to pH 6.5 using
less than 5 ml of 0.1 M KOH or 0.1 M HC1 per liter of solution. Test
medium osmotic potential should be greater than -3 bars to avoid osmotic
effects which can retard root elongation and seed germination. Osmolarity
cannot be adjusted except by dilution with water. No more than one percent
organic solvent (usually dimethylsulfoxide) should be present in the
test sample.
Negative controls should be run using distilled water as the test medium.
For a.positive control, it is recommended that cadmium chloride be used
at a concentration of 15 mg/1 for all seeds except wheat, for which 75 mg/1
should be used.
Extended Dose Range Test. The extended range-finding test consists of
two control tanks, two tanks of 100-percent effluent, and one tank each
of 50, 25, 10, 1, 0.1 and 0.01 percent effluent.
A species need not be tested further if both tanks containing 100 percent
effluent had mean root lengths of at least 65 percent of control and at
least 10 of 15 seeds in one control, 8 of 15 seeds in the second control
and 8 of 15 seeds in both 100-percent effluent tanks germinated. Also,
in this situation it is not necessary to examine the plates containing
this species in the 10- to 0.01-percent tanks. If one or more of the
species show mean root lengths less than 50 percent of the control at
even the most dilute concentrations, it may be desirabli; to repeat the
test.
Procedure for planting seed. Whatman 3MM chromatography-filter-paper
rectangles (5-1/8 x 6 in.) are soaked in the test solution in a shallow
tray for a minimum of 5 minutes to saturate. One sheet of filter paper
is removed from the test solution, allowed to drain, and placed on a
glass plate to which the paper adheres. Trapping air bubbles between
the filter paper and the glass plate should be avoided. Using forceps,
15 seeds from one species are placed on the filter paper substrate in a
row, equally spaced, across the top of the plate, 1 in. down from the
top edge. Seeds are placed with the radicle end toward the bottom of
the plate and, in the case of wheat, with embryo side of the seed up. A
narrow strip (1-2 cm wide) of previously cleaned single-p.ly tissue is
placed over the row of seeds to hold them in place and, if necessary,
sprayed with just enough fine, distilled-water mist to cause the tissue
to cling to the seeds and filter paper. Test solution, usually 500 ml,
is poured into the rectangular glass tank fitted with glass peg guides
(empty tank if glass rack is used). The glass plate holding seed and
substrate is inserted in the glass tank between the glass peg guides or
in the glass rack to support the plates at a 67° angle (Figure 5.4).
The lower end of the plate opposite the seeds should be immersed in the
test solution with a minimum of 2 cm, but not more than 3 cm, of the
plate and filter paper in the solution. Normally, volumes less than
97
-------
Figure 5.4 Glass Tank With Glass Plates in Position Between Pegs
98
-------
500 ml are not tested, however, samples with less than 500 ml can be
tested if needed if clean inert glass beads or marbles are added to the
solution to displace and raise the liquid level. This procedure, with
one plate of each seed type (cucumber, lettuce, radish, red clover, wheat)
per container, is repeated with each test concentration and control.
Incubation. The glass tank, containing five plates with 15 seeds of each
species per plate and the test solution, is enclosed in a heavy plastic
bag and tied shut. The enclosed tank is placed in the dark, 25°C control-
led chamber. A tank is prepared for each test solution of sample effluent
and for the distilled water controls and positive controls.
Measurement of root length. Measurement of root length is made at 115
hours from the start of dark incubation. It is important to measure
each plate as nearly as possible to 115 hours (rvot to exceed ± 30 minutes).
To measure root length, a plate is removed from the tank and placed on a
flat surface. The lengths of all roots are measured to the nearest mil-
limeter and entered on the data sheet. Measurement is from the transition
point between hypocotyl and root to the tip of the root (Figure 5.5).
At the transition between the hypocotyl and the primary root, the axis
may be slightly swollen, contain a slight crook or change noticeably in
size (radish, lettuce, cucumber, red clover). In wheat, the single longest
primary or seminal root is measured from the point of attachment to the
root tip. Additional descriptions and photographs, helpful in making
root measurements, are presented in References 45, 68 and 69.
5.3.4 Results and Data Interpretation
Assay acceptance criteria. To estimate accurately the EC™ (the concentra-
tion which reduces root elongation,:by 50 percent), the following criteria
must be met for each of the species except for any species which showed
no effect with 100 percent effluent.
Criteria
1. At least 10 of 15 seeds on one negative control plate and 8 of
15 seeds on the other negative control plate must germinate.
And
2. Each effluent concentration in a series must be at least 50
percent as strong as the next concentration, except for controls.
And At Least One Of The Following
3. There must be at least one effluent concentration for which
mean root length was above 65 percent of the control and one
concentration for which it was below 35 percent of the control;
both of these effluent concentrations must have eight or more
seeds germinate. In addition, any concentration more dilute
than "above 65 percent" concentration must have a mean root
99
-------
Figure 5.5 Seedlings Showing Method of Measuring Roots
TOO
-------
length above 50 percent of the control and any concentration
less dilute than the "below 35 percent" concentration must
have a mean root length below 50 percent of control.
Or
4. All conditions required in criterion 3 (preceding) must be
satisfied with the sole exception that eight or more seeds
need not germinate at the "below 35 percent" concentration.
However, there must also be one concentration stronger than
the "below 35 percent" concentration for which fewer than 8 of
15 seeds germinated.
5. There must be two concentrations each of which have mean root
lengths at least 65 percent of the control -at the lower con-
centration ten or more seeds must have germinated and at the
higher concentration five or fewer seeds must have germinated.
In addition, there must be one concentration higher than the
"five or fewer" concentration for which seven or fewer seeds
germinate.
Since most effluents affect root elongation at lower concentrations than
germination, criterion 3 will usually be used to satisfy the requirements
of the test in addition to criteria 1 and 2. However, in cases where
germination is inhibited at lower concentrations than elongation, it
may be necessary to use criterion 4 or 5 in place of 3. If a species
fails to satisfy criteria 1, 2 and one of 3, 4 or 5, the extended dose
range test, must be repeated for that species.
Calculation of an EC5Q for Root Elongaton by Graphical Interpolation.
Provided criteria 1, 2 and 3 are met in the definitive test, the EC™
can be estimated in the following manner. For each species which satis-
fied these three criteria, plot on semi-log paper effluent concentration
on the logarithmic axis and percent control mean root length on the arith-
metic axis. Draw a straight line between the two effluent concentrations
used to satisfy criterion 3. Mean root length will be above 65 percent
of control for one of these concentrations and will be below 35 percent
of control for the other. The concentration at which this line crosses
the 50-percent point for root length is the EC™ for root elongation.
If no effects were seen with the 100 percent effluent, or if criterion 3
could not be met due to germination inhibition (criteria 4 or 5 instead);'
it is not possible to estimate an EC™ for root elongation.
Reporting. For each of the species either the concentration in (a) or
(b) or the quantities in (c) must be calculated and reported.
(a) If the species satisfied criteria 1, 2 and 3, report an esti-
mated EC™ for root elongation. Use graphical interpolation
to estimate the EC™ and rank the test sample using evaluation
criteria in Table 5.4.
101
-------
TABLE 5.4 ROOT ELONGATION TEST EVALUATION CRITERIA3
Aqueous Liquids
Toxicity (EC5Q in Percent Effluent)
High . <0.01
Moderate 0.01-1
Low; 1-100
Not Detectable >100
Criteria for aqueous liquids are tentative and still under evaluation.
Criteria for other sample forms (such as soluble solids and aqueous
leachates of insoluble solids) are under development.
(b) If the species satisfied criteria 1 and 2 but not criterion 3
(criterion 4 or 5 used instead), report the lowest concentra-
tion for which fewer than 8 of 15 seeds germinated. The EC™
cannot be estimated for root elongation or inhibition of seea
germination from data in this category. Currently, test samples
are not ranked from data of this type.
(c) If the range-finding test showed 100-percent effluent had no
effect on a species, report the number of seeds which germinated
and percent control mean root length for the two 100-percent
effluent tanks. The test sample is ranked as having nondetect-
able (ND) toxicity.
5.4 INSECT TOXICITY ASSAY
5.4.1. Introduction and Rationale
Drosophila melanogaster is a common insect species in nature; this organism
possesses many features which make it attractive as an in vivo test system
for detecting environmental toxicants. Among these features are: 1) a
short life-cycle time of 12-14 days; 2) minimal space, monetary and man-
power requirements to maintain stocks; 3) ability to detect toxic effects
at specific life-cycle stages (adult, germinal and developmental stages);
4) a well-defined genetic system which makes the detection of specific
genetic end points possible, and 5) ability to biotransform genotoxic
chemicals via i_n vivo metabolic enzyme systems. Treatment methods which
are normally encountered in animal studies (such as feeding, aerosol
inhalation and test-article injection) can also be applied to Drosophila,
although feeding is the most common route of exposure.
102
-------
Drosophila is included in EPA Level 1 environmental assessment testing
because its application as a toxicity screening test can be expanded
into testing for additional end points. For Level 1 applications,
Drosophila will be used to assess acute toxicity in the adults and repro-
ductive capacity among surviving flies after exposure to or treatment
with environmental samples. The acute toxicity data will be used for
the ranking of effluent streams and identifying sources for further
testing. The fertility response will be recorded and may be used to
provide possible direction for testing at Level 2.
5.4.2 Materials and Methods
Stocks. Drosophila melanogaster can be easily found in nature but pedi-
greed stocks may be obtained fronrthe Drosophila Stock Center*. One-day-
old wild-type Oregon R (Ore R) male flies are used for exposure to or
treatment with environmenta1~samples. One-day-old Ore R virgin female
flies are used for mating and egg production in fertility tests. Instant
Drosophila mediumt (Formula 4-24 without dyes) is used in preparing culture
bottles and vials. The flies are maintained on the instant medium in
8-dram glass vials plugged with nonabsorbant cotton. Stock cultures
should be placed on new medium every two or three weeks. The flies can
be immobilized with C02 or ether. The biology and handling of Drosophila
melanogaster are described in References 70 and 71.
5.4.3 Experimental Design
Feeding study. Drosophila may ingest particles suspended in liquid but
generally are exposed to liquid or solid samples in solution. Liquid
environmental samples are tested with various solvents such as water,
dimethylsulfoxide (DMSO) and ethanol to determine the most suitable solvent
or feeding medium. Water-soluble samples are dissolved directly in a
one-percent-sucrose feeding solution. Liquid samples that are not soluble
in water are solubilized in ethanol or DMSO and then added to a sucrose
feeding solution. The feeding solution is applied evenly to filter paper
at appropriate concentrations. The filter papers are allowed to dry and
are then placed in a dosing vial as shown in Figure 5.6. Liquid samples
not soluble in water, ethanol or DMSO are mixed into a thick paste made
with yeast extract and one percent-sucrose feeding solution to form an
emulsion mixture which is fed to the flies by applying it to the filter
paper liner.
Solid samples are dissolved in one percent-sucrose feeding solution if
soluble. Otherwise they are processed into fine particles no larger .
than 5 urn (Section 2.3.1) and mixed with one percent-sucrose solution to
form a suspension. Both solutions and suspensions are applied to filter-
paper liners for feeding.
*Drosophila Stock Center, Bowling Green, KY 42101.
tCarolina Biological Supply Company, Burlington, NC 27215.
103
-------
Glass Culture Vial
Plugged With Cotton Plug
Filter Paper Liner[Solublllzed compound
is added , spread evenly, and dried.]
Vial Prepared For Exposure. [Fifty flies are added to the vial for 24 hours.
Figure 5.6 PREPARATION OF TREATMENT CHAMBERS FOR DROSOPHILA
104
-------
Flies are maintained in dosing vials for 24 hours. The number of flies
surviving treatment at the end of the exposure period are counted and
living flies are transferred to new vials with medium to measure repro-
ductive capacity (Fertility Test).
Range finding. For feeding experiments, groups of 50 male flies which
have been starved for two hours, are placed in vials containing 1.5 ml
of feeding solution on filter paper at test-agent concentration levels
of 0, 0.1, 1, 10 and 50 mg/ml for solids and 0, 1, 10, 100 and 500 ul/ml
for liquids, or containing 5 g of yeast paste with test agent concentration
levels of 0, 0.1, 1, 10 and 50 mg/g or 0, 1, 10, 100 and 500 ul/g. The
vials are scored for dead flies immediately after the 24-hour exposure
period and a range of concentrations for LDcg evaluation is developed:
including levels of the test sample which produce some lethality. :
Definitive test for LD5Q determination. For feeding studies, the ID™
determination is performed by using five equally spaced concentration
levels derived from the information obtained from the range-finding test.
A total of 150 starved male flies, 50 per culture vial, are fed the test
sample at each specified concentration level for 24 hours.
Dead flies are counted immediately at the end of the feeding or exposure
period and the 24-hour LD5Q and its upper and lower limits are calculated
according to Litchfield ana Wilcoxin (20).
Optional fertility test. Twenty-five to fifty male flies obtained from
each dose level of the toxicity tests are individually mated to three
virgin females in food vials. After four days, the adults are discarded.
Vials are examined daily for five to eight days after the exposure for
evidence of larval activity. Absence of larvae or lack of normal -larval
activity as compared with control flies will indicate a decrease in ferti-
lity of the sample-treated group. In situations where the difference in
larval activity is only marginal, the number of emerged flies per vial
will be counted after another seven days. Fertilities of various dose
levels are reported for each sample. A more detailed study using a brood-
pattern analysis to determine germ-cell stage specificity may be used as
follow-up study if the sample shows significant reduction in fertility.
Fertility studies provide information on the toxic effects of chemicals
on the egg or sperm cells of exposed adults and may be a more sensitive
indicator of toxicity than lethality in the adult. The effects of chemi-
cals on fertility can occur at any of several distinct phases including
hatching of eggs, larval activity, or metamorphosis.
5.4.4 Results and Data Interpretation
Using the maximum applicable dose (MAD) concept developed for Level 1
screening bioassays, the response of chemicals in the Drosophila melano-
gaster test may be ranked as high, moderate, low or not detectable.
Table 5.5 gives MAD and response ranges for the Drosophila Level 1 Insect-
Toxicity Assay of environmental samples.
105
-------
TABLE 5.5 DROSOPHILA INSECT TOXICITY ASSAY EVALUATION CRITERIA3
Activity
Measured
Lethality (LD5Q)
Solid Samples
Liquid Samples
Fertility (EC5Q)
Solid Samples
Liquid Samples
MAD3
50
500
50
500
Response Ranges
High
<0.5
<5
<0.5
<5
Medium
0.5-5
5-50
0.5-5
5-50
Low
5-50
50-500
5-50
50-500
Not Detectable
>50
>500
>50
>500
Concentrations are in mg/ml for solids and slurries and (jl/ml for liquid
samples tested as solutions and suspensions. Insoluble liquids are reported
as mg or ul per gram of yeast extract paste.
Assay evaluation criteria are tentative and under evaluation.
106
-------
CHAPTER 6
LEVEL 1 DATA FORMATTING AND ANALYSIS
6.1 INTRODUCTION
Data formatting as described here is a guide to organizing data from all
Level 1 bioassays into a uniform evaluation format to aid in the use and
interpretation of the data. This format is structured so that data can
be converted from the conventional bioassay output into four levels of
response: Nondetectable (ND), Low (L), Moderate (M) and High (H).
The approach .is based on the following rationale:
Biological activity measured by lethality has sufficient common
phenomena to produce valid comparisons. The Ames Salmonella
assay measures gene revertants rather than toxicity, but its
results can be grouped in a similar fashion.
Each assay, regardless of the type of response measured, has a
maximum applicable dose above which the test data are virtually
impossible to interpret because of nonspecific responses of
the test organisms to the gross quantities of substance added.
A structure is needed for data formatting and analysis that
can categorize toxicity and mutagenicity data from diverse
sources into a series of similar comparative categories.
The categories (nondetectable, low, moderate and high) are
sufficiently broad to allow for normal variability and species
differences, yet are narrow enough to provide data upon which
decisions can be made.
The data from Level 1 testing are directed towards ranking of
streams from the sampling site for further studies and decisions
on applicable control technology; they are not intended for
making human-risk estimates.
To ensure uniform data recording and translation of raw data into the
final summarized form, standard data recording and data transition forms
have been developed. The forms for recording original data are dis-
cussed in the three separate sections of Reference 9 for health, aquatic
ecological and terrestrial ecological effects tests. Data transition
forms are used in sequence for data summary and analysis. The critical
data values determined for each assay, MEC, EC™, LD5Q or LC5Q; are recorded
on critical data summary forms. Health effects test oata are summarized
using the Health Effects Critical Data Summary Form (Figure 6.1) while
aquatic test data are summarized using the Aquatic Ecological Effects
Critical Data Summary Form (Figure 6.2). A standard critical data summary
form for the terrestrial effects bioassays is under development. Test
materials are then ranked, using the critical data values reported in
107
-------
Contract No.
Sample Identification
FIGURE 6.1 •'•-.
HEALTH EFFECTS CRITICAL DATA SUMMARY FORM^
.Technical Directive or Project No -- ; _ Site Sampled.
RAM Cytotoxiclty (EC50]C
Ames CHO Clonal I
Mutageniclty Toxlcity Viability
[MEC]b [ECSO]c Viability Index
ATP/ Per
ATP 106 Cells
o
CO
Rodent Toxlclty
Toxic Signs0
aThe assays, observed parameters and evaluation criteria are presented in IERL-RTP Procedures Manual; Level 1^ Environmental Assessment
Biological Tests,
bMEC: Minimum Ellecllve Concentration - Lowest concentration for any tester strain giving a mutagenic response.
CEC50: El lee live concentration that reduces the observed parameter to 50 percent of the appropriate negative control.
dLDso: The dose lethal to 50 percent of treated animals.
"Toxic signs are identified in a numbered list in the Level 1 manual. Only the number is reported here.
LBI-0798 1/81
-------
FIGURE 6.2
AQUATIC ECOLOGICAL EFFECTS CRITICAL DATA SUMMARY FORM8
Contract No.
o
V£>
Fresh Water Marine
8 Fish
.
Sample Identification
Daphnia Algae Fish
96-hr LCsob TSC 48-hr ECsod 120-hr ECsod
' 96-hr LC5ob TSC
Mysid Algae
96-hr ECsod 96-hr ECsod
aThe assays, observed parameters and evaluation criteria are presented in IERL-RTP Procedures Manual; Level 1 Environmental
Assessment Biological Tests.
bLCso: The concentration lethal to 50 percent of the treated animals.
CTS: Toxic signs are Identified in a numbered list in the Level 1 manual. Only the number is reported here.
dEC5Q: Effective concentration that reduces the observed parameter to SO percent ol the appropriate negative control.
LBI-08084/8t "'.
-------
the summary forms as the basis for the evaluation. Evaluation criteria
are presented in the Results and Data Interpretation section for each
assay. The ranking of each sample in each test performed is then recorded
in the Bioassay Summary Table (Figure 6.3).
6.2 DEGREE OF SENSITIVITY OF LEVEL 1 BIOASSAYS
An understanding of the results and interpretation of bioassay data is
dependent on a knowledge of the level of sensitivity of the bioassays.
The minimal levels of detection indicate the amount of toxicants that
might exist undetected in a mixture.
The concept of a minimal detectable level is amenable to bioassays with
dichotomous responses (+) or (-), such as the Ames Salmonella assay, but
is not readily applied to bioassays with continuous responses, such as
those measuring an LC,-Q. The limit of detection of an LC,-n is dependent
on the sample size ana other test parameters.
A recent EPA report (73) discusses some of the calculated minimum detect-
able levels of pure chemicals in several Level 1 health effects bioassays.
110
-------
FIGURE 6.3
BIOASSAY SUMMARY TABLE
Technical Directive or Project No.
Contract No.
Sample Identification
[Health Effects Tests! Ecological Effects Tests
[ 1 Aquatic | Terrestrial
' /Fresh Water/ Marine /
MSM/MM
•:•'
•
^ /
^//
$/
'//
/ Notes
ND = No Detectable Toxicity
L = Low Toxicity
M = Moderate Toxicity
H = High Toxicity
LBI-0468 R 11/80
111
-------
CHAPTER 7
LEVEL 1 QUALITY CONTROL AND QUALITY ASSURANCE REQUIREMENTS
7.1 INTRODUCTION
If Level 1 assessments are to be used as a basis for decisions regarding
further bioassay assessment, it will be necessary to ensure the quality
of the test data. Quality control standardization and standardized data
documentation will contribute significantly to ensurance of test quality
and reproducibility. The documentation for test-quality verification
consists of: ;>
1. Detailed description of the work to be performed, individuals
responsible for the study, sampling location, and approximate
dates of the sampling and the analysis.
2. Standard Operating Procedures (SOPs) which outline the details
of specific laboratory operations and quality control procedures
(including positive and negative controls).
3. Complete set of raw data including name of the individual col-
lecting the data and the date on which the data was recorded.
4. Report of the study which includes analysis and interpretation
of all data.
All aspects of the protocol, raw data and report should be consistent;
any deviations require justification and detailed explanation..
The procedures described above are consistent with the intent and the
requirements of the FDA Good Laboratory Practice (GLP) Regulations (74).
7.1.1 General Quality Control Required For Level 1 Bioassay Performance
A separate set of documents outlining recommended quality control and
quality assurance procedures has been prepared and are available as a
guide for laboratories conducting Level I bioassays (9). These guides
outline the basic steps involved in the Level 1 procedures and provide
sample quality control recording forms for sample data collecting. The
quality control/ quality assurance documents will be especially helpful
to laboratories beginning to conduct Level 1 testing.
7.2 REQUIREMENTS FOR QUALITY ASSURANCE
7.2.1 Quality Assurance Samples
In order to ensure the quality of test results from biological laboratories
involved in the environmental assessment program, audit samples have
been made available, either as blind samples during analysis of assess-
ment samples, or separately as coded unknown samples.
Preceding page blank 113
-------
Coded laboratory assessment samples have been prepared to submit to labora-
tories wishing to ensure Level 1 testing proficiency. The audit substances
have the following characteristics:
1. Physical properties similar to those of natural samples, but
defined compositions to ensure reproducible preparation.
2. Stability when stored under normal ambient conditions.
3. A full range of bioassay responses from no detectable toxicity
(or mutagenicity) to high toxicity (or mutagenicity).
4. Prepared in sets with different levels of difficulty assigned
to each set, permitting different levels of discrimination in
the quality assurance or certification process.
At the present time several coded audit samples which have been developed
with the characteristics described above are available through the Process
Measurements Branch, Industrial Environmental Research Laboratory, U.S.
EPA, Research Triangle Park, NC 27711. The results of the tests conducted
by the requesting laboratory will be evaluated against the results estab-
lished for each sample by a reference laboratory with an extensive data
base for test results with the audit samples in the appropriate Level 1
bioassays. The reports submitted by the requesting laboratory will also
be evaluated for compliance with Level 1 protocols and reporting require-
ments. Tables 7.1 and 7.2 provide examples of items reviewed in the
auditing process in addition to the actual test data. When the audit is
completed, a full report will, be prepared by the auditing laboratory and
submitted to the laboratory which conducted the Level 1 testing.
TABLE 7.1 AMES SALMONELLA/MICROSOME MUTAGENESIS ASSAY
QUALITY ASSURANCE AUDIT
Study Design Quality Control Report
Cell Maintenance Cell Maintenance Results
Preparation of Strains Preparation of Strains Protocol
Culture Media Culture Media Tables
Metabolic Activation Metabolic Activation Graphs
Preparation of Test Sample Preparation of Test Sample Summary
Assay Conditions Data Acceptance Criteria Organization
Data Evaluation Criteria Data Evaluation Criteria
114
-------
TABLE 7.2 CHO CLONAL TOXICITY ASSAY QUALITY ASSURANCE AUDIT
Study Design
Qua Iity Control
Report
Cell Maintenance
Prep, of Test Cultures
Prep, of Test Sample
Treatment of Cells
Data Evaluation
Cell Maintenance
Prep, of Test Cultures
Prep, of Test Sample
Treatment of Cells
Data Acceptance Criteria
Data Evaluation Criteria
Results
Protocol
Tables
Graphs
Summary
Organization
7.3 REQUIREMENTS FOR QUALITY CONTROL
In addition to audit samples, the quality control documents referenced
in 7.1 are also designed to define the level of documentation required
to comply with the proposed FDA GLP regulations. From time to time it
will also be necessary to review final bioassay reports for consistency
and completeness based on the suggestions in this manual and the three
sections of the quality control manual (9).
115
-------
CHAPTER 8
ENVIRONMENTAL ASSESSMENT BEYOND LEVEL 1
Level 1 environmental assessment should provide an accurate ranking
of emissions from stationary sources with respect to their potential
toxicity. Moreover, the ranking should ensure that the toxicity is from
the emissions as released into the environment. Level 1 assessment should
also generate information concerning rate of effluent discharge into the
environment and environmental fate of the emission.
A composite of summarized bioassay and chemical analysis data will provide
a measure of toxicity and the potential for damage to the environment.
This concept is outlined in the scheme in Figure 8.1. Data from this
scheme could be used in several ways.
1. It may be used to conclude that there are no detectable toxic
emissions generated at the particular site and that there is
no need for further testing.
2. The Level 1 chemical and bioassay results may be adequate to
rank streams for further assessment and to provide sufficient
guidance for control technology to individual process streams.
In this case, Level 1 tests may be used to monitor the effec-
tiveness of the control procedures over both long periods of
time and varying process conditions.
3. The data may warrant initiation of a full Level 2 assessment.
The results of the Level 2 studies will confirm Level 1 results
and indicate if additional assessment or monitoring should be
undertaken.
Therefore, it should be emphasized that a reasonable amount of flexibility
can be introduced when using Level 1 analysis techniques for applications
beyond Level 1 environmental assessment. The decision to go into Level 2
analysis should be developed as comprehensively as possible, as this
level may require additional tests beyond those specified in this manual
for the minimum Level 1 bioassay testing matrix.
Preceding page blank 117
-------
ASSESSMENT OF-
ENVIRONMENTAL
FATE
BIOACCUMULATION
MEASUREMENT
EMISSION
SITE
00
MEASUREMENT AND
DOCUMENTATION _
OF RELEASE RATE
SAMPLE COLLECTION
[DEFINE CONDITIONS]'
LEVEL 1
.CHEMICAL.
ANALYSIS*
.DISCHARGE.
SEVERITY
WEIGHTED
- DISCHARGE -
SEVERITY [WDS]
QUANTITATIVE
- RANKING OF
TOXICITY
PRETEST
"PROCESSING"
LEVEL 1
BIOASSAYS
TOXICITY
DEFINITION
•Defined In IERL-RTP Procedures Manual:
Level 1 Environmental Assessment [Second Edition] [Reference 1]
DATA
NORMALIZATION
L*-FROM PRETEST -
PROCESSING
Figure 8.1 PROPOSED SCHEME FOR A SECOND STAGE EVALUATION OF LEVEL 1 RESULTS.
-------
REFERENCES
1. Lentzen, D.E., et al. "IERL-RTP Procedures Manual: Level 1 Environmental
Assessment (Second~Tdition)," EPA-600/7-78-201, (NTIS PB 293795),
Research Triangle Institute, Research Triangle Park, NC, October
1978, 279 pp.
2. Hamersma, J.S. and Reynolds, S.L. "Field Test Sampling/Analytical
Strategies and Implementation Cost Estimates; Coal Gasification and
Flue Gas Desulfurization," EPA-600/2-76-093b, (NTIS PB 254166), TRW,
Inc., Redondo Beach, CA, April, 1976.
3. Dorsey, J., e_t ah "Guidelines for Environmental Assessment Sampling and
.Analysis Programs - Level 1." U.S. Environmental Protection Agency,
Research Triangle Park, NC, March 1976, 35 pp. '
4. Hamersma, J.W. and Reynolds, S.L. "Tentative Procedures for Sampling
and Analysis of Coal Gasification Processes," EPA Contract No. 68-02-
1412, Task Order No. 3, TRW, Inc., Redondo Beach, CA, March 1975, 150 pp.
5. Harris, J.C., et al. "Evaluation of Solid Sorbents for Water Sampling,"
EPA-600/2-80-193.TNTIS PB 81106585), Arthur D. Little, Inc., Cambridge,
MA, July 1980, 80 pp.
6. Ames, B., McCann, J. and Yamasaki, E. "Methods for Detecting Carcino-
gens and Mutagens with the Salmonella/Mammalian-Microsome Mutagenicity
Test," Mutation Res., Vol. 31, 1975, pp. 347-364.
7. Brusick, D.J. "Bacterial Mutagenesis and Its Role in the Identification
of Potential Animal Carcinogens," Carcinogens: Identification and
Mechanics of Action (A.C. Griffin and G.R Shaw, eds.), Raven Press,
New York, 1979, pp. 95-107.
8. Ames, B.N. "Identifying Environmental Chemicals Causing Mutations
and Cancer," Science, Vol. 204, 1979, pp. 587-593.
9. Brusick, D.J. , et al_. "Procedures for Quality Control and Quality
Assurance of the Level 1 Health Effects Bioassays," EPA Contract
No. 68-02-2681, Technical Directive 403, Litton Bionetics, Inc.,
Kensington, MO. February 1982, 148 pp.
10. Yahagi, T. , et a]_. "Mutagenicities of N-nitrosamines on Salmonella,"
Mutation Res., Vol. 48, 1977, pp. 121-130. ...... ...
11. Waters, M.D., et al_. "Screening Studies on Metallic .Salt's Using
the Rabbit Alveolar Macrophage, in vitro," Environ. Res., Vol. 10,
1974, p. 342.
12. Garrett, N.E. "Development and Implementation of Cellular Toxicity Assays
for Particulate Matter Using the Alveolar Macrophage and Chinese
Hamster Ovary Cell," EPA Contract No. 68-02-2566, Northrop Services, Inc.,
Research Triangle Park, NC, August 1980, 172 pp.
119
-------
13. Mahar, H. "Evaluation of Selected Methods for Chemical and Biological
Testing of Industrial Particulate Emissions," EPA-600/2-76-137 (NTIS
PB 257912), The Mitre Corp., McLean, VA, May 1976.
14. Coffin, D.L., et al_. "Influence of Ozone on Pulmonary Cells," Arch.
Environ. Health, Vol. 16, 1968, pp. 633-636.
15. Gardner, D.E., et a]_. "Technique for Differentiating Particles that
are Cell Associated or Ingested by Macrophages," Appl. Microbiol.,
Vol. 25, 1975, p. 471.
16. Garrett, N.E. and Stack, H.F. "Cellular Toxicology Data Analysis
Using a Programmable Calculator," Comput. Biol. Med., Vol. 10, No. 3,
1980, pp. 153-167. .;
17. Hsie, A.W., et al. "Quantitative Mammalian Cell Genetic Toxicology:
Study of the Cytotoxicity and Mutagenicity of Seventy Individual
Environmental Agents Related to Energy Technologies and Three
Subfractions of a Crude Synthetic Oil in the CHO/HGPRT System,"
Application of Short-Term Bioassays i_n the Fractionation and Analysis
o_f Complex Environmental Mixtures. EPA 600/9-78-027.U.S. Environmental
Protection Agency, Research Triangle Park, NC, 1978, pp. 292-315.
18. Wininger, M.T., Kulik, F.A. and Ross, W.D. "In Vitro Clonal Cytotoxicity
Assay Using Chinese Hamster Ovary Cells (CHO-K1) for Testing Environmental
Chemicals," In Vitro, Vol. 14, 1978, p 381.
19. "The Toxic Substance List, 1974 Edition," (Christensen, H.E., Luginbyhl,
T.T. and Carroll, B.S., eds.), HEW Contract No. HSM 99-73-45, HEW
(NIOSH) 74-134, Tracor Jitco, Inc., Rockville, MD, June 1974,.904 pp.
20. Litchfield, J.T. and Wilcoxin, F. "A Simplified Method of Evaluating
Dose-Effect Experiments," J. Pharmac. Exp. Ther., Vol. 96, 1949,
pp. 99-113.
21. Committee on Methods for Toxicity Tests with Aquatic Organisms.
"Methods for Acute Toxicity Tests with Fish, Macroinvertebrates and
Amphibians." EPA-660/3-75-009, (NTIS PB 242105), 1975.
22. Duke, K.M., Davis, M.E. and Dennis, A.J. "IERL-RTP Procedures Manual:
Level 1 Environmental Assessment, Biological Tests for Pilot Studies,"
EPA-600/7-77-043 (NTIS PB 268484), Battelle, Columbus, OH, April
1977.
23. Davis, H.G. "Culture of Disease of Game Fishes." Univ. of California
Press, Berkeley, CA, 1953.
24. Hoffman, G.L. and Meyer, F.L. "Parasites of Freshwater Fishes,"
TFH Publications, Inc. Neptune City, NJ.
25. Reichenback-Klinke, H. and Elkan, E. "The Principal Diseases of
Lower Vertebrates," Academic Press, New York, 1965.
120
-------
26. "A Symposium on Diseases of Fishes and Shell-fishes," (Snieszko, S.F.,
Ed.) Spec. Publ. 5, American Fisheries Society, Washington, D.C.,
1970.
27. Van Duijn, C. "Diseases of Fishes," (3rd ed.) Charles C. Thomas.
Springfield, IL, 1973.
28. Mount, D.I. and Brungs, W.A. "A Simplified Dosing Apparatus for
Fish lexicological Studies," Water Res., Vol. 1, 1967, pp. 21-29.
29. American Public Health Association. "Standard Methods for the
Examination of Water and Wastewater," 14th ed. New York, 1975.
30. Thurston, R.U., Russo, R.C.. and Emerson, K. "Aqueous Ammonia Equi-
librium Calculations,"• Tech. Rep. No. 74-1. Fisheries Bioassay
Laboratory, Montana State University, Bozeman, MT, 1974..
31. U.S. EPA, National Environmental Research Center, Methods Develop-
ment and Quality Assurance Research Lab. "Methods for Chemical
Analysis of Water and Wastes," EPA-625/6-74-003, EPA, Office of
Technology Transfer, Washington, DC, 1974.
32. Andrew, R.W. and Glass, G.E. "Amperometric Methods for Determining
Residual Chlorine, Ozone, and Sulfite," U.S. EPA, National Water
Quality Laboratory, in press.
33. Marking, L.L. and Dawson, V.K. "Toxicity of Quinaldine Sulfate to
Fish," Invest. Fish. Control No. 48. U.S. Fish Wild!. Sen/.,
Washington, D.C., 1973.
34. Stephan, C.E. "Methods for Calculating an LCcn," Aquatic Toxicology
and Hazard Evaluation, ASTM STP 634 (F.L. Mayif and J.L. Hamelink, .
Eds.), American Society for Testing Materials, 1977.
35. Miller, W.E., Green, J.C. and Shiroyama, T. "The Selenastrum
capricornutum Printz Algal Assay Bottle Test," EPA-600/9-78-018,
EPA, Environmenta1 Research Lababoratory, Corvallis, OR, 1978.
36. Dunnett, C.W. "A Multiple Comparison Procedure for Comparing Several
Treatments With a Control," J. Amer. State Assoc., Vol. 50, 1955,
p. 1096.
37. Dunnett, C.W. "New Table for Multiple Comparison With a Control,". , .
Biometrics, Vol. 20, 1964, p. 482.
38. Williams, D.A. "A Test for Differences Between Treatment Means When
Several Dose Levels are Compared With a Zero Dose Control," Bionetics,
Vol. 27, 1971, p. 103.
39. Williams, D.A. "The Comparison of Several Dose Levels With a Zero
Dose Control," Biometrics, Vol. 28, 1972, p. 519.
121
-------
40. "Bioassay Procedures for Ocean Disposal Permit Program," EPA-600/
9-78-010, (NTIS PB 278631), EPA, Environmental Research Laboratory,
Gulf Breeze, FL, March 1978.
41. Nimmo, D.R., et al_. "Mysidopsis bahia: An Estuarine Species Suit-
able for Life-Cycle Toxicity Tests to Determine the Effects of a
Pollutant," ASTM STP 634 (F.L. Mayer and J.L. Hamelink, eds.)
American Society for Testing and Materials, 1977.
42. Schimmel, S.C., Hansen, D.J. and Forester, J. "Effects of Aroclor
1254 on Laboratory-Reared Embryo and Fry of Sheepshead Minnows
(Cyprinodon variegatus)." Trans. Amer. Fish. Soc., Vol. 3, 1974,
pp. 582-586.
43. Borthwick, P.W. "Methods for Acute Static Toxicity Tests with Mysid
Shrimp (Mysidopsis bahia)." Bioassay Procedures for Ocean Disposal
Permit Program. EPF^OO/9-78-010 (NTIS PB 278631), EPA, Environmental
Research Laboratory, Gulf Breeze, FL, March 1978.
44. Federal Register, Vol. 43, No. 243, "Bioaccumulation Potential Tests,"
Part IV, Environmental Protection Agency, Hazardous Wastes, Proposed
Guidelines and Regulations and Proposal on Identification and Listing.
December 18, 1978, pp. 59025-57027.
45. Brusick, D.J., et a_K "Workbook for Level 1 Terrestrial Ecological
Tests," EPA Contract No. 68-02-2681, Technical Directive 404, Litton
Bionetics, Inc., Kensington, MD, October 1981, 84 pp.
46. Abeles, F.B. "Stress Ethylene," Ethylene in Plant Biology. (F.B.
Abele, Ed.), Academic Press, New York, 1973, pp. 87-102.
47. Salveit, M.E. and Dilley, D.R. "Rapidly Induced Wound Ethylene from
Excised Segments of Etiolated Pi sum sativum L., cv. Alaska," Plant
Physio!., Vol. 61, 1978, pp. 447-450.
48. Bressan, R.A., et a_l_. "Emission of Ethylene and Ethane by Leaf Tissue
Exposed to InjufTous Concentrations of Sulfur Dioxide or Bisulfite
Ion," Plant Physio!., Vol. 63, 1979, pp. 924-930.
49. Guinn, G. "Nutritional Stress and Ethylene Evolution by Young Cotton
Bolls," Crop Science, Vol. 16, 1976, pp. 89-91.
50. Wilson, L.G. , et al_. "Bisulfite Induced Ethylene and Ethane Produc-
tion: Dependence on Light and Photosynthetically Compete/it Tissue,"
Plant Physiol., Vol. 61S, 1978, p. 93.
51. Abeles, A.L. and Abeles, F.B. "Biochemical Pathway of Stress-Induced
Ethylene," Plant Physiol., Vol. 50, 1972, pp. 496-498.
52. Lieberman, M. "Biosynthesis and Action of Ethylene," Ann. Rev. Plant
Physiol., Vol. 30, 1979, pp. 533-591.
122
-------
53. Heck, W.W., Dunning, J.A. and Johnson, H. "Design of a Simple Plant
Exposure Chamber," EPA-APTD-68-6 (NTIS PB 195151), U.S. Department of
Health, Education and Welfare, National Center for Air Pollution Control,
Cincinnati, OH, June 1967, p. 13.
54. Downs, R.J. and Hellmer, H. "Environment and the Environmental Control
of Plant Growth," Academic Press, New York, 1975.
55. Erickson, R.O. and Michelini, F.J. "The Plastochron Index," Am. J.
Botany, Vol. 44(4), 1957, pp. 297-305.
56. Maksymoywch, R. "Analysis of Leaf Development," Cambridge at the
University Press, Cambridge, MA, 1973, pp. 3-7.
57. .Tingey, D.T., Pettit, N. and Bard, L. "Effect of Chlorine on Stress
Ethylene Production," Environ. Exp. Botany, Vol. 18, 1978, pp. 61-66.
58. Horowitz, M. "Application of Bioassay Techniques to Herbicide Inves-
tigations," Weed Research, Vol. 15, 1976, pp. 209-215.
59. Santelman, P.W. "Herbicide Bioassay," Research Methods in Weed Science,
.. ..Weed Sci. Sop.., USA, 1972, pp. 91-101.
60. Imai, I. and Siege!, S.M. "A Specific Response to Toxic Cadmium
Levels in Red Kidney Bean Embryos," Physiol. Plant, Vol. 29, 1973,
pp. 118-120.
61. Walley, K., Kahn, M.S.I, and Bradshaw, A.D. "The Potential for
Evolution of Heavy Metal Tolerance in Plants. I. Copper and Zinc
Tolerance in Agrostis tenuis," Heredity, Vol. 32, 1974, pp. 309-319.
62. Ourrant, M.J., Draycott, A.P. and Payne, P.A. "Some Effects of Nad
on Germination and Seedling Growth of Sugar Beet," Ann. Bot., Vol. 38,
1974, pp. 1045-1051.
63. Neiman, R.H. and Poulsen, L.L. "Plant Growth Suppression on Saline
Media: Interactions with Light," Bot. Gaz. , Vol. 132, 1971, pp. 14-19.
64. Hikino, H. "Study on the Development of the Test Methods for Evalua-
tion of the Effects of Chemicals on Plants," Chemical Research Report
No. 4, Office of Health Studies, Environmental Agency, Japan, 1978.
65. Rubins-tern, R. et a]_. "Test Methods for Assessing the Effect of
Chemicals on Plants," EPA-560/5-75-008 (NTIS PB 248198), Franklin
Institute Research Laboratories, Philadelphia, PA, June 1975,
246 pp.
66. .. Asplund, R.D. "Some Quantitative Aspects of the Phytotoxicity of
Monoterpenes," Weed Sci., 1969, pp. 454-455.
67. Muller, W.H. "Volatile Materials Produced by Sal via leucophylla:
Effects on Seedling Growth and Soil Bacteria," Bot. Gaz., Vol. 126(3),
1965, pp. 195-200.
123
-------
68. U.S.D.A. "Manual for Testing Agricultural Horticultural Seeds,"
Agriculture Handbook No. 30. Washington, DC, 1952.
69. Wellington, P.S. "Handbook for Seedling Evaluation," Cambridge,
Eng., National Institute of Agricultural Botany, 1961, 149 pp.
70. Demerec, M. and Kaufman, B.P. "Drosophila Guide, Introduction to the
Genetics and Cytology of Drosophila melanogaster, (8th edition)
Carnegie Institution of Washington, Washington, DC, July 1967, 45 pp.
71. Wurgler, F.E., Sobels, F.H. and Vogel, E. "Drosophila as an assay
system for detecting genetic changes," Handbook of Mutagenicity
Test Procedures, (Kilbey, B.J., Legator, M. and Nichols, W., Eds.),
Elsevier/North Hoi 1 and Biomedical Press, Amsterdam, 1977, pp. 335-373.
72. Brusick, D.J. "Level 1 Biological Testing Assessment and Data
Formatting," EPA-600/7-80-079 (NTIS PB 80-184914), Litton Bionetics,
Inc., Kensington, MD., April 1980, 100 pp.
73. Brusick, D.J. "Level 1 Bioassay Sensitivity," EPA-600/7-81-135,
Litton Bionetics, Inc., Kensington, MD, August 1981, 58 pp.
74. DHEW Food and Drug Administration. "Nonclinical Laboratory Studies.
Good Laboratory Practice Regulations," Fed. Regist. Dec. 22, Part II,
1978, pp. 59986-60020.
75. Stetka, D.G. "Sister Chromatid Exchange in Animals Exposed to Mutagenic
Carcinogens - An Assay for Genetic Damage in Humans," Proceeding of the
9th Annual Conference on Environmental Toxicology. March 28,29, and
. 30. 1979.AMRL-TR-79-68, University of California, Irvine, CA, August
1979, 381 pp.
76. Terasima, T. and Tolmach, L.J. "Changes in X-ray Sensitivity of
HeLa Cells During the Division Cycle," Nature, Vol. 190, 1961,
pp. 1210-1211.
77. Perry, P. and Wolff, S. "New Giemsa Method for the Differential
Staining of Sister Chromatids," Nature, Vol. 251, 1974, pp. 156-158.
78. Goto, K., et al. "Factors Involved in Differential Giemsa-staining of
Sister ChromatTds," Chromosoma, Vol. 66, 1978, pp. 351-359.
79. Hollander, M. and Wolfe, D.A. "Nonparametric Statistical Methods,"
John Wiley and Sons, New York, 1973.
124
-------
APPENDIX A
SISTER CHROMATID EXCHANGE IN CHINESE HAMSTER OVARY CELLS
A. 1.1 INTRODUCTION AND RATIONALE
The objective of this i_n vitro assay is to evaluate the ability of a
test sample to induce sister chromatic! exchange (SCE) in Chinese hamster
ovary (CHO) cells. This test is recommended to supplement the standard
Level 1 health effects bioassays and is designed either to be run con-
currently with the CHO clonal toxicity assay or, subsequently, after
cytotoxicity information has been collected.
The frequency of sister chromatid exchanges is a very sensitive indicator
of exposure of the genetic material in mammalian cells to chemical muta-
gens. The SCE test simply involves treating cultured cells with a test
compound, growing the cells with 5-bromo-deoxyuridine (BrdU) for approxi-
mately 24 hours (two cell cycles) and making chromosome preparations
that are stained for detection of SCE.
The chromosomes of dividing cells consist of two identical halves, or
sister chromatids. To see exchanges between these (SCEs), a staining
technique to differentiate between the chromatids is employed. This is
achieved by growing cells in the presence of BrdU: after two cell cycles,
one chromatid contains twice as much BrdU as the other and reacts differ-
ently to certain stains. Now one chromatid will stain intensely while
its pair, or sister, is pale. Figure A.I illustrates the formation of
SCEs.
A.1.2 MATERIALS AND METHODS
Indicator cells. A cell line originally derived from Chinese hamster
ovarian tissue and designated CHO-K1 is used for this assay. This cell
type spontaneously transformed to a stable, hypodiploid line of rounded,
fibroblastic cells with unlimited growth potential. Monolayer cultures
have a fast doubling time of 11 to 14 hours and normally can be cloned
at 80 percent or greater efficiency. Permanent stocks are maintained in
liquid nitrogen and laboratory cultures are maintained by serial subpassage
(not exceeding 15 passages). Laboratory cultures are periodically checked
by culturing methods for the absence of mycoplasma contamination.
The cell line used is the same as in the rodent cell (CHO) clonal toxicity
assay (Section 3.4).
Medium and cell cultures. CHO cells for this assay are grown in McCoy's
5a medium supplemented with 10-percent fetal bovine serum (FBS), L-glut-
amine, penicillin and streptomycin. Cultures are set up approximately
24 hours prior to treatment by seeding 8 x 10s cells per 75 cm2 plastic
flask in 10 ml of fresh medium.
125
-------
VISUALIZATION OF SISTER CHROMATID EXCHANGE [75]
Sister Chromatlds
—Centromere
ONA
Chromosome Call
'
r
Division
f
Synthesis
with BrdU
r
\
"• r
"
JL
*
i
DNA Exchange
Call
Division
or
DNA
Synthesis with BrdU
or
or
: :
: :
: tf • :•
Sister Chromatid
Exchange [SCE]
SCORING SCEs
Stained Chromosome
With No Exchanges
SCE
SCE
SCE
Stained Chromosome
With 5 Exchanges
Figure A1 SISTER CHROMATID EXCHANGE [SCE]
126
-------
Test material. This assay is compatible with test material of all sample
forms except for nonparticulate-laden gases or vapors. Pretest sample
processing will be as for the CHO clonal toxicity test, summarized in
Table 2.4. If the CHO clonal toxicity test and the SCE test in CHO cells
are run concurrently, the same stock dilutions of test material should
be used for both experiments.
Immediately before use, a stock solution of the test compound is prepared
in a suitable solvent such as culture medium, distilled water, dimethyl-
sulfoxide, acetone or absolute ethanol. Serial dilutions are prepared
in the same solvent to achieve desired final concentrations by addition
of 0.1 ml of test solution to each 10 ml of culture, unless limited
solubility requires use of a larger volume. ;
A.1.3 EXPERIMENTAL DESIGN
Dose, selection. Cells will be tested by being exposed to the same range
of concentrations of the test article as used in the CHO clonal toxicity
test (Section 3.4). Selection of dose levels to process further and to
score is based upon the concentration of test material which causes a
reduction in the colony-forming ability of CHO cells by 50 percent (EC™),
as determined in the CHO test. Cells will be scored from the dose level
closest to the EC™ and at two lower dose levels to include one non-toxic
dose, if possible: If the EC™ is previously determined, doses selected
to initiate the SCE test shouia include the EC™ concentration and four
or five lower concentrations, ranging to non-toxic levels.
A second criterion for selection of the high dose for scoring is that a
sufficient number of M2 cells (described below) is recovered to score.
Negative and solvent controls. The same sets of negative and solvent
controls are ued as described in the CHO clonal toxicity test (Section 3.4).
Positive controls. A known direct-acting mutagenic and chromosome-breaking
agent is used.Triethylenemelamine (TEM) is dissolved in water immediately
before use and added to the culture medium. The final concentration is
0.025 ug/ml.
Cell treatment. In addition to the cells previously used for the CHO
clonal toxicity test, approximately 3 x 10s cells are treated with the
test article for 1 hour for the SCE test. Then, 5-bromodeoxyuridine
(BrdU; 10 uM final, concentration) is added to the culture tubes and incu-
bation continued in the dark for 26 to 30 hours. If the test compound
produces a marked amount of precipitate that would interfere with fixation
and chromosome preparations, cells are washed with saline and fresh medium
containing BrdU added, just prior to addition of colcemid. Colcemid is
added for the last two to three hours of incubation (2 x 107M final con-
centration), and metaphase cells are collected by mitotic shake-off (76).
The. cells are swollen.with 0.075 M KC1 hypotonic solution, then washed
three times in a fixative (methanol:acetic acid, 3:1), dropped onto slides
and air-dried. Cell suspensions may be stored in fixative at 4°C until
suitable dose levels for scoring have been selected.
127
-------
Test for cell-cycle delay and repeated harvests. Because many compounds
cause cell-cycle delay, a technique is used for assessing this and, if
necessary, for performing later harvests on the same cultures. After
two to three hours' incubation with colcemid, cells are harvested by
mitotic shake-off and centrifuged to collect as a pellet. The superna-
tant medium can then be returned to appropriate flasks and reincubated
at 37°C. After fixation of cells, test slides are made and stained for
10 minutes in Hoechst 33258* (0.5 ug/ml in phosphate buffer, pH 6.8),
rinsed in water and mounted in the same buffer. These slides may be
examined by fluorescence microscopy to assess the frequency of cells
that have completed two cell cycles in BrdU. If there is significant
delay, the same cultures may be harvested repeatedly until an adequate
yield of cells showing complete differentiation between chroinatids is
obtained.
Staining and scoring of slides. Once dose levels for scoring are selected,
slides are prepared and stained. Selection of dose levels for scoring
is made (as described above) based upon the EC™ for clonal toxicity and
the presence of sufficient M2 cells in each dose.
Staining for detection of SCE is accomplished by a modified fluorescence-
pi us-Giemsa (FPG) technique described by Perry and Wolff (77) and Goto
(78).. Slides are stained for 10 minutes with Hoechst 33258 (5 ^g/ml) in
phosphate buffer (pH 6.8), mounted in the same buffer and exposed at
55°-65°C to "black-light" from 15-Watt tubes for the amount of time
required for differentiation between chromatids. Finally, slides are
stained with 5-percent Giemsa for 10 to 20 minutes and air dried.
A. 1.4 RESULTS AND DATA INTERPRETATION
M2 cells will be scored for the frequency of SCE per cell. Fifty cells
will be scored per dose. Figure A.I presents an example of scoring.
If an increase in SCE is observed, one of the following criteria must
normally be met to assess the compound as positive:
Two-fold increase: Approximately a doubling in SCE frequency over
the "background" (solvent and negative control) levels, at a minimum
of two doses.
Dose response: A positive assessment may be made in the absence of
a^doubling if there is a statistically significant increase at a
minimum of two doses and evidence for a positive dose response.
In some cases, statistically significant increases are observed with
neither a doubling nor a dose response. These results are assessed
according to repeatablity, magnitude of the response and proportion
of the dose levels affected.
^Supplied by Calbiochem-Behringer Corporation, LaJolla, CA 92037.
128
-------
Statistical analysis employs a Wilkoxon Rank Sum Test (79) to compare
SCE frequencies in treated cultures with negative and positive controls;
the Jonkheere's test is used for a dose relation (79).
Evaluation criteria have not been developed at this point for ranking
test samples based upon their ability to induce SCE in CHO cells. It
will be sufficient to report a sample as positive or negative and the
minimum concentration at which a positive response is observed.
129
-------
GLOSSARY
Artemia: An aquatic arthropod, available dried, used as food for other
aquatic organisms.
9AA: 9-aminoacridine; chemical used in the Ames assay as a positive
control for strain TA-1537.
alveolar macrophage: Migratory, phagdcytic cells located in the lungs
which engulf and remove particulates, bacteria and foreign cells
lodged in the lung.
Ames: Shortened name for Ames Salmonel1 a/microsome mutagenesis assay;
one of the Level 1 health effects tests.
ANTH: 2-anthramine; a mutagenic chemical used as a positive control in
the Ames assay for all strains with activation.
Aroclor 1254: A polychlorinated biphenyl preparation used to induce
liver enzymes in rats prior to the preparation of the S-9 fraction.
ATP: Adenosine triphosphate. The chemical in living cells that provides
the primary source of energy. Cellular ATP levels are used in the
RAM assays as an indicator of cell viability.
auxotrophy: The condition, under genetic control, in which a cell can
not synthesize an essential nutrient; that nutrient must be supplied
in the culture medium to allow growth.
base pair substitution: The substitution of a nucleotide base pair for
the original base pair in double stranded DMA, which results in a
change in the information content of the DMA.
bioaccumulation: The biological process by which organisms concentrate
ambient chemicals in tissues and/or organs.
BrdU: 5-bromodeoxyuridine; a chemical analog of thymidine which the
cell incorporates into DNA in place of thymidine.
caudal peduncle: In a fish, the base of the tail (caudal) fin (generally
the area where the tail narrows).
CHO: Acronym for a cell line derived from Chinese hamster ovary tissue.
CHO also used to identify the Level 1 cytotoxicity test which uses
CHO cells.
chromosome: A complex unit of deoxyribonucleic acid (DNA), ribonucleic
acid (RNA) and proteins that replicates during cell division and is
usually constant in number in the cells of any one kind of plant or
animal.
130
-------
clonal toxicity: A measure of toxicity based on the reduction in the
colony-forming ability of single cells exposed to a test material.
This forms the basis for the CHO clonal toxicity assay.
Coulter Counter: Electronic particle counting and sizing device manu-
factured by Coulter Electronics, Inc., Hialeah, FL.
Cucumis sativus: One of the test organisms used in the RE test. The
common name is cucumber.
Cyprinodon variegatus: Test organism used in the Level 1 marine aquatic
ecological effects bioassay using fish. The common name is the
sheepshead minnow.
Daphnia magna: Freshwater invertebrate used in the Level 1 freshwater
aquatic ecological effects bioassay using macroinvertebrates. Common
name for Daphnia is the water flea.
DMSO: Dimethylsulfoxide. A common laboratory solvent used to dissolve
or suspend water-insoluble samples. DMSO, at low concentrations, .
is compatible with test organisms used in most Level 1 tests.
dose-response: The relationship between a biological response (an assay
parameter) and the applied concentration of a test material.
Drosophila melanogaster: Insect used in the Level 1 Insect Toxicity Test
as part of the terrestrial ecological effects bioassays. Common
name for Drosophila is the fruit fly.
EC50: Effective concentration; the estimated or calculated concentration -
of test material that causes a reduction in an observed parameter
by 50 percent relative to the appropriate control.
EDTA: Ethylenedinitrilotetraacetic acid; a chelating agent.
EMEM: Eagle's Minimum Essential Medium with Earle's salts. The standard
culture medium for the cultivation of rabbit alveolar macrophage
cells.
ephippial eggs: Specialized eggs usually produced for overwintering.
Ephippia are produced in response to adverse growth conditions.
eukaryotic: A cell type typical of higher plant and animal forms.
Eukaryotic cells have specilized traits such as nuclear membrane
and DNA organized into chromosomes.
FBS: Fetal bovine serum. A required growth supplement for culture media
used in the RAM and CHO cytotoxicity assays.
femtogram: Unit of weight equal to 10-15 gram, used to measure ATP con-
centration in RAM cells.
131
-------
foliar injury: Visable injury to the leaf surfaces of a plant.
FPG: Fluroescence-plus-Giemsa technique for staining cells for sister
chromatic! exchange.
frame-shift mutation: A quantitative change (addition or deletion) in
the number of nucleotide pairs, in a DMA molecule, which alters the
informational content of the DNA.
fugitive emission: Any emission transmitted to the environment without
passing through some stack, duct, pipe or channel designed to direct
or control their flow. The sample may be gaseous, parti cul ate/aerosol
- or liquid.
gavage: To introduce material into the stomach by a tube.
Giemsa stain: A histological stain used for staining blood and other
cells.
GLP: Good Laboratory Practices. A set of regulations developed by the
U.S. FDA to define standards for nonclinical health effects studies.
GRAV: Acronym for the gravimetric method for determining the content of
nonvolatile compounds in liquid sample. Determined by weighing the
residue of a known volume of liquid.
hemocytometer: A glass chamber of precise volume, divided by grid lines
into defined areas, used in conjunction with a microscope, to deter-
mine the number of cells per unit volume of fluid.
histidine: An amino acid (CeHgNsQ^) essential to the growth of the strains
• • of Salmonella typhimurium used for the Ames mutagenesis assay. The
.biochemical pathway for the biosynthesis of histidine is under genetic
control and reversions to prototrophy for this pathway form the
basis for detecting mutagens.
HPLC: High-performance liquid chromatography.
hypocotyl: Part of the stem below the cotyledons (primary leaves) in
the embryo of a plant.
IERL-RTP: Industrial Environmental Research Laboratory, U.S. Environmental
Protection Agency, Research Triangle Park, NC 27711.
incubation: The maintenance of living organisms (e.g., bacteria, mammalian
cells in culture) in favorable conditions to promote growth.
instar: Developmental stages between molts in Daphnia. and other related
animals.
IT: Insect toxicity test. One of the Level 1 terrestrial ecological
effects bioassays.
132
-------
jji vitro: Outside the living body; tests or test conditions involving
living cells maintained in an artificial (laboratory) environment.
j_n vivo: In the living body; tests or test conditions involving intact
plants or animals.
Lactuca sativa L.: One of the test organisms used in the RE test. The
common name is lettuce.
LC5Q: Lethal concentration; the estimated or calculated concentration
of a test material that is lethal to 50 percent of the test organisms
during coutinuous exposure for a specified period of time.
LD5Q: Lethal dose; the estimated or calculated dose of test material
that is lethal to 50 percent of the test organisms following exposure
to a single dose of test material.
M2 cells: Cells which have proceeded through a second cell division
after a defined point such as following addition of 5-bromodeoxyuri-
dine or addition of test material.
MAD: Maximum applicable dose. The highest concentration recommended
for routine Level 1 testing for a given test.
manometer: A U-shaped instrument used for measuring air or gas pressure.
MEC: Minimum effective concentration. The lowest concentration giving
a positive response according to the evaluation criteria in the
Ames assay. .
microsome: A cellular fraction consisting of membrane fragments and
organelles that contain the enzymatic activities that biotransform
chemicals. Liver cells are a good source of microsomes that contain
enzymes associated with drug and chemical metabolism.
MOPS: Morpholinopropanesulfonic acid. The chemical used as a buffering
system in the analysis of ATP content in rabbit alveolar macrophage
cells. •'• •*•••••
mutation: Alteration of a heritable characteristic of a living organism
usually resulting from a molecular change in the organism's deoxyri-
bonucleic acid (DNA).
Mycoplasma: A genus of bacteria which do not contain a true cell wall.
They are occasional contaminants of mammalian cells in culture.
Mysidopsis bahia: Marine invertebrate used in the Level 1 marine aquatic
ecological effects bioassay using macroinvertebrates.
NF: 2-nitrofluorene; a mutagen used as a positive control in the Ames
assay for strain TA-98 without activation.
133
-------
NOEC: No observed effect concentration. The maximum concentration of
test material which produces no observable deleterious effect by
any criteria under study.
PBS: Phosphate buffered saline. A physiological saline used for proce-
dures (such as washing) with mammalian cells in culture.
PCB: Polychlorinated biphenyl. See Arclor 1254.
phagocytosis: The process of ingestion and destruction of particulate
matter, microorganisms or foreign cells by certain cells known as
phagocytes. RAM cells are considered to be phagocytic.
Phaseolus vulgaris L.: Test organism used in the PSE test. The common
name is the bush bean.
Pimephales promelas: Test organism used in the Level 1 fresh water aquatic
ecological effects bioassay using fish. The common name is the
fathead minnow.
plastochron index: Numerical index of the developmental status of plants
derived from leaf length.
PMB: Process Measurements Branch. The branch in the Industrial Environ-
mental Research Laboratory, U.S. EPA, Research Triangle Park, NC
responsible for the development and validation of the Level 1 environ-
mental assessment testing program.
POM: Polycyclic organic material.
prokaryotic: Cell type characteristic of simple organisms such as bac-
teria, viruses and some blue-green algae, in which the genetic
material is arranged essentially into one chromosomal complex not
separated by a membrane from the rest of the cell.
prototrophy: The condition, under genetic control, where a cell can
synthesize an essential nutrient. The cell can grow in culture
medium devoid of that essential nutrient.
PSE: Plant stress ethylene test; one of the Level 1 terrestrial ecological
effects bioassays.
quanta! test: In Level 1 jjn vivo rodent toxicity testing, the preliminary
all-or-nothing test at the maximum applicable dose level -to determine
if a test material is toxic.
quantitative test: In Level 1 testing, the definitive portion of the i_n
vivo rodent toxicity test using multiple dose levels to determine
the LDgQ of a test material.
radicle: The lower part of the axis of an embryo seeding which will
form the root.
134
-------
RAM: Rabbit alveolar macrophage cells or the Level 1 cytotoxicity test
which uses these cells. See alveolar macrophage.
Raphanus sativus L.: One of the test organisms used in the RE test.
The common name is radish.
RE: Root elongation test; one of the Level 1 terrestrial ecological
effects bioassays.
reverse mutation: A mutation that restores the original genotypic (or
phenotypic) condition of a cell that was lost by an initial mutation.
rotameter: A calibrated flow meter used to measure flow rate cf a gas.
S9 mix: The liver homogenate preparation (S-9) derived from Aroclor 1254-
induced Sprague Dawley rats combined with several cofactors to main-
tain hepatic enzyme activity. The term S-9 refers to the supernatant
(which contains the microsomes) obtained after centrifuging the
liver homogenate at 9000 x g. Used to biotransform test chemicals
in the Ames assay to detect mutagenic activity.
Salmonella typhimurium: Bacterial test organism used in the Ames Salmo-
nella/microsome mutagenesis assay. Four different histidine requiring
strains are used in the Level 1 assay; TA-98, TA-100, TA-1535 and
TA-1537.
SA: Sodium azide; a mutagenic chemical used as a positive control in
the Ames assay for strains TA-1535, and TA-100 without activation.
SASS: Source Assessment Sampling System. Sampling system for sampling
particulate laden gases developed by IERL-RTP and manufactured by
Aerotherm Corporation, 485 Clyde Avenue, Mt. View, CA.
SC^Q: Stimulatory concentration; the calculated concentration of a test
material that causes a stimulation in growth of 20 percent relative
to the appropriate control during continuous exposure for a specified
period of time.
SCE: Sister chromatid exchange or the test which detects SCE. Microscopi-
cally visible exchange of portions of chromatid arms of the same
chromosome. Elevated levels of SCE are indicative of damage to the
genetic material.
Selenastrum capricornutum: Test organism used in the Level 1 -freshwater
aquatic ecological effects bioassay using algae. The organisms is
a unicellular, non-motile chlorophyte.
Skeletonema costatum: Test organism used in the Level 1 marine aquatic
ecological effects bioassay using algae.
SOP: Standard Operating Procedures. SOPs are explicit test procedures
followed by personnel in a given laboratory.
135
-------
TCO: Acronym for the total chromatographable organics method for deter-
mining the volatile organic content in a liquid sample. Determined
by a chromatographic analysis of samples collected and stored under
conditions that prevent volatilization of organics.
TEM: Tri ethyl enemel ami ne; a direct- acting mutagenic and chromosome-
breaking chemical used as a positive control in the SCE test,
TrrUcum aesti'vum L. : One of the test organisms used in the RE test.
The common name is wheat. ' ;.
Biological strain used for diluting and staining cells for.
cell density and cell viability counting. Live cells exclude the
dye and remain colorless while dead cells are stained blue.
trypsin: Chemical (enzyme) that digests extracellular proteins produced
by mammalian cells that attach cells to one another and to substrates.
WAT: Acute jrn vivo toxicity test in rodents (whole animal test); one of
the Level 1 health effects tests.
/
Irj£glJJjni ££atenj>e L. : One of the test organisms used in the RE test.
the common name is red clover.
weanling mice: Mice that have recently been weaned. The age of weanling
mice for the jj\ vivo rodent toxicity assay is 21 to 28 days.
Wrights stain: A histoTogic stain, developed for staining blood cells.
XAO-2: A porous sorbent resin used in sampling gaseous streams with tne
SASS train technique and in concentrating organic material from
aqueous samples. This resin has a high affinity for non-polar species
and a low affinity for polar species. XAD-2 is manufactured by
Rohm and Haas Co. , Philadelphia, PA.
XE-347: A porous sorbent resin used in concentrating organic material
from aqueous samples. This resin has a high affinity for polar
species. XE-347 is manufactured by Rohm and Haas Co., Philadelphia,
PA.
136
------- |